Spatial overlap of peptide hotspots and

canonical drug pockets in a model enzyme

Walraj S. Gosal June 2015

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In collaboration with:

With funding from:

From molecular display peptides to small molecule inhibitors.

| partnerships in biologics discovery

fold into precise 3D structures

chemical reactions of life through the precise arrangement & dynamics of atoms

Primary seq. 100 amino acid protein = 1x10130 sequences

Display technology is pivotal for biologics discovery

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Humira FAB

TNFα"

Approved: RA, UC, CD

•  Humira (monoclonal antibody) is the biggest selling drug worldwide ($13bn in sales).

•  Computational approaches are showing evermore promise (e.g. Rosetta)1,2.

Design

Crystal structure

1Kuhlman,B. et al. Science (2003). 2Fleishman,S.J. et al. Science (2011).

Display technology is pivotal for the emergence of biologics •  Recent emergence of over a dozen scaffolds ranging in size (~ 20 to 170 amino acids)1,2.

1Lofblom,J. et al. Current Opinion in Biotechnology (2011). 2Gronwall, C. et al. Journal of Biotechnology (2009).

Humira FAB

TNFα"

Approved: RA, UC, CD

Knottin

e.g. Ziconotide (Approved: pain relief)

DARPin e.g. Abicipar pegol (Phase III:

macular degeneration)

e.g. SOBI002 (Phase I: inflammation)

Affibody

Anticalin e.g. PRS-050 (Phase I:

angiogenesis)

Adnectin / Centyrin e.g. Angiocept (phase II

glioblastoma)

Display technology is pivotal for biologics discovery

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•  First described by George Smith in 1985 using bacterial filamentous phage (phage display)1.

•  Basic idea is to enrich phenotypes in a library and be able to read genotypes. RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed protein

target

random library

random library

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed proteintarget

In vitro transcription & translation

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed protein

target

random library

random library

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed proteintarget

Incubation with target

1Smith,G.P. Science (1985). 2Odegrip,R. et al. PNAS (2004).3Eldridge,B. et al. Protein Eng. Des. Eng. (2009). 4Patel,S. et al. Protein Eng. Des. Eng. (2013).

CIS Display2-4

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed protein

target

random library

random library

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed proteintargetPCR

amplification of binders

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed protein

target

random library

random library

RepA

CISori

RepA

genotypephenotypelink

peptideseq.

Displayed proteintarget

Can molecular display be used to inform small molecule discovery?

Protein target

3. Structure determination(NMR or X-ray)

1. CIS display& NGS

3D structure:Protein-Peptide complex

2. Peptide hit validation(ELISA, truncation,binding, activity,epitope mapping)

Allostericsitebinders

Activesitebinders

RepA

CISori

genotypephenotypelink

peptideseq.

peptide

target

Target-binding peptide candidates

selection, amplifiction & NGS

random peptidelibrary

VLSEGEWQLVLHVWAKVEADV

AQGAMNKALELFRKDIAAKYK105

1

Small target-binding peptides

site II

site I

Protein target

3. Structure determination(NMR or X-ray)

1. CIS display& NGS

3D structure:Protein-Peptide complex

2. Peptide hit validation(ELISA, truncation,binding, activity,epitope mapping)

Allostericsitebinders

Activesitebinders

RepA

CISori

genotypephenotypelink

peptideseq.

peptide

target

Target-binding peptide candidates

selection, amplifiction & NGS

random peptidelibrary

VLSEGEWQLVLHVWAKVEADV

AQGAMNKALELFRKDIAAKYK105

1

Small target-binding peptides

site II

site I

CIS display & NGS

3D structure:Protein-Peptide complex

Allostericsitebinders

Activesitebinders

X-ray / NMR

3D structure:Protein-Peptide complex

Allostericsitebinders

Activesitebinders

4. Residue hotspots & field calculation(mutational analysis, Rosetta, & XED force field)

5. Virtual smallmolecule screen

(>107 small molecules)

6. Focused experimental screen(~100 small molecules)

7. Validation & ‘hit to lead’ optimisation (inhibitionmodality, Kd, Ki, X-ray Crystallography) Lead compound

Screening hits

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1

h1

h2

h3

h4

Peptide field map

hydrophobic

+ve/HBA

VdW surface

-ve/HBD

Screening candidates

100

1O

NH

O

OH

NH

HN NH2

N

N

N

HN

O

NH2

N

N

S O

O

O

NH

NH2

Cl

NH

O

O

NH

NH2

NH

O

O

NH

Computation & screening

Target of choice: thrombin - a serine protease

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•  40 year race in the drug industry to replace anticoagulants - Warfarin (1953) & Heparin (1937)1,2.

•  Large body of public information that covers all major discovery platforms (fragment screening, structure-

based design & HTS).

1Gustafsson,D. Nature Reviews Drug Discovery 3 649-659 (2004). 2Nar, H. Trends in Pharm. Sci. 33 279-268 (2012). 3Huntington, J. A. Thromb. Haemost., 111 583-9 (2014).

Substrate: FPA

S1

S2

S4

Dabigatran Melagatran

S1

S2 S4

S1

S2

S4

s-variegin3 (32 aa) Tropical bont tick

Hirudin3 (65aa) Medicinal leech

Anophelin3 (31 aa) Mosquito

•  Unique structure based solutions from Nature3.

Summary & Acknowledgements

Contact Walraj S. Gosal walraj.gosal@isogenica.com www.isogenica.com

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1.  In a retrospective study, CIS display peptide ‘hot-spots’ spatially overlap with known

drug pockets in thrombin, although novel pockets are induced.

2.  Peptides cover the “chemical solution space” for the primary S1 pocket.

3.  Field patterns based on peptides can be used to find new inhibitors.

Tom Blundell Jim Huntington Ty Adams

Robert Scoffin Andy Vinter Mark Mackey

Steve Gardner Dirk Gewert Peter Campbell

Chris Ullman Neil Cooley Kevin Matthews Gabriela Ivanova Amanda Hallott Shabana Vohra

With funding from:

Gordon Woodrow Stephan Krapp