Expanded ensemble predictions of absolute binding free energies in the SAMPL9 host-guest challenge.

As part of the SAMPL9 community-wide blind host-guest challenge, we implemented an expanded ensemble workflow to predict absolute binding free energies for 13 small molecules against pillar[6]arene. Notable features of our protocol include consideration of a variety of protonation and enantiomeric states for both host and guests, optimization of alchemical intermediates, and analysis of free energy estimates and their uncertainty using large numbers of simulation replicates performed using distributed computing. Our predictions of absolute binding free energies resulted in a mean absolute error of 2.29 kcal mol-1 and an R2 of 0.54. Overall, results show that expanded ensemble calculations using all-atom molecular dynamics simulations are a valuable and efficient computational tool in predicting absolute binding free energies.

Allosteric inhibitors of the main protease of SARS-CoV-2.

SARS-CoV-2 has raised the alarm to search for effective therapy for this virus. To date several vaccines have been approved but few available drugs reported recently still need approval from FDA. Remdesivir was approved for emergency use only. In this report, the SARS-CoV-2 3CLpro was expressed and purified. By using a FRET-based enzymatic assay, we have screened a library consisting of more than 300 different niclosamide derivatives and identified three molecules JMX0286, JMX0301, and JMX0941 as potent allosteric inhibitors against SARS-CoV-2 3CLpro, with IC50 values similar to that of known covalent inhibitor boceprevir. In a cell-based antiviral assay, these inhibitors can inhibit the virus growth with EC50 in the range of 2-3 µM. The mechanism of action of JMX0286, JMX0301, and JMX0941 were characterized by enzyme kinetics, affinity binding and protein-based substrate digestion. Molecular docking, molecular dynamics (MD) simulations and hydration studies suggested that JMX0286, JMX0301, JMX0941 bind specifically to an allosteric pocket of the SARS-CoV-2 3CL protease. This study provides three potent compounds for further studies.

Markov State Model of Lassa Virus Nucleoprotein Reveals Large Structural Changes during the Trimer to Monomer Transition.

Lassa virus contains a nucleoprotein (NP) that encapsulates the viral genomic RNA forming the ribonucleoprotein (RNP). The NP forms trimers that do not bind RNA, but a structure of only the NP N-terminal domain was co-crystallized with RNA bound. These structures suggested a model in which the NP forms a trimer to keep the RNA gate closed, but then is triggered to undergo a change to a form competent for RNA binding. Here, we investigate the scenario in which the trimer is disrupted to observe whether monomeric NP undergoes significant conformational changes. From multi-microsecond molecular dynamics simulations and an adaptive sampling scheme to sample the conformational space, a Markov state model (MSM) is constructed. The MSM reveals an energetically favorable conformational change, with the most significant changes occurring at the domain interface. These results support a model in which significant structural reorganization of the NP is required for RNP formation.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Catalytic Domains of Phosphodiesterase 5, 6, and 5/6 Chimera Display Differential Dynamics and Ligand Dissociation Energy Barriers.

The enzyme phosphodiesterase 6 (PDE6) is a critical component of the visual signaling pathway and functions to convert cGMP to GMP. The ability of PDE6 to affect cellular cGMP levels leads to deactivation of cGMP-gated ion channels in both rod and cone cells. PDE6 has been difficult to structurally characterize experimentally, though the structures of the closely related PDE5 and a PDE5/6 chimera have been determined by X-ray crystallography. In this work, we employ a computational approach to study the dynamics of the catalytic domains of PDE6, PDE5, and the PDE5/6 chimera. Through equilibrium molecular dynamics (MD) simulations, we identify a region of PDE6 (α12) to be correlated to distal regions of the enzyme (H- and M-loops), which surround the catalytic center. These correlations are not observed for PDE5, and we speculate that these unique motions in PDE6 may relate to the high catalytic efficiency of PDE6 and the requirement for an endogenous inhibitory subunit (Pγ). We further investigate the ligand binding pathways and energetics by enhanced sampling simulations (metadynamics) using the inhibitor sildenafil and GMP. The energetics and pathways of ligand binding are consistent with the high efficiency of PDE6 and further implicate the α12 region as an important regulatory element for PDE6 functional dynamics.

Folding a viral peptide in different membrane environments: pathway and sampling analyses.

Flock House virus (FHV) is a well-characterized model system to study infection mechanisms in non-enveloped viruses. A key stage of the infection cycle is the disruption of the endosomal membrane by a component of the FHV capsid, the membrane active γ peptide. In this study, we perform all-atom molecular dynamics simulations of the 21 N-terminal residues of the γ peptide interacting with membranes of differing compositions. We carry out umbrella sampling calculations to study the folding of the peptide to a helical state in homogenous and heterogeneous membranes consisting of neutral and anionic lipids. From the trajectory data, we evaluate folding energetics and dissect the mechanism of folding in the different membrane environments. We conclude the study by analyzing the extent of configurational sampling by performing time-lagged independent component analysis.

Influence of RNA Binding on the Structure and Dynamics of the Lassa Virus Nucleoprotein.

Lassa virus protects its viral genome through the formation of a ribonucleoprotein complex in which the nucleoprotein (NP) encapsidates the single-stranded RNA genome. Crystal structures provide evidence that a conformational change must occur to allow for RNA binding. In this study, the mechanism by which NP binds to RNA and how the conformational changes in NP are achieved was investigated with molecular-dynamics simulations. NP was structurally characterized in an open configuration when bound to RNA and in a closed form in the absence of RNA. Our results show that when NP is bound to RNA, the protein is highly dynamic and the system undergoes spontaneous deviations away from the open-state configuration. The equilibrium simulations are supported by free-energy calculations that quantify the influence of RNA on the free-energy surface, which governs NP dynamics. We predict that the globally stable states are qualitatively in agreement with the observed crystal structures, but that both open and closed conformations are thermally accessible in the presence of RNA. The free-energy calculations also provide a prediction of the location of the transition state for RNA binding and identify an intermediate metastable state that exhibits correlated motions that could promote RNA binding.