fragments for drug discovery and chemical biology

1

Fragments for drug discovery and chemical biology

Rod Hubbard

YSBL, York,

Vernalis, Cambridge

Warwick, Sep 2016

For slides – [email protected]

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Finding small molecule hits

• Trying to find compounds that bind to target

• Compounds need to have required shape and chemistry

• High Throughput Screening

• Compounds decorated in the wrong way

• Particularly a problem with new target classes

Target

Hit

3

• Hits from fragments

• Find small parts that bind – usually biophysical screen

• Then grow or merge fragments to create hit compound

• Usually structure-guided design

Why fragments?

Hit

Target

Screen Structure

guided

design

4

Why fragments?

• Hits from fragments

• Find small parts that bind

• Then grow or merge fragments to create hit compound

• Can also generate ideas

• Deconstruct other hits to optimise key interaction motifs

• Suggest interactions to exploit in hit / lead optimisation

• Scaffold hopping

Hit

Target

5

Overview

• Requirements for fragment-based discovery

• Examples of drug discovery • Hsp90

• Examples of chemical biology • Using fragments to explore binding

• Enzyme activators

• Probing the bacterial replisome

Warwick, Sep 2016

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Why are fragments different?

• A fragment is just a small weak hit

• Requires assay(s) that can detect binding reliably

• Methods for evolving fragments (libraries and/or design)

• Design of library includes constraints of assay / evolution

Affinity

10mM 1mM 100mM 10mM 1mM

Fragments MW 110-250

Scaffolds MW 250-350

Lead Compounds

Hit Compound MW 250-500

Warwick, Sep 2016

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Screening fragment libraries

• Different experimental approaches have different strengths and limitations

Warwick, Sep 2016

Affinity 10mM 1mM 100mM 10mM 1mM

Fragments MW 110-250

Scaffolds MW 250-350

Lead Compounds

X-Ray crystallography

Ligand-observed NMR

Surface Plasmon Resonance (SPR)

Enzyme / binding assays (HCS)

Isothermal Titration Calorimetry (ITC)

Hit Compound MW 250-500

Protein-observed NMR

Differential scanning fluorimetry (DSF / TSA)

Hubbard & Murray (2011), Meth Enzym, 493: 509; Meiby et al (2013) Anal Chem 85: 6756

Mass spectrometry (MS)

Weak Affinity Chromatography (WAC)

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Optimise fragment Fragment to hit : SAR by catalog off-rate screening

-5

0

5

10

15

20

25

30

-50 0 50 100 150 200 250 300

RU

Resp

on

se

Tim e s

Cycle: 95 VER-00082099i 50 nM

Fitted Cycle: 95 VER-00082099i

Cycle: 96 VER-00082099i 500 n

Fitted Cycle: 96 VER-00082099i

Cycle: 97 VER-00082099i 5000

Fitted Cycle: 97 VER-00082099i -4

-2

0

2

4

6

8

-100 -50 0 50 100 150 200 250 300

RU

Resp

on

se

Tim e s

Cycle: 103 VER-00055030l 50 n

Fitted Cycle: 103 VER-00055030l

Cycle: 104 VER-00055030l 500


Cycle: 105 VER-00055030l 5000


Characterisation

X-ray or NMR guided model

The process

Warwick, Sep 2016

Hubbard et al (2007), Curr Topics Med Chem, 7: 1568 Hubbard and Murray (2011), Methods Enzym, 493: 509

Target

Optimise fragment

Hits

Competitive NMR screen Fragment Library

~ 1500 compounds Ave MW 190

Design, Build & Test

N

N SNH2

O

NH

Cl

Cl

ON

NO

OH

OH

O

NH

N O

NN

NH2

OOMe

NN

NH2

SNH

O

N

N

N

NH2

Cl

ClN

SNNH2

NH2

N

NH2

O

OEt

Virtual screen; literature; library screen Screen by SPR, DSF, WAC,

biochem assay, Xray

Drug?

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Finding fragments

• Finding fragments that bind is not difficult • A good way of assessing target “ligandability”

• Low hit rate can indicate difficult to progress • See also Hajduk (2005) J Med Chem, 48, 2518

• Low hit rates from catenin, Pin1 and Hsp70 ATP site

Updated from Chen & Hubbard (2009), JCAMD, 23: 603

0%

1%

2%

3%

4%

5%

6%

7%

8%

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Dscore

Cla

ss 1

hit

s ra

tes

Low hit rates (< 2%) High hit rates (> 2%)

Kinases

high hit rate

1 Calculate druggability

Val

idat

ed h

it r

ate

Ligandability calculated from structure (DScore)

Validated

hit rate

from a

ligand-

observed

NMR

screen

protein-protein interaction

targets – varying hit rates

Poor targets

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Overview






Warwick, Sep 2016

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Pre-Clinical Clinical Trials

Drug Discovery

Warwick, Sep 2016

Discovery

I II III

Patient

Target Hit ID

H2Ls

Lead

Optimisation

Good Idea

Hit Compound

Lead Compound

Clinical Candidate

Drug

Medicine

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Hsp90 – the Target

• Hsp90 function is complex involving protein dimerisation and

association to other chaperones and co-factors

GM Geldanamycin – a fungal natural product

Hsp90 requires ATP to function (via hydrolysis to ADP)

Kamal et al (2004) Trends in Mol Med 10: 283

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Hsp90: Fragment screen

• Targetting the N-terminal domain – an ATPase

• FBLD programme began in early 2002 • - screened library of 729 fragments by NMR

• 17 fragments identified • Crystal structures for most fragments binding to Hsp90

Amide Amino-pyrimidine Second site binder

Resorcinol

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Using fragments

• Finding fragments that bind is not difficult

• The challenge is knowing what to do with the hits • Link, grow or merge

Screen GROW

Warwick, Sep 2016

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Hsp90 – AUY922 story

Starting fragment Hit from SAR by

Catalogue (also MTS and VS)

• GI50 in HCT116 colon cell line

FP IC50 = 0.28mM

GI50 = 6mM

FP IC50 = 0.009mM

GI50 = 0.014mM

FP IC50 = ~1mM

D93 G97

K58

F138

L107

rCat N

O

O

O

O

Phase II Candidate

(Novartis)

D93

G97

K58

F138

L107

Brough et al (2008) J Med Chem 51,196-218 Roughley et al (2012) Top Curr Chem , 317, 61

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Using fragments

• Finding fragments that bind is not difficult

• The challenge is knowing what to do with the hits • Link, grow or merge

Known Ligands

Virtual Screening hits Screen

Detailed Design

17

NN

NH2

OOMe

VER-26734

FP IC50>5mM

NN

NH2

SNH

OVER-52959

FP IC50=535mM

Fragment Evolved fragment

HSP90 – BEP800 story

N

N

N

NH2

Cl

ClN

NO

OH

OH

O

NH

N O

luminespib

Vernalis Phase II candidate (FBLD

/ SBDD derived)

VER-41113

FP IC50=1.56mM

Virtual Screening Hit

VER-45616

FP IC50=0.9mM

SNNH2

NH2

N

NH2

O

OEt


Brough et al (2009) J Med Chem 52,4794-4809 Roughley et al (2012) Top Curr Chem 317, 61

D93 G97 K58

F138 L107

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N

N SNH2

O

NH

Cl

Cl

ON

NN

NH2

OOMe

VER-26734

FP IC50>5mM

NN

NH2

SNH

OVER-52959

FP IC50=535mM

Fragment Evolved fragment

HSP90 – BEP800 story

N

N

N

NH2

Cl

ClN

VER-82576

NVP-BEP800

FP IC50=0.058mM

KD = 0.9nM (SPR)

HCT116 GI50=0.161mM

BT474 GI50=0.057mM

NO

OH

OH

O

NH

N O

luminespib

Vernalis Phase II candidate (FBLD

/ SBDD derived)

VER-41113

FP IC50=1.56mM


VER-45616

FP IC50=0.9mM

SNNH2

NH2

N

NH2

O

OEt


Brough et al (2009) J Med Chem 52,4794-4809 Roughley et al (2012) Top Curr Chem 317, 61

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Drug leads from fragments

• Vernalis have disclosed leads for: • kinases such as Chk1, DYRK1A, PDPK1, Pim1, Pak1, Pak4,

STK33 and PDHK

• ATPases such as DNA gyrase, HSP70 and HSP90

• protein-protein interaction targets Pin1, Mcl-1 and Bcl-2

• FAAH and tankyrase

• In recent years – clinical candidates for: • Chk1, FAAH, Hsp90, (Bcl-2 – inspired by), Mcl-1

• Many other examples published • Small and large pharma

• List on http://practicalfragmentsblogspotcom/.

• Some recent reviews and books

Warwick, Sep 2016

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Overview






Warwick, Sep 2016

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Drug Discovery

Warwick, Sep 2016

Discovery

I II III

Patient

Target Hit ID

H2Ls

Lead

Optimisation

Good Idea

Hit Compound

Lead Compound

Clinical Candidate

Drug

Medicine

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Chemical Biology

Warwick, Sep 2016

Discovery

I II III

Patient

Target Hit ID

H2Ls

Lead

Optimisation

Good Idea

Hit Compound

(Lead Compound)

Clinical Candidate

Drug

Medicine

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Overview






Warwick, Sep 2016

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Warwick, Sep 2016 The TolB project

• A protein involved in bacterial attack / defence

• Structures determined of TolB in complex with various proteins (collaboration with Colin Kleanthous, now in Oxford)

• Exact mechanism of action still unclear

• Crystal structure with peptide bound

• Fragment screen (thermal shift) identified fragments that bind to the tryptophan pocket

• But also new site

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Warwick, Sep 2016 The TolB project

• A protein involved in bacterial attack / defence

• Structures determined of TolB in complex with various proteins (collaboration with Colin Kleanthous, now in Oxford)

• Exact mechanism of action still unclear

• Crystal structure with peptide bound

• Fragment screen (thermal shift) identified fragments that bind to the tryptophan pocket

• But also new site

Sequence is conserved in this region

(not noticed before)

Mutation affects activity

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Warwick, Sep 2016 Overview






•Warwick, Sep 2016

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Warwick, Sep 2016 Bacterial orthologue – BtGH84

• Family 84 glycoside hydrolase

• 40% identity with human OGA domain

• Crystal structure available

• Low micromolar (class-wide unselective)

inhibitors developed previously

• tool compounds

• Fragment screen of 91 fragments

• Ligand observed NMR

• PUGNAc as competitor

• Investigated both competitive and non-competitive inhibitors

PUGNAc Thiamet G

Darby et al Angew Chemie (2014) 53:13419

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Warwick, Sep 2016

• Non-competitive fragment M1E05 enhances BtGH84 activity

• Confirmed with alternative substrate, many controls

• Activation is concentration dependent

• Increases the Vmaxapp and reduces the Km

app - 2.5 mM to 1.3 mM

BtGH84 is activated by a fragment

M1E05


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Warwick, Sep 2016 BtGH84 is activated by a fragment






• Assay, NMR and ITC confirm that M1E05 affects PUGNAc binding

M1E05


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Warwick, Sep 2016 BtGH84 is activated by a fragment






• Assay, NMR and ITC confirm that M1E05 affects PUGNAc binding

• Higher activity (200µM) compounds identified (see paper)

• Non-essential activator kinetics suggests mechanism

• Compounds stabilise folding of loop required for catalysis

• Better compounds required for probing GlcNAc status in cells

• But possibilities for industrial / bioprocessing enzymes

• Investigating covalent tethering of activators (it works !!)

M1E05


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Warwick, Sep 2016 Overview






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Warwick, Sep 2016 Fragnet

• EU ITN funded from March 2016

• 15 advanced PhD students employed across Europe to be trained in the methods of fragment based discovery

• Students in academic beneficiaries: VUA, Amsterdam, University of York, University of Barcelona, Hungarian Academy of Sciences

• Students in beneficiaries: Novartis, Switzerland, Vernalis, UK; Beactica, Sweden; Zobio, Holland

• Partners: GSK, UK, Iota, UK, Roche, Switzerland, Servier, France

• About 60% of the projects are developing methods

• Computational, novel synthesis, covalent targetting

• A couple of the applications are antimicrobial

• One of which is the bacterial replisome work at York

•

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Warwick, Sep 2016 Bacterial replisome

• A collection of proteins / complexes that together replicate DNA

in bacteria

• Peter McGlynn at York able to reconstitute in vitro

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Warwick, Sep 2016 Bacterial replisome

• A collection of proteins / complexes that together replicate DNA

in bacteria

• Peter McGlynn at York able to reconstitute in vitro

• Screen of 1000 member York library for inhibition of DNA

synthesis – a pseudo phenotypic screen

• Identified DNA intercalators through topoisomerase assay

• Now running functional assays to identify possible mechanisms

• Crystal structures available for many of the components / sub-

complexes

• Target identification underway