Predicting modulating sites on proteins: a new route to drug discovery?

Joe GreenerBioinformatics London Meetup

28th April 2016

Aims

Introduce allostery in proteins

Describe two computational methods for allosteric site discovery: AlloPred Distance geometry method

Briefly mention BioJulia

Allosteric modulation

Conventionally, drugs bind to the active site orthosteric regulation

Modulators that bind to a site separate from the active site are allosteric regulators

Advantageous: Unexplored drug space Highly specific Modulation up or down

The protein ensemble Proteins exist in an ensemble of conformational states An allosteric modulator changes the occupancies of the states, leading to a functional change

Allosteric examples

Adenylate cyclase (left) with a modulator (green) bound to an allosteric site and a substrate (orange) bound at the active site

Some allosteric drugs available, e.g. cinacalcet and maraviroc for GPCRs

Allosteric modulators found for targets as diverse as the GABA receptor, hepatitis C virus polymerase and RNA

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

Normal mode analysis

The structural fluctuations of a protein around an equilibrium conformation are decomposed into harmonic orthogonal modes

Decent results with backbone only, and even C-alpha only Low frequency modes associated with long-range communication

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

Perturbation method

Modulator (M) binding suppresses vibration at the potential modulator binding site (P)

Modulator binding is approximated by an increase in spring constant k at the potential modulator binding site

The effect of the change in the normal modes is measured at the active siteGreener, JG and Sternberg, MJE.

BMC Bioinformatics (2015) 16:335

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

AlloPred

Calculate normal modes of the protein

Predict pockets on the protein using Fpocket

Introduce normal mode perturbation at each pocket

Combine effect of perturbation with pocket features in a support vector machine (SVM)

Rank pockets in terms of allosteric character

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

Validation on known allosteric sites

Set of 40 known allosteric proteins from the AlloStericDatabase

AlloPred ranks an allosteric pocket top in 23 of 40 cases and top two in 28 of 40 cases

Performance similar and complementary to existing methods

AlloPred AlloSite

PARS

4 2

2

6

12

11

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335

AlloPred web server

Results table JSmol visualisation

Greener, JG and Sternberg, MJE. BMC Bioinformatics (2015) 16:335http://www.sbg.bio.ic.ac.uk/allopred/home

Distance geometry methodObtain two structures (e.g. active and inactive) for a protein

Extract distance constraints from protein structures these arise from bonds, angles, hydrophobic interactions etc.

Use an interative process stochastic proximity embedding to generate structures from constraints

Determine new constraints arising from a predicted modulator and generate structures with these additional constraints

Compare the two sets of structures to see if that modulator is predicted to be allosteric

CDK2 ensemble generation

PC1 /

PC2 /

Active

InactiveActive crystal structureInactive crystal structure5 generated structures

Projection of structures onto principal components of ensemble

CDK2 allosteric site prediction

PC1 / PC1 /

PC2 / PC2 /

Active

Inactive

Pocket 1Pocket 3 is similar

Pocket 2Pockets 4-8 are similar

Julia language

New programming language developed at MIT from 2012 Syntax like Matlab or Python Very fast approaches C and Fortran speeds when coded properly Just-in-time (JIT) compiled Seamless calls to C Currently at v0.4 not yet stable and under heavy development Currently lacking package ecosystem to rival R and Python

BioJulia

Bioinformatics and computational biology infrastructure for Julia Modules:

Biological sequences: Seq Biological sequence alignment: Align Intervals and annotations: Intervals Molecular structures: Structure

http://biojulia.github.io/Bio.jl/

Conclusions

Allostery is an important biological process Allosteric drugs have much potential AlloPred can be used to predict allosteric sites on proteins Distance geometry methods can be used to explore allostery Next steps test experimentally

Acknowledgements

Structural Bioinformatics Group, Imperial College London Mike Sternberg David Mann, Alan Armstrong Ioannis Filippis BBSRC

http://www.sbg.bio.ic.ac.uk