DXMSguided Design of Small Molecules that Target Protein Protein Interaction Surfaces

Development of small molecule inhibitors of protein-protein binding interactions has been notoriously difficult. The tremendous investment that the pharmaceutical industry has made in the development and marketing of whole protein therapeutics such as monoclonal antibodies and recombinant proteins is testament to the strength of the belief that small molecule replacements for these ''expensive to produce and administer'' biologics will not soon be forthcoming. Multiple noncovalent interactions between a protein and a ligand are required for sufficient affinity and specificity. Typical ''druggable'' proteins usually have identifiable cavities or crevices on their surfaces that allow direction of multiple interactions to a resident small molecule ligand, that is typically less than or equal to 500 Da in size. In protein-protein binding, these multiple interactions do not need to be focused to a single small area, but typically are spread across a broad, fairly flat binding surface that is devoid of identifiable ''druggable'' cavities [65].

Fortunately, it has been found that many protein-protein binding surfaces contain a small number of amino acid residues (binding surface ''hot spots'') that predominantly contribute to the binding energy between partners. This was first demonstrated by Wells and collaborators in studies of the complex formed between hGH and its cellular receptor protein [66, 67]. Unfortunately, the methodology that demonstrated this phenomenon (site-directed mutagenesis-induced perturbation of binding affinity) has not proven to be a robust guide to small-molecule development to binding surfaces. One reason for this failure is suggested by the observation that small, but important, highly localized conforma-tional changes are induced in apparently bland interaction surfaces when protein ligands bind to each other [65, 68]. Protein-protein binding may induce the formation of localized topography that focuses binding energy by way of induced crevices. Mutagenesis approaches may not allow these small conformational changes to be induced or localized with sufficient precision. These inducible crevices, if they could be reliably localized, might serve as targets for small molecule design efforts to protein binding surfaces. DXMS analysis can provide precisely the information required to identify protein-protein binding surface hot spots, and then guide the design of small molecules that precisely target such ''hot spot'' regions, all without the use of mutant proteins.

A United States patent describes how this can be accomplished [69]. First, DXMS analysis is performed on the interacting proteins, separately and com-plexed to each other. Measurement of the magnitude of exchange slowing (protection factor) in the complexed versus unbound state, for each of the amides participating in the protein-protein binding surface, allows direct identification of ''hot spots''. The hot spots are the areas of the binding surface with the highest protection factors. Protein-protein binding surfaces are not rigid structures, but undergo continuous flexible movement, as does the entire protein. Proteinprotein binding surfaces are bound together most tightly at their thermodynamic hot spots, and the bulk solvent has little opportunity to interact with the amides in such tightly bound regions. Indeed, the amide hydrogens in the hot spots serve as highly localized sensors of binding-induced free energy change, where binding free energy (AG) at such amides is related to specific measured amide protection factors according to Eq. (5).

Once binding hot spots are identified by DXMS analysis, combinatorial libraries can be generated, based in part on structural information available that may suggest the nature of the peptidic features of the interacting proteins that

12.8 Optimal Formulation and Quality Control of Whole Potein Therapeutics with DXMS | 393

are present at the exchange-localized hot spot. Libraries are screened against the protein target, not with conventional binding or activity assays, but by performing repeat high-throughput DXMS studies in which the target protein is functionally deuterated in the presence of an excess of each test compound. Small molecules that, by virtue of their protein binding, can induce exchange slowing selectively at the previously identified protein-protein binding surface hot spots are identified. These are then selected for further combinatorial perturbation, and repeat DXMS screening against the target protein. In this manner, DXMS analysis provides an almost real-time guide to the identification of library elements in each round of selection that are capable of binding to the protein-protein interaction-defined binding hot spots.

An analogy can be made to oil well drilling. Initial DXMS analysis of a clinically important protein-protein interaction provides the equivalent of a seismic map of an oilfield, showing where the oil may be located (hot spots). Further high-throughput DXMS analysis of protein-small molecule mixtures at each round of selection is analogous to having a sensor in the oil drilling rig that measures the proximity of the drill tip to the targeted oil deposit in real time. The numerous whole protein therapeutics that have proven to be great successes in the clinic make tempting targets for this small-molecule design strategy

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