Understanding Cryptic Pocket Formation in Protein Targets by Enhanced Sampling Simulations

Abstract

Cryptic pockets, that is, sites on protein targets that only become apparent when drugs bind, provide a promising alternative to classical binding sites for drug development. Here, we investigate the nature and dynamical properties of cryptic sites in four pharmacologically relevant targets, while comparing the efficacy of various simulation-based approaches in discovering them. We find that the studied cryptic sites do not correspond to local minima in the computed conformational free energy landscape of the unliganded proteins. They thus promptly close in all of the molecular dynamics simulations performed, irrespective of the force-field used. Temperature-based enhanced sampling approaches, such as Parallel Tempering, do not improve the situation, as the entropic term does not help in the opening of the sites. The use of fragment probes helps, as in long simulations occasionally it leads to the opening and binding to the cryptic sites. Our observed mechanism of cryptic site formation is suggestive of an interplay between two classical mechanisms: induced-fit and conformational selection. Employing this insight, we developed a novel Hamiltonian Replica Exchange-based method "SWISH" (Sampling Water Interfaces through Scaled Hamiltonians), which combined with probes resulted in a promising general approach for cryptic site discovery. We also addressed the issue of "false-positives" and propose a simple approach to distinguish them from druggable cryptic pockets. Our simulations, whose cumulative sampling time was more than 200 μs, help in clarifying the molecular mechanism of pocket formation, providing a solid basis for the choice of an efficient computational method.

Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: Conformational analysis and binding mode of multisite inhibitors

“…The results presented in this study support the reliability of combining extended MD simulations with the analysis of collective dynamics to disclose “floppy” pockets hidden in highly flexible regions and to exploit these secondary pockets for the design of novel drugs hitting cryptic pockets in challenging targets [60–62], such as BACE-1. Within the plethora of the therapeutically useful proteins codified by the “druggable genome”, β -secretase (BACE-1) has been classified in the subset of the target receptors considered of “difficult” ligandability through small molecules [63].…”

Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition

“…Consequently, as found here, the recognition ability of nanoparticles is crucially related to their ability to form pockets with the proper structure and lifetime. Fascinatingly, the series of events listed able also suggests an interplay between conformational selection and induced fit, 46 well mimicking the recognition and binding process for protein-ligand complex formation.…”

Discovery of Rab1 binding sites using an ensemble of clustering methods

“…Thorough knowledge of their common and unique features is highly critical, especially in the earliest stages of drug discovery pipelines, including the target and lead identification stages. Additionally, knowledge of the binding site residues will facilitate placement of probes (e.g., small organic compounds) that are potential in opening transient and/or cryptic binding sites through procedures such as probe‐based molecular dynamics simulations and Hamiltonian replica exchange techniques …”

“…Additionally, knowledge of the binding site residues will facilitate placement of probes (e.g., small organic compounds 81 ) that are potential in opening transient and/or cryptic binding sites through procedures such as probe-based molecular dynamics simulations 82 and Hamiltonian replica exchange techniques. 83…”