Research

Greg Craven

Kinetic Template-Guided Tethering for Antiviral Therapy: Identification of Inhibitors of Viral Cyclin/Cdk Protein-Protein Interactions

Greg Craven - Writing up

Abstract

All cellular processes rely on protein-protein interactions (PPIs) and modulation of such events would provide a powerful means of intervening in pathophysiology for either experimental or therapeutic purposes. This project aims to build on our exciting proof of concept work generating a novel, general approach, ‘Kinetic Template-Guided Tethering’ (KTGT, Chem. Med. Chem. 2012, in press: http://dx.doi.org/10.1002/cmdc.201200404), for identifying chemical fragments that bind to a particular protein at a particular location (in our case, a protein-protein interaction interface). For KTGT, one partner protein involved in the PPI of interest must have a cysteine (either natural or engineered) in the vicinity of the desired binding surface. The protein is then incubated with mixtures of acrylamide-modified fragments. If a fragment in the mixture binds non-covalently to the desired pocket, it concomitantly brings the acrylamide group into close proximity with the local cysteine thiol resulting in covalent capture of the fragment by an irreversible Michael addition. On the timescale of this assay, capture at non-templating cysteines is not observed. Crude mixtures of fragments can be analysed directly using electrospray mass spectrometry with bound adducts readily identified by mass changes. We have published a preliminary proof-of-concept using the model protein thymidylate synthase for which a known positive control ligand was selected from a small library of acrylamide-containing fragments.

This project will build on our proof of concept work by (a) developing minimal linkers to prevent fragments affecting the intrinsic reactivity of the acrylamide to ensure binding is dependent only on fragment templating; (b) developing methods for random introduction of a linker/acrylamide into a fragment to maximise library diversity from a fixed number of fragments; (c) constructing an appropriately functionalised fragment library incorporating novel stereodefined 3D-structures including new substituted small-ring heterocycles (e.g. oxetanes, azetidines) and spiro/bicyclic amines. This important latter goal will allow exploration of new regions of 3D-chemical space, likely to be necessary for intervening in PPIs. With these new fragments available, KTGT will be applied to the discovery of fragments that interact with the cdk binding site of the cyclin encoded by Human Herpesvirus 8 (K cyclin of HHV8) with the aim of disrupting K cyclin/cdk interactions. HHV8 is the causative agent of several lymphoproliferative cancers (Kaposi’s Sarcoma, Multicentric Castelman’s Disease and Primary Effusion Lymphoma) that are particularly prevalent in immunosuppressed individuals (e.g. AIDS sufferers). The viral cyclin is thought to help drive the associated cell proliferation by interacting with normal human cdk6 to form a constitutively active kinase. There is currently no specific drug targeted at this virus and disruption of the K cyclin/cdk interaction may be sufficient to block virally induced cell cycle deregulation.