WO2008156363A1

WO2008156363A1 - Selective inhibitors of the nuclear factor of activated t-cells

Info

Publication number: WO2008156363A1
Application number: PCT/NL2008/050402
Authority: WO
Inventors: Haixiang Yu; Erik Anna Leonardus Biessen; Theodorus Josephus Cornelis Van Berkel; Karen Sliedregt-Bol; Herman Steven Overkleeft
Original assignee: Stichting Voor De Technische Wetenschappen; Universiteit Leiden
Priority date: 2007-06-19
Filing date: 2008-06-19
Publication date: 2008-12-24
Also published as: NL1034004C2

Abstract

It has been found to inhibit NFAT by providing a dually active compound comprising a side which is capable of binding to cysteine (possibly Cys 266) within the catalytic domain of calcine urin, and a side which is capable of binding to the docking site for the peptide H2N-MAGPHPVIVITGPHEE- COOH in calcineurin. Preferably, these are conjugates of (a) a peptide of the VTVIT type and (b) an INCA compound, at least an inhibitor of the association between NFAT and calcineurin. The conjugates found can be used in the manufacture or the development of medicinal products for the treatment of inflammations.

Description

Title: SELECTIVE INHIBITORS OF THE NUCLEAR FACTOR OF ACTIVATED T-CELLS

Field of the Invention

The invention relates to a concept for inhibiting the nuclear factor of activated T-cells, typically referred to by the abbreviation NFAT (Nuclear Factor of Activated T-cells), and to NFAT inhibitors. The invention further relates to pharmaceutical compositions comprising such inhibitors, and to the use thereof in manufacturing medicinal products for the treatment of inflammations and cardiovascular disorders.

Background of the Invention

The calcineurin/NFAT cascade coordinates inflammatory responses in various inflammatory cells and cells involved in cardiovascular disorders and is as such an attractive candidate for intervention in pathological immune responses. Thus, various inhibitors of this cascade, including classic immunosuppressives such as cyclosporine A and FK506, currently- used on a large scale in clinics to prevent rejection responses after transplantation, and for the treatment of chronic inflammatory responses and autoimmune diseases. The current generation of immunosuppressives is particularly directed to calcineurin activity rather than NFAT, which results in NFAT- independent processes being affected as well. This results in a range of side effects, which, according to current insight, would not take place with selective inhibition of NFAT. One class of NFAT inhibitors are the so-called INCA compounds, INCA being the abbreviation of Inhibitor of the NFAT-Calcineurin Association. See for instance Kang.S., Li₁H-, Rao,A. & Hogan,P.G. J. Biol. Chem. 280, 37698-37706 (2005) and Roehrl,M.H. et al. Proc. Natl. Acad. Sd. U. S. A 101, 7554-7559 (2004).

Another class of NFAT inhibitors are VIVIT peptides and analogs (here, VTVIT stands for the amino acid sequence Valine - Isoleucine — Valine - Isoleucine - Threonine). See for instance Aramburu.J. et al. Science 285, 2129-2133 (1999), Yu₁H. et al. Arterioscler. Thromb. Vase. Biol. 26, 1531-1537 (2006), and Huiming Ld et al, JMB 2004 1659. Both classes of NFAT inhibitors have the property to inhibit the interaction of NFAT with calcine urin, but are insufficiently potent to qualify for therapeutic use. In addition, the INCA compounds have the tendency to be too highly toxic for therapeutic application.

Summary of the Invention

In one aspect, the invention contemplates providing a new concept for inhibiting NFAT. In another aspect, the invention contemplates providing NFAT inhibitors which are more potent than the INCA compounds and VTVIT analogs, and with less toxicity than the INCA compounds. More in particular, in a further aspect, the invention contemplates providing NFAT inhibitors which are not only more potent, but also more selective. The invention also contemplates providing NFAT inhibitors which retain their inhibitory activity for a longer period of time after administration.

In order to provide one or more of these objects, the invention is based on a new concept, based on the insight to create a dual compound which is capable of simultaneously binding to two calcineurin docking sites. It has now surprisingly been found that this insight enables the creation of NFAT inhibitors which are not toxic, and which selectively inhibit NFAT with low nanomolar potency, without affecting calcineurin phosphatase activity.

The invention further relates to the use of such dual compounds as lead compounds for the development of medicinal products which suppress the immune system. The invention also relates to an assay for determining whether compounds are suitable as selective NFAT inhibitors, where it is determined whether the compound binds to the two calcineurin docking sites involved, and the use of this assay for finding suitable substances.

More in particular, the invention relates to NFAT inhibitors comprising a conjugate of (a) a peptide comprising the amino acid sequence

HPxIyIT, in which H stands for histidine, P for proline, I for isoleucine, T for threonine, and x and y each independently stands for an amino acid, and

(b) an INCA compound. Preferably, x and y are both valine, and more preferably the peptide satisfies the structural formula H2N-MAGPHPVIVITGPHEE-COOH, in which the one-letter abbreviations of the other amino acids involved stand for methionine (M), alanine (A), glycine (G), and glutamic acid (E).

More preferably, the VTVIT peptide is bound to the INCA compound via the proline which is directly linked to the VIVIT portion. This link preferably goes via a spacer, which preferably has a length of about 4 to about 55 angstrom, and more preferably of about 8 to about 28 angstrom. The conjugates according to the invention can be included in pharmaceutical compositions. More in particular, the conjugates according to the invention can be used in the manufacture of medicinal products for the treatment of disorders which benefit from inhibition of NFAT, and more in particular inhibition of the interaction between NFAT and calcineurin. Preferably, the conjugates according to the invention are used in the treatment of inflammations, more in particular of chronic inflammatory responses and autoimmune diseases, of rejection symptoms, more in particular after organ transplantation, and in the treatment of cardiovascular disorders, more in particular cardiovascular hypertrophy, heart failure, restenosis, and with vein-graft disease.

Detailed Description of the Invention

The dual inhibitors

While theory should not be considered binding herein, the inventors are convinced that the key to the effective NFAT inhibition resides in the recognition that a cysteine (possibly Cys 266) is adjacent to the VIVIT docking site within the catalytic domain of calcineurin, which possibly covalently binds with INCA compounds. It is part of the invention that this insight is translated into the possibility to create compounds which are dually capable of binding to the above-mentioned two different docking sites. In this connection, the invention relates to an NFAT inhibitor comprising a dually active compound, comprising a side capable of binding to a thiol group within the catalytic domain of calcineurin, and more in particular to cysteine 266 within the catalytic domain of calcineurin, and a side capable of binding to the docking site for the peptide H2N-MAGPHPVIVITGPHEE-COOH in calcineurin.

Insofar as such compounds in themselves do not already have the suitability to serve for therapeutic use, these compounds are at least suitable as lead compounds for the development of medicinal products inhibiting NFAT, and more in particular suppressing the immune system. In this connection, the invention also relates to an assay for determining whether the compounds are suitable as selective NFAT inhibitors, where it is determined whether the compound binds to the two calcineurin docking sites involved. This assay may, for instance, be carried out as a combination of assays known in the field for each of the two bindings. The invention also relates to compounds found with this assay.

Such dual compounds which are, according to the invention, in themselves already suitable as a therapeutic, and which are preferred, are conjugates comprising at least the following structural portions to be discussed in more detail: a VIVIT peptide, optionally a spacer and a reactive unit of the INCA type (INCA group for short).

The VWIT peptide

The VIVIT peptide, and analogs thereof, are typically peptides of 7 to 16 amino acids, which in any case comprise the heptapeptide portion

HPxIyIT, in which x and y each separately stand for an amino acid. On the basis of current insight, this is the domain of the "VIVIT" peptide essential for the NFAT/calcineurin interaction. Preferably, the VIVIT peptide actually comprises a VTVIT sequence, i.e. in above-mentioned heptapeptide, x and y then stand for valine.

The VIVIT peptide may be limited to above-mentioned heptapeptide, but may also contain one or more further amino acids on one or both binding sides thereof. Preferably, the heptapeptide is part of a peptide with 8 to 20 amino acids. For x and y, it is preferred to choose substantially aliphatic amino acids, such as Alanine, Valine, Isoleucine, Leucine or an amino acid variant or mimetic with an aliphatic side group.

More preferably, the peptide also satisfies the structural formula H2N-MAGPHPVIVITGPHEE-COOH.

Instead of only traditional amino acid moieties, the VTVIT peptide may also comprise peptidomimetics in the peptide backbone. They then preferably have the same side groups as the above-mentioned amino acids, irrespective of the nature of the backbone. D-peptides or retro-inverβo peptides may be used as well (where, for the retro-inverso peptide, the INCA group on the C-terminal side is to be conjugated for the sake of the correct spatial orientation). The conjugation with the INCA group is preferably via an amino acid, more in particular proline, directly linked with the VIVIT portion of the peptide.

The spacer

The spacer is a bond, or an essentially pharmacologically inactive, flexible group or chain.

Preferably, the spacer is an essentially pharmacologically inactive, flexible group comprising a chain of 4 to 40 atoms which can be seen as a backbone.

The term "essentially pharmacologically inactive" means that the spacer comprises no atoms or groups which display pharmacological activity in themselves in the dose in which the conjugates according to the invention are used. As a result, with therapeutic use of the conjugates, the spacer will not cause any perceptible pharmacological side effects.

The spacer may either comprise stiff elements or partly or wholly consist of a flexible chain. The chain may be an aliphatic hydrocarbon, linear or branched, optionally comprising one or more cycloaliphatic or aromatic groups. The spacer may comprise heteroatoms such as oxygen, nitrogen, sulfur.

Although the spacer is preferably essentially pharmacologically inactive, according to the invention, a structural part of the spacer can contribute directly or indirectly to the calcineurin binding of the conjugate. Without wishing to be bound to this theory, it is possible that such a contribution originates with an aromatic group, more in particular a phenyl group, in the spacer, particularly on the side of the INCA group.

For an, according to current insight, optimal action of both the INCA portion and the VTVIT portion of the conjugate, the spacer preferably has a length of about 4 to about 55 angstrom, more preferably of about 8 to about 28 angstrom, and still more preferably around 11 to 15 angstrom. Such a spacer length is suitably realized by, for instance, a phenylbυtyl structure as shown in the formula below.

Structures which are preferred as spacers are, for instance, phenylbutyl, preferably 4-phenylbutyl, or cyclohexylbutyl, preferably 4-cyclohexylbutyl. There are no insuperable difficulties for an average skilled person to provide other suitable spacers, preferably with a similar length.

The INCA group

It will be readily apparent to a skilled person that, as an INCA group, a structure is chosen which has the property to inhibit the association or interaction between calcineurin and NFAT in the calcineurin/NFAT cascade, but not by interaction with the VIVIT binding domain.

The INCA group may comprise a wide range of structures, which have in common that the compound is capable of forming a covalent bond with a thiol group in calcineurin, more in particular the thiol group Cys 266 of calcineurin, the point of binding for INCA compounds according to current insight. Examples of INCA compounds are known. Such compounds typically bind via a maleimide group, but other groups such as iodoacetamide and derivates of maleimide are suitable as well.

INCA groups which are preferred are, for instance, provided by the INCA compounds described in Kang.S., Li₁H., Rao.A. & Hogan.P.G. J. Biol. Chem. 280, 37698-37706 (2005).

The invention is expressly not limited to these examples. The conjugates according to the invention can be manufactured in a manner known per se for the manufacture of peptide conjugates. Optionally, after being made suitable for conjugation in a pre-step, the polypeptide is allowed to react with the INCA compound, linked to the spacer. General synthetic methods for the manufacture of bioconjugates are described in "Bioconjugate Techniques" by Greg T. Hermanson, 1996, Academic Press. The invention further relates to the use of the above-described dual compounds in the treatment of disorders which benefit from inhibition of NFAT, and more in particular inhibition of the interaction between NFAT and calcineurin. More in particular, the invention relates to treatment methods of inflammations and cardiovascular disorders where an effective dose of a dual NFAT inhibitor as described hereinabove is administered to a patient who needs it. At least, the invention relates to the use of dual NFAT inhibitors as described hereinabove in the manufacture of medicinal products for the purpose of such a treatment method.

The dose of the NFAT inhibitors according to the invention can be determined in manners known in the field to determine effective doses and will preferably be between 0.0001 and 1 mg per kg of body weight, more preferably 0.001 -0.1 mg per kg of body weight.

For use as a medicinal product, the above-described dual NFAT inhibitors may be provided in a usual manner in a form suitable for administration as a medicinal product. Pharmaceutical administration routes, such as oral, mucosal, intravenous, transdermal, subcutaneous, rectal, nasal, pulmonary, intravaginal, etc. are known and require no further explanation. For such administration routes, dosage forms are known, such as tablets, capsules, injection preparations, suppositories, implants, band-aids, ointments and oils, vaporizers, vaginal rings, etcetera, which require no further explanation either in the framework of the invention. In such dosage forms, common non-effective additives are included such as carrier materials, diluents, fillers, lubricants, lubricating jellies, disintegration agents, adhesives, stabilizers, antioxidants, anti- discoloring agents, etcetera. In order to manufacture suitable pharmaceutical composition, a skilled person has many handbooks at his disposal, for instance Gennaro et al., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.

The invention will be explained hereinafter on the basis of the following examples and Figures not delimiting the invention.

Description of the Figure Fig. 1 shows a diagram in which a standard luciferase activity determination (vertical axis) is plotted against a number of conjugates (horizontal axis).

Example 1

Synthesis of conjugates

A core sequence FmocHN His(Trt)-Pro-Val-Ile-Val-Ile-Thr(tBu)-Wang resin is manually synthesized with the aid of standard Fmoc chemistry.

Preloaded Fmoc-Thr(tBtu)-Wang resin (load 0.6 mmol/g, Novabiochem) is deprotected and linked with other amino acids in the presence of HOBt(I- hydroxybenzotriazole)/TBTU2-(lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium tetrafluoroborate)/Dipea (4, 4, and 8 eq., respectively). After linking, the resin is washed with DMF, iso-propanol, methanol and DCM₁ and then dried. The N-terminal Fmoc group of sequence 2 is removed by means of 20% piperidine in DMF. After washing with NMP, 3-MBA-OSu is added (4eq, for the synthesis of MCV2) or SMPB (4eq, for the synthesis of MCVl) together with DIPEA (8eq), and the reaction is monitored by means of the Kaiser test until a negative signal is obtained. The resin is washed with DMF, iso-propanol, methanol and DCM and dried. After removal of the solvent, the INCA conjugates are isolated from the resin by means of a mixture of trifluoroacetic acid, triisopropylsilane, and water (95:2.5:2.5, v/v/v). The crude peptides are purified on a preparative Ciβ RP-HPLC column (All tech). The purified peptides are characterized by means of LC- MS and are found to be at least 70% pure. Each sample is dissolved in anhydrous dimethyl sulfoxide (DMSO) and stored at -20⁰C. Peptides and results

Name Peptide NFAT inhibition

L-Hl HPVIVIT -H-

D-Hl HPVIVIT +

RD-H l TΓVTVPH -H-

L-MCVl MPB-HPVIVIT I I I I I

D-MCVl MPB-HPVIVIT not determined

RD-MCVl MPB-TIVIVPH not determined

RD-MCVIl TIVIVK-MPB not determined

RD-MCV12 TIVIVPK-MPB not determined

RD-MCV13 TIVIVPK-MBA not determined

Determination

See Figure 1. RAW 264.7 macrophages are temporarily transfected with two plasmids containing the gene for firefly luciferase under control of an NFAT activity-sensitive promoter (pNFAT-luc) and for an inert reference luciferase (Renilla luciferase; pRL-TK), respectively, are stimulated for 12 hours with PMA (200 nmol/L) and ionomycin (500 nmol/L) for 12 hours, in the presence or absence of FK506, HPVIVIT, whole D-HPVIVIT or retro-inverso-whole D- HPVIVIT (D-TIVIVPH) at the indicated concentrations. Cell lysates are prepared en tested for dual luciferase activity. Firefly luciferase activity is normalized for Renilla luciferase activity. The values shown represent average SD of a threefold experiment (* P<0.05, ** P<0.01 compared to

PMA/ionomycin-stimulated cells). All D-HPVIVIT conjugates selectively and potently inhibit the calcineurin-mediated NFAT activity.

Claims

1. An inhibitor of the nuclear factor of activated T -cells, abbreviated NFAT, comprising a dually active compound, comprising a side which is capable of binding to cysteine 266 within the catalytic domain of calcineurin, and a side which is capable of binding to the docking site for the peptide H2N-MAGPHPVIVITGPHEE-COOH in calcineurin.

2. An NFAT inhibitor according to claim 1, comprising a conjugate of (a) a peptide of the VIVIT type and (b) an INCA group, at least an inhibitor of the association between NFAT and calcineurin.

3. An NFAT inhibitor according to claim 1 or 2, wherein the peptide comprises 8 to 20 amino acids.

4. An NFAT inhibitor according to any one of the preceding claims, wherein the VTVlT peptide comprises the heptapeptide portion HPxIyIT.

5. An NFAT inhibitor according to claim 4, wherein x and y each independently is a substantially aliphatic amino acid, such as alanine, valine, isoleucine, leucine, or an amino acid variant or mimetic with an aliphatic side group.

6. An NFAT inhibitor according to claim 5, wherein the peptide satisfies the structural formula H2N-MAGPHPVTVITGPHEE-COOH.

7. An NFAT inhibitor according to any one of the preceding claims, wherein the INCA group comprises a functional group chosen from the group consisting of maleimide, derivates of maleimide, and iodoacetamide.

8. An NFAT inhibitor according to claim 7, wherein the INCA group is an INCA compound 1, 2, 6 or 12 in accordance with the introduction to the specification.

9. An NFAT inhibitor according to any one of claims 2-9, wherein the peptide is linked with the INCA group via the proline directly bound to the

VrVIT portion.

10. An NFAT inhibitor according to any one of the preceding claims, wherein, between the peptide and the INCA compound, a spacer is present, preferably with a length of about 4 to about 55 angstrom.

11. An NFAT inhibitor according to claim 10, wherein the spacer is phenylbutyl, preferably 4-phenylbutyl, or cyclohexylbutyl, preferably 4- cyclohexylbutyl.

12. An assay for determining whether a compound is suitable as a selective NFAT inhibitor, wherein it is determined whether the compound binds to both cysteine 266 within the catalytic domain of calcine urin, and the docking site for the peptide H2N-MAGPHPVIVITGPHEE-COOH in calcineurin.

13. Compounds found by means of use of an assay according to claim 12.

14. Use of an NFAT inhibitor according to any one of claims 1-11 in the manufacture of a medicinal product for the treatment of inflammations, more in particular of chronic inflammatory responses and autoimmune diseases, of rejection symptoms, more in particular after organ transplantation, and in the treatment of cardiovascular disorders, more in particular cardiovascular hypertrophy, heart failure, restenosis, and with vein-graft disease.