STS

1
Steroid Sulfatase Inhibitors Potential New
Drugs for the Treatment of Acne and Cancer
Peter Nussbaumer Novartis 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 diseasesacne, androgenetic
  alopecia, hirsutism, cancer (prostate)
• Estrogen-dependent diseasescancer (breast,
  endometrium)

5
Steroid Sulfatase (STS, aryl sulfatase C, E.C.
3.1.6.2)

• 65 kDa 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

IC50 gt 100 µM
EMATE, IC50 53 nM KI 670 nM
Howarth, Purohit, Reed, PotterJ. 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

Side chain
Linker
Aryl sulfamate
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 BMCL 2003, 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 11
  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
gt 100
9
gt 100
50
13
gt 100
6.2
gt 100
gt 100
0.08
Caveat in cellular system substantially less
active
Nussbaumer, Geyl, Horvath, Lehr, Wolff, Billich
BMCL 2003, 13, 3673
36
From Sulfonylureas to Acylsulfonamides as
Reversible STS Inhibitors
37
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 BMCL 2005, 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 cancer was achieved
  with a prototype inhibitor

43
Clinical PoC of an STS Inhibitor in Breast Cancer
M. 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