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WO1992017182A1 - Procedes et composes d'inhibition de l'activite de rnase h de la transcriptase inverse - Google Patents

Procedes et composes d'inhibition de l'activite de rnase h de la transcriptase inverse Download PDF

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Publication number
WO1992017182A1
WO1992017182A1 PCT/US1992/002770 US9202770W WO9217182A1 WO 1992017182 A1 WO1992017182 A1 WO 1992017182A1 US 9202770 W US9202770 W US 9202770W WO 9217182 A1 WO9217182 A1 WO 9217182A1
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WO
WIPO (PCT)
Prior art keywords
metal material
rnase
reverse transcriptase
activity
concentration
Prior art date
Application number
PCT/US1992/002770
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English (en)
Inventor
Jay F. Ii Davies
Zuzana Hostomska
Zdenek Hostomsky
Steven R. Jordan
David A. Matthews
Richard C. Ogden
Michael D. Varney
Stephen E. Webber
Original Assignee
Agouron Pharmaceuticals, Inc.
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Application filed by Agouron Pharmaceuticals, Inc. filed Critical Agouron Pharmaceuticals, Inc.
Publication of WO1992017182A1 publication Critical patent/WO1992017182A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/242—Gold; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/241—Lead; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof

Definitions

  • the present invention relates to metal materials that can be used to inhibit RNase H activity of reverse transcriptase.
  • HIV HIV
  • HIV replication pathway One suggested target is the HIV replication pathway, and in fact, reverse transcription has been suggested as one target in that pathway (Mitsuya et al., Science. 249;1533-1544 (1990)).
  • Retroviruses and hepadnaviruses Reverse transcription of viral genomic RNA is an essential step in replication of human
  • HIV immunodeficiency virus
  • hepadnaviruses nonlimiting examples include HIV-1, HIV-2, HTLV-1, HBV, FeLV, or SIV. This process is catalyzed by reverse
  • RT transcriptase
  • RNA-dependent DNA polymerase RNA-dependent DNA polymerase
  • DNA-dependent DNA polymerase DNA-dependent DNA polymerase
  • RNase H ribonuclease H
  • RNA- and DNA-dependent DNA polymerase and RNase H activities are required (Gilboa et al., Cell, 18:93-100 (1979)). Owing to its ability to selectively cleave phosphodiester bonds in the RNA moiety of the RNA/DNA heteroduplex intermediate (Crouch et al., Eds. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982), p. 211; R. J . Crouch, New Biologist, 2 : 771 (1990)), RNase activity is indispensable at several stages of this complex process.
  • RNase H (1) degrades the RNA template during synthesis of minus-strand DNA from the tRNA primer, (2) generates primer for the synthesis of plus-strand DNA, and (3) specifically removes both primers via an endonucleolytic mechanism (Omer et al., Cell, 30:797 (1982); Rattray et al., J. Virol.,
  • RT and the RNase H domain of RT are prime targets for antiretroviral and antihepdnaviral therapy, especially in connection with HIV infections and AIDS (Mitsuya et al., supra).
  • An object of the present invention is to provide materials that inhibit RNase H activity of reverse transcriptase.
  • Another object according to certain preferred embodiments is to provide materials that bind to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.
  • Another object is to provide methods of inhibiting RNase H activity of reverse transcriptase by exposing the RNase H domain of reverse transcriptase to materials that inhibit RNase H
  • Yet another object is to provide methods of inhibiting RNase
  • RNase H activity by exposing the RNase H domain of reverse transcriptase to materials that bind to at least a portion of the RNase H domain active site, such that RNase H activity is inhibited.
  • Still another object is to inhibit viral replication by exposing the RNase H domain of reverse transcriptase to materials that inhibit RNase H activity. According to certain preferred embodiments, those materials bind to at least a portion of the RNase H domain of reverse transcriptase, such that RNase H
  • metal materials that are capable of inhibiting RNase H activity of reverse transcriptase of viruses that are reverse transcriptase dependent.
  • the metal materials are provided in an amount that is sufficient to inhibit the RNase H activity of reverse transcriptase of viruses that are reverse transcriptase dependent.
  • those metal materials bind to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.
  • Figure 1 is a schematic representation of the subunits in the heterodimer of HIV-1 RT. Relative positions of DNA polymerase and RNase H domains are indicated. The shaded area defines the C- terminal portion of the p66 subunit.
  • Figure 2 is a stereo drawing of seven invariant residues in retroviral and bacterial RNases H. These conserved residues (Asp443, Glu478, Asp498, Ser499, His539, Asn545, and Asp549) are clustered at one edge of the molecule. The loop containing His539 is disordered in the structure of the HIV-1 RNase H domain. The histidine (yellow) is positioned by analogy with its location in the E. coli RNase H structure. Positions for the other side chains are from refined coordinates of the native structure.
  • Figure 3 depicts the substrate which can be used for
  • Figure 4 is a picture of the gel after electrophoresis that is further described in Example I below. Lane 1 was the substrate as depicted in Fig. 3 without reverse transcriptase. The
  • lanes 2 through 8 included the substrate as depicted in Fig. 3 with reverse transcriptase and the following concentrations of K 3 UO 2 F 5 : lane 2, 0 ⁇ M; lane 3, 10 ⁇ M; lane 4, 40 ⁇ M; lane 5, 60 ⁇ M; lane 6, 100 ⁇ M; lane 7, 125 ⁇ M; lane 8, 150 ⁇ M.
  • the arrow shows the 28 nucleotides long reaction product.
  • Figure 5 is a response curve generated from the data obtained in the experiment described in Example I below for the analysis of RNase H activity of HIV-1 reverse transcriptase in the presence of different concentrations of K 3 UO 2 F 5 .
  • Figure 6 is a response curve generated from the data obtained in the experiment described in Example II below for the analysis of RNase H activity of HIV-1 reverse transcriptase in the presence of different concentrations of copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt.
  • RT reverse transcriptase
  • RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred embodiments, encompasses RT of any strain of
  • the present inventors contemplate the RT of other retroviruses and hepadnaviruses, and certain nonlimiting examples include HTLV-1, HBV, Feline Leukemia virus (FeLV) or SIV.
  • an enzyme in general, includes a binding site where the enzyme binds to the substrate, and includes a catalytic site responsible for the activity of the enzyme. Those sites can be separate or overlapping, but are both required for the enzyme to display its activity on the substrate.
  • the generic term "active site" encompasses the binding site and/or the catalytic site, because the binding of the metal materials according to the present invention to either site will inhibit RNase H activity.
  • the metal materials according to the present invention inhibit RNase activity of RT, and according to certain preferred embodiments bind to at least a portion of the active site of the RNase H domain of RT.
  • the metal can be part of an inorganic or organometallic compound.
  • Metal materials that should inhibit RNase H activity and may do so by binding to at least a portion of the active site of the RNase H domain of RT include the following materials and
  • phthalocyanine tin phthalocyanine, silver phthalocyanine, nickel phthalocyanine, lead phthalocyanine, iron phthalocyanine,
  • phthalocyanine dichlorogermanium phthalocyanine, flourochromium phthalocyanine, chloroaluminum phthalocyanine, phthalocyanine green, alcian blue, solvent blue, copper phthalocyanine
  • the present inventors have for the first time known to them determined the crystal structure of the RNase H domain of RT of HIV-1 (Hostomska et al., "Proteolytic Release and Crystallization of the RNase H Domain HIV-1 Reverse Transcriptase", J. Biol. Chem.. 266:14697-14702 (1991); and Davies et al., "Crystal Structure of the RNase H Domain of HIV-1 Reverse Transcriptase", Science, 252:88-95
  • HIV-1 RT forms a heterodimer composed of two subunits, p66 and p51 ( Figure 1) (Di Marzo Veronese et al., Science, 231:1289-1291 (1986); Lightfoote et al., J. Virol., 60:771-775 (1986)), which have identical N-termini.
  • Figure 1 Di Marzo Veronese et al., Science, 231:1289-1291 (1986); Lightfoote et al., J. Virol., 60:771-775 (1986)
  • the presence of several domains in HIV-1 RT has been deduced from sequence homology studies (Johnson et al., Proc.
  • HIV-1 RT corresponds to the DNA polymerase domain while the C-terminal portion shows homology with RNase H of E. coli as well as with the RNase H domain of RT from Moloney murine leukemia virus (MoMuLV) and other retrovirusea.
  • the p51 subunit of HIV-1 RT apparently results from proteolytic processing of p66, during which the C-terminal 120 amino acid residues are removed (Le Grice et al.,
  • the C-terminal portion of p66 as an isolated domain (p15) is necessary but not sufficient for RNase H activity.
  • the C-terminal domain of the p66 subunit of HIV-1 RT was expressed separately but, in contrast with MoMuLV, it is not sufficient for RNase H activity.
  • the RNase H activity of HIV-1 RT can be reconstituted in vitro by combining isolated p15 with the purified p51 domain (Hostomsky et al., "Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 Reverse Transcriptase", Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991)).
  • RNase H domain in a structural rather than a functional sense, to describe the C-terminal domain of HIV RT that shows sequence homology with other known RNase H domains.
  • the present inventors were able to analyze the RNase domain active site.
  • the present inventors analyzed the active site using the UO 2 F 5 3- anion.
  • the present inventors discovered that the UO 2 F 5 3- anion binds to the active site of the RNase domain of RT and is an RNase H inhibitor. Residues that form the heavy atom binding site are among seven amino acids conserved in all analyzed bacterial and retroviral RNase H sequences (Doolittle et al., The Quarterly
  • crystals are grown at 4oC in hanging drops equilibrated against a reservoir solution containing 0.15 M sodium potassium tartrate, 20 percent
  • the starting drops are composed of equal volumes of stock protein solution, i.e., the
  • RNase H domain of RT protein at 10 mg/ml, 25 mM potassium phosphate pH 7.0
  • Inhibitor (metal material) binding to RNase H is studied using x-ray diffraction data from a crystal soaked in reservoir solutions containing the inhibitor of interest. A difference map calculated with
  • An assay which can be used includes incubating RT with the metal material being tested and with a test RNA/DNA hybrid
  • RNase H domain will produce a characteristic pattern of degradation products. Inhibition of such activity can thus be detected by variations from that characteristic pattern.
  • the concentration range of metal material could be from about 10 -3 M to about 10 -12
  • Substrate for detection of RNase H activity (See Figure 3) A portion of the gag region of HIV-1 [nucleotides 629-694 in the nucleotide sequence of BH-10 (Ratner et al., Nature. 313:277-284 (1985)), shown to contain a cluster of HIV-1 RNase H cleavage sites numbered I-VI (Mizrahi, Biochemistry, 28:9088-9094 (1989)), is cloned in the plasmid pTZ18R (Pharmacia). Uniformly labeled runoff transcripts (boldface) of this region are prepared from th resulting plasmid with an RNA synthesis kit (Stratagene) and UTP[ ⁇ 35 S]. The 3' ends of these transcripts are generated by Pvu
  • transcripts are gel purified and hybridized with complementary synthetic oligodeoxyribonucleotide, the sequence of which is shown in italics. Analysis of RNase H activity
  • Determinations on inhibition are then made based on the data generated.
  • RNase H activity is essential for viral replication of viruses that are RT dependent. (Mitsuya, supra; Tisdale et al., supra; and Repaske et al., supra).
  • the present invention is also directed to inhibition of viral replication that is dependent on RT for replication.
  • PBMCs PBMCs with HIV-1
  • Ficoll/Hypaque-isolated PBMCs are stimulated for 3 days in RPMI/FCS containing phytohemagglutinin (5 ⁇ g/ml). The cells are washed and suspended at 10 cells per ml in RPMI/FCS, and HIV-1 LAV is added at the multiplicity of 0.005 TCID 50 per cell. After a 2 hour adsorption period, the volume is raised 20-fold with RPMI/FCS supplemented with 10% (vol/vol) interleukin 2-containing
  • the cells are seeded in 24-well tissue culture plates plus the "test" metal material additions (2.5 ⁇ 10 5 cells/1.25 ⁇ 10 3 TCID 50 of HIV LAV in a total volume of 1 ml per well).
  • the metal material could be tested in concentrations from 10 -3 M to 10-12 M, although that concentration range should in no way be considered limiting, and according to certain preferred embodiments the concentration could be less than 10 -12 M.
  • the cells are diluted 1:2 in fresh metal material-containing medium. At 6 days, the supernatants are harvested and analyzed for HIV-1p24 and RT activity.
  • the relative numbers of viable cells are determined by the
  • MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] oxidation procedure, which has been shown to correlate well with the trypan blue exclusion assay (Berger et al., AIDS Res. Hum.
  • Relative cell numbers are expressed as percent of the MTT value of control wells that receive an equal number of cells but no virus or metal material.
  • the metal materials could be administered in a concentration range of about 10 -3 M to about 10 -12 M, and according to certain preferred embodiments the concentration could be less than 10 -12 M.
  • RNA/DNA hybrid substrate The RNase H activity of HIV-1 reverse transcriptase was assayed with RNA/DNA hybrid substrate in the presence of K 3 UO 2 F 5 .
  • the RNA/DNA substrate is described in detail in Fig. 3 (Hostomsky et al., Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991).
  • K 3 UO 2 F 5 represents 100% RNase H activity. (See lane 1 of Figure 4). Based on the generated data, 50% inhibition of RNase H activity (IC 50 ) was observed at 50 ⁇ M. (See Figure 5). The protein forms visible precipitation at concentrations above 400 ⁇ M K 3 UO 2 F 5 .
  • the RNase H activity of HIV-1 reverse transcriptase was assayed with RNA/DNA hybrid substrate in the presence of copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt.
  • RNA/DNA substrate is described in detail in Fig. 3 (Hostomsky et al., Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991).

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Abstract

On décrit une quantité suffisante d'un matériau métallique pour inhiber l'activité de RNase H de la transcriptase inverse des virus qui sont transcriptase inverse dépendants. On décrit également des procédés d'inhibition de cette activité par l'exposition de ces virus à une quantité suffisante du matériau métallique.
PCT/US1992/002770 1991-04-03 1992-04-03 Procedes et composes d'inhibition de l'activite de rnase h de la transcriptase inverse WO1992017182A1 (fr)

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US68006891A 1991-04-03 1991-04-03
US680,068 1991-04-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998009622A1 (fr) * 1996-09-03 1998-03-12 University Of Iowa Research Foundation Composes contenant du gallium en tant qu'inhibiteurs de pathogenes intracellulaires
WO2003061579A3 (fr) * 2002-01-18 2003-12-04 Univ Emory Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine
WO2007033444A3 (fr) * 2005-09-21 2007-12-21 Leuven K U Res & Dev Nouveaux traitements antiviraux

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989011277A2 (fr) * 1988-05-23 1989-11-30 Georgia State University Foundation, Inc. Preparations antivirales a base de porphyrine et de phtalocyanine
JPH03264530A (ja) * 1990-03-14 1991-11-25 Tsumura & Co 抗レトロウイルス剤

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989011277A2 (fr) * 1988-05-23 1989-11-30 Georgia State University Foundation, Inc. Preparations antivirales a base de porphyrine et de phtalocyanine
JPH03264530A (ja) * 1990-03-14 1991-11-25 Tsumura & Co 抗レトロウイルス剤

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Patent Abstracts of Japan, vol. 16, no. 71 (C-913)[5114], 21 February 1992; & JP,A,3264530 (AKIHISA KATO) 25 November 1991, see abstract *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998009622A1 (fr) * 1996-09-03 1998-03-12 University Of Iowa Research Foundation Composes contenant du gallium en tant qu'inhibiteurs de pathogenes intracellulaires
US5997912A (en) * 1996-09-03 1999-12-07 University Of Iowa Research Foundation Method for inhibiting growth of P. aeruginosa using gallium-containing compounds
US6203822B1 (en) 1996-09-03 2001-03-20 University Of Iowa Research Foundation Gallium-containing compounds for the treatment of infections caused by intracellular pathogens and pathogens causing chronic pulmonary infection
WO2003061579A3 (fr) * 2002-01-18 2003-12-04 Univ Emory Compositions pharmaceutiques a base de phtalocyanine et de porphyrazine
WO2007033444A3 (fr) * 2005-09-21 2007-12-21 Leuven K U Res & Dev Nouveaux traitements antiviraux
US8193157B2 (en) 2005-09-21 2012-06-05 K.U.Leuven Research & Development Antiviral therapies

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