Taylor, Jonathan D.; (2005) Exploitation of NMR in the analysis and screening of fragment ligands for an Sh2 domain. Doctoral thesis , UCL (University College London).

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Abstract

A fragment-based approach to drug design has recently emerged in which small chemical groups that bind to adjacent sites on a receptor are identified, optimised, and linked together to generate a high-affinity lead compound. Due to its sensitivity, nuclear magnetic resonance (NMR) is a particularly useful technique for the detection of low-affinity fragment binding. An aim of this project was to explore further the utility of NMR with regards to fragment ligand binding using a well- understood model system - the Src Homology 2 (SH2) domain from v-Src kinase. This choice was supported by a large body of structural, functional, and thermodynamic data, coupled with a decade of pharmaceutical investigation into several SH2 domains. Our ongoing biophysical studies of v-Src SH2 required determination of the apo solution structure of this domain. A high quality structural ensemble was obtained using standard NMR techniques and a combination of manual and automated assignment methods. The internal hydrogen bonding, ionisation states, and backbone dynamics of the apo v-Src SH2 domain was also explored using NMR. The perturbation of v-Src SH2 backbone chemical shifts and dynamics by interaction with fragment ligands yielded insights into SH2 domain binding behaviour and specificity. Computational approaches were used to identify potential fragment ligands for v-Src SH2. A small library of these molecules were screened in vitro using a recently-proposed 19F-NMR competition screening approach, which was optimised for the detection of low-affinity fragments. Follow-up NMR and calorimetry experiments confirmed the screening results and provided further characterisation of the novel fragment ligands. Such compounds may be useful as phosphotyrosine mimetics in SH2-related drug design. A novel 31P-based screening experiment was also proposed. These studies have furthered our understanding of the SH2 domain, in terms of its binding specificity and drug design, and of the NMR screening approaches useful for fragment-based lead discovery.

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