Fragment Based Approaches to Drugging Proteases · Fragment Based Approaches to Drugging Proteases 4th RSC-BMCS Fragment Based Drug Discovery Meeting STFC Rutherford Appleton Laboratory,

Fragment Based Approaches to Drugging Proteases4th RSC-BMCS Fragment Based Drug Discovery MeetingSTFC Rutherford Appleton Laboratory, Harwell, Oxfordshire UK

Steven J. Taylor

Agenda

01 March 2013PLEASE INSERT Presentation title 2

1. Overview of 3 Fragment Based Strategies the Boehringer Ingelheim Leverages for Identification and Optimization of Chemical Matter

2. Vignettes

• Chymase• MMP-13

3. Summary and Conclusions

Evolution of Fragment Based Drug Discovery at BIPI


2000

-Small Focused group, separate from project

teams -Chemistry and

structural research FTE’s embedded1-2 Projects Max

2013

-No Dedicated FTE’s-Efforts Driven entirely

by project team-All projects that are structurally enabled.

Skeptics Believers

Fragment Hit to LeadThree Possible Strategies

GrowExtend the fragment

hit into adjacent pockets to gain

potency

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LinkJoin adjacent fragment

hits to gain potency

ReplaceExchange regions of

a lead associated with a liability (e.g. PK) with fragment

hit

A fragment hit will generally not be sufficiently potent to be considered a “lead”

A fragment hit having high “ligand efficiency” can be leveraged to drive chemistry using several strategies

Fragment-Based Screening Confidential

Chymase

Chymase as a Target for Heart Failure and Fibrosis

6

• Chymase is a serine protease that catalyzes the peptide cleavage and conversion of angiotensin I to active angiotensin II independent of ACE

• Chymase contributes to heart failure by —Inducing fibrosis through enhancement of collagen and

ECM deposition in key cells—Stimulating remodeling via MMP activation—Activating inflammatory mediators• Chymase inhibitors demonstrated efficacy in heart

failure animal models • Lead ID campaign around the literature compound TPC-

806 identifies a new chemical series and non-covalent inhibitor; specificity against Cathepsin G is desired

Chymase IC50 70 nMCat G IC50 2030 nM

Chymase IC50 22nMCat G IC50 50nM

NN

O

S O

OHNN

S

S

O

OH

Scaffold Hop

Lead has undesirable drug properties


tPSA < 75 tPSA > 75logP > 3 2.4 (85) 0.41 (38)logP < 3 1.1 (27) 0.39 (57)

• tPSA < 75 and logP >3 space is 6-times as likely to have in vivo tox signal @ 10 M.

Odds of in vivo toxicity at 10 M

Crystal structure of Inhibitor bound Chymase

Chymase IC50 70 nMCat G IC50 2030 nMLogP = 4.3tPSA = 64Forms reactive metabolites

NN

O

S O

OH

8

Fragment Hit to Lead Strategy



potency

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hit


•Strong correlation between logP of P1 substituent and potency against the protease.

•Can this trend be disrupted by FBS?

Chymase Fragment Based ScreeningScreening and Hit Triaging Summary

10

NMR80

FA13

SEC-MS46

11

12

2024

770 fragments screened206 Total Fragment Hits

NMR9/35

FA3/10

SEC-MS4/16

4/5

3/7

7/1113/16

95/206 hits screened by X-Ray41/95 fragments yield co-structure

• X-ray success rate is low (~25-30%) when hits unique to a single screening method is pursued.

• Hit confirmation by at least two techniques consistently improves the X-Ray success rate.

• Overlap hits from all the three primary techniques have high probability to yield co-structures and used in prioritizing fragments for X-Ray follow up.Most fragments bind to the S1 ‘hot spot’ site of Chymase

Overlay of fragment structures bound to Chymase

S1

Fragment Hit and Analoging

11

S1

Asp102

His57

Lys192

Val227

Ser214

Arg217

Ser195

NH

O

Cl

Chymase IC50 470 M LE 0.42CatG IC50 > 1000 M

S1

• Polar fragment binds to lipophilic S1 pocket and hosts water mediated interactions to network with protein

• Fragment SAR is used to probe the nature of interactions and the stability of binding mode

Crystal structure of fragment bound Chymase

Overlay of fragment analogs bound Chymase

NH

ClO

Chymase IC50>500 M

12

Structure Based Inhibitor DesignThe “Replace” Approach to Fragment HTL

1.3Å

Ser195

S1

Asp102

His57

Lys40

Lys192

NH

O

Cl

Chymase IC50 470,000 nM

NN

OS O

O

H

Chymase IC50 70 nMCatG IC50 2,030 nM

Ser195S1

Asp102

His57 Lys40

Lys192

2.7Å

3.2Å

• Co-structure overlay of fragment with inhibitor shows 4-position to be most suitable for linking.

• Polar substituent is allowed in the S1 Pocket and binds as deep as the fragment hit.• Selectivity over Cathepsin G is achieved via polar substituent on P1 moiety

NH

O

Cl

NN

O

O

OH

Chymase IC50 3,800 nMCatG IC50 > 10,000 nM

NH

O

Br

NN

O

O

OH

Chymase IC50 50 nMCatG IC50 >10,000 nM

13

Understanding of Unanticipated Cathepsin G Selectivity

• Understanding role of waters in the binding site is key for modulating potency and selectivity

• Gain of selectivity is conferred to the negative interaction with E226 and polar P1 substituent

Overlay of Chymase inhibitor with its calculated water dipole structure from apo

Docking of Chymase inhibitor to CatG and CatG water dipoles

MMP-13



Pierre-Auguste Renoir


MMP-13 as an Rheumatoid Arthritis TargetRationale

• Inhibition of MMP-13 (the most proficient catalyst of collagen II) predicted to reduce cartilage degradation associated with the progression of RA. Reduced inflammation response predicted as a secondary effect.

• MMP-13 associated with osteoclast attachment and maturation on bone surfaces leading to bone erosion.

• MMP-13 implicated in invasion of synovial fibroblast cells.

• Adenovirus over expression of MMP-13 in joints produces an RA-like phenotype.

• MMP-13 -/- mouse shows ~40-50% AbCIA efficacy (Poster report – Takaishi/D’Armiento Groups)

Non-selective MMP programs have failed in the clinic principally due to MSS

LI program goals: Generate two series having required potency, selectivity and drug-like properties. Demonstrate support of drug concept. 18

MMP-13 Structural BiologyProgram Starting Point

Selectivity loop S1´ pocket

S1´ *

CatalyticZinc

N

O

NH

O

NH

O

O O

O

O

NH

O

NH

N

NN FO

OH

Alantos

O

N

NOO

OH

PfizerEX 75,470

PfizerEX 75,484

N

O

NH

O

NH

F F

Aventis1XUD

BI chemistry focused on developing non zinc-binding inhibitors accessing the S1`* pocket of MMP13 to gain selectivity over

Literature crystal structures of both zinc-chelating and non-zinc chelating inhibitors

available

19

Fragment Based ScreeningPrimary Screening Summary

Functional Assay NMR STD Binding SEC MS

Prioritization for Fragment Crystallography

20

Starting points for a medicinal chemistry

optimization campaign

NH

O

O

NH2

O

MMP-13 IC50 = 42 MMMP-14/MMP-2 IC50 = 500/60 MLE = 0.35

MMP-13 Indole series initial FBS “Hit”Co-Structure: Initial SAR

• Low potency and no selectivity, can this be elaborated into potent selective inhibitor?• Can the ester be replaced?• Novel interactions as well as chemical motif and LE make indole fragment an attractive SP

Key Issues & Context

ONH

ONH

OH

ONH

W

ONH

O

-O

N

O

OO

N

H

HH

W

WO

NH

OH

F241

G237

E223

T245

T247 T245

F241 Zn

G237

T247

E228MMP13 Specificity Loop

Co-Structure of MMP-13 with Indole

I. Mugge, A. Padyana, B. Co

Strategy to increase potency of initial fragment hit




potency

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hit

NH

O

O

NH2

O

MMP-13 IC50 = 42 M MMP-13 IC50 = <0.001M

?

Co- Crylstallography provides roadmap for optimization


NH

O

O

NH2

O N

O

NH

O

NH

F F

Are hybrid literature/fragment inhibitors possible?Key Issue: potency & selectivity

NH

N

Hydrogen bonding: opportunities for heterocycles: H20-M253, or T247

N

NH

NN

•Multiple methyl-substituted heterocycles can be used to gain potency and selectivity by accessing the P252 pocket which is specific for MMP-13•Heterocycles as opposed to other linkers, provides a defined trajectory for accessing the S1` * pocket

42°

60°

NH

O

ONH2

O

N

N

NH

O

O

NH

OR

MMP-13 IC50 42 M

65°

MMP-13 IC50 82 M

MMP-13 IC509.7 (+/- 2) M

MMP-13 IC50 56 M

MMP-13 IC503.9 (+/- 3) M

MMP-13 IC502.5 (+/- 0.5) M

P252PocketP252Pocket

<2X selective over MMP-2

>10X selective over MMP-2

Comparison of cores and their effect on potency of elaborated molecules

NH

O

O

NN

NH

ON

NH O

NH

O

O

N

N

NH

O

O

NNH

NH

ON

NH

O

O

NN

OO

O O

NH

O

O

N

NH

O

O

NNH

OO

pyridylMMP-13 IC50 (nM): 2800

imidazoleMMP-13 IC50 (nM): 40

pyrazoleMMP-13 IC50 (nM): 2600

Elaboration

Fragments

Elaborated imidazoleMMP-13 IC50 (nM): 1.9

Elaborated pyridylMMP-13 IC50 (nM): 120

Elaborated pyrazoleMMP-13 IC50 (nM): 12022X improvementover fragment

20X improvementover fragment

21X improvementover fragment,

O

OH

N

THR245

•Structure guided fragment elaboration leads to low nanomolar, potent selective MMP-13 inhibitors•Flexibility in the core heterocycle provides opportunities for adjusting physiochemical

RHS Ester ReplacementsKey Issue: Potency and Microsomal Stability

Ester, although potent presents a potential metabolic alert via oxidative metabolism or plasma esterase activity:

NNNH

O

O

OH NH

R

O

O

Ether 220 nM 75 % Qh

Ester1 nM 80 % Qh

Alcohol2,100 nM 37 % Qh

Unsubstituted26,000 nM 25 % Qh

O

OHAcid1,650 nM <24% Qh

•Replacement of ester with a moiety that retains potency but is stable to esterase activity should significantly increase half life of this series

NNNH

O

O

OH NH

O

O

In Vitro Metabolite ID of Ester

98% of metabolism is ester hydrolysis

O

H

OH

A. Abeywardan

Strategy to remove metabolic liability




potency

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hit

NNNH

O

O

OH NH

O

O

NNNH

O

O

OH NH

RPotent

Low metabolic stabilityPotent

High metabolic stability

Opportunities for ester replacements by Fragment Merging

NH

O

O

N

N

Indole analogMMP13 IC50: 2,500 nM

NNNH

O

O

OH NH

N

HybridMMP13 IC50:<1 nM

N

NNS

DI 603,051 MMP13 IC50: 190 M

A challenge for the team was to remove the “metabolic liability” of the ethyl ester of the original hit.Proof that this could be accomplished was provided by the binding mode of BI 644,577Fragment-Based Screening Confidential

NNNH

O

O

OH NH

N

MMP-13 Potency and Metabolic Stability Strategy Methods to identify an ester replacement

1. Replacement from fragment “merging”

N

NNS

190,000 nM 2,900 nM

0.13 nM

N O

N

6.3 nM

O

N2. Replacement from fragment SAR

<1 nM

NH

NH

RO

0.8 nM

57,000 nM 160,000 nM 150,000 nM

H

>20k nM

>20k nM

•Despite steep SAR, equipotent ester replacements can be identified from fragment merging and from SAR done on fragment starting points, independent of the elaborated molecule

NH

R

NNNH

O

O

OH

NH

O

ON

N

O

O

Co-Structure of DI 603,051 overlaid with Co-structure of BI 661,404

N. Farrow, A. Abeywardane, Z. X

Further optimization of potency


NH

N

NO

NNH

NH

OR

From Fragment Hit to Prospective Lead SeriesElaboration of ALI hit

MMP13 IC50: 42,000 nM (LE 0.35)MMP-2/14 IC50: 77 />500 M

Fragment virtual screening hit (IM)

MMP13 IC50: 120 nM (LE 0.32)MMP-2/14 IC50: >500 M

First fully elaborated fragment that access S1`*

NH

O

O

NH

N

NH

O

O

N

N

MMP13 IC50: 56,000 nM (LE 0.31)MMP-2/14 IC 50: 68/>500 M

Provides defined Trajectory to S1`*

NH

O

O

NH2

O

NH

O

O

NN

NH

ON

MMP13 IC50: 2,500 nM (LE 0.38)MMP-2/14 IC50: >500/>500 M

MMP- 1/8/9 IC50: >500/500/64 MAccesses Pro pocket, provides potency

and selectivity

NH

O

O

NNH

NH

ON

NH

NNH

NH

O

N

NO

O

OHMMP13 IC50: 1.8 nM (LE 0.39)

MMP-2/14 IC50: >250 MCore change increase potency 20x

MMP13 IC5o: 0.27 nM (LE 0.40)MMP-2/14 IC50: >250 M

Bioisosteric ester replacements identified

>150,000 fold potency improvement over starting point - Increased ligand efficiencyFragment-Based Screening Confidential

32

MMP-13 ChemistryComparative Progression of HTS and Fragment Hit series

Compound Count

Low

est

K d(n

M)

Number of Co-StructuresIndole 8HTS Series 2 4IHTS Sereis1 3

HTS Series 1IC50 10µM LE 0.24

HTS Series 1(Best Potency)

IC50: 2.4nM LE: 0.32

HTS Series 2IC50 230nM LE 0.23

HTS Series 2(Best Potency)

IC503nM LE 0.35

NH

O

O

NH2

O

NH

NH

N

NH

O

N

NNO

Fragment SeriesIC50 41µM LE 0.35

Fragment SeriesIC500.45nM LE 0.41


Synthesis of key indole intermediates

01 March 2013Fragment-Based Screening Confidential 34

OO

OO

NH2

NH

NN

OHO

O

NN

BrO

O

POBr3

NH

BO

O

R1

NH

NN

OO

R1NH

NN

NH

OR2

R1

+1. 0 C, 1 h 2. 120 C 72% 50%

1. Pd Catalyst15-75%

NH

OH

O

Br

NH

Br N

NO N

B N

NO

O

O

OON

NH2

OH

1.CDI 96%2. Microwave 98%

1. Pd, pinacol Borane 96%2. (BOC)2O 80 %

N

NHBr

N

NBr

SEM

O

O

N

NN

O

O

ON

NO

O

SEM1. SEM-Cl, 90%2. LDA ethylchloroformate 70%

Pd Catalyst

45-60%

In Vivo Proof of Concept for MMP-13 InhibitionMurine Collagen Induced Arthritis Model

01 March 2013Fragment-Based Screening Confidential 35

NATURE PROTOCOLS |VOL. NO.52007 1269

AbCIA BID Dosing Groups – AbCIA Response

0

2

4

6

8

10

12

14

16

3 4 5 6 7 8 9 10 11 12 13 14

Experiment Day

Mea

n A

rthr

itic

Scor

e (+

/- SE

)

1%CMC, 0.015%Tween 80, 10ml/kg bidEX00075470 BS 1, 100mpk bidBI00644394 SE 3, 100mpk bidBI00644569 BS, 100mpk bidEX00075490 SE 2, 100mpk bid

0

2

4

6

8

10

12

14

16

Top BI compound showed 69% inhibition (Mann-Whitney non-parametric test on AUC).

BI 644,569 dosed from day4,all others dosed

prophylactically Veh

icle

Com

petit

or 1

Com

petit

or 2

Top

BI

Com

poun

d

Ear

ly B

I C

ompo

und

Summary

Fragment based screening and optimization can provide a complementary method to HTS for identifying attractive chemical matter

LE should be tracked and used to help asses progress of an optimization campaign in parallel to potency and physicochemical properties

It is important to keep a focus on the Patients and why we as Scientists got into this business, after all we are saving and improving lives of those with few options.


Acknowledgements

Medicinal ChemistryChuck CywinAmy GaoDan GoldbergAlexander Heim-RietherKen MeyersNeil MossAnthony ProkopowiczLana Keenan-SmithHidenori TakahashiZhaoming XiongYang YuMichael Zhang

Fragment Based ScreeningAsitha AbeywardaneBrandon CollinsSandy FarmerKathy HavertyXiang LiShuang LiangAnil PadyanaJohn ProudfootSteven Taylor

Inflammation and ImmunologyLaura AmodeoJun LiJerry NaboznyMark PanzenbeckDon SouzaJohn Xiang LiLily Zuvela-Jelaska

Drug Discovery SupportWalt CaoRyan FryerPaul HarrisonSuzanne-Nodop MazurekRaj NagarajaHani Zaher

High Throiughput ChemistryJuergen MackDieter WiedenmeyerBernd Wellenzohn

Structural Research Ingo MüggeQiang Zhang

ToxicologyRay KemperJames Tarca

38

Backup Slides


Difference between Fragment hits and HTS hits VS Drugs

01 March 2013Nature Reviews Drug Discovery 3, 660-672 (August 2004) 40

HTS Hit

Fragment Hit

Lower MW fragment hits provide more room for SAR optimization

Fragment Based ScreeningComparison to uHTS

Typical uHTS campaigns screen 106 drug like compounds against a targetTypically, hits are defined as having IC50s of < 20 M

Typical Fragment based campaigns run 103-104 MW <270 compounds against a target Typically, hits are defined as having IC50s in the M - mM range

Makeup of Screening Libraries

HTS Library

A collection on ~1 million compounds collected from multiple sources MW ≤ 700 purity > 50%

Generic Fragment Library

A collection of ~1,500 highly characterized compounds satisfyingMW ≤ 270; Nacc ≤ 3; Nrot ≤ 4; Nfused_rings ≤ 3; 3/2.75 ≤ X/ClogP ≥ 0;

N ≤ 2Fragment-Based Screening Confidential

Ligand Efficiency Definition

Ligand Efficiency is a measure of how effective a compound is at binding its target.

Generally, it is possible to increase binding by increasing the MW of a compound – however Lipinski’s Rules shows that a desirable MW for a drug is < 500

Ideally a HTL program would like to start with a small highly potent compound – a compound with high Ligand Efficiency (LE)

LE = -RT log(IC50)/N

N = Number of heavy atoms (rule of thumb N = MW/13.1)

The nature of fragments is such that even though they bind in the mM-M range they are highly ligand efficient as a result of their low MW

A Fragment Hit to Lead (FHTL) program will seek to increase potency and build in other properties (e.g. selectivity) while maintaining LE.


Documents

Fragment Based Approaches to Drugging Proteases · Fragment Based Approaches to Drugging Proteases 4th RSC-BMCS Fragment Based Drug Discovery Meeting STFC Rutherford Appleton Laboratory,