Developing NMR and Biophysical Strategies to Study Protein-Ligand Interactions and Reveal Interesting Compound Properties

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There is a widely held view that the study of ligands binding to macromolecules should be simple and straightforward. In practice, however, the available tools are sparse and compromised by artifacts. Moreover, binding involves many events, some of which have yet to be fully revealed. This research program aims to develop strategies to enable the elucidation of various binding events and to expose relevant free-state properties of small-molecule ligands. The research will be executed within the context of mounting fragment-based ligand discovery (FBLD) efforts in my laboratory where the binding of thousands of compounds to a variety of protein types will be systematically evaluated. Briefly, FBLD will involve the screening of libraries (e.g. ~1000-8000 members) of fragment compounds (~120-300 Da) in search for binders (Kd ~ 100-1000 uM) that will then be subjected to follow-up characterization. *** We will begin by building libraries of compounds using prioritization filters based on theoretical and experimental attributes. Cheminformatics will serve to rank fragments that have desirable calculated physicochemical properties, functional group diversity and three-dimensionality. New NMR and biophysical profiling methods will be implemented to identify insoluble compounds and characterize those that tumble as lone molecules in the free-state versus those that self-assemble into aggregates or nano-entities. Compounds that self-assemble will be extensively characterized in follow-up studies to establish structure-aggregate-property relationships and to explore design opportunities for nano-biotechnology purposes. *** Theses libraries will then be screened to identify compounds that bind to protein targets. A set of comprehensive NMR screening techniques will be developed to detect binding via changes in NMR resonance shifts (HSQC, DLB, 19F NMR), relaxation (CPMG, NOESY), saturation (STD) and diffusion (DOSY). These and other biophysics methods will help to reveal and elucidate the various types of free-state properties and binding modes (e.g. specificity, stoichiometries, promiscuity, stability at various time-scales). The evaluation of large amounts of data generated will require the assistance and development of software. Follow-up biophysical and structure-activity relationships will then be established to best characterize the binding modes and identify correlations with properties. New protocols will also be implemented to enable efficient screening in various buffers, biologically-relevant media, and cell-based environments, and to screen atypical targets such as intrinsically disordered proteins.*** In summary, this research will establish new strategies, expose and exploit fundamental chemical properties, and define new FBLD methods to improve our understanding of the molecular properties that define protein-ligand interactions.**