TY - JOUR
T1 - Small Glycols Discover Cryptic Pockets on Proteins for Fragment-Based Approaches
AU - Bansia, Harsh
AU - Mahanta, Pranjal
AU - Yennawar, Neela H.
AU - Ramakumar, Suryanarayanarao
N1 - Funding Information:
The authors thank CSIR-UGC and the Indian Institute of Science (IISc) for providing financial support to H.B., UGC for Emeritus Fellowship to S.R., X-ray Facility at the Molecular Biophysics Unit (IISc), Bengaluru, India, and European Synchrotron Radiation Facility (ESRF), Grenoble, France for X-ray data collection, staff for providing access to and support on the beamline BM14 at ESRF, DBT for funding the trip to ESRF, DST sponsored computational facility in the Department of Physics (IISc), and Supercomputer Education and Research Center (IISc) for providing access to CRAY XC40-“SAHASRAT” supercomputer and other computational resources for MD simulations and Dr. V.S. Reddy and Dr. Amit Bharadwaj for providing protein samples of RBSX and RBSX-W6A. The authors thank Julia Fecko for the ITC binding study. The ITC work was supported by the NIH grant S10OD025145 to Dr. Yennawar for the TA Instruments Low Volume Auto Affinity ITC, housed in the Automated Biological Calorimetry Core Facility at the Penn State, Huck Institutes of the Life Sciences.
Publisher Copyright:
©
PY - 2021/3/22
Y1 - 2021/3/22
N2 - Cryptic pockets are visible in ligand-bound protein structures but are occluded in unbound structures. Utilizing these pockets in fragment-based drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, they are difficult to identify. Here, we show that small glycols find cryptic pockets on a diverse set of proteins. Initial crystallography experiments serendipitously revealed the ability of ethylene glycol, a small glycol, to identify a cryptic pocket on the W6A mutant of the RBSX protein (RBSX-W6A). Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with the exposed state of the cryptic pocket (ethylene glycol removed) revealed closure of the pocket reiterating that the exposed state of cryptic pockets in general are unstable in the absence of ligands. Also, no change in the pocket was observed for simulations of RBSX-W6A with the occluded state of the cryptic pocket, suggesting that water molecules are not able to open the cryptic pocket. "Cryptic-pocket finding"potential of small glycols was then supported and generalized through additional crystallography experiments, explicit-cosolvent MD simulations, and protein data set construction and analysis. The cryptic pocket on RBSX-W6A was found again upon repeating the crystallography experiments with another small glycol, propylene glycol. Use of ethylene glycol as a probe molecule in cosolvent MD simulations led to the enhanced sampling of the exposed state of experimentally observed cryptic sites on a test set of two proteins (Niemann-Pick C2, Interleukin-2). Further, analyses of protein structures with validated cryptic sites showed that ethylene glycol molecules bind to sites on proteins (Bcl-xL, G-actin, myosin II, and glutamate receptor 2), which become apparent upon binding of biologically relevant ligands. Our study thus suggests potential application of the small glycols in experimental and computational fragment-based approaches to identify cryptic pockets in apparently undruggable and/or difficult targets, making these proteins amenable to drug-design strategies.
AB - Cryptic pockets are visible in ligand-bound protein structures but are occluded in unbound structures. Utilizing these pockets in fragment-based drug-design provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, they are difficult to identify. Here, we show that small glycols find cryptic pockets on a diverse set of proteins. Initial crystallography experiments serendipitously revealed the ability of ethylene glycol, a small glycol, to identify a cryptic pocket on the W6A mutant of the RBSX protein (RBSX-W6A). Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with the exposed state of the cryptic pocket (ethylene glycol removed) revealed closure of the pocket reiterating that the exposed state of cryptic pockets in general are unstable in the absence of ligands. Also, no change in the pocket was observed for simulations of RBSX-W6A with the occluded state of the cryptic pocket, suggesting that water molecules are not able to open the cryptic pocket. "Cryptic-pocket finding"potential of small glycols was then supported and generalized through additional crystallography experiments, explicit-cosolvent MD simulations, and protein data set construction and analysis. The cryptic pocket on RBSX-W6A was found again upon repeating the crystallography experiments with another small glycol, propylene glycol. Use of ethylene glycol as a probe molecule in cosolvent MD simulations led to the enhanced sampling of the exposed state of experimentally observed cryptic sites on a test set of two proteins (Niemann-Pick C2, Interleukin-2). Further, analyses of protein structures with validated cryptic sites showed that ethylene glycol molecules bind to sites on proteins (Bcl-xL, G-actin, myosin II, and glutamate receptor 2), which become apparent upon binding of biologically relevant ligands. Our study thus suggests potential application of the small glycols in experimental and computational fragment-based approaches to identify cryptic pockets in apparently undruggable and/or difficult targets, making these proteins amenable to drug-design strategies.
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U2 - 10.1021/acs.jcim.0c01126
DO - 10.1021/acs.jcim.0c01126
M3 - Article
C2 - 33570386
AN - SCOPUS:85101624214
SN - 1549-9596
VL - 61
SP - 1322
EP - 1333
JO - Journal of Chemical Information and Modeling
JF - Journal of Chemical Information and Modeling
IS - 3
ER -