All-atom simulations of the trimeric spike protein of SARS-CoV-2 in aqueous medium: Nature of interactions, conformational stability and free energy diagrams for conformational transition of the protein.

The spike protein of SARS-CoV-2 exists in two major conformational states, namely the 'open' and 'closed' states which are also known as the 'up' and 'down' states, respectively. In its open state, the receptor binding domain (RBD) of the protein is exposed for binding with ACE2, whereas the spike RBD is inaccessible to ACE2 in the closed state of the protein. In the current work, we have performed all-atom microsecond simulations of the full-length trimeric spike protein solvated in explicit aqueous medium with an average system size of ~0.7 million atoms to understand the molecular nature of intra- and inter-chain interactions, water-bridged interactions between different residues that contribute to the stability of the open and closed states of the protein, and also the free energy landscape for transition between the open and closed states of the protein. We have also examined the changes of such interactions that are associated with switching from one state to the other through both unbiased and biased simulations at all-atom level with total run length of 4 µs. Interestingly, after about 0.8 µs of unbiased molecular dynamics run of the spike system in the open state, we observed a gradual transition of the monomeric chain (B) from open to its partially closed or down state. Initially the residues at the interface of chain B RBD in the open state spike protein were at non-hydrogen-bonding distances from the residues of chain C RBD. However, the two RBDs gradually came closer and finally the residue S459 of the RBD of chain B made a hydrogen bond with F374 of chain C in the last 200 ns of the simulation along with formation of a few more hydrogen bonds involving other residues. Since no transition from closed to the open state of the protein is observed in the present 1 µs unbiased simulation of the closed state protein, the current study seems to suggest that the closed conformational state is preferred for the spike protein of SARS-CoV-2 in aqueous medium. Furthermore, calculations of the free energy surface of the conformational transition from open (up) to the closed (down) state using a biased simulation method reveal a free energy barrier of ~3.20 kcal/mol for the transition of RBD from open to the closed state, whereas the barrier for the reverse process is found to be significantly higher.

Analogue and structure based approaches for modelling HIV-1 integrase inhibitors.

A set of 220 inhibitors belonging to different structure classes and having HIV-1 integrase activity were collected along with their experimental pIC50 values. Geometries of all the inhibitors were fully optimized using B3LYP/6-31 + G(d) level of theory. These ligands were docked against 4 different HIV-1 integrase receptors (PDB IDs: 4LH5, 5KRS, 3ZSQ and 3ZSV). 30 docked poses were generated for all 220 inhibitors and ligand interaction of the first docked pose and the docked pose with the highest score were analysed. Residue GLU170 of 4LH5 receptor shows the highest number of interactions followed by ALA169, GLN168, HIS171 and ASP167 residues. Hydrogen bonding and stacking are mainly responsible for the interactions of these inhibitors with the receptor. We performed Molecular Dynamics (MD) simulation to observe the root-mean-square deviation (RMSD), for measure the average change of displacement between the atoms for a particular frame with respect to a reference and The Root Mean Square Fluctuation (RMSF) for characterization of local changes along the protein chain of the docked complexes. Analogue based models were generated to predict the pIC50 values for integrase inhibitors using various types of descriptors such as constitutional, geometrical, topological, quantum chemical and docking based descriptors. The best models were selected on the basis of statistical parameters and were validated by training and test set division. A few new inhibitors were designed on the basis of structure activity relationship and their pIC50 values were predicted using the generated models. All the designed new inhibitors a very high potential and may be used as potent inhibitors of HIV integrase. These models may be useful for further design and development of new and potent HIV integrase inhibitors.Communicated by Ramaswamy H. Sarma.

All-Atom Simulations of Human ACE2-Spike Protein RBD Complexes for SARS-CoV-2 and Some of its Variants: Nature of Interactions and Free Energy Diagrams for Dissociation of the Protein Complexes.

The spike protein of SARS-CoV-2 is known to interact with the human ACE2 protein via its receptor binding domain (RBD). We have investigated the molecular nature of this interprotein interaction and the associated free energy diagrams for the unbinding of the two proteins for SARS-CoV-2 and some of its known variants through all-atom simulations. The present work involves generation and analysis of 2.5 µs of unbiased and 4.2 µs of biased molecular dynamics trajectories in total for five explicitly solvated RBD-ACE2 systems at full atomic level. First, we have made a comparative analysis of the details of residue-wise specific interactions of the spike protein with ACE2 for SARS-CoV-1 and SARS-CoV-2. It is found that the average numbers of both direct interprotein and water-bridged hydrogen bonds between the RBD and ACE2 are higher for SARS-CoV-2 than SARS-CoV-1. These higher hydrogen bonded interactions are further aided by enhanced nonspecific electrostatic attractions between the two protein surfaces for SARS-CoV-2. The free energy calculations reveal that there is an increase in the free energy barrier by ∼1.5 kcal/mol for the unbinding of RBD from ACE2 for SARS-CoV-2 compared to that for SARS-CoV-1. Subsequently, we considered the RBDs of three variants of SARS-CoV-2, namely N501Y, E484Q/L452R, and N440K. The free energy barrier of protein unbinding for the N501Y variant is found to be ∼4 kcal/mol higher than the wild type SARS-CoV-2 which can be attributed to additional specific interactions involving Tyr501 of RBD and Lys353 and Tyr42 of ACE2 and also enhanced nonspecific electrostatic interaction between the protein surfaces. For the other two mutant variants of E484Q/L452R and N440K, the free energy barrier for protein unbinding increases by ∼2 and ∼1 kcal/mol, respectively, compared with the wild type SARS-CoV-2, which can be attributed to an increase in the number of interprotein hydrogen bonds for the former and also to enhanced positive electrostatic potential on the RBD surfaces for both of the variants. The successive breaking of interprotein hydrogen bonds along the free energy pathway of the unbinding process is also found out for all five systems studied here.

In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance.

In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2 The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches.

OBJECTIVE: Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. METHODS: In the present study, we targeted the nucleotide-binding site in the "on" and "off" state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. RESULTS: Interestingly, the designed compounds exhibit a binding preference for the "off" state over "on" state conformation of K-Ras protein. Moreover, the designed compounds' interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski's rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. CONCLUSION: Thus, through the current study, we propose targeting only "off" state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein.