Alchemical Absolute Protein-Ligand Binding Free Energies for Drug Design

The recent advances in relative protein-ligand binding free energy calculations have shown the value of alchemical methods in drug discovery. Accurately assessing absolute binding free energies, although highly desired, remains a challenging endeavour, mostly limited to small model cases. Here, we demonstrate accurate first principles based absolute binding free energy estimates for 128 pharmaceutically relevant targets. We use a novel rigorous method to generate protein-ligand ensembles for the ligand in its decoupled state. Not only do the calculations deliver accurate protein-ligand binding affinity estimates, but they also provide detailed physical insight into the structural determinants of binding. We identify subtle rotamer rearrangements between apo and holo states of a protein that are crucial for binding. When compared to relative binding free energy calculations, obtaining absolute binding free energies is considerably more challenging in large part due to the need to explicitly account for the protein in its apo state. In this work we present several approaches to obtain apo state ensembles for accurate absolute ΔG calculations, thus outlining protocols for prospective application of the methods for drug discovery.

Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake

Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo–electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

We have performed multicanonical molecular dynamics (McMD) based dynamic docking simulations to study and compare the binding mechanism between two medium-sized inhibitors (ABT-737 and WEHI-539) that bind to the cryptic site of Bcl-xL, by exhaustively sampling the conformational and configurational space. Cryptic sites are binding pockets that are transiently formed in the apo state or are induced upon ligand binding. Bcl-xL, a pro-survival protein involved in cancer progression, is known to have a cryptic site, whereby the shape of the pocket depends on which ligand is bound to it. Starting from the apo-structure, we have performed two independent McMD-based dynamic docking simulations for each ligand, and were able to obtain near-native complex structures in both cases. In addition, we have also studied their interactions along their respective binding pathways by using path sampling simulations, which showed that the ligands form stable binding configurations via predominantly hydrophobic interactions. Although the protein started from the apo state, both ligands modulated the pocket in different ways, shifting the conformational preference of the sub-pockets of Bcl-xL. We demonstrate that McMD-based dynamic docking is a powerful tool that can be effectively used to study binding mechanisms involving a cryptic site, where ligand binding requires a large conformational change in the protein to occur.

Molecular insights on CALX-CBD12 inter-domain dynamics from MD simulations, RDCs and SAXS

ABSTRACTNa+/Ca2+ exchangers (NCX) are secondary active transporters that couple the translocation of Na+ with the transport of Ca2+ in the opposite direction. The exchanger is an essential Ca2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca2+-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca2+-sensor called CBD12. Binding of intracellular Ca2+ to CBD12 activates the NCX but inhibits the Na+/Ca2+ exchanger of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant inter-domain flexibility in the Apo state, but assume rigid inter-domain arrangements in the Ca2+-bound state. However, detailed structure information on CBD12 in the Apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the Apo state, we applied molecular dynamics (MD) simulations, NMR (1H-15N RDCs) and Small-Angle X-Ray Scattering (SAXS) data in a combined modelling strategy that generated atomistic information on the most representative conformations. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, while the wide-open inter-domain arrangement characteristic of the Ca2+-bound state is less frequently sampled. These results are consistent with the view that Ca2+ binding shifts the CBD12 conformational ensemble towards extended conformers, which could be a key step in the Na+/Ca2+ exchangers’ allosteric regulation mechanism. The present strategy, combining MD with NMR and SAXS, provides a powerful approach to select representative structures from ensembles of conformations, which could be applied to other flexible multi-domain systems.SIGNIFICANCEThe conformational ensemble of CALX-CBD12, the main Ca2+-sensor of Drosophila’s Na+/Ca2+ exchanger, was characterized by a combination of MD simulations with SAXS and NMR data using the EOM approach. This analysis showed that this two-domain construct experiences opening-closing motions, providing molecular information about CALX-CBD12 in the Apo state. Ca2+-binding shifts the conformational ensemble towards extended conformers. These findings are consistent with a model according to which Ca2+ modulation of CBD12 plasticity is a key step in the Ca2+-regulation mechanism of the full-length exchanger.

Antibiotic binding releases autoinhibition of the TipA multidrug-resistance transcriptional regulator

Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6–α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6–α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance.

High-Resolution Mining of SARS-CoV-2 Main Protease Conformational Space: Supercomputer-Driven Unsupervised Adaptive Sampling

We provide a new unsupervised adaptive sampling strategy capable of producing microsecondtimescale molecular dynamics (MD) simulations using many-body polarizable force fields (PFF) on modern supercomputers. The global exploration problem is decomposed into a set of separate MD trajectories that can be restarted within an iterative/selective process to achieve sufficient phase-space sampling within large biosystems, while accurate statistical properties can be obtained through debiasing. With this pleasingly parallel setup, the Tinker-HP package can be powered by an arbitrary large number of GPUs (Graphics Processing Unit) cards available on pre-exascale supercomputers, reducing to days explorations that would have taken years. We applied the approach to the urgent problem of the modeling of the SARS–CoV–2 Main protease (Mpro) dimer. A 15.14 microsecond high-resolution all-atom simulation (AMOEBA PFF) of its apo state is provided and compared to other available long-timescale non-PFF data. Noticeable differences are found between clustering analysis of the simulations, the AMOEBA adaptive results exhibiting a richer conformational space. Overall, our high-resolution AMOEBA structural analysis captures key experimental observations concerning the stability of the oxyanion hole, a marker of activity through the stability of different stacking and salt bridge interactions. A dissymmetry is found between the enzyme protomers that exhibit different volumes. One of them appears fully inactive while the other is "activable", exhibiting some partial activity features. This activity evaluation can be further traced back to the large flexibility of the C terminal domain, fully captured by AMOEBA but not seen in X-rays due to insufficient electron densities related to the domain high mobility. The C–terminal region of the fully inactive protomer is shown to oscillate between several states, one of them interacting with the other protomer active site, therefore potentially modulating down its activity. Overall, these results reinforce the experimental hypothesis of a full inactivation of the apo state and clearly capture the asymmetric nature of protomers. Additional analysis show that the cavities volumes of the active and distal sites are found to be larger in the most active protomer with AMOEBA. To a larger extend, the PFF finds significantly larger cavities than those obtained with classical, non-polarizable simulations. The consequences on druggability are discussed as additional potential druggable cryptic pockets are found. All data produced within this research are fully accessible to the community for further analysis.

High-Resolution Mining of SARS-CoV-2 Main Protease Conformational Space: Supercomputer-Driven Unsupervised Adaptive Sampling

We provide a new unsupervised adaptive sampling strategy capable of producing microsecondtimescale molecular dynamics (MD) simulations using many-body polarizable force fields (PFF) on modern supercomputers. The global exploration problem is decomposed into a set of separate MD trajectories that can be restarted within an iterative/selective process to achieve sufficient phase-space sampling within large biosystems, while accurate statistical properties can be obtained through debiasing. With this pleasingly parallel setup, the Tinker-HP package can be powered by an arbitrary large number of GPUs (Graphics Processing Unit) cards available on pre-exascale supercomputers, reducing to days explorations that would have taken years. We applied the approach to the urgent problem of the modeling of the SARS–CoV–2 Main protease (Mpro) dimer. A 15.14 microsecond high-resolution all-atom simulation (AMOEBA PFF) of its apo state is provided and compared to other available long-timescale non-PFF data. Noticeable differences are found between clustering analysis of the simulations, the AMOEBA adaptive results exhibiting a richer conformational space. Overall, our high-resolution AMOEBA structural analysis captures key experimental observations concerning the stability of the oxyanion hole, a marker of activity through the stability of different stacking and salt bridge interactions. A dissymmetry is found between the enzyme protomers that exhibit different volumes. One of them appears fully inactive while the other is "activable", exhibiting some partial activity features. This activity evaluation can be further traced back to the large flexibility of the C terminal domain, fully captured by AMOEBA but not seen in X-rays due to insufficient electron densities related to the domain high mobility. The C–terminal region of the fully inactive protomer is shown to oscillate between several states, one of them interacting with the other protomer active site, therefore potentially modulating down its activity. Overall, these results reinforce the experimental hypothesis of a full inactivation of the apo state and clearly capture the asymmetric nature of protomers. Additional analysis show that the cavities volumes of the active and distal sites are found to be larger in the most active protomer with AMOEBA. To a larger extend, the PFF finds significantly larger cavities than those obtained with classical, non-polarizable simulations. The consequences on druggability are discussed as additional potential druggable cryptic pockets are found. All data produced within this research are fully accessible to the community for further analysis.

High-Resolution Mining of SARS-CoV-2 Main Protease Conformational Space: Supercomputer-Driven Unsupervised Adaptive Sampling

We provide a new unsupervised adaptive sampling strategy capable of producing microsecondtimescale molecular dynamics (MD) simulations using many-body polarizable force fields (PFF) on modern supercomputers. The global exploration problem is decomposed into a set of separate MD trajectories that can be restarted within an iterative/selective process to achieve sufficient phase-space sampling within large biosystems, while accurate statistical properties can be obtained through debiasing. With this pleasingly parallel setup, the Tinker-HP package can be powered by an arbitrary large number of GPUs (Graphics Processing Unit) cards available on pre-exascale supercomputers, reducing to days explorations that would have taken years. We applied the approach to the urgent problem of the modeling of the SARS–CoV–2 Main protease (Mpro) dimer. A 15.14 microsecond high-resolution all-atom simulation (AMOEBA PFF) of its apo state is provided and compared to other available long-timescale non-PFF data. Noticeable differences are found between clustering analysis of the simulations, the AMOEBA adaptive results exhibiting a richer conformational space. Overall, our high-resolution AMOEBA structural analysis captures key experimental observations concerning the stability of the oxyanion hole, a marker of activity through the stability of different stacking and salt bridge interactions. A dissymmetry is found between the enzyme protomers that exhibit different volumes. One of them appears fully inactive while the other is "activable", exhibiting some partial activity features. This activity evaluation can be further traced back to the large flexibility of the C terminal domain, fully captured by AMOEBA but not seen in X-rays due to insufficient electron densities related to the domain high mobility. The C–terminal region of the fully inactive protomer is shown to oscillate between several states, one of them interacting with the other protomer active site, therefore potentially modulating down its activity. Overall, these results reinforce the experimental hypothesis of a full inactivation of the apo state and clearly capture the asymmetric nature of protomers. Additional analysis show that the cavities volumes of the active and distal sites are found to be larger in the most active protomer with AMOEBA. To a larger extend, the PFF finds significantly larger cavities than those obtained with classical, non-polarizable simulations. The consequences on druggability are discussed as additional potential druggable cryptic pockets are found. All data produced within this research are fully accessible to the community for further analysis.

High-Resolution Mining of SARS-CoV-2 Main Protease Conformational Space: Supercomputer-Driven Unsupervised Adaptive Sampling

We provide a new unsupervised adaptive sampling strategy capable of producing microsecondtimescale molecular dynamics (MD) simulations using many-body polarizable force fields (PFF) on modern supercomputers. The global exploration problem is decomposed into a set of separate MD trajectories that can be restarted within an iterative/selective process to achieve sufficient phase-space sampling within large biosystems, while accurate statistical properties can be obtained through debiasing. With this pleasingly parallel setup, the Tinker-HP package can be powered by an arbitrary large number of GPUs (Graphics Processing Unit) cards available on pre-exascale supercomputers, reducing to days explorations that would have taken years. We applied the approach to the urgent problem of the modeling of the SARS–CoV–2 Main protease (Mpro) dimer. A 15.14 microsecond high-resolution all-atom simulation (AMOEBA PFF) of its apo state is provided and compared to other available long-timescale non-PFF data. Noticeable differences are found between clustering analysis of the simulations, the AMOEBA adaptive results exhibiting a richer conformational space. Overall, our high-resolution AMOEBA structural analysis captures key experimental observations concerning the stability of the oxyanion hole, a marker of activity through the stability of different stacking and salt bridge interactions. A dissymmetry is found between the enzyme protomers that exhibit different volumes. One of them appears fully inactive while the other is "activable", exhibiting some partial activity features. This activity evaluation can be further traced back to the large flexibility of the C terminal domain, fully captured by AMOEBA but not seen in X-rays due to insufficient electron densities related to the domain high mobility. The C–terminal region of the fully inactive protomer is shown to oscillate between several states, one of them interacting with the other protomer active site, therefore potentially modulating down its activity. Overall, these results reinforce the experimental hypothesis of a full inactivation of the apo state and clearly capture the asymmetric nature of protomers. Additional analysis show that the cavities volumes of the active and distal sites are found to be larger in the most active protomer with AMOEBA. To a larger extend, the PFF finds significantly larger cavities than those obtained with classical, non-polarizable simulations. The consequences on druggability are discussed as additional potential druggable cryptic pockets are found. All data produced within this research are fully accessible to the community for further analysis.

High-Resolution Mining of SARS-CoV-2 Main Protease Conformational Space: Supercomputer-Driven Unsupervised Adaptive Sampling

We provide a new unsupervised adaptive sampling strategy capable of producing microsecondtimescale molecular dynamics (MD) simulations using many-body polarizable force fields (PFF) on modern supercomputers. The global exploration problem is decomposed into a set of separate MD trajectories that can be restarted within an iterative/selective process to achieve sufficient phase-space sampling within large biosystems, while accurate statistical properties can be obtained through debiasing. With this pleasingly parallel setup, the Tinker-HP package can be powered by an arbitrary large number of GPUs (Graphics Processing Unit) cards available on pre-exascale supercomputers, reducing to days explorations that would have taken years. We applied the approach to the urgent problem of the modeling of the SARS–CoV–2 Main protease (Mpro) dimer. A 15.14 microsecond high-resolution all-atom simulation (AMOEBA PFF) of its apo state is provided and compared to other available long-timescale non-PFF data. Noticeable differences are found between clustering analysis of the simulations, the AMOEBA adaptive results exhibiting a richer conformational space. Overall, our high-resolution AMOEBA structural analysis captures key experimental observations concerning the stability of the oxyanion hole, a marker of activity through the stability of different stacking and salt bridge interactions. A dissymmetry is found between the enzyme protomers that exhibit different volumes. One of them appears fully inactive while the other is "activable", exhibiting some partial activity features. This activity evaluation can be further traced back to the large flexibility of the C terminal domain, fully captured by AMOEBA but not seen in X-rays due to insufficient electron densities related to the domain high mobility. The C–terminal region of the fully inactive protomer is shown to oscillate between several states, one of them interacting with the other protomer active site, therefore potentially modulating down its activity. Overall, these results reinforce the experimental hypothesis of a full inactivation of the apo state and clearly capture the asymmetric nature of protomers. Additional analysis show that the cavities volumes of the active and distal sites are found to be larger in the most active protomer with AMOEBA. To a larger extend, the PFF finds significantly larger cavities than those obtained with classical, non-polarizable simulations. The consequences on druggability are discussed as additional potential druggable cryptic pockets are found. All data produced within this research are fully accessible to the community for further analysis.