2- and 4-Halopyridines were previously identified as a novel class of protein modifiers. These weakly electrophilic fragment sized molecules were shown to act as quiescent affinity labels that selectively and covalently modify some protein thiols. Covalent modification can occur when a carboxylate side chain is found at a certain distance and orientation from a cysteine thiol. This arrangement of residues catalyzes covalent bond formation by stabilizing the active, protonated form of the pyridine, which allows subsequent attack by the cysteine thiol. This need for catalysis imparts a unique selectivity that sets 2- and 4-halopyridines apart from most electrophilic fragments since efficient modification is not only derived from inherent reactivity and proximity to the target nucleophile, but also by way of a protonation ‘switch’ catalyzed by two residues at the binding site of the target protein. The inherent selectivity of these fragment sized molecules and their relatively straightforward derivitization at multiple positions on the pyridine ring have prompted further study to characterize their unique features as a class of covalent protein modifiers and to explore their utility to engage therapeutic targets.
In this work, the features that set 4-halopyridines apart from other fragment-sized electrophiles are identified and characterized. These studies revealed the protonation ‘switch’ allows modulation of halopyridine reactivity through catalysis whereas the non- enzymatic reactivity remains low an effect not obtainable with other electrophilic fragments. After definition of the general requirements for protein modification and identification of methods to alter reactivity, additional protein targets that meet the requirements for 4-halopyridine modification are identified and validated. Modification of one of the identified targets for 4-halopyridines, V-Ki-ras2 Kristen rat sarcoma viral oncogene (KRas), is characterized in detail. It is demonstrated that the 4-halopyridine fragment represents a starting point to selectively modify this historically difficult drug target in cells.
In a separate project, enzyme inhibition is studied and applied in a different way. The utility of a substrate-inactivatable Pseudomonas aeruginosa dimethylarginine dimethylaminiohydrolase (PaDDAH) mutant enzyme, PaDDAH T165L, for the quantitative measurement of asymmetric dimethylarginine (ADMA) is evaluated. The catalytic partitioning between normal turnover and self-inactivation enables a low cost quantitative means for measurement of the clinically-relevant biomarker ADMA.Biochemistr
