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Steroid Sulfatase Inhibitors: Potential New Drugs for the Treatment of Acne and Cancer

Steroid Sulfatase Inhibitors: Potential New Drugs for the Treatment of Acne and Cancer. Peter Nussbaumer Novartis Institutes for BioMedical Research Vienna. Agenda. Introduction Steroid sulfatase pathway Potential indications for inhibitors Enzyme characteristics

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Steroid Sulfatase Inhibitors: Potential New Drugs for the Treatment of Acne and Cancer

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  1. Steroid Sulfatase Inhibitors: Potential New Drugs for the Treatment of Acne and Cancer Peter NussbaumerNovartis Institutes for BioMedical Research Vienna

  2. Agenda • Introduction • Steroid sulfatase pathway • Potential indications for inhibitors • Enzyme characteristics • Approaches to STS Inhibition (IC50, rIC50, KI) • Irreversible inhibitors and associated issues • Reversible inhibitors: discovery and optimisation • Clinical Proof of Concept

  3. Production of Androgens and Estrogens in Peripheral Tissues post menopause tumor growth ER sebum production tumor growth AR

  4. Potential Indications for STS Inhibitors • Androgen-dependent diseases:acne, androgenetic alopecia, hirsutism, cancer (prostate) • Estrogen-dependent diseases:cancer (breast, endometrium)

  5. Steroid Sulfatase(STS, aryl sulfatase C, E.C. 3.1.6.2): • 65kDa membrane-bound (ER) protein • catalyzes hydrolysis of steroid sulfates (e.g., estrone, DHEA) • ca. 30 % homology to aryl sulfatase A and B (structures solved by X-ray), but different substrates • 3D structure only available since 2004 • over-expressed in breast tumours, acne lesions, dermal papilla • features unusual AA oxoalanine (posttranslational modification of cysteine) in the active site

  6. Approaches to STS Inhibition • transition state analogues:not feasible because of trigonal-bipyramidal transition state • substrate analogues • active-site directed inhibition • irreversible inhibitors • novel types of inhibitors by rational designby HTScreening • structure-based design (X-ray, homology modeling) oxo-Ala essential for catalysis X-ray: sulfate is bound

  7. 3D Structure of Steroid Sulfatase active site membrane anchor Hernandez-Guzman, Higashiyama, Pangborn, Osawa, Ghosh: J. Biol. Chem. 2003, 278, 22989

  8. Active Site of STS with Docked Substrate

  9. Profiling of STS Inhibitors Primary Screen:IC50 on purified human STS All inhibitors: IC50 in CHO cells over-expressing STS Potential candidates:STS inhibition in: fibroblasts, keratinocytes, monocytes; human skin homogenate Specificity testing: arylsulfatase A and B; species selectivity; HaCaT cell proliferation Stability & solubility Penetration studies: pig and human skin Development candidate: In vivo testing in pig: STS inhibition Pharmacokinetic studies after topical application (skin and systemic levels)

  10. Substrate-Based Inhibitors • non-cleavable substrate analogues • active site-directed inactivation IC50= 0.17 - 52 µM IC50= 75 µM Nussbaumer, Billich: Med. Res. Rev. 2004, 24 (4), 529

  11. Active Site-Directed Inhibition • aryl sulfamates as irreversible inhibitors EMATE, IC50= 53 nM KI = 670 nM IC50> 100 µM Howarth, Purohit, Reed, Potter:J. Med. Chem. 1994, 37, 219 • other functional groups do not work, except: aryl formates IC50= 420 nM Schreiner, Billich: BMCL 2004, 14, 4999

  12. EMATE as Lead Molecule Woo, Purohit, Reed, Potter: J. Med. Chem. 1996, 39, 1349 Woo, Purohit, Malini, Reed, Potter: Chem. Biol.2000, 7, 773 Li, Milano, Kluth, Rhodes: J. Steroid Biochem. Mol. Biol.1996, 59, 41

  13. (Thio)Chromenone-BasedInhibitors

  14. (Thio)Chromenone-Based Inhibitors Nussbaumer, Lehr, Billich: J. Med. Chem. 2002, 45, 4310

  15. Synthesis of 6-Hydroxychromenones

  16. Synthesis of (Thio)Chromenones and Sulfamates

  17. SAR for Non-Steroidal Aryl Sulfamates: Aryl sulfamate Linker Side chain no substitution allowed optional space aryl IC50 = 0.3 nM bulky aliphatic group linker should contain hetero atom,bicyclic ring structures preferred Nussbaumer, Billich: Med. Res. Rev.2004, 24 (4), 529

  18. Inhibition of DHEAS Metabolism by STS Inhibitor Metabolism of DHEAS in human skin in vitro blue trace: no inhibitor added red trace: incubation in the presence of 10 nM inhibitor

  19. Inhibition of STS: Problem Solved? Remaining issues: • Estrogenicity • EMATE has strong estrogenic activity in vivo • Aryl sulfamates are produgs of phenols • Phenols have to be considered as well • Clastogenic potential • Chemical instability

  20. Estrogenicity Issue Solved! non-estrogenic arylsulfamates + phenols: 2-methoxy EMATE C17-subst. E2 sulfamates 667COUMATE Nussbaumer, Winiski, Billich: J. Med. Chem. 2003, 46, 5091

  21. SAR for Estrogenicity of Chromenone Sulfamates Nussbaumer, Winiski, Billich: J. Med. Chem. 2003, 46, 5091

  22. Inhibition of STS: Problem Solved? Remaining issues: • Estrogenicity • EMATE has strong estrogenic activity in vivo • Aryl sulfamates are produgs of phenols • Phenols have to be considered as well • Clastogenic potential • Chemical instability

  23. Micronucleus Test Screen of Sulfamates Clastogenic Weakly clastogenic Clean VAC527

  24. Synthesis & X-ray Structure of VAC527 Overall yield: 45% No chromatographic purification involved Schreiner, Billich: BMCL2003, 13, 4313

  25. VAC527 – In Vitro and In Vivo Profile

  26. In Vivo Activity after Topical Application to Pigs inhibition of STS activity in skin at 6 hrs post treatment skin concentration of inhibitor Billich, Meingassner, Desrayaud, Nussbaumer, Lam, Schreiner: J. Steroid Biochem. Mol. Biol.2004, 92, 29

  27. VAC527 Induces Atrophy of the Sebaceous Gland Minipig; topical treatment Dosage: 1% in isopropanol/propylene glycol 1:1 for 2 weeks Control animal (treated with placebo) Animal treated with VAC527 VAC527 was well tolerated and did not cause treatment-related histopathological findings in other organs.

  28. Stability Profile of VAC527 • VAC527 was abandoned due to insufficient stability for standard topical development!

  29. Inhibition of STS: Problem Solved? Remaining issues: • Estrogenicity • EMATE has strong estrogenic activity in vivo • Aryl sulfamates are produgs of phenols • Phenols have to be considered as well • Clastogenic potential • Chemical instability

  30. Chemical (In)Stability of Aryl sulfamates • Aryl sulfamates are stable in the solid state • In solution aryl sulfamates are degraded to the corresponding phenols: T1/2= 24 hrs to 8 days at pH 7.5 / 37 °C • Limited stability in polar solvents, e.g. DMSO, PEG 400 • Susceptibility to hydrolysis is an inherent property of the aryl sulfamates • No correlation between chemical reactivity (hydrolysis) and enzyme inhibitory activity

  31. Chemical Stability of Aryl Sulfamates Relative inhibitory activity of test compounds against STS vs. rate of hydrolysis

  32. Search for Non-sulfamate Inhibitors“Rational approach“ → stable inhibitors lead Potent, stable, reversible STS inhibitor: purified STS: Ki = 0.5 µM ... but only poorly active in cells! Horvath et al.: J. Med. Chem. 2004, 47, 4268

  33. Reversible STS Inhibitors from (HT)Screening

  34. Sulfonylureas as Reversible Inhibitors High-througput screening hit:

  35. SAR of Sulfonylurea-Type Inhibitors IC50 = 0.9 µM IC50 [µM] 16.6 > 100 9 > 100 50 13 > 100 6.2 > 100 > 100 0.08 Caveat: in cellular system substantially less active Nussbaumer, Geyl, Horvath, Lehr, Wolff, Billich: BMCL2003, 13, 3673

  36. From Sulfonylureas to Acylsulfonamides as Reversible STS Inhibitors

  37. 1000 500 starting point - 154 100 50 10 5 - 2.4 • lipophilic meta-substituentspreferred • acids with 1,4 orientation preferred Parallel Synthesis to Optimize the Aryl Moiety and to Evaluate 3 Central Scaffolds Ki = 16.1 µM rIC50 Ki = 0.22 µM Lehr, Billich, Wolff, Nussbaumer: BMCL2005, 15, 1235

  38. Further Refinement of the Central ScaffoldIsonipecotic, 4-piperidinylacetic, 4-piperidinylenacetic acid-type R2 R1

  39. Highly Potent, Reversible STS Inhibitors – ProfilesSelected compounds Superior in human Skin! VAC624

  40. Profile of VAC624  Clinical Candidate

  41. Synthesis of VAC624 Overall yield: 40%

  42. Summary • Based on EMATE as lead, we discovered highly potent, non-estrogenic, irreversible STS blockers featuring a (thio)chromenone scaffold, but weak clastogenicity is an unacceptable safety risk • Benzoxazole-type inhibitors (VAC527) were found to be non-clastogenic and sufficiently potent in vivo, but the intrinsic instability of aryl sulfamates in topical formulations was a “no go” for further development • HTScreen delivered lead-like N-sulfonyl ureas as reversible inhibitors • Scaffold morphing and subsequent optimisation led to the discovery of a clinical candidate (VAC624) for the indication acne • While clinical PoC for the indication acne is still pending, PoC in breast cancerwas achieved with a prototype inhibitor

  43. Clinical PoC of an STS Inhibitor in Breast CancerM. Reed et al: Clin. Cancer Res. 2006, 12, 1585 • PhI: STX64 (= 667COUMATE) • 9 (5mg) + 5 (20 mg) patients (postmenopausal women with BC) • Preliminary results: • well tolerated, only minor drug-related adverse effects • 98 % inhibition of STS in PBLs 99 % inhibition of STS in breast tumor tissue • significant reduction serum levels of estrone, estradiol, androstenediol, and DHEA • clinical evidence of stable disease in 4 subjects who previously progressed on aromatase inhibitor treatment

  44. Acknowledgements Chemistry: A. Horvath, P. Lehr, P. Nussbaumer, E. Schreiner Biology: A. Billich, J. Meingassner, A. Winiski, B. Wolff-Winiski Modeling: A. Aszodi, A.Berces

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