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WO2002007780A2 - Procedes d'identification de cibles therapeutiques destinees a traiter des maladies infectieuses - Google Patents

Procedes d'identification de cibles therapeutiques destinees a traiter des maladies infectieuses Download PDF

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Publication number
WO2002007780A2
WO2002007780A2 PCT/US2001/023095 US0123095W WO0207780A2 WO 2002007780 A2 WO2002007780 A2 WO 2002007780A2 US 0123095 W US0123095 W US 0123095W WO 0207780 A2 WO0207780 A2 WO 0207780A2
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WIPO (PCT)
Prior art keywords
enzymes
enzyme
information
iecta
target
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PCT/US2001/023095
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English (en)
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WO2002007780A3 (fr
Inventor
Michael H. Shepard
David B. Lackey
Brian E. Cathers
Maria V. Sergeeva
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Newbiotics, Inc.
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Priority to AU2001277093A priority Critical patent/AU2001277093A1/en
Publication of WO2002007780A2 publication Critical patent/WO2002007780A2/fr
Publication of WO2002007780A3 publication Critical patent/WO2002007780A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/556—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells enzyme catalyzed therapeutic agent [ECTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • the present invention relates to the field of Enzyme Catalyzed Therapeutic Activation (ECTATM) therapy and in particular, ECTA therapies targeting intrinsic and unique enzymes in pathogenic microorganisms or in host cells.
  • ECTATM Enzyme Catalyzed Therapeutic Activation
  • the combination of drugs often works toward inhibition of separate enzyme targets.
  • trimethoprim and sulfomethoxazole is used to simultaneously inhibit dihydrofolate reductase and dihydroopterate synthetase, respectively.
  • Similar approaches are used for the treatment of viral infections and cancer.
  • anti-HIN therapy the combination of reverse transcriptase and viral protease inhibitors is commonly employed.
  • cocktail that include a fluoropyrimidine and methotrexate inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, are often used.
  • Pathogens can also enzymatically modify a therapeutic so that it cannot bind to its target (as seen with aminoglycosides and chloramphenicol). In both of the cases outlined above, an enzyme encoded by the pathogen mediates resistance. (II). Membrane permeability. Pathogens adapt and change their cell wall (the porin structures) to prevent drug entry. This can occur in response to almost any antibacterial agent.
  • Pathogens adapt and change membrane transport proteins (also an enzyme family), such that they operate with increased efficiency toward the antibiotic. This is an important mechanism of resistance to tetracycline.
  • Target mutation Pathogens mutate the therapeutic target thereby preventing activation of the antibiotic. Common mutations occur in the penicillin binding protein, which prevents activation of the antibiotic.
  • the most recent worrisome resistance traits to emerge include plasmid-mediated resistance to imipenem and to third- generation cephalosporins among nosocomial gram-negative bacteria, and the acquisition of resistance to vancomycin by enterococci.
  • Methicillin- resistant staphylococci continue to be a problem, with about 75% of clinical strains found to be resistant to the penicillin-related drugs, and increasingly resistant to numerous other agents.
  • the most important resistance traits seen in community-acquired organisms include beta-lactam resistance in Streptococcus pneumoniae and combined ampicillin and chloramphenicol resistance in Haemophilus influenzae. Shigellae resistant to essentially all commonly used oral agents are also a problem, particularly in developing countries (Reviewed by Murray, B. (1997)).
  • This invention provides a vertically integrated drug discovery program that Applicant has utilized to identify therapeutic enzyme targets and which can be used to identify prodrugs which act by a unique mechanism of action (termed “Enzyme Catalyzed Therapuetic Activation” or "ECTA”TM).
  • the invention provides systems and methods to identify enzyme targets in silico.
  • the invention provides a method to design potential prodrugs activated by the enzyme targets.
  • in vitro and in vivo assays are provided. The assays and prodrugs also are useful to test potential therapeutics. Further provided are methods to inhibit the growth of target organisms, cells, or host cells using the prodrugs of this invention. Methods to treat or alleviate the symptoms of selected diseases are further provided using the prodrugs of this invention.
  • the in silico methods comprise selecting from a suitable database an enzyme or list of enzymes expressed by a target organism, by an infectious agent or in an infected host cell, or by or in a pathological cell. The results of this search are compared against a search of expressed enzymes in or by a suitable control The method selects for enzymes expressed in one cell type or organism but not in another.
  • Various embodiments of this aspect are provided herein. For example, one embodiment identifies enzymes expressed by a pathogen or on in a pathogen- infected cell but not expressed in the host or uninfected host cell.
  • target enzymes for a novel ECTA approach to treat a variety of diseases including bacterial, fungal, parasitic and viral infections.
  • conventional therapies rely on the use of inhibitors of enzymes critical for target viability and/or proliferation.
  • the prodrug compounds of this invention do not act as enzyme inhibitors but undergo enzyme catalyzed transformation by target enzymes resulting in the generation of cytotoxic reaction product(s).
  • the formation of cytotoxic species is achieved by engineering unique substrates (ECTA prodrugs or compounds) which are transformed into toxins by the target enzymes.
  • the target enzymes of this invention are pathogen- specific enzymes that are only expressed by pathogens, e.g., bacterial and fungal pathogens or in virally infected cells.
  • pathogens e.g., bacterial and fungal pathogens or in virally infected cells.
  • the pharmaceutical and agricultural industries have focused on development of inhibitors of selected target enzymes for the development of anti-infectives, insecticides and herbicides (Shaner and Singh (1997) and Papamichael (1999)). This approach has suffered from several issues: (1) the presence of salvage pathways which allow specific enzyme inhibition to be circumvented; (2) mutation of the enzyme so that it no longer binds inhibitor, but can still metabolize substrate; and (3) inhibitor-associated enzyme overexpression leading to resistance.
  • protease inhibitors used in HIV treatment have been shown to affect glucose control, lipid metabolism, and body fat distribution (Mulligan (2000)).
  • This invention defines a new ECTA approach that targets intrinsic enzymes ("iECTA" approach) which overcomes the limitations and problems associated with prior art therapies.
  • Applicant's approach is distinguished from prior approaches because iECTA enzymes are NOT endogenous enzymes for the host cell and are not necessarily related to drug resistance. In other words, only pathogens or pathogen-infected cells express the iECTA enzymes.
  • the prodrug compounds which are designed to be selectively activated by the iECTA enzymes also avoid side effects by achieving alternative, more selective therapies that preferentially affect diseased cells with little or no effect on healthy tissue. To the best of Applicant's knowledge, this approach has not been described or utilized previously. Therapeutics designed and generated using iECTA technology supplement and complement present day enzyme inhibitor-based treatments.
  • the present method can be applied to identify target enzymes other than iECTA enzymes by searching a first suitable data structure (database) to obtain a first set of information relating to one or more enzymes associated with a target organism.
  • the enzyme is overexpressed or selectively expressed as compared to a control counterpart.
  • a search also is conducted on one or more other suitable data structures (databases) to obtain one or more additional sets of information relating to one or more expressed enzymes associated with one or more additional class of organisms or by the same organism growing under in a different environment or in a different host.
  • the first set of information is compared to the one or more additional sets of information to identify enzymes in the first set of information that are not present in the one or more additional sets of information.
  • ECTA prodrugs While each prodrug is selectively activated by a specific target enzyme counterpart, there are some general features of ECTA prodrugs.
  • Figures 2A and 2B describe general characteristics of ECTA prodrugs that distinguish them from conventional therapeutics.
  • One feature of an ECTA enzyme/ECTA compound combination is the absence of irreversible inhibition or inactivation of the target enzyme by the ECTA compound, intermediates or products of the reaction.
  • iECTA compounds or prodrugs which are selectively activated by yet to be identified iECTA enzymes using the methods of this invention.
  • This invention further provides iECTA compounds or prodrugs activated by infectious agents or in host cells, e.g., the enzymes listed in Figure 7A and 7B and their biological equivalents.
  • infectious agents or in host cells e.g., the enzymes listed in Figure 7A and 7B and their biological equivalents.
  • a "biological equivalent" is defined infra.
  • the iECTA compounds are provided alone or in combination with a liquid or solid carrier.
  • Compositions comprising at least one iECTA compound or its biological equivalent in combination with an additional therapeutic is further provided by this invention.
  • an assay for an iECTA compound that selectively inhibits the growth of an infectious agent in a target cell or an infected cell.
  • the iECTA prodrug is contacted with its target enzyme in a cell-free system under suitable conditions. Activation by the target enzyme is monitored by methods well known in the art.
  • the iECTA enzyme is contacted with a pathogen or host cell containing or expressing the target enzyme.
  • the host cell and the prodrug are contacted under conditions that that favor incorporation of the compound into the host cell.
  • the pathogens or host cells are assayed for inhibition of growth or killing of the infectious agent or the host cell.
  • Control systems and/or cells can be contacted with the prodrug and assayed.
  • This invention also provides a method for inhibiting the growth or proliferation of an infectious agent or a host cell by contacting the infectious agent or host cell with an effective amount of an ECTA prodrug, e.g., an iECTA prodrug.
  • a method for determining whether a subject will be suitably treated by an ECTA prodrug such as an iECTA prodrug is provided by this invention.
  • an iECTA compound is delivered to an infected cell under suitable conditions such that the growth of the infectious agent or infected cell is inhibited or the agent is killed.
  • a different and/or additional enzyme target can be assayed against the same iECTA prodrug or a different and/or additional prodrug can be assayed against the same target enzyme.
  • Prior art therapeutics or therapeutic methods can be combined with the use of the iECTA prodrugs to enhance or modify the biological activity of the iECTA prodrug. These methods can also be modified by varying the amount of the iECTA prodrug and/or additional therapeutic or alternatively or in combination, the order of the prodrugs and or therapies can be modified, e.g., simultaneous or sequential.
  • the sequential order can further be modified. These methods are fiirther modified for prophylactic use.
  • kits for determining whether a pathogen or pathogen-infected cell will be suitably treated by an iECTA therapy is also provided by this invention.
  • the kit comprises an effective amount of at least one compound of this invention and instructions for use.
  • Figure 1 shows how ECTA technology preferentially targets selected cells.
  • Figures 2 A and 2B are the process for successful identification of one embodiment of this invention, the identification of iECTA target enzymes and iECTA compounds.
  • Figure 2C is a flowchart for a process for identifying enzymes for designing ECTA compounds in accordance with an embodiment of the present invention.
  • Figure 2D is a schematic diagram of an illustrative system capable of executing the process for identifying enzymes for designing ECTA compounds set forth in Figure 2C in accordance with an embodiment of the present invention.
  • Figure 3 shows the results of one embodiment of the method of this invention that utilizes .BLAST alignment for the identification of a "favorable reaction type" iECTA. Shown in the figure is a .BLAST alignment of Pseudomonas aeruginosea acetolactate synthase large subunit amino acid sequence with the human expressed sequence tag database (translated in all six possible reading frames). The low "Expect" (E) values indicate that it is extremely unlikely that any of these alignments could occur by chance alone. Only the ten best E values and the best alignment are shown.
  • Figure 5 is a proposed mechanism of AcLS ECTA.
  • Figure 6 is a comparison of 2-oxobutyrate metabolism in humans and E. coli.
  • Figure 7A is a list of the Enzyme Commission Numbers representing intrinsic and unique enzymes for the following organisms:
  • 'Neurospora crassa 24. 'Neisseria meningitidis ser. B " 25. 'Neisseria meningitidis ser. A " 26. 'Neisseria gonorrhoeae” 27. 'Mycoplasma pneumoniae” 28. 'Mycoplasma genitalium” 29. 'Mycobacterium tuberculosis” 30. 'Mycobacterium leprae" 31. 'Mycobacterium bovis” 32. 'Klebsiella pneumoniae" 33. 'Helicobacter pylori” 34. 'Helicobacter pylori J99" 35. 'Haemophilus influenzae" 36.
  • Figure 7B is an abbreviated list consisting of all the EC number descriptions, but listing only one occurrence for each organism and consists of the 673 enzymes.
  • Figure 8 illustrates an illustrative system with a plurality of components in accordance with one embodiment of the present invention.
  • FIG. 9 illustrates a representative hardware environment in accordance with one embodiment of the present invention.
  • Figure 10 shows chemical structures representative of different chemical classes of AcLS inhibitors currently used as herbicides.
  • Figure 11 shows the synthetic pathway for valine and leucine.
  • Figure 12 shows the synthetic pathway for iso leucine.
  • compositions and methods include the recited elements, but do not exclude others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • An "infectious agent” is intended to be synonymous with “pathogen” and includes, but is not limited to bacteria, parasites, rickettesia, virus, and fungus.
  • any of the terms “toxin”, “toxoid”, “prototoxophore”, “toxophore”, “Tox”, or “TOX” are synonymous and intend any molecule or functional group that is released or unmasked (revealed) upon the action of the enzyme resulting in toxicity to the pathogen, pathological cell or in an infected host cell.
  • the toxin or toxoid will vary with the target enzyme, the pathogen, the host cell and the subject being treated.
  • toxins include, but are not limited to anthracyclins, vinca alkaloids, mitomycins, bleomycins, penicillins, cephalosporins, oxacillins, carbopenems, tetracyclins, chloramphenicols, macrolides, cycloserines, fluoroquinolones, glycopeptides, aminoglycosides, peptide antibiotics, oxazolidinones, quinolones, sulfonamides, cytotoxic nucleosides, pteridine family, nitrogen mustards, polyhalogenated biphenyls, diynenes, podophillotoxins, taxoids, alkylating agents.
  • Some of the useful representatives of these classes include doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophillotoxin, etoposide, etoposide phosphate, melphalan, vindesine, vinblastine, vincristine, leurosidine, leurosine, bis-(2- chloroethyl)amine, trichlorcarban, trichlorocarbanilide, tribromosalicylanilide, sulphamethoxazole, chloramphenicol, cycloserine, trimethoprim, chlorhexidine, hexachlorophene, fentichlor, 5-chloro-2-(2,4- dichlorophenoxy)phenol, 4-chloro-2-(2,4-dich
  • a “prodrug” is s a precursor or derivative form of a pharmaceutically active agent or substance that is less cytotoxic to target or hyperproliferative cells as compared to the drug metabolite and is capable of being enzymatically activated or converted into the more active form (see Connors, T.A. (1986) and Connors, T.A. (1996)).
  • the toxicity of the agent is directed to cells that are producing the converting enzyme in an amount effective to produce a therapeutic concentration of the cellular toxin in the diseased cell.
  • a “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • a pharmaceutical carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, Remington's Pharm. Sci., 15th Ed.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • the term “effective amount” is to include therapeutically or prophylactically effective amounts.
  • the term also refers to an amount effective in treating or preventing an infection in a patient or an infestation in a plant either as monotherapy or in combination with other agents.
  • prophylactically effective amount refers to an amount effective in preventing infection in a subject or plant infestation.
  • linker indicates a spacer or connector between two parts of a single molecule such that when a particular bond is severed between the two parts of the molecule separate.
  • “Inhibiting the growth" of a microorganism or infected cell means reducing by contact with an agent, the rate of proliferation of such a microorganism or infected cell, in comparison with a control microorganism of the same species not contacted with this agent or as compared to an uninfected cell.
  • treating refers to any of the following: the alleviation of symptoms of a particular disorder in a patient; the improvement of an ascertainable measurement associated with a particular disorder; or a reduction in microbial number.
  • a "subject,” “individual” or “patient” or “host” is used interchangeably herein and refers to plants, avians, fish and animals, e.g., a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative”.
  • a “suitable control” is variable and depends in part on one or more of the following criteria: the target pathogen, the target enzyme, expression level of the target enzyme, the host cell, the subject or host as well as the specific genotype or phenotype of each.
  • a suitable control can be one or more of a normal counterpart cell, a counterpart cancer cell that has not undergone any therapy or been exposed to an inducing agent or a different therapy or a cell that has been treated in a different environment or microenvironment, e.g., in vitro versus in vivo.
  • the control cell can be one that been or will be treated with a known therapeutic or therapeutic method.
  • the object of the invention is to identify intrinsic ECTA target enzymes expressed by pathogens or in pathogen-infected cells
  • the control counterpart can be one or more of a pathogen that has not been exposed to an inducing agent or one that is not infected with the pathogen.
  • an “inducing agent” includes any agent (chemical, physical or mechanical) which alters the genotype or phenotype of a pathological cell or infectious agent or infected cell. Examples include prior chemotherapy (in the case of cancer), prior treatment with one or more antibiotics (in the case of pathogens and pathogen-infected cells) or prior exposure to another organism resulting in the exchange of genetic material, e.g. plasmids that confer antibiotic resistance to a host cell. Additional examples include, but are not limited to exposure to radiation, chemicals, ultra-violet light, metals or genetic manipulation.
  • “expressed at elevated levels” in the context of infectious disease is intended to include any amount over the base line or control as compared with host cells (e.g., an uninfected or normal cell) taking into consideration the sensitivity of the detection system and statistical variation in the computation methods.
  • “expressed at elevated levels” is intended to include any amount that is more than an amount over the base line or control (e.g., a normal counterpart cell) taking into consideration the sensitivity of the detection system and statistical variation in the computation methods. In some aspect, it is at least 2X, or more than 3X or preferably more than 4X than that expressed in a normal cell.
  • pathological cells, “target cells”, “host cells” and “hyperproliferative cells” in the context of cancer encompass cells characterized by the activation by genetic mutation or the endogenous overexpression of an intracellular enzyme which may confer resistance to the inhibitory or cytotoxic effects of chemotherapy . Overexpression of the enzyme can be related to loss of tumor suppressor gene product function drug resistance or the genetic instability associated with a pathological phenotype.
  • infectious agents encompass cells infected with or containing an infectious agent as defined herein.
  • infectious agents encompass cells overexpressing an enzyme which confers resistance to the cytotoxic effects of the antibiotic.
  • Sequence comparison is used to compare character strings representing proteins or fragments of DNA to gather evidence for common function or biological origin. Proteins which are thought to have a common ancestor are called homologous.
  • the process of evolution introduces mutations in DNA which may take the form of: the substitution of one or more nucleic acids for another; the deletion of one or more nucleic acids; or the insertion of one or more nucleic acids.
  • s[i] for the fth character of _, where i is between 1 and ⁇ s ⁇ , the length of 5.
  • the goal of pairwise sequence alignment algorithms is to find a high-scoring alignment of a given pair of sequences (or subsequences of those sequences), according to some prescribed alignment scoring method.
  • “Classic dynamic programming algorithms” are thought of as exact, in the sense that they are guaranteed to compute the best possible alignment of the two strings under the supported alignment scoring system.
  • the scoring systems usually prescribes a method of scoring character pairings, and the total score for a particular alignment is the sum of the character pair scores.
  • a scoring function S: A ' x A ' — > R is symmetric in its two arguments.
  • mismatches may be assigned different scores depending on the severity of the mismatch (for example, two amino acids that have similar chemical properties may be substituted for one another without greatly affecting the function of the resulting protein, and matching one with the other may be almost as good as a perfect match and be awarded a positive score. On the other hand, two amino acids may have very different chemical properties and their mismatch may be awarded a negative score).
  • Gaps are usually awarded negative scores. In the simplest case, the penalty for a gap in the completed alignment is proportional to its length, and the scoring function may be represented as a symmetric matrix. However, there are biological reasons for penalizing small gaps more heavily than larger ones, and popular implementations usually use affine gap penalties of the form I + R*(k-1) for a gap of length k; this requires a minor change in representation for the scoring function.
  • MfiJ is interpreted as the score of the best alignment of the subsequences t[l..i] and sfl.J] that ends by pairing tfi] with s[jj.
  • the zeroth column and row represent leading gaps, and are assigned negative scores according to the gap scoring regimen.
  • the fundamental notion in all these algorithms is that the value of MfiJ] must be the best (maximum) of
  • Tracing back from element MfiJ] involves recomputing the scores for the extensions of prefix alignments as above, then selecting one that equals MfiJ], and then tracing back from the corresponding element Mfi-lJ], Mfi-lJ-l], or MfiJ-1] (Of course, it could be that two or all three of the prefix alignments lead to the same score; in this case, there is usually some policy on selecting one type of alignment over another.
  • the traceback starts at the maximum element in the last row together with the last column ofM, and ends when the zeroth row or column is reached.
  • the algorithm described above is similar to the Needleman-Wunsch global alignment algorithm.
  • the algorithm is called a "global" alignment algorithm because it tries to find the best alignment over the whole strings s and t.
  • the same technique can be used to find the best local alignment between s and t, that is, the highest-scoring (global) alignment of substrings sfipAj and tfji.JJ.
  • the changes required are:
  • MfiJ] 0 is encountered. This is the Smith- Waterman local alignment algorithm. Of the classic dynamic programming methods, it is the most commonly used. For biological reasons one may wish to not penalize gaps that occur at the beginning or end of an alignment. These variations are easily accommodated by changing the initialization of the zeroth row and column ofM.
  • the classic algorithm as presented requires 0(]s ⁇ * ⁇ t ⁇ ) (quadratic) time and space.
  • the matrix M is normally filled in row-by-row or column-by- column, and it is never necessary to have more than two rows or columns of the matrix in memory at once.
  • the actual alignment may not be necessary, and only the maximum score over all possible alignments may be required.
  • the algorithm uses only linear (0(min( ⁇ s ⁇ , ⁇ t ⁇ ))) space.
  • the "FAST” algorithm is a heuristic approach that tries to approximate the best (local) alignment and score while reducing the computational expense of the Smith- Waterman algorithm. Strictly speaking, it is a database search algorithm: we have a query string q, and wish to compare it against every string in a database of strings. Typically, it is best to report the best n scores and corresponding alignments, where n is much smaller than the database size.
  • the computation for each database string 5 is a local alignment with q, and proceeds in four stages: 1) the strings are rapidly scanned for exact substring matches of length ktup. ktup is usually quite small, only 1 or 2.
  • a table of ⁇ q ⁇ + ⁇ s ⁇ - 1 counters is maintained, one for each diagonal in a table, similar to the dynamic programming matrix M; for a match of a &-tuple with starting points qfi] and sfj], the i-jth counter is incremented.
  • the table of counters gives the number of hits for each diagonal. 2) for each diagonal with more than one hit, hits are merged into regions. These regions may contain mismatches, but since everything is in the same diagonal, there are no gaps. 3) the five best regions are rescored using a protein substitution matrix (for example, PAM120, PAM250, or, more recently, a BLOSUM matrix).
  • a protein substitution matrix for example, PAM120, PAM250, or, more recently, a BLOSUM matrix.
  • the best of these scores is reported for the sequence pair, and is called the initial score.
  • the matches in the database are ranked according to their initial scores. 4) the pairs with the n best initial scores are then re-examined using a modified Smith- Waterman alignment algorithm that is restricted to a band 64 diagonals wide centered around the best diagonal. This new score is called the optimized score.
  • both initial and optimized scores are listed; often very good matches have a dramatically better optimized score than the initial score.
  • the FAST package includes a program for testing the statistical significance of high-scoring matches. It works by scrambling one of the strings and running the Smith- Waterman algorithm on the new pair; this is repeated many times. If the score reported for the original pair is sufficiently far from the mean score for the alignments on the scrambled strings, the match is considered significant (unlikely to be due to chance). Recent versions of FASTA evaluate the statistical significance of scores using a theory based on extreme value distributions.
  • BLAST jBasic ocal flignment Search 7bol
  • FAST BLAST
  • BLAST jBasic ocal flignment Search 7bol
  • a hit is extended by adding characters to the front and back of each of the two substrings until a maximal score (under the same substitution matrix as above) is reached: dropping or adding a pair of characters at either end lessens the score. In practice, the pair is discarded if the score falls a prescribed distance below the best score reported for the same- length extension so far.
  • the best extension scores (or maximal segment pair (MSP) scores) are used to rank the database strings.
  • the process for DNA is similar, except that the scoring is simpler (there are no substitution matrices), and the values of the parameters are different.
  • BLAST attempts to estimate the statistical significance of the MSP scores based on a statistical theory of how MSP scores should be distributed for random strings.
  • a method that identifies ECTA enzymes.
  • a first suitable data structure is searched to obtain a first set of information relating to one or more enzymes associated with a target organism.
  • the enzyme can be one that is expressed, overexpressed or selectively expressed. This search provides a first enzyme list.
  • a search also is conducted on one or more other suitable data structures to obtain one or more additional sets of information relating to one or more expressed enzymes associated with one or more controls.
  • the first set of information is compared to the one or more additional sets of information to identify enzymes in the first set of information that are not present (or absent) in the first search, but not the second.
  • These identified enzymes are targets for ECTA compounds.
  • Examples of data structures include, but are not limited to databases of genetic or expressed genetic information relating to enzymes.
  • the information may be in the form of DNA, RNA or protein and may include, where appropriate information relating to quantitative expression of the enzyme.
  • the information may be organized in any manner. In one aspect, the information is restricted to the pathogen or host cell expressing it. In another aspect, the information is organized by tissue distribution, e.g., enzymes expressed in cancer cells, enzymes expressed in normal, noncancerous cells, enzymes overexpressed as a result of prior therapy (e.g., antibiotic or chemotherapy), or enzymes expressed in a specified tissue type (e.g., breast versus liver).
  • the organism is selected from the group consisting of an animal, a vertebrate, an avian, a mammal, a human patient, a pet, a farm animal, a plant, and a plant root.
  • the target enzyme is present in the pathogen or in the infected cell but normally absent in the host or in uninfected host cells.
  • databases include, but are not limited to commercially available genomic and protein databases (e.g., LifeSeq® available from Incyte Genomics, Inc.).
  • Examples of public domain databases containing information that can be processed according to the invention can be accessed at a number of internet locations or Web sites.
  • WIT a world wide web based system to support the curation of functional assignments made to genes, now "ERGO” maintained by the Argonne National Laboratory of the University of Chicago.
  • WIT a world wide web based system to support the curation of functional assignments made to genes, now "ERGO”
  • Another such database is located at a web site called KEGG (Kyoto Encyclopedia of Genes and Genomes) currently maintained by the Institute for Chemical Research at Kyoto University, Japan.
  • the actual URL (universal resource locator) used to access WIT can change, but has recently been used as http://wit.mcs.anl.gov/WIT2.
  • the KEGG site http://www.blast.genome.ad.jp/kegg/kegg2.html can be used.
  • the databases are searched for enzymes using their respective Enzyme Commission Numbers ("EC"). ECs uniquely identify individual enzymes and are interpretable in terms of the reaction mechanism of each enzyme so named. Thus, these numbers can be useful for sorting through large numbers of candidate enzyme entries in a variety of databases.
  • the method requires selecting from a database an enzyme that is expressed by an infectious agent or in an infected cell and comparing these results with a database of expressed enzymes in at least one different class of organisms. In one aspect, these results are further compared to a database comprising enzymes expressed by yet a different class of organisms to identify an enzyme that is expressed in at least one class of organisms but not expressed in another class of organisms. For example, the method is useful to identify target enzymes present in a pathological organism but absent in an uninfected subject host such as enzymes present in pathogenic bacteria but not in human cells.
  • a list of the identified enzymes may also be outputted.
  • the identified enzymes may further be organized into a first set of enzymes capable of being placed into metabolic pathways and a second set of enzymes not capable of being placed into metabolic pathways. The first and second sets of enzymes may then be displayed such that the first set of enzymes is distinguishable from the second set of enzymes.
  • a third data structure may be queried to organize the identified enzymes.
  • biologically equivalent iECTA enzymes are characterized by possessing at least 75%, or at least 80%, or at least 90% or at least 95% amino acid sequence homology as determined using a sequence alignment program under default parameters correcting for ambiguities in the sequence data, changes in nucleotide sequence that do not alter the amino acid sequence because of degeneracy of the genetic code, conservative amino acid substitutions and corresponding changes in nucleotide sequence, and variations in the lengths of the aligned sequences due to splicing variants or small deletions or insertions between sequences that do not affect function.
  • a "biological equivalent” intends a protein sequence identified by BLAST search using our the iECTA sequence as input and that results in "hits” having E values indicating that the probability that the "hit” is due to chances is less than 1 in 1000, or 1 in 100, or 1 in 10. This identifies any protein that is related at all, even if the sequence similarity by alignment is less than 10%.
  • Catalytically equivalent enzymes have been identified by BLAST search in this way, even when the % similarity is on the order of a few percent. Human telomerase is a good example of this, because it was identified by BLAST search using a protein sequence obtained from the corresponding enzyme of the ciliate Euplotes.
  • iECTA enzymes having one or more of the following characteristics: 1) enzyme is expressed only by the pathogen of interest or in the cell infected with the pathogen; 2) enzyme is expressed by the pathogen of interest but not by the host organism; 3) enzyme is part of a critical biochemical pathway for the pathogen or cell infected by the pathogen; or 4) enzyme is or is analogous to an enzyme present in a "favorable reaction type" in the pathogen or in a cell infected by the pathogen.
  • pathogen-specific enzymes include drug resistance enzymes expressed by those organisms.
  • examples include resistance plasmid-encoded drug-modifying enzymes (e.g., chloramphenicol acetyl transferase and other plasmid- or chromosomally-encoded enzymes like beta- lactamases, Table 1, Part C).
  • Intrinsic ECTA targets differ from resistance ECTA targets only in that the intrinsic enzymes (e.g., viral encoded protease) are present or expressed in na ⁇ ve or untreated pathogens. Resistance enzymes are typically only expressed or expressed at elevated levels as a result of challenge by therapeutic agents such as enzyme inhibitors.
  • the method of this invention identifies enzymes that occur in one class of organisms, but NOT in another class.
  • the "class" can be defined by the user. It is likely that, contained in the output list of enzymes, some enzymes will be more amenable than others to development for iECTA.
  • the described technique allows for an examination of the original output list for enzymes with unique mechanisms of action (analogous to the enzymes described in Table 1, below).
  • the methods can be practiced using local alignment search algorithms (i.e., BLAST, FASTA) or by directly searching various genome sequence databases, see for example, Figures 3 and 4.
  • This method can be applied to any target organism for which DNA sequence information is available.
  • These databases include microbial genome databases, human genome databases, and expressed sequence tag databases.
  • This invention provides a way of querying databases using genome sequence information to identify potential iECTA enzymes. For example, an open reading frame (ORF) amino acid sequence is obtained for each target using a search program to determine which of these represents an enzyme (EC number) according to current annotation.
  • ORF open reading frame
  • EC number enzyme
  • a local alignment algorithm such as BLAST, the amino acid sequence of the candidate enzyme is compared with each sequence of a database consisting of human expressed sequence tags.
  • the result obtained by these comparisons can be interpreted as a probability that an enzyme represented by sequence data is expressed in human cells. This would indicate that the target organism shared a common ancestor with humans and that the enzyme from humans and the target organism are related. If the enzymes are so related, they may share traits such as similar mechanism of action and similar substrate specificity and this might counter indicate the usefulness of related enzymes as iECTA targets.
  • the methods of this invention identify enzymes and metabolic pathways present in the pathogenic organisms, but absent in the host, and as such, are a source of selectivity. For example, some pathways, as well as the enzymes involved, have only been found in bacteria, fungi and plants and not in mammalian cells.
  • One example is the synthesis of "essential" amino acids - amino acids that animals cannot synthesize and must ingest with food (see Table 2 and Nelson and Cox (1972)).
  • This invention also provides a means of uncovering potential enzyme targets in pathways that are common only in biochemical outcome but differ in route taken.
  • cysteine is not an essential amino acid, but many pathogenic microbes synthesize cysteine in a fashion different from humans and other higher organisms.
  • the enzyme cysteine synthase (EC 4.2.99.8) is not found in humans, drosophila, or mus muscalus according to our search algorithm and is therefore a potential ECTA target.
  • Part B is required for specific cleavage of virus-encoded gpl60 to yield gpl20, which is necessary for virus maturation (Markowitz and Ho (1996)).
  • Protease inhibitors have been used for patient treatment, and inhibitor- resistant mutants of the enzyme have been described (Shirasaka, et al. (1995) and Venturi, et al. (2000)).
  • a possible iECTA compound is based on the structure of a pharmacophore derived from the natural gpl60 cleavage site (Kirkpatrick, et al. (1999); Bohocek and Martin (1997); and Ekins, et al.
  • HIV protease is present only in virus-infected cells, only those cells will be affected following exposure to an HIV protease iECTA compound.
  • virally encoded iECTA targets include essential viral-specific replication enzymes like reverse transcriptase encoded by retroviruses (e.g., HIV), and RNA-dependent RNA polmerase encoded by flaviviruses (e.g., HCV).
  • retroviruses e.g., HIV
  • RNA-dependent RNA polmerase encoded by flaviviruses e.g., HCV
  • herpes virus-encoded thymidine kinase (Coen (1996) and Oram, et al. (2000)). For this reason, the herpes virus-encoded thymidine kinase is not included as a preferred target in Table 2, while essential enzymes like reverse transcriptase, RNA-dependent RNA polymerase and virally-encoded proteases are included. Pathogen specific enzymes are listed in Table 1 (Part C).
  • the methods of this invention operate on a typical computer system.
  • the computer system can include various input devices such as a keyboard.
  • the computer system also includes a processor such as CPU and internal memory.
  • the processor may be a special purpose processor with database processing capabilities or it may be a general-purpose processor.
  • the memory may comprise various types of memory, including RAM, ROM, and the like.
  • the computer system also includes external storage that includes devices such as disks, CD ROMs, ASICs, external RAM, external ROM and the like.
  • the present invention can be implemented as part of the processor or as a program residing in memory and external storage and running on processor or as a combination of program and specialized hardware.
  • the program can be in a RAM, a ROM, an internal or external disk, a CD ROM, an ASIC or the like.
  • the program when implemented as a program or in part as a program, can be encoded on any computer-readable medium or combination of computer- readable media, including but not limited to a RAM, a ROM, a disk, an ASIC, a PROM and the like.
  • the computer system also includes a display and, optionally, an output device such as a printer.
  • the computer system can run any operating system and can be implemented in any computer programming language or combination of computer programming languages, although preferably it is implemented, at least in part, in a language which is suitable for database access and manipulation.
  • this invention provides a system for identifying enzymes for designing Enzyme Catalyzed Therapeutic Activation (ECTA) compounds, comprising logic for searching a first data structure to obtain a first set of information relating to one or more enzymes associated with a target organism that are expressed in a pathological cell or by a infectious agent or in an infected cell as compared to a suitable control and logic for searching one or more other data structures to obtain one or more additional sets of information relating to one or more expressed enzymes associated with one or more additional classes of organisms that are expressed respective class.
  • ECTA Enzyme Catalyzed Therapeutic Activation
  • the system also comprises logic for comparing the first set of information to the one or more additional sets of information to identify enzymes in the first set of information that are not present in the one or more additional sets of information, wherein the identified enzymes are capable of being used to design ECTA compounds.
  • the enzymes are overexpressed as compared to a suitable control.
  • the overexpressed enzyme is the result of prior treatment, e.g., antiobiotic or chemotherapy.
  • the system further comprises logic for outputting a list of the identified enzymes.
  • the system comprises logic for organizing the identified enzymes into a first set of enzymes capable of being placed into metabolic pathways and a second set of enzymes not capable of being placed into metabolic pathways; and logic for displaying the first and second sets of enzymes such that the first set of enzymes are distinguishable from the second set of enzymes.
  • a third data structure is queried to organize the identified enzymes.
  • the system can be part of a network that is utilized to search at least one of the first data structure and the second data structure.
  • suitable networks include, but are not limited to, a network capable of communicating utilizing TCP/IP or IPX protocols.
  • the information relating to the one or more enzymes of the organism includes information about Enzyme Commission (EC) numbers of the one or more enzymes.
  • the one or more additional sets of information relating to the one or more expressed enzymes associated with one or more classes of organisms includes information about Enzyme Commission (EC) numbers of the one or more expressed enzymes.
  • This invention further provides a computer program product for identifying enzymes for designing Enzyme Catalyzed Therapeutic Activation (ECTA) compounds, comprising computer code for searching a first data structure to obtain a first set of information relating to one or more enzymes associated with a pathological cell, by an infectious agent or in an infected cell and computer code for searching one or more other data structures to obtain one or more additional sets of information relating to one or more expressed enzymes associated with one or more additional classes of organisms that are express.
  • the program product also contains computer code for comparing the first set of information to the one or more additional sets of information to identify enzymes in the first set of information that are not present in the one or more additional sets of information, wherein the identified enzymes are capable of being used to design ECTA compounds.
  • Further additions include, but are not limited to computer code for outputting a list of the identified enzymes, computer code for organizing the identified enzymes into a first set of enzymes capable of being placed into metabolic pathways and a second set of enzymes not capable of being placed into metabolic pathways; and computer code for displaying the first and second sets of enzymes such that the first set of enzymes are distinguishable from the second set of enzymes.
  • a third code is supplied to query and optionally organize the information.
  • the information regarding enzyme expression can be organized according to ECC number.
  • the system can work on a stand alone computer system or be a component of a network.
  • the network is capable of communicating utilizing TCP/IP or IPX protocols.
  • Figure 2C is a flowchart for process 240 for identifying iECTA enzymes for designing iECTA compounds in accordance with an embodiment of the present invention.
  • a first data structure is searched to obtain a first set of information relating to one or more enzymes associated with a target organism that are expressed at an elevated level in a pathological cell as compared to a normal counterpart cell or host cell.
  • one or more other data structures are searched to obtain one or more additional sets of information relating to one or more expressed enzymes associated with one or more additional classes of organisms that are expressed at elevated levels in the respective class.
  • the first set of information is compared to the one or more additional sets of information in operation 246 to identify enzymes in the first set of information that are not present in the one or more additional sets of information.
  • a network may be utilized to search the first data structure and/or the second data structure.
  • the network may be capable of communicating utilizing TCP/IP and or IPX protocols.
  • the information relating to the one or more enzymes of the target organism may include information about
  • Enzyme Corrimission (EC) numbers of the one or more enzymes may also include information about Enzyme Commission (EC) numbers of the one or more expressed enzymes.
  • the identified enzymes may be capable of being used to design iECTA compounds.
  • operations 244 and 246 may be executed sequentially for each additional database. In other words, operations 244 and 246 may be repeated for each additional database searched. For example, operations 244 and 246 may be executed for a first additional database (i.e., a second database) to obtain a first output which identifies enzymes in the first set of information that are not present in the information obtained from the first additional database. Operations 244 and 246 may then be executed utilizing the first output and a second additional database (i.e., a third database) to obtain a second output which identifies enzymes in the first set of information that are not present in the information obtained from the second additional database, and so on.
  • a first additional database i.e., a second database
  • Operations 244 and 246 may then be executed utilizing the first output and a second additional database (i.e., a third database) to obtain a second output which identifies enzymes in the first set of information that are not present in the information obtained from the second additional database, and so on.
  • FIG. 2D is a schematic diagram of an illustrative system 250 capable of executing the process 240 for identifying enzymes for designing ECTA compounds set forth in Figure 2C in accordance with an embodiment of the present invention.
  • a user's computer 252 is connected via a network 254 (e.g., a LAN or a WAN such as the Internet) to a plurality of databases 256, 258, 260 (i.e., data structures).
  • a network 254 e.g., a LAN or a WAN such as the Internet
  • databases 256, 258, 260 i.e., data structures
  • each database 256, 258, 260 may be hosted by a separate server 262, 264, 266 connected to the network.
  • the databases may be hosted all on one server or on two servers, or even more than three servers.
  • One of the databases of the system 250 may contain information relating to one or more enzymes associated with a target organism that are expressed at an elevated level in a pathological cell as compared to a normal counterpart or host cell.
  • a second of the databases may contain information relating to one or more expressed enzymes associated with another class of organisms that are express at elevated levels in the particular class.
  • the user's computer 242 may be utilized to compare the information obtained from the databases.
  • FIG. 8 illustrates an exemplary system 1200 with a plurality of components 1202 in accordance with an embodiment of the present invention.
  • such components include a network 1204 which take any form including, but not limited to a local area network, a wide area network such as the Internet, etc. Coupled to the network 1204 is a plurality of computers which may take the form of desktop computers 1206, laptop computers 1208, hand-held computers 1210, or any other type of computing hardware/software.
  • the various computers may be connected to the network 1204 by way of a server 1212 which may be equipped with a firewall for security purposes. It should be noted that any other type of hardware or software may be included in the system and be considered a component thereof.
  • Figure 9 illustrates a typical hardware configuration of a workstation in accordance with one embodiment having a central processing unit 1310, such as a microprocessor, and a number of other units interconnected via a system bus 1312.
  • a central processing unit 1310 such as a microprocessor
  • the workstation shown in the figure includes a Random Access Memory (RAM) 1314, Read Only Memory (ROM) 1316, an I/O adapter 1318 for connecting peripheral devices such as disk storage units 1320 to the bus 1312, a user interface adapter 1322 for connecting a keyboard 1324, a mouse 1326, a speaker 1328, a microphone 1332, and/or other user interface devices such as a touch screen (not shown) to the bus 1312, communication adapter 1334 for connecting the workstation to a communication network 1335 (e.g., a data processing network) and a display adapter 1336 for connecting the bus 1312 to a display device 1338.
  • RAM Random Access Memory
  • ROM Read Only Memory
  • I/O adapter 1318 for connecting peripheral devices such as disk storage units 1320 to the bus 1312
  • a user interface adapter 1322 for connecting a keyboard 1324, a mouse 1326, a speaker 1328, a microphone 1332, and/or other user interface devices such as a touch screen (not shown) to the
  • the workstation typically has resident thereon an operating system such as, for example: the Microsoft Windows NT or Windows 95/98/2000 Operating System (OS), the IBM OS/2 operating system, the MAC OS, or UNIX operating system.
  • OS Microsoft Windows NT or Windows 95/98/2000 Operating System
  • IBM OS/2 operating system the IBM OS/2 operating system
  • MAC OS the MAC OS
  • UNIX operating system a trademark of Lucent Technologies Inc.
  • OOP Object oriented programming
  • An embodiment may be written using JAVA, C, and the C++ language and utilizes object oriented programming methodology or any other means.
  • Object oriented programming (OOP) has become increasingly used to develop complex applications.
  • OOP moves toward the mainstream of software design and development, various software solutions require adaptation to make use of the benefits of OOP.
  • OOP is a process of developing computer software using objects, including the steps of analyzing the problem, designing the system, and constructing the program.
  • An object is a software package that contains both data and a collection of related structures and procedures.
  • OOP Since it contains both data and a collection of structures and procedures, it can be visualized as a self-sufficient component that does not require other additional structures, procedures or data to perform its specific task. OOP, therefore, views a computer program as a collection of largely autonomous components, called objects, each of which is responsible for a specific task. This concept of packaging data, structures, and procedures together in one component or module is called encapsulation.
  • OOP components are reusable software modules which present an interface that conforms to an object model and which are accessed at run-time through a component integration architecture.
  • a component integration architecture is a set of architecture mechanisms which allow software modules in different process spaces to utilize each others capabilities or functions. This is generally done by assuming a common component object model on which to build the architecture. It is worthwhile to differentiate between an object and a class of objects at this point.
  • An object is a single instance of the class of objects, which is often just called a class.
  • a class of objects can be viewed as a blueprint, from which many objects can be formed.
  • OOP allows the programmer to create an object that is a part of another object.
  • the object representing a piston engine is to have a composition-relationship with the object representing a piston.
  • a piston engine comprises a piston, valves and many other components; the fact that a piston is an element of a piston engine can be ⁇ logically and semantically represented in OOP by two objects.
  • OOP also allows creation of an object that "depends from” another object. If there are two objects, one representing a piston engine and the other representing a piston engine wherein the piston is made of ceramic, then the relationship between the two objects is not that of composition.
  • a ceramic piston engine does not make up a piston engine. Rather it is merely one kind of piston engine that has one more limitation than the piston engine; its piston is made of ceramic.
  • the object representing the ceramic piston engine is called a derived object, and it inherits all of the aspects of the object representing the piston engine and adds further limitation or detail to it.
  • the object representing the ceramic piston engine "depends from" the object representing the piston engine. The relationship between these objects is called inheritance.
  • the object or class representing the ceramic piston engine inherits all of the aspects of the objects representing the piston engine, it inherits the thermal characteristics of a standard piston defined in the piston engine class.
  • the ceramic piston engine object overrides these ceramic specific thermal characteristics, which are typically different from those associated with a metal piston. It skips over the original and uses new functions related to ceramic pistons.
  • Different kinds of piston engines have different characteristics, but may have the same underlying functions associated with it (e.g., how many pistons in the engine, ignition sequences, lubrication, etc.). To access each of these functions in any piston engine object, a programmer would call the same functions with the same names, but each type of piston engine may have different/overriding implementations of functions behind the same name.
  • polymo ⁇ hism This ability to hide different implementations of a function behind the same name is called polymo ⁇ hism and it greatly simplifies communication among objects.
  • an object can represent just about anything in the real world.
  • one's logical perception of the reality is the only limit on determining the kinds of things that can become objects in object-oriented software.
  • Objects can represent physical objects, such as automobiles in a traffic-flow simulation, electrical components in a circuit-design program, countries in an economics model, or aircraft in an air-traffic- control system.
  • Objects can represent elements of the computer-user environment such as windows, menus or graphics objects.
  • An object can represent an inventory, such as a personnel file or a table of the latitudes and longitudes of cities.
  • An object can represent user-defined data types such as time, angles, and complex numbers, or points on the plane.
  • OOP allows the software developer to design and implement a computer program that is a model of some aspects of reality, whether that reality is a physical entity, a process, a system, or a composition of matter. Since the object can represent anything, the software developer can create an object which can be used as a component in a larger software project in the future. If 90% of a new OOP software program consists of proven, existing components made from preexisting reusable objects, then only the remaining 10% of the new software project has to be written and tested from scratch. Since 90% already came from an inventory of extensively tested reusable objects, the potential domain from which an error could originate is 10% of the program. As a result, OOP enables software developers to build objects out of other, previously built objects.
  • C++ is an OOP language that offers a fast, machine-executable code.
  • C++ is suitable for both commercial-application and systems-programming projects.
  • C++ appears to be the most popular choice among many OOP programmers, but there is a host of other OOP languages, such as Smalltalk, Common Lisp Object System (CLOS), and Eiffel. Additionally, OOP capabilities are being added to more traditional popular computer programming languages such as Pascal.
  • Encapsulation enforces data abstraction through the organization of data into small, independent objects that can communicate with each other. Encapsulation protects the data in an object from accidental damage, but allows other objects to interact with that data by calling the object's member functions and structures.
  • Subclassing and inheritance make it possible to extend and modify objects through deriving new kinds of objects from the standard classes available in the system. Thus, new capabilities are created without having to start from scratch.
  • Polymo ⁇ hism and multiple inheritance make it possible for different programmers to mix and match characteristics of many different classes and create specialized objects that can still work with related objects in predictable ways.
  • Class hierarchies and containment hierarchies provide a flexible mechanism for modeling real-world objects and the relationships among them.
  • class libraries This framework is more complex and consists of significant collections of collaborating classes that capture both the small scale patterns and major mechanisms that implement the common requirements and design in a specific application domain. They were first developed to free application programmers from the chores involved in displaying menus, windows, dialog boxes, and other standard user interface elements for personal computers. Frameworks also represent a change in the way programmers think about the interaction between the code they write and code written by others.
  • event loop programs require programmers to write a lot of code that should not need to be written separately for every application.
  • the concept of an application framework carries the event loop concept further. Instead of dealing with all the nuts and bolts of constructing basic menus, windows, and dialog boxes and then making these things all work together, programmers using application frameworks start with working application code and basic user interface elements in place. Subsequently, they build from there by replacing some of the generic capabilities of the framework with the specific capabilities of the intended application.
  • Application frameworks reduce the total amount of code that a programmer has to write from scratch.
  • the framework is really a generic application that displays windows, supports copy and paste, and so on, the programmer can also relinquish control to a greater degree than event loop programs permit.
  • the framework code takes care of almost all event handling and flow of control, and the programmer's code is called only when the framework needs it (e.g., to create or manipulate a proprietary data structure).
  • a programmer writing a framework program not only relinquishes control to the user (as is also true for event loop programs), but also relinquishes the detailed flow of control within the program to the framework.
  • a framework basically is a collection of cooperating classes that make up a reusable design solution for a given problem domain. It typically includes objects that provide default behavior (e.g., for menus and windows), and programmers use it by inheriting some of that default behavior and overriding other behavior so that the framework calls application code at the appropriate times.
  • Class libraries are essentially collections of behaviors that you can call when you want those individual behaviors in your program.
  • a framework provides not only behavior but also the protocol or set of rules that govern the ways in which behaviors can be combined, including rules for what a programmer is supposed to provide versus what the framework provides.
  • • Call versus override With a class library, the programmer instantiates objects and calls their member functions. It is possible to instantiate and call objects in the same way with a framework (i.e., to treat the framework as a class library), but to take full advantage of a framework's reusable design, a programmer typically writes code that overrides and is called by the framework.
  • the framework manages the flow of control among its objects.
  • a preferred embodiment of the invention utilizes HyperText Markup Language (HTML) to implement documents on the Internet together with a general-pu ⁇ ose secure communication protocol for a transport medium between the client and the Newco.
  • HTTP or other protocols could be readily substituted for HTML without undue experimentation.
  • Information on these products is available in T. Berners-Lee, D. Connoly, "RFC 1866: Hypertext Markup Language - 2.0" (Nov. 1995); and R. Fielding, H, Frystyk, T. Berners-Lee, J. Gettys and J.C. Mogul, "Hypertext Transfer Protocol ⁇ HTTP/ 1.1: HTTP Working Group Internet Draft" (May 2, 1996).
  • HTML is a simple data format used to create hypertext documents that are portable from one platform to another.
  • HTML documents are SGML documents with generic semantics that are appropriate for representing information from a wide range of domains. HTML has been in use by the World-Wide Web global information initiative since 1990. HTML is an application of ISO Standard 8879; 1986 Information Processing Text and Office Systems; Standard Generalized Markup Language (SGML). To date, Web development tools have been limited in their ability to create dynamic Web applications which span from client to server and interoperate with existing computing resources. Until recently, HTML has been the dominant technology used in development of Web-based solutions. However, HTML has proven to be inadequate in the following areas: • Poor performance;
  • UI User Interface
  • Custom “widgets” e.g., real-time stock tickers, animated icons, etc.
  • client-side performance is improved.
  • Java supports the notion of client-side validation, offloading appropriate processing onto the client for improved performance.
  • Dynamic, real-time Web pages can be created. Using the above-mentioned custom UI components, dynamic Web pages can also be created.
  • Sun's Java language has emerged as an industry-recognized language for "programming the Internet.”
  • Sun defines Java as: "a simple, object- oriented, distributed, inte ⁇ reted, robust, secure, architecture-neutral, portable, high-performance, multithreaded, dynamic, buzzword-compliant, general-pu ⁇ ose programming language.
  • Java supports programming for the Internet in the form of platform- independent Java applets.”
  • Java applets are small, specialized applications that comply with Sun's Java Application Programming Interface (API) allowing developers to add "interactive content” to Web documents (e.g., simple animations, page adornments, basic games, etc.).
  • API Java Application Programming Interface
  • Applets execute within a Java-compatible browser (e.g., Netscape Navigator) by copying code from the server to client.
  • Java's core feature set is based on C++.
  • Sun's Java literature states that Java is basically, "C++ with extensions from Objective C for more dynamic method resolution.”
  • Another technology that provides similar function to JAVA is provided by Microsoft and ActiveX Technologies, to give developers and Web designers wherewithal to build dynamic content for the Internet and personal computers.
  • ActiveX includes tools for developing animation, 3-D virtual reality, video and other multimedia content. The tools use Internet standards, work on multiple platforms, and are being supported by over 100 companies.
  • the group's building blocks are called ActiveX Controls, small, fast components that enable developers to embed parts of software in hypertext markup language (HTML) pages.
  • HTML hypertext markup language
  • ActiveX Controls work with a variety of programming languages including Microsoft Visual C++, Borland Delphi, Microsoft Visual Basic programming system and, in the future, Microsoft's development tool for Java, code named "Jakarta.” ActiveX Technologies also includes ActiveX Server Framework, allowing developers to create server applications. One of ordinary skill in the art readily recognizes that ActiveX could be substituted for JAVA without undue experimentation to practice the invention.
  • the method requires comparing the results of a database search of enzymes expressed in an infected cell or by an infectious agent with a database search for enzymes expressed by a different class of organisms to identify an enzyme that is expressed in at least one class of organisms but not expressed in another class of organisms.
  • additional organism can be searched.
  • the enzyme is overexpressed in the first class of organism as compared to the second class of organism or vice versa.
  • Beta-lactamase is an enzyme expressed by bacteria and its expression renders them resistant to beta-lactam antibiotics (Schaechter et al., 1993). Applicant previously identified this enzyme as an ECTA enzyme based on its overexpression as result of prior antibiotic therapy, see PCT Application No. PCT/US98/27493. Thus, in one aspect, beta-lactamase and peptide deformylase are specifically excluded as an iECTA enzyme.
  • beta- lactamase as an iECTA target enzyme.
  • pathogen- specific, drug resistance enzymes include resistance plasmid-encoded drug- modifying enzymes (e.g. chloramphenicol acetyl transferase and other plasmid- or chromosomally-encoded enzymes (Table 1, Part C).
  • Table 1 Parts B and C provides examples of enzyme targets for ECTA technology which suggest the utility of the ECTA approach in treating other diseases characterized by expression of pathogen specific enzymes.
  • the invention provides a method of selecting iECTA targets by identifying pathogen encoded enzymes that catalyze favorable reaction types. This was accomplished by first selecting a specific enzyme that has been shown to be effective at metabolizing an ECTA substrate and then using Enzyme Commission numbers to identify enzymes that catalyze a similar reaction in another microorganism. Comparison of the sequences of these pathogen encoded enzymes with a human gene index or by comparison of EC numbers with EC numbers (enzymes) found in humans or other higher organisms was then performed to select individual target enzymes that are not present in human cells.
  • the International Enzyme Commission has developed a classification scheme that assigns each enzyme a unique number that specifies which of approximately 4500 distinct reaction types is catalyzed by the enzyme. This method is based on dividing enzyme catalyzed reactions into six classes, then further subdividing each of these classes, and so on through four levels of classification.
  • ECTA enzymes it is desirable in identifying ECTA enzymes to search for specific reaction types or similar reaction types in annotated databases of microbial genomes, for example.
  • the Enzyme Commission numbering system provides a way of automating these searches.
  • beta-lactamase EC 3.5.2.6
  • similar enzymes can be identified in target organisms by selecting EC numbers with varying degrees of similarity to find enzymes catalyzing similar reactions, i.e., the hydrolysis of cyclic amides.
  • FIGS. 7A and 7B list iECTA target enzymes. The enzymes are organized according to EC number. Enzymes that share the first 3 numbers carry out chemical reactions in a very similar fashion, they just use different substrates. Substrate prodrugs have been designed based on the "natural" generic substrate. The prodrugs were designed by evaluating the enzyme mechanism to determine chemically the best position to substitute the natural substrate with an ECTA prototoxophore. The prototoxophore is chosen based upon the enzyme active site and how the natural substrate binds this site. The prototoxophore can be a simple leaving group appended onto the natural substrate, but it does not necessarily resemble or mimic any or part of the natural substrate.
  • the prototoxophore can be a reactive analogue of a natural fragmentation product that is released (unmasked) only after enzyme activation (for example see EC 4.1.3.27 anthranilate synthase).
  • the prototoxophore can be a small chemical change to the natural substrate that takes advantage of the natural movement of electrons to create a highly reactive and toxic product that resembles the natural product.
  • "unspecified” is intended to encompass all possible substituents, limited only by the laws of chemistry and physics and by what is tolerated by the ECTA enzyme target.
  • the Enzyme Commission (EC) numbers define a specific enzyme reaction and therefore dictate the basic scaffold or substrate molecule to which substituents are added to create ECTA substrates or prodrugs.
  • toxin or toxoid is a specified substituent
  • Applicant intends that the toxin or toxoid be substituted at any appropriate atom on the compound, provided that the function of the compound is retained for its intended pu ⁇ ose.
  • Oxidoreductases All enzymes catalysing oxido-reductions belong to this class.
  • the substrate oxidized is regarded as hydrogen or electron donor.
  • the classification is based on 'donor: acceptor oxidoreductase'.
  • the recommended name is 'dehydrogenase', wherever this is possible; as an alternative, 'acceptor reductase' can be used.
  • 'Oxidase' is used only where 02 is an acceptor. Classification is difficult in some cases, because of the lack of specificity towards the acceptor. A lack of specificity for the acceptor can be a major advantage when making unnatural (ECTA) substrates.
  • transketolases and transaldolases EC 2.2.1.1 transketolase EC 2.2.1.2 transaldolase EC 2.2.1.3 formaldehyde transketolase EC 2.2.1.4 acetoin-ribose-5-phosphate transaldolase
  • Lyases are enzymes cleaving C-C, C-O, C-N and other bonds by means other than by hydrolysis or oxidation. They differ from other enzymes in that two substrates are involved in one reaction direction, but only one in the other direction. When acting on the single substrate, a molecule is eliminated and this generates either a new double bond or a new ring.
  • the systematic name is formed according to 'substrate group-lyase'. In recommended names, expressions like decarboxylase, aldolase, etc. are used. ' Dehydratase' is used for those enzymes eliminating water. In cases where the reverse reaction is the more important, or the only one to be demonstrated, 'synthase' may be used in the name.
  • Biological Confirmation - Enzyme Assays Also provided by this invention is a cell-free assay to confirm the efficacy of iECTA prodrugs by contacting the prodrug and enzyme in a cell-free system under conditions that favor activation of the prodrug by the enzyme.
  • the enzymes and methods for expression of enzyme nucleic acids are known in the art, and therefore need not be reproduced herein. For example, all enzyme sequence information and reaction conditions are available online at one or more of the following sites: www./Brenda.bc.uni-koeln.de/ and www.expasy.ch/enzyme.
  • coding sequences for bacterial or fungal AcLS and KARI are cloned as described (Pang and Duggleby
  • This invention provides a method for confirming therapeutic potential for the treatment of infectious disease.
  • the agent is considered a potential therapeutic agent if proliferation and/or replication of the infectious agent or the host cell are reduced relative to the cells in a control sample.
  • the infectious agent is killed by the agent.
  • Infected cells can be procaryotic (bacterial such as E. coli) or eucaryotic.
  • the cells can be mammalian or non-mammalian cells, e.g., yeast cells, murine cells, rat cells, avian cells, human cells.
  • This invention also provides a quick and simple screening assay that will enable initial identification of compounds with at least some of the desired characteristics.
  • the assay requires two cell types, the first being a control cell in which the target enzyme is not expressed or does not contain the infectious agent, or is expressed at a low level.
  • the second cell type is the test cell, in which the target enzyme is expressed at a detectable level, e.g., a high level or a sample that contains the infectious agent.
  • a counte ⁇ art genetically modified to differentially express the target enzyme, or enzymes (containing the appropriate species of target enzyme) is used.
  • More than one species of enzyme can be used to separately transfect separate host cells, so that the effect of the candidate drug on a target enzyme can be simultaneously compared to its effect on another enzyme or a corresponding enzyme from another species.
  • a third target cell is used as a control because it receives an effective amount of a compound, such as, for example, the compounds shown below, which have been shown to be potent prodrugs. This embodiment is particularly useful to screen for new agents that are activated by iECTA enzymes.
  • the in vitro assays are confirmed in animal or plant models infected with a pathogen expressing the target enzyme to determine in vivo efficacy.
  • a potential prodrug will be successful if microbial load is reduced or the symptoms of the infection are ameliorated, each as compared to an untreated, infected animal. It also can be useful to have a separate negative control group of cells or animals which has not been infected, which provides a basis for comparison.
  • the candidate prodrug is administered or delivered to the animal in effective amounts.
  • the term "administering" for in vivo and ex vivo pu ⁇ oses means providing the subject with an effective amount of the candidate prodrug effective to reduce microbial load.
  • the agent or prodrug may be administered with a pharmaceutically acceptable carrier.
  • the agents, prodrugs and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • Another aspect of this invention is a method for treating a subject or alleviating the symptoms of an infection by a pathogen in a subject, wherein the pathogen or a pathogen infected cell expresses an iECTA enzyme by delivering to the subject an effective amount of an iECTA prodrug compound that is converted to a toxin by the iECTA enzyme. Further provided is a method of treating a disease associated with an infection with a pathogen expressing an iECTA enzyme, or an infected host cell expressing an iECTA enzyme, by delivering to the subject an effective amount of an iECTA prodrug compound that is converted to a toxin by the iECTA enzyme.
  • iECTA expressing pathogens examples include iECTA expressing pathogens and the corresponding diseases and symptoms caused by infection by these microorganisms, are provided in Table 3, below.
  • a method for producing a medicament to treat a subject as indicated above comprising combining an effective amount of a suitable iECTA prodrug and a pharmaceutically acceptable carrier.
  • This invention also provides a method for treating or protecting plants from infection by applying an effective amount of the iECTA prodrug compound to the foliage, roots or the soil surrounding the plants or roots.
  • isolated compounds can be combined with known pesticides or insecticides.
  • Compounds within the present invention when used to treat or protect plants from infections they can be formulated as wettable powders, granules and the like, or can be microencapsulated in a suitable medium and the like.
  • suitable formulations include, but are not limited to soluble powders, wettable granules, dry flowables, aqueous flowables, wettable dispersible granules, emulsif ⁇ able concentrates and aqueous suspensions.
  • Other suitable formulations will be known to those skilled in the art.
  • This invention further provides a method for administering the prodrug compound to fish in an amount effective to either prevent or treat an infection.
  • the compound may be administered by inco ⁇ orating the compound into the food supply for the fish. Alternatively, the compound may be added to the water in which fish live, or are contained within. Finally, the compound may be administered to the fish as a suitable pharmaceutical preparation.
  • Other suitable formulations will be known to those skilled in the art.
  • the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • Animal models that can be used to test utility of candidate iECTA compounds set forth below have been described in the literature. Examples include animal models of infection by Staphyloccus aureus (Josefsson and Tartowski (1999) and Totsuka, et al. (1999)), Pneumocystis carinii (Tamburrini, et al. (1999)), enterococci (Zimbelman, et al. (1999)), multimicrobial peritonitis (Montravers, et al. (1999)), and fungal infections (Louie, et al. (1999)).
  • the candidate iECTA compound is compared with an antibiotic currently used to treat the disease.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the pu ⁇ ose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
  • the agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to a compound of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • a compound of the formula of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • a suitable dose for each of the above-named compounds is in the range of about 1 to about 100 mg per kilogram body weight of the recipient per day, preferably in the range of about 1 to about 50 mg per kilogram body weight per day and most preferably in the range of about 1 to about 25 mg per kilogram body weight per day.
  • all weights of active ingredient are calculated as the parent compound of the formula of the present invention, for salts or esters thereof, the weights would be increased proportionately.
  • the desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • sub-doses may be administered in unit dosage forms, for example, containing about 1 to about 100 mg, preferably about 1 to above about 25 mg, and most preferably about 5 to above about 25 mg of active ingredient per unit dosage form. It will be appreciated that appropriate dosages of the compounds and compositions of the invention may depend on the type and severity and stage of the disease and can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
  • the prodrug should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the prodrug, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the prodrug may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
  • prodrug ingredient While it is possible for the prodrug ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers, therefore, and optionally other therapeutic agents.
  • Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, topical
  • formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil.
  • a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, about 0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w and most preferably about 0.5 to about 10% w/w.
  • the prodrug may be employed with either a paraffmic or a water-miscible ointment base.
  • the prodrug ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3- diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances abso ⁇ tion or penetration of the prodrug ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilize ⁇ s) make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.
  • suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the prodrug ingredient.
  • the prodrug ingredient is preferably present in such formulation in a concentration of about 0.5 to about 20%, advantageously about 0.5 to about 10% particularly about 1.5% w/w.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as suppositories, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the prodrug ingredient, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the prodrug ingredient.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more tissues.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of a prodrug ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
  • Prodrugs and compositions of the formula of the present invention may also be presented for the use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art.
  • this invention also provides a composition
  • a composition comprising the compound of this invention and a carrier, such as a solvent or agriculturally suitable carrier.
  • the composition includes at least one chemical or biological pesticide, or both, as is conventionally used in the art.
  • the formulations can be processed into a formulation selected from the group consisting of a wettable powder, an aqueous suspension, an emulsifiable concentrate and a microencapsulated formulation.
  • the compounds of this invention can be used in a method for protecting or treating a plant or plant root from pathogenic infestations by applying an effective amount of the compound to the plant or root.
  • the method further comprises applying at least one chemical or biological pesticide.
  • the query method involves:
  • Bioinformatics Institute currently at http://srs6.ebi.ac.uk/srs6bin/cgi- bin/wgetz?-page+top+-newId.
  • Use the SRS interface to query a database representing enzymes expressed in humans.
  • the BRENDA database can be downloaded in this way by querying for [Organism] Human
  • the resulting list of EC numbers is most conveniently saved as a text file, opened in Microsoft Word (or similar word processing program) and processed as in steps 3) through 6) above; save the final text file as human_ec_num. 8.
  • a list of enzymes occurring in the target organism, but not in humans, or other species or combination of species can be obtained by running a computer program written in PERL or other computer language.
  • the program can be re-run to delete enzymes present in any number of databases by re-applying step 8) using another database.
  • the SwissProt database human enzymes can be subtracted as well as the BRENDA database.
  • the SwissProt and BRENDA lists can be combined, and the program run just once.
  • the following illustrative PERL program was used to obtain the list of enzymes set forth in step 8: To delete known human enzymes, as represented by enzyme commission (EC numbers) from lists of enzyme commission numbers comprising a number of pathogenic microorganisms.
  • ECTA EC numbers are: ⁇ n"; #print the list to the screen open (OUT,”>target_ec_numbers”); #save the list to a file foreach (@target_ec_list) ⁇ print (OUT $ J; print (OUT " ⁇ n”); print "$_ ⁇ n”;
  • the output of the program in step 8 lists all ECTA targets, whether or not they are part of a recognized metabolic pathway; enzymes present in BRENDA (Homo sapiens and other mammals, in this instance) and SwissProt (Homo sapiens) can be indicated.
  • a list of enzymes organized into metabolic pathways can be obtained from the resulting total target_ec_num_ list by pasting this list into the KEGG website http://www.blast.genome.ad.ip/kegg/kegg2.html, selecting the organism homo sapiens, selecting Display EC/Compound/Gene(s) NOT found in the search, and clicking execute.
  • ECTA enzymes that cannot be placed in a metabolic pathway by KEGG will be listed apart from those organized into metabolic pathways.
  • @ARGV ("Yersinia pseudotubercul”, “Yersinia pestis”, “Vibrio cholerae El Tor N 16961”, “Ureaplasma urealyticum”, “Treponema pallidum”, “ Streptomyces coelicolor”, “ Streptomyces coelicolor”, “Streptococcus pyogenes”, “Streptococcus pneumonia”, “Streptococcus mutans”, “Streptococcus equi”, “Staphylococcus aureus”, “Salmonella typhimurium”, “Salmonella typhi”, “Salmonella paratyphi”, “Salmonella enteritidis”,
  • %seen (); foreach $item (@target_ec_list) ⁇ push (@uniq, $item) unless $seen ⁇ $item ⁇ ++;
  • Figure 7A which lists the EC numbers and descriptions of ECTA enzymes for each target organism.
  • An abbreviated list consisting of all the EC number descriptions, but listing only one occurrence for each organism is shown in Figure 7B, and consists of the 673 enzymes indicated by the following PERL script:
  • %seen (); foreach $item (@target_ec_lis.) ⁇ push (@uniq, $item) unless $seen ⁇ $item ⁇ ++;
  • $b $a; open (PATHOGEN_EC_LIST, "KITTY EC LIST TEXT copy"); while ( ⁇ PATHOGEN_EC_LIST>) ⁇ if ($a eq $b) ⁇ if ( ⁇ b$a ⁇ b/) ⁇
  • a new data set is generated with useful properties that may contain enzymes or enzyme types that are present in pathogenic or undesirable microorganisms, but not present in uninfected or host cells.
  • the method makes use of an existing data set that returns enzyme names and EC numbers in response to a search tool, the use of a computer algorithm to return all enzyme names which identify potential iECTA enzyme targets, by EC number, in pathogenic organisms is an innovation that can be broadly applied for identifying pathogen or species targets for therapeutics development, or other applications (e.g., discriminating between yeast and bacteria, and pathogenic vs. nonpathogenic bacteria, plant pests vs. food plants).
  • the method has been illustrated by examples applicable to iECTA, the method is not limited to iECTA.
  • databases of enzymes elevated or expressed only in human cancer cells as compared to normal cells can be identified in an analogous fashion.
  • target enzymes for ECTA in cancer are expressed at elevated levels in tumor tissue as compared to normal tissue. Examples of such enzymes are given in Table 1(A). The difference in target enzyme expression between normal and tumor tissue allows for a positive therapeutic index to be achieved with ECTA compounds.
  • the ECTA compound NB 1011 (See U.S. Patent No. 6,245,750) targets the enzyme thymidylate synthase (TS) which is overexpressed in cancer cells.
  • TS thymidylate synthase
  • Cvtotoxicity of NB1011 is proportional to TS protein levels in model cell-based systems.
  • TS inhibitors such as 5- fluorouridine have the reverse cytotoxicity profile since they are more toxic to the cells which express low amounts of the enzyme (Copur et al., 1995).
  • In vivo studies have demonstrated efficacy against colon and breast cancer in animal models with little or no toxicity to the host.
  • This map illustrates an aspect of ECTA enzyme selection, i.e. that it is desirable for the ECTA target enzyme to be connected to the network in such a way that there are no enzymes occurring in humans that are connected to the substrate (in this case, 4-imidazolone 5-propionate). This ensures that any ECTA substrate is unlikely to interact with a human enzyme.
  • This condition is met in the above example, since 4.2.1.49 and 1.14.13- are both represented by unfilled boxes.
  • EC numbers 4.3.1.3; 4.2.1.49; 1.14.13-; 3.5.2.7; 3.5.1.68 and 3.5.3.8 have been identified with open reading frames in P. aeruginosa according to the WIT database, while only 4.3.1.3 was also found in humans.
  • methyl transferase Another example of selection of an intrinsic ECTA target by identification of an enzyme that catalyzes a favorable reaction type is methyl transferase.
  • the methyl transferase enzyme thymidylate synthase has been shown to be amenable to development of ECTA substrates.
  • a search of the WIT database for alternative related enzymes identified 2- demethylmenaquinone methyl transferase, EC 2.1.1.- as a potential intrinsic ECTA target.
  • the S-adenosylmethionine dependent 2- demethylmenaquinone methyl transferase catalyzes a step in the biosynthesis of menaquinone, or vitamin K 2 .
  • a number of pathogenic bacteria express this enzyme, including Escherichia coli, Enterococcus faecalis, Haemophilus influenza, Mycobacterium leprae, Mycobacterium tuberculosis,
  • the reaction catalyzed by this enzyme involves the transfer of a methyl group, and is similar in this respect to thymidylate synthase, EC 2.1.1.45.
  • a tBLASTn search indicates that there is no human gene in the TIGR (The Institute for Genomic Research) human gene index that has a statistically significant degree of similarity to the S-adenosylmethionine dependent 2-demethylmenaquinone methyl transferase. This result is also consistent with the pathway data obtained from the Kyoto Encyclopedia of Genes and Genomes.
  • Enzyme EC 2.1.1.- is present in the target organism (Pseudomonas aeuruginosa), but not in humans. 2.5.1.- represents an enzyme that is present in humans. The pathway has no branches, thus the substrate 2-demethyl menaquinone is not expected to be a substrate for any human enzymes and is a useful target for development of ECTA compounds.
  • Experiment # 3 Designing iECTA Compounds for Bacterial and Fungal Infections Using Enzymes in the Branched Chain Amino Acids Pathway
  • acetolactate synthase AcLS
  • ketol-acid reductoisomerase KARI
  • iECTA Branched Chain Amino Acid
  • BCAA Branched Chain Amino Acid
  • Acetolactate synthase (AcLS) is the first enzyme in the pathway of branched chain amino acid (BCAA) synthesis.
  • the active enzyme is present in bacteria, fungi, and plants, but not in mammals (Shaner and Singh (1997)).
  • the absence of AcLS in animals allows effective use of AcLS inhibitors in herbicides, while avoiding toxicity to humans and animals (Shaner and Singh (1997) and Grandoni, et al. (1998)).
  • Selectivity of enzyme function between disease causing organisms and animal or plant hosts can be used for designing iECTA compounds to fight bacterial and fungal infections.
  • the product(s) may include toxins or antimetabolites that are preferentially generated by the bacteria or fungi.
  • Acetolactate synthase is an ⁇ 2 ⁇ 2 oligomer that consists of four subunits: two catalytic subunits with molecular weight of 60 kD and two regulatory subunits with molecular weight of 10-17 kDa (Pang and Duggleby (1999)).
  • the enzyme catalyzes two similar reactions: the condensation of two pyruvate molecules to yield 2-acetolactate ( Figure 11), and the condensation of pyruvate with 2-oxobutyrate (2-OB, 2-ketobutyrate) to yield 2-aceto-2-hydroxybutyrate ( Figure 12).
  • Thiamine pyrophosphate TPP is a cofactor in the reaction.
  • Branched chain amino acids inhibit the enzyme by an allosteric mechanism since they do not occupy the substrate binding site, but rather a distinct site between the two subunits (Shaner and Singh (1997)).
  • AcLS inhibitors are effective as herbicides at low concentrations and have little toxicity to humans (Whitcomb (1999)).
  • KARI follows AcLS in the pathway of branched chain amino acid synthesis. It catalyzes isomerization of 2-acetolactate or 2-aceto-2- hyrdoxybutyrate with concomitant hydride transfer. The products of the reaction are 2,3-dihydroxy-isovalerate and 2,3-dihydroxy-3-methyl-valerate, respectively. The mechanism of the reaction is known in the art (Aulabaugh and Schloss (1990)).
  • KARI inhibitors include analogs of the transition state of the reaction (Halgand, et al. (1999)). Because the crystal structure of KARI is known (Halgand, et al. (1999)), this information can be used to aid the design of KARI iECTA compounds using simulated docking technology (Kirkpatrick, et al. (1999)).
  • Such iECTA compounds will have therapeutic antimicrobial (and possible herbicidal) properties.
  • Proposed pathways of metabolism for AcLS iECTA compounds derived from 2-OB are compared for humans and bacteria as shown in Figure 6.
  • cystathionine-2-lyase catalyzes 2-oxobutyrate conversion to L-homoserine and L-cystathionine.
  • two additional enzymatic reactions can occur. These are reactions of 2-OB with 1-aminocyclopropane-l-carboxylate deaminase and acetolactate synthase.
  • the structure of a potential AcLS iECTA compound is disclosed infra.
  • the AcLS iECTA product may:
  • KARI Bind to KARI and be converted to the rearranged and reduced product. This product could be toxic, or become transformed to a toxin following a subsequent reaction. 2. It may not bind to KARI, but rather accumulate as a "dead-end" product and eventually starve the cells of pyruvate. 3. Be inco ⁇ orated into cellular polypeptides, thereby leading to the formation of dysfunctional proteins.
  • the design of candidate KARI iECTA compounds is based upon the same rationale as the design of AcLS iECTA compounds. In this case, the proposed scaffold is 2-aceto-2-hydroxybutyrate.
  • Either AcLS or KARI will utilize substrates and convert them to antimetabolites targeting multiple enzyme pathways.
  • This invention also provides compounds useful as AcLS and KARI iECTA compounds.
  • the compounds have the structure:
  • n is 0 or an integer from 1 to 6, more preferably n is 0, 1, or 2, most preferably n is 0;
  • X is H or halogen, more preferably halogen
  • Y is H or halogen, more preferably halogen
  • Z is any of H; halogen; CF 3 ; aliphatic group; substituted or unsubstituted aromatic or heteraromatic ring, more preferably phenyl ring; substituted or unsubstituted aromatic carbonyl or heteraromatic carbonyl, more preferably substituted or unsubstituted benzoyl.
  • R H, a pharmaceutically acceptable cation, or an aliphatic substituent, more preferably methyl or ethyl.
  • the compounds have the structure:
  • A is a substituted or unsubstituted phenyl ring or a substituent having the structure:
  • n is 0 or an integer from 1 to 6, and more preferably 0, 1 or 2, and most preferably 0; wherein X is H or halogen, and more preferably halogen; wherein Y is H or halogen, and more preferably halogen; wherein Z is H; halogen; CF 3 ; aliphatic group; substituted or unsubstituted aromatic or heteraromatic ring, and more preferably phenyl ring; substituted or unsubstituted aromatic carbonyl or heteraromatic carbonyl, more preferably substituted or unsubstituted benzoyl.
  • B is a substituted or unsubstituted phenyl ring or a substituent having the structure:
  • n is 0 or an integer from 1 to 6, and more preferably 0, 1 or 2, and most preferably 0; wherein X is H or halogen, and more preferably halogen; wherein Y is H or halogen, and more preferably halogen; wherein Z is H; halogen; CF 3 ; aliphatic group; substituted or unsubstituted aromatic or heteraromatic ring, and more preferably phenyl ring; substituted or unsubstituted aromatic carbonyl or heteraromatic carbonyl, more preferably substituted or unsubstituted benzoyl.
  • R is H, a pharmaceutically acceptable cation, or an aliphatic substituent, more preferably methyl or ethyl.
  • Biological activity similar to iECTA compounds for AcLS and KARI has not been ascribed to any known drugs.
  • the literature has provided a synthetic protocol for a possible candidate-compound. (Wakselman and Tordeuz (1982)). This paper describes synthesis of 3,3,3- trifluoropropriaonic and 4,4,4-trifluoro-2-ketobutyric acids. This synthetic protocol does not describe the synthesis of all compounds of the class identified above, but is easily adapted by those of skill in the art for this pu ⁇ ose. EC 3.1.3.15 Histidinol Phosphatase
  • Histidinol phosphatase is found on the histidine biosynthetic pathway and is found in bacteria and yeast, but not in mammals. Mechanism is simply water hydrolysis of a phosphate group.
  • a toxophore can be substituted for the phosphate group in the substrate to create an ECTA substrate.
  • the DNA polymerase inhibitor phosphonoformate is used as an example of a toxophore.
  • DHAD Dihydroxyacid dehydratase
  • DHAD has been shown to be the target for the bacteriostatic effects of 4,7-dicyanobenzofurazan (See Takabatake et al.)
  • the DHAD ECTA substrate shown below is designed to generate a very reactive alkylating agent upon activation by the enzyme.
  • Chorismate is the branch point for the biosynthesis of several natural products.
  • the reaction shown above is the first step down the tryptophan biosynthetic pathway where chorismate is converted to anthranilate and pyruvate.
  • halogens for one or both hydrogens as shown above, a very potent alkylating agent can be produced, di- or mono-halo- pyruvate. This can be the basis for several other ECTA substrates.
  • the synthesis of a chorismate based ECTA substrate is shown below:
  • Hexamethyldisilazane (0.350 g, 2.16 mmol) is added to a suspension of oil free NaH (0.048 g, 2.00 mmol) (pre-washed with petroleum ether) in 5 mL of anhydrous DMF.
  • a solution of lactone A-1 (See Ganem et al.) (0.536 g, 2.00 mmol) in 5 mL DMF is added.
  • a solution of trifluoromethyl iodide (0.800 g, 4.10 mmol) in 5 mL DMF is added, and the reaction is allowed to proceed at room temperature for 3 hours.
  • reaction is then poured into saturated NaCl solution and extracted with ethyl acetate (2 x 25 mL). The combined organic layers are washed with saturated NaCl (2 x 25 mL), dried over anhydrous Na 2 SO , and the solvent evaporated under reduced pressure to furnish crude A-2, which is processed as is in the next step.
  • the reaction is then acidified with dilute HCl, and the product is obtained by extraction with two 25 mL portions of ethyl acetate.
  • the ethyl acetate fractions are combined, dried with anhydrous Na 2 SO 4 , fitered and the solvent is removed under reduced pressure.
  • the product 1-3 is purified by chromatography on silica gel using EtOAc/Hexane/HOAc.
  • the reaction shown above is on the phenylalanine metabolism pathway.
  • the enzyme converts a stable vinyl-halo ester into a very reactive (alkylator) allyl halide species.
  • the reaction shown above is the final step in folic acid biosynthesis where a glutamate is conjugated to the dihydropteroic acid substrate.
  • Most microorganisms produce their own folic acid, whereas it is an essential vitamin for humans because we lack this biosynthetic pathway right up through this step.
  • Antifolates have been used for both cancer chemotherapy as well as for microbial infections, but they are only potent after glutamate conjugation (See Goodman and Gillman (1996) THE PHARMACOLOGICAL
  • the 5,10-dideazapteroic acid is a known compound, and its synthesis has been published.
  • the 5,10-dideazafolate is an experimental antifolate (Id.) (See also Degraw et al. and Taylor et al.).
  • Salmonella typhimurium, Escherichia coli or other bacteria or fungi are used as test cells. Two phenotypes are employed. One strain is normal for acetolactate synthase (AcLS) and the other is deficient. Such strains have been previously described, e.g., Shaw, et al. (1980) and Weinstock, et al. (1992) and can be obtained from the American Type Culture Collection, the E. coli Genetic Stock Center (Yale University), the Salmonella Genetic Stock Center (University of Calgary, Canada), and other sources. The AcLS- negative strains are generally referred to as ilv " because they are dependent upon added isoleucine and valine for growth.
  • the ilv " mutant strains will be compared to the normal parent strains (ilv + ) for sensitivity to candidate compounds. Strains that express the active form of AcLS will be able to transform an AcLS iECTA compound into a cytotoxic moiety. For this reason, the normal strains will be more sensitive to a successful AcLS compound than will the mutant ilv " strains. Similar assays can also be performed on mammalian cells to determine the degree of specificity for AcLS-producing bacteria or fungi.
  • Assays are performed on agar plates or in liquid media containing the appropriate nutrients (Miller (1972)). Inhibition of growth of ilv + strains is measured by decreased colony formation on agar plates containing a potential AcLS activated prodrug, or decreased growth rate in liquid culture containing the candidate drug (Minimal Inhibitory Concentration, MIC.) Utility is further demonstrated by performing these assays comparing the candidate AcLS iECTA compounds with known antibiotics versus pathogens. Similar growth assays can be performed to test the utility of potential compounds on yeast and other potential pathogens using methods appropriate for these eukaryotic organisms, as described by Spector, et al. (1998). Additional tests are performed to demonstrate minimal toxicity vs. normal animal or human cells.
  • the compound When the enzyme is a member of the subgroup 1.1, the compound has the structure: R-CHOH-X-Toxin, wherein X is selected from the group consisting of O, S, and NH.
  • the prodrug is a compound of the structure: RlR2-CH-CHR3-CH2-X-Toxin, wherein Rl, R2 and R3 are unspecified, and wherein X is O or S.
  • the prodrug is a compound of the structure:
  • the prodrug is a compound having either structure:
  • Y is selected from the group consisting of O, S, Se, and NR; wherein R is unspecified; wherein X is selected from the group consisting of C and N; and wherein each of Rl, R2, R3 and R4 is independently the same or different and is a toxoid, or is unspecified.
  • the "toxoid” is directly linked toxoid or connected through a linker (i.e., self-immolative).
  • the prodrug is a compound of the structure:
  • Toxoid is either toxoid or linker-toxoid.
  • the prodrug is a compound of the structure:
  • A, X, and Y are independently the same or different and is selected from the group consisting of a toxoid, CH OH or CH 2 OPO 3 ; wherein Z is selected from the group consisting of CH2, N, NR, O, S and Se; and wherein R is unspecified.
  • the prodrug is a compound of the structure:
  • Rl, R2, R3 are independently the same or different and are unspecified; and wherein Z is a linker and/or a toxoid.
  • the prodrug is a compound of the structure:
  • X is a halide or hydrogen; and wherein Y is a linker-toxoid or a toxoid.
  • the prodrug is a compound of the structure:
  • each of Rl and R2 are independently the same or different and is selected from the group consisting of an amino acid, an amino acid side chain, a toxoid, a linker, or a peptide; and wherein Z is selected from the group consisting of a toxoid which may have RNA like structure or be RNA or a nucleic acid analog.
  • the prodrug is a compound of the structure:
  • R2 is a fatty acid; wherein X, Y and Z are independently the same or different and is selected from the group consisting of a toxoid, toxoid linker, O, S, and NR, wherein R is unspecified; and wherein A and B are independently the same or different and is selected from the group consisting of O, S, and NR wherein R is unspecified.
  • the prodrug is a compound of the structure: wherein A and Z are independently the same or different and is selected from the group consisting of toxoid, toxoid-linker, a halogen and a heteroatom.
  • the prodrug is a compound of the structure:
  • Z is selected from the group consisting of toxoid and toxoid- linker.
  • the prodrug is a compound of the structure:
  • Z is selected from the group consisting of toxoid and toxoid- linker; and wherein X is selected from the group consisting of O, S, and NR, wherein R is unspecified.
  • the prodrug is a compound of the structure: wherein RS is Coenzyme A ("CoAS") or a variable thiol including smaller analogs of CoAS; wherein A is selected from the group consisting of O, S, and NR, wherein R is unspecified; and wherein X is CI, Br, I and F.
  • CoAS Coenzyme A
  • thiol variable thiol including smaller analogs of CoAS
  • the prodrug is a compound of the structure:
  • X is H or a toxoid; wherein A is selected from the group consisting of O, S, and NR;and wherein R is selected from the group consisting of H, a halomethyl and a toxoid.
  • the prodrug is a compoimd of the structure:
  • R is selected from the group consisting a simple or complex thiol and ACP or acyl carrier protein; wherein A is O, S, and NR, wherein R is unspecified; and wherein Z is selected from the group consisting of a toxoid, a toxoid- linker, and a fatty acid analog having antibacterial/antifungal/antimicrobial properties.
  • the enzyme is a member of the subgroup 3.1.3.10
  • the prodrug is a compound of the structure:
  • A, B, X and Y are independently the same or different and each is selected from the group consisting of toxoid, CH2OH, CH20PO3 and H; wherein Z is selected from the group consisting of CH2, N, O, S, SE or NR, wherein R is unspecified; and wherein D and E are independently the same or different and is selected from the group consisting of OH, NHCH 2 CH 2 C1 and SCH 2 CH 2 C1.
  • the prodrug is a compound of the structure:
  • Z is selected from the group consisting of a toxoid, H and a toxoid-linker; and wherein X and Y are independently the same or different and is selected from the group consisting of OH, NHCH 2 CH 2 C1 and SCH 2 CH 2 C1.
  • the prodrug is a compound of the structure:
  • the prodrug is a compound of the structure:
  • Z is selected from the group consisting of H, a toxoid and toxoid-linker.
  • the prodrug is a compound of the structure:
  • the prodrug is a compound having the structure:
  • the base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; and wherein Z is a toxoid.
  • the prodrug is a compound having the structure:
  • X is H or OH; and wherein Tox is a toxoid.
  • the prodrug is a compound having the structure:
  • R and Rl is a toxoid or linker-toxoid; and wherein Tox is a toxoid.
  • the prodrug is a compound having the structure:
  • Tox is a toxoid
  • the prodrug is a compound having the structure:
  • base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein Base 1 is a toxoid; wherein Z is selected from the group consisting of toxoid and toxoid- linker; and wherein X is OH or a phosphate.
  • the prodrug is a compound having the structure: wherein Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein B is a phosphate or a DNA small oligonucleotide; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein A and B are independently the same or different and are selected from the group consisting of a phosphate and a deoxyribonucleic acid small oligonuleotide; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein A and B are independently the same or different and are selected from the group consisting of a phosphate and a deoxyribonucleic acid small oligonuleotide; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • the base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein A and B are independently the same or different and is a phosphate or a ribonucleic acid small oligonuleotide; and wherein Z is a toxoid or toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein A and B are independently the same or different and are selected from the group consisting of a phosphate and a ribonucleic acid oligonuleotide; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • Base is selected from the group consisting of adenine, tyrosine, guanine, cytosine and uracil; wherein Basel is a toxoid; wherein X is OH or a phosphate; wherein Y is H or OH; and wherein Z is a toxoid or a toxoid-linker.
  • the prodrug is a compound having the structure:
  • R is H and Rl is a glucose polymer of the formula (glucose)n, wherein n is an integer from 1 to ; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R and Rl are the same or different and is repeating beta-(l,4)- glucose in cellulose; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R and Rl are the same or different and are repeating beta-D- glucans containing 1-3 or 1-4 linkages; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R is H or an oligosaccharide; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein R is CO 2 H; wherein Rl and R2 is polygalacturonic acids linked alpha 1-4; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is H or beta-glucosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is phosphate or 6-phospho-beta-glucosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein Rl is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid or a glucose derivative.
  • the prodrug is a compound having the structure:
  • each of Rl, R2, R3, R4, R5 and R6 are independently the same or different and is selected from the group consisting of H or a saccharide; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is H; wherein R2 is P0 3 ; wherein R 3 is H; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is agarose; wherein R2 and R3 are each H; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R3 is a carrageen polymer; wherein Rl is SO 3 " ; wherein R2 is OH; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is an arabinogalactan polymer
  • R2 and R3 are independently the same or different and is a H or an arabinogalactan polymer
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • TOX is a toxoid that may alternatively have a saccharide structure.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • Base is a purine; wherein X and Y are independently the same or different and is OH or a purine nucleosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Base is adenine; wherein X is CH 3 S; wherein Y is OH or a methylthioadenosine nucleosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Base is a 3-methylated adenine; wherein X is deoxyribonucleic acid; wherein Y is H or DNA-3-methyladenine glycosidase I; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein Base is ring-opened N7-methylguanine; wherein X is deoxyribonucleic acid; wherein Y is H or formamidopyrimidine-DNA glycosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Base is adenine; wherein X is OPO 3 ; wherein Y is OH or AMP nucleosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Base is adenine; wherein X is S-homocysteine; wherein Y is OH or S-adenosylhomocysteine nucleosidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • Rl is a leucyl side chain; wherein R2 is any amino acid; wherein X is an oligopeptide or a leucyl aminopeptidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is a proline side chain; wherein R 2 is any amino acid; wherein X is an oligopeptide or a proline iminopeptidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein Rl is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R2, R3 and R4 are unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl and R2 is a D-Ala amino acid side chain; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is unspecified; wherein R2 is a glutamate side chain or glutamate carboxypeptidase; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl and R2 are unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • R2 is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is a lysyl side chain; wherein R2 is unspecified, and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is unspecified or endopeptidase LA wherein R2 is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is pro in immunoglobulin A or IgA-specific serine endopeptidase; wherein R2 is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is Ala84 in reprecursor lex A or repressor lexA peptidase; wherein R2 is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is an N-terminal leader sequence in a signal peptide or signal peptidase I; wherein R2 is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein Rl and R2 are independently the same or different and is a hydrophobic side chain or muco ⁇ epsin; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is unspecified; wherein R2 is a cysteinyl side chain or signal peptidase II; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • Rl is arginine; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl is glycine or alanine; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • Rl, R3, R4 and R5 are unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure: wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • Al and A2 are independently the same or different and is unspecified; and wherein TOX is a toxoid.
  • the prodrug is a compound having the structure: wherein TOX is a toxoid.
  • the prodrug is a compound having the structure:
  • TOX is a toxoid
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • X is selected from the group consisting of CI, Br, I and F.
  • the prodrug is a compound having the structure: wherein X is selected from the group consisting of CI, Br, I and F.
  • the prodrug is a compound having the structure:
  • X is selected from the group consisting of CI, Br, I and F.
  • the prodrug is a compound having the stmcture:
  • X is selected from the group consisting of CI, Br, I and F.
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • the prodrug is a compound having the structure:
  • X is selected from the group consisting of CI, Br, F or I.
  • the prodrug is a compound having the structure: wherein X is selected from the group consisting of CI, Br, F, and I.

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Abstract

L'invention concerne des procédés et des systèmes d'identification d'enzymes qui agissent en tant qu'activateurs thérapeutiques catalysés par des enzymes et des enzymes identifiées au moyen de ces procédés. L'invention concerne également des composés activés par ces enzymes, ainsi que des compositions contenant ces composés.
PCT/US2001/023095 2000-07-20 2001-07-20 Procedes d'identification de cibles therapeutiques destinees a traiter des maladies infectieuses WO2002007780A2 (fr)

Priority Applications (1)

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AU2001277093A AU2001277093A1 (en) 2000-07-20 2001-07-20 Methods for identifying therapeutic targets

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US21959800P 2000-07-20 2000-07-20
US60/219,598 2000-07-20
US24495300P 2000-11-01 2000-11-01
US60/244,953 2000-11-01
US27672801P 2001-03-16 2001-03-16
US60/276,728 2001-03-16

Publications (2)

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WO2002007780A2 true WO2002007780A2 (fr) 2002-01-31
WO2002007780A3 WO2002007780A3 (fr) 2003-02-20

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AU (1) AU2001277093A1 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1499318A2 (fr) * 2002-04-18 2005-01-26 Celmed Oncology (USA), Inc. Promedicaments actives par peptide deformylase

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1307421C (zh) * 1997-08-08 2007-03-28 塞米得肿瘤学美国公司 克服生物及化学治疗抗性的方法与组合物
EP1045897B1 (fr) * 1998-01-23 2002-01-30 Newbiotics Inc. Agents therapeutiques obtenus par catalyse enzymatique
AU775601B2 (en) * 1999-07-22 2004-08-05 Celmed Oncology (Usa) Inc. Enzyme catalyzed therapeutic activation
CA2379834A1 (fr) * 1999-07-22 2001-02-01 Newbiotics, Inc. Agents therapeutiques anti-infectieux catalyses par des enzymes
KR20020059341A (ko) * 1999-07-22 2002-07-12 뉴바이오틱스 인코퍼레이티드 치료법-내성 종양의 치료 방법
AU2001241878A1 (en) * 2000-02-28 2001-09-12 Newbiotics, Inc Anti-infective ECTA
AU2001257490A1 (en) * 2000-05-02 2001-11-12 Newbiotics, Inc. Improved beta-lactam antibiotics

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1499318A2 (fr) * 2002-04-18 2005-01-26 Celmed Oncology (USA), Inc. Promedicaments actives par peptide deformylase
EP1499318A4 (fr) * 2002-04-18 2006-07-19 Celmed Oncology Usa Inc Promedicaments actives par peptide deformylase

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Publication number Publication date
WO2002007780A3 (fr) 2003-02-20
AU2001277093A1 (en) 2002-02-05

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