Most pharmaceuticals in clinical use target proteins and the number of potential protein targets has dramatically increased with the availability of the sequences of all human proteins. There is a large gap, bridged only with much investment in effort and time, between the discovery of a protein target for potential therapeutic intervention in a disease and the development of small molecules that can modulate the protein's activity in a clinically useful manner. A protein's high-resolution three-dimensional structure is typically determined by crystallographic means, its binding partners identified (other proteins/metabolites/signaling small molecules), and the precise manner in which these several entities bind to each other and modify each other's activity and conformation characterized. Small-molecule therapeutic candidates must then be produced and iteratively refined for their ability to interfere with or promote these activities.

While the well established protein structure determination methods of X-ray crystallography and nuclear magnetic resonance (NMR) are being pushed to meet the challenge, both technologies have limitations in their applicability and throughput. Crystallization is a major obstacle for X-ray analysis: most eukaryotic proteins are difficult to crystallize without lengthy experimentation, or may be inherently noncrystallizable. NMR has size limitations and sensitivity issues. X-ray crystallography can study a protein only in the solid state and NMR requires that the study protein be at high concentration. Both techniques require that the protein be highly purified.

Enhanced hydrogen/deuterium (H/D) exchange mass spectrometry (DXMS) has emerged as an effective tool free of these limitations, which promises to speed many of the steps from therapeutic target discovery to the development of drugs ready for clinical evaluation. In the early 1990s, Zhang and Smith described a methodology, in which H/D exchange reactions were followed by steps involving proteolysis, HPLC separation, and mass spectrometric (MS) analysis [1]. In this approach, medium-resolution information could be obtained by mea suring the deuterium incorporation within each proteolytically generated peptide fragment. Since then, improvements to their fundamental method have been used to study protein structure/dynamics [1-15], protein-ligand interactions [16-19], and protein-protein interactions [20-29]. Reflecting the increased activity and advances in this field, several comprehensive reviews have been published [4, 30-33].

DXMS can now be used to dramatically improve the crystallizability of proteins for structure determination, characterize the binding interactions between the target protein and its binding partners, and rapidly determine the conformational changes that often accompany such binding events. Most importantly, DXMS has considerable potential to guide the development of clinically useful small-molecule therapeutics that can target protein-protein interaction surfaces - a notoriously difficult task. In this review, the experimental methodologies of enhanced DXMS technology are described, and examples given of its ability to speed the steps of the translational process.

Was this article helpful?

0 0

Post a comment