Drugging the Undruggable…

Protein-Protein Interactions

Professor Robin LeatherbarrowHead of Biological Chemistry

Department of Chemistry

What is a “typical” Drug?

• Modulates a specific biological process• Enzyme inhibitor

Reversible competitive inhibitor• e.g. Viagra

Irreversible enzyme inhibitor• e.g. Penicillin, aspirin

• Small molecular weight compound Obeys Lipinski “rule of 5”

• Less than 500 molecular weight etc Orally available

The ‘Druggable’: Inhibiting Enzyme-Substrate Interactions

PPEnzyme

Enzyme with a defined substrate-binding site

Substrate binds and is converted to product(s)

Inhibitor blocks substrate binding

The ‘Undruggable?’: Inhibiting Protein-Protein Interactions

Protein1

Protein responsible for biological effect

Effect modulated by protein-protein interaction

Inhibitor regulates effect by blocking protein-protein interaction

Protein2

I

Biological Effect

Human genome: 30,000 drug targets

Disease-related targets: 10%

Druggable 20-50%Undruggable 50-80%

What proportion of drug targets are druggable?

Figures from Hopkins and Groom (2002) Nature Rev Drug Disc 1, 727

Why Target Protein-Protein Interactions?

• Account for the majority of biological control points

• Are implicated in all areas of medicine Wide ranging impact Many therapeutic areas

• …BUT ARE DIFFICULT TO TARGET

Typical Enzyme-Substrate Interaction Typical Protein-Protein Interaction

Well-defined binding pocket Relatively flat surface

Relatively small contact area (300-1000 Å2)

Relatively large contact area (1500-3000 Å2)

Intrinsic interaction is relatively weak, so easy to block

Intrinsic interaction is relatively strong…

Screening involves looking for compounds that affect enzyme activity—assay is easy

No enzyme assay for easy screening

Substrate structure gives “clues” towards inhibitor design

No such information available

Inhibitors the size of the substrate are still likely to be small (Rule of 5 compliant)

Inhibitors of comparable size to the interacting surface will be too large

There are MANY examples of successful drugs that target enzymes

There are VERY FEW examples of any drugs that target Protein-Protein interactions

Problems…

Protein 1 Protein 2SEnzyme

Examples to date

• There are a few examples of successful drug leads that are targeted at Protein-Protein interfaces

• However, there are currently NO marketed drugs that work this way…

Review: Wells & McClendon (2007) Nature 450, 1001

1. Bcl-XL binders

• B-cell lymphoma (Bcl) 2 family proteins are important regulators of apoptotic cell death and form homodimers with other family members

Bcl-XL (grey) bound to partner protein via alpha helical region

Bound small molecule inhibitor of this interaction

2. IL-2 binders

• Interleukin-2 is a cytokine that has a key role in activation of T cells and in the rejection of tissue grafts, by binding to IL-2 receptor

IL-2 (grey) bound to partner protein

Bound small molecule inhibitor of this interaction

3. HPV E2 binders

• Human papilloma virus (HPV) causes warts and some cervical cancers. The interaction between HPV transcription factor E2 and helicase E1 is vital for the viral life cycle

HPC E2 (grey) bound to EPV E1 Bound small molecule inhibitor of this interaction

Issues

• Where do we start? Fragment screening? Peptidomimetic approaches? Allosteric modulation?

• How do we assay? Throughput / sensitivity?

• How do we optimise leads? Starting points not “drug-like”?

Trypsin – Trypsin Inhibitor interactions

Trypsin Trypsin Inhibitor

Protein (BBI)

Interacting region

Constrained Peptides as Functional Motifs

Trypsin Inhibitor Protein (BBI)

Protease

Synthetic interacting motif

Ki = 9 nM

• Discrete liquid droplets are encapsulated by a carrier fluid

• Droplets: are isolated and form the

dispersed phase in which reactions may occur

can be dosed with varying amounts of input reagents

can be generated at kHz frequencies

Assay: Droplet-based Microfluidics

Andrew de Mello, Imperial College

ANG-AF647 (nM)0 20 40 60

EF

RE

T

0.0

0.2

0.4

0.6

0.8

1.0

• Angiogenin – anti-Angiogenin

Measuring Protein-Protein Interactions in Microdroplets

KD = 6.4 nM

Monpichar Srisa-Art, Dong-Ku Kang, Jongin Hong, Hyun Park, Robin J. Leatherbarrow, Joshua B. Edel, Soo-Ik Chang, and Andrew J. deMello; ChemBioChem 2009

Conclusions

• Protein-protein interactions are potentially extremely useful drug targets

• They are far more difficult than “traditional” drug targets

• They offer new therapeutic possibilities that should become exploited in coming years