Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

Inhibitory effects of Ephedra alte on IL-6, hybrid TLR4, TNF-α, IL-1ß, and extracted TLR4 receptors: in silico molecular docking.

Inflammation is a physiological reaction of the immune system required to remove the presence of pathogenic germs. Many herbal-derived extracts and phytoconstituents show anti-inflammatory effects. Among these natural phytoconstituents is Ephedra alte (E. alte), which shows pepsin enzyme inhibitory, antibacterial, and antioxidant activities. In this work, molecular docking study is conducted on five major human anti-inflammatory cytokines receptors (IL-6, hybrid TLR4, TNF-α, IL-1ß, and extracted TLR4) to explore the molecular recognition process and complex ligand-receptor interactions of E. alte phytoconstituents. Human TLR4 receptor has been computationally extracted, for the first time, from the hybrid TLR4 human and VLRB inshore hagfish. Among E. alte phytoconstituents, only ß-Sitosterol and Androstan-3-one have better LBE (Lowest Binding Energy) scores with inhibition constant (K i) values than those of other tested compounds. The ß-Sitosterol and Androstan-3-one results indicate that these compounds could be efficient inhibitors of inflammation and reduce the oxidative stress by interfering with the activity of the five studied proteins.

Most Probable Druggable Pockets in Mutant p53-Arg175His Clusters Extracted from Gaussian Accelerated Molecular Dynamics Simulations.

p53, a tumor suppressor protein, is essential for preventing cancer development. Enhancing our understanding of the human p53 function and its modifications in carcinogenesis will aid in developing more highly effective strategies for cancer prevention and treatment. In this study, we have modeled five human p53 forms, namely, inactive, distal-active, proximal-active, distal-Arg175His mutant, and proximal-Arg175His mutant forms. These forms have been investigated using Gaussian accelerated molecular dynamics (GaMD) simulations in OPC water model at physiological temperature and pH. Our observations, obtained throughout [Formula: see text] of production run, are in good agreement with the relevant results in the classical molecular dynamics (MD) studies. Therefore, GaMD method is more economic and efficient method than the classical MD method for studying biomolecular systems. The featured dynamics of the five human p53-DBD forms include noticeable conformational changes of L1 and [Formula: see text]-[Formula: see text] loops as well as [Formula: see text]-[Formula: see text] and [Formula: see text]-[Formula: see text] turns. We have identified two clusters that represent two distinct conformational states in each p53-DBD form. The free-energy profiles of these clusters demonstrate the flexibility of the protein to undergo a conformational transition between the two clusters. We have predicted two out of seven possible druggability pockets on the clusters of the Arg175His forms. These two druggability pockets are near the mutation site and are expected to be actual pockets, which will be helpful for the compound clinical progression studies.

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study.

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.

Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs), which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM) and COOH-terminal tail (C-terminal tail). Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and provides new clues to cancer mutations that alter the canonical modes of ErbB kinase regulation.

Computational studies of gramicidin permeation: an entry way sulfonate enhances cation occupancy at entry sites.

The impact on the cation-transport free-energy profile of replacing the C-terminal ethanolamine in the gramicidin A channel with a taurine residue is studied using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl saline solution. The potential of mean force for ion transport is obtained by umbrella sampling. The presence of a negatively charged sulfonate group at the entrance of the gramicidin channel affects the depth and the location of the binding sites, producing a strong attraction for the cations in the bulk. The potential of mean force by the sulfonate acting directly through electrostatics and van der Waals interactions on the test ion is highly modulated by indirect effects (i.e., sulfonate effects on other components of the system that, in turn, affect the ion free-energy profile in the channel). Because the "entry" sites are located symmetrically at both entry and exit of the channel, the deeper free-energy wells should inhibit exit. Given that the channel has increased conductance experimentally, the simulation results suggest that the channel conductance is normally entry limited.

Free-energy profiles for ions in the influenza M2-TMD channel.

M(2) transmembrane domain channel (M(2)-TMD) permeation properties are studied using molecular dynamics simulations of M(2)-TMD (1NYJ) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. This study allows examination of spontaneous cation and anion entry into the selectivity filter. Three titration states of the M(2)-TMD tetramer are modeled for which the four His(37) residues, forming the selectivity filter, are net uncharged, +2 charged, or +3 charged. M(2)-TMD structural properties from our simulations are compared with the properties of other models extracted from NMR and X-ray studies. During 10 ns simulations, chloride ions occasionally occupy the positively-charged selectivity filter region, and from umbrella sampling simulations, Cl(-) has a lower free-energy barrier in the selectivity-filter region than either Na(+) or NH(4) (+), and NH(4) (+) has a lower free-energy barrier than Na(+). For Na(+) and Cl(-), the free-energy barriers are less than 5 kcal/mol, suggesting that the 1NYJ conformation would probably not be exquisitely proton selective. We also point out a rotameric configuration of Trp(41) that could fully occlude the channel.

The gramicidin channel ion permeation free-energy profile: direct and indirect effects of CHARMM force field improvements.

A revised CHARMM force field for tryptophan residues is studied as well as a new grid-based correction algorithm, called CMAP, using molecular dynamics simulations of gramicidin A (1JNO) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. The conformational stability of the interfacial side chains is studied, which shows good stability on the 10 ns time scale. The revised force field for the tryptophan side chain produces, in the decomposition, a Na(+) PMF(Trp) profile that is consonant with the prediction from the experimental results, analyzed with rate theory by Durrant et al. (2006), but in stark contrast to the prediction of the original CHARMM force field, version 22. However, the effect is diluted in the PMF profile due to indirect effects mediated by other components of the system (polypeptide, lipid molecules, ions, and water molecules). CMAP corrections to the L-amino acids help reduce the excessive translocation barrier. Decomposition demonstrates that this effect is due to effects on the K(+) PMF(H(2)O) profile rather than on the K(+) PMF(gA) profile. The results have been confirmed to be robust using an alternative umbrella-potential method. Further force field balancing efforts (direct and indirect) are required for future studies to evaluate whether these effects give rise to predictions that are consistent with those observables extracted from real experiments.