Efficient detection of cryptic pockets on challenging drug targets using MD-free co-vibrational modes

Abstract

Allosteric and cryptic binding sites provide unique opportunities in drug discovery for achieving selectivity, reducing toxicity and unlocking challenging therapeutical targets. Yet, cryptic pockets are particularly elusive in that they are difficult to capture with experimental methodologies, and therefore often remain undetected. Computational approaches for flexible cryptic site detection traditionally involve extensive unbiased molecular dynamics simulations, or more efficient but computationally still highly demanding biased MD alternatives that make use of enhanced sampling along low-frequency motions of the protein structure. Here we utilize a new variant of the recently introduced and orders of magnitude faster, MD-free COVIB algorithm to identify cryptic pockets efficiently in two challenging targets, the KRAS oncoprotein and the N-terminal domain of the STAT5B transcription factor. Using experimental and AI-modelled structures, respectively, we identify numerous cryptic sites, some of which have not yet been reported. The sites are prioritized and validated with our pharmacophore optimized photoaffinity-tagged fragment library via MS-based peptide mapping and NMR spectroscopy. Besides identifying new, targetable regions on the surface of these important oncotargets, we have established a protocol combining COVIB-based cryptic pocket detection and photoaffinity-based experimental peptide mapping as a robust approach for exploring unique and tractable cryptic pockets.