Computational Insights into SARS-CoV-2 Main Protease Mutations and Nirmatrelvir Efficacy: The Effects of P132H and P132H-A173V.

Nirmatrelvir, a pivotal component of the oral antiviral Paxlovid for COVID-19, targets the SARS-CoV-2 main protease (Mpro) as a covalent inhibitor. Here, we employed combined computational methods to explore how the prevalent Omicron variant mutation P132H, alone and in combination with A173V (P132H-A173V), affects nirmatrelvir's efficacy. Our findings suggest that P132H enhances the noncovalent binding affinity of Mpro for nirmatrelvir, whereas P132H-A173V diminishes it. Although both mutants catalyze the rate-limiting step more efficiently than the wild-type (WT) Mpro, P132H slows the overall rate of covalent bond formation, whereas P132H-A173V accelerates it. Comprehensive analysis of noncovalent and covalent contributions to the overall binding free energy of the covalent complex suggests that P132H likely enhances Mpro sensitivity to nirmatrelvir, while P132H-A173V may confer resistance. Per-residue decompositions of the binding and activation free energies pinpoint key residues that significantly affect the binding affinity and reaction rates, revealing how the mutations modulate these effects. The mutation-induced conformational perturbations alter drug-protein local contact intensities and the electrostatic preorganization of the protein, affecting noncovalent binding affinity and the stability of key reaction states, respectively. Our findings inform the mechanisms of nirmatrelvir resistance and sensitivity, facilitating improved drug design and the detection of resistant strains.

Identification of and Mechanistic Insights into SARS-CoV-2 Main Protease Non-Covalent Inhibitors: An In-Silico Study.

The indispensable role of the SARS-CoV-2 main protease (Mpro) in the viral replication cycle and its dissimilarity to human proteases make Mpro a promising drug target. In order to identify the non-covalent Mpro inhibitors, we performed a comprehensive study using a combined computational strategy. We first screened the ZINC purchasable compound database using the pharmacophore model generated from the reference crystal structure of Mpro complexed with the inhibitor ML188. The hit compounds were then filtered by molecular docking and predicted parameters of drug-likeness and pharmacokinetics. The final molecular dynamics (MD) simulations identified three effective candidate inhibitors (ECIs) capable of maintaining binding within the substrate-binding cavity of Mpro. We further performed comparative analyses of the reference and effective complexes in terms of dynamics, thermodynamics, binding free energy (BFE), and interaction energies and modes. The results reveal that, when compared to the inter-molecular electrostatic forces/interactions, the inter-molecular van der Waals (vdW) forces/interactions are far more important in maintaining the association and determining the high affinity. Given the un-favorable effects of the inter-molecular electrostatic interactions-association destabilization by the competitive hydrogen bond (HB) interactions and the reduced binding affinity arising from the un-compensable increase in the electrostatic desolvation penalty-we suggest that enhancing the inter-molecular vdW interactions while avoiding introducing the deeply buried HBs may be a promising strategy in future inhibitor optimization.

Mechanistic Origin of Different Binding Affinities of SARS-CoV and SARS-CoV-2 Spike RBDs to Human ACE2.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (RBDCoV2) has a higher binding affinity to the human receptor angiotensin-converting enzyme 2 (ACE2) than the SARS-CoV RBD (RBDCoV). Here, we performed molecular dynamics (MD) simulations, binding free energy (BFE) calculations, and interface residue contact network (IRCN) analysis to explore the mechanistic origin of different ACE2-binding affinities of the two RBDs. The results demonstrate that, when compared to the RBDCoV2-ACE2 complex, RBDCoV-ACE2 features enhanced dynamicsand inter-protein positional movements and increased conformational entropy and conformational diversity. Although the inter-protein electrostatic attractive interactions are the primary determinant for the high ACE2-binding affinities of both RBDs, the significantly enhanced electrostatic attractive interactions between ACE2 and RBDCoV2 determine the higher ACE2-binding affinity of RBDCoV2 than of RBDCoV. Comprehensive comparative analyses of the residue BFE components and IRCNs between the two complexes reveal that it is the residue changes at the RBD interface that lead to the overall stronger inter-protein electrostatic attractive force in RBDCoV2-ACE2, which not only tightens the interface packing and suppresses the dynamics of RBDCoV2-ACE2, but also enhances the ACE2-binding affinity of RBDCoV2. Since the RBD residue changes involving gain/loss of the positively/negatively charged residues can greatly enhance the binding affinity, special attention should be paid to the SARS-CoV-2 variants carrying such mutations, particularly those near or at the binding interfaces with the potential to form hydrogen bonds and/or salt bridges with ACE2.

Exploring the Cold-Adaptation Mechanism of Serine Hydroxymethyltransferase by Comparative Molecular Dynamics Simulations.

Cold-adapted enzymes feature a lower thermostability and higher catalytic activity compared to their warm-active homologues, which are considered as a consequence of increased flexibility of their molecular structures. The complexity of the (thermo)stability-flexibility-activity relationship makes it difficult to define the strategies and formulate a general theory for enzyme cold adaptation. Here, the psychrophilic serine hydroxymethyltransferase (pSHMT) from Psychromonas ingrahamii and its mesophilic counterpart, mSHMT from Escherichia coli, were subjected to µs-scale multiple-replica molecular dynamics (MD) simulations to explore the cold-adaptation mechanism of the dimeric SHMT. The comparative analyses of MD trajectories reveal that pSHMT exhibits larger structural fluctuations and inter-monomer positional movements, a higher global flexibility, and considerably enhanced local flexibility involving the surface loops and active sites. The largest-amplitude motion mode of pSHMT describes the trends of inter-monomer dissociation and enlargement of the active-site cavity, whereas that of mSHMT characterizes the opposite trends. Based on the comparison of the calculated structural parameters and constructed free energy landscapes (FELs) between the two enzymes, we discuss in-depth the physicochemical principles underlying the stability-flexibility-activity relationships and conclude that (i) pSHMT adopts the global-flexibility mechanism to adapt to the cold environment and, (ii) optimizing the protein-solvent interactions and loosening the inter-monomer association are the main strategies for pSHMT to enhance its flexibility.

The interactive effects of ketamine and ethanol on dopamine expression in the ventral tegmental area of rats.

BACKGROUND: A number of studies have demonstrated the significant and rapid antidepressant effects of ketamine, which is also known as a neurotoxic and illicit drug. Ketamine and alcohol are increasingly used together in clubs by teenagers and young adults. Previous studies have proven that chronic ketamine consumption induces a delayed and persistent activation of the dopamine (DA) system. However, the rewarding properties of recreational ketamine abuse remain unclear, and the underlying mechanisms of the effects on the DA system after administration of ketamine with ethanol are yet to be explored. METHODS: Here, we evaluated the effects of two different doses of ketamine (30 mg/kg and 60 mg/kg) with and without ethanol (0.3156 g/kg) on DA concentration in the rat's ventral tegmental area (VTA), a vital region in the reward and motivation system. We explored the effects of the combined drug treatment on the expression profiling of the DA metabolism genes, tyrosine hydroxylase, dopa decarboxylase, vesicular monoamine transporter 2, and synaptosomal-associated protein 25, as well as protein expression level of brain-derived neurotrophic factor in the rat's VTA. RESULTS: We found that administration of ketamine with ethanol led to a significant increase of DA in the VTA associated with differential regulation of mRNA levels of the four DA metabolism genes and protein levels of brain-derived neurotrophic factor. Moreover, the rewarding properties of coadministration of ketamine and ethanol were related to dopaminergic neuron activation in the VTA. CONCLUSION: These results indicated the possibility that combined drug treatment might positively affect the mesencephalic DA reward system.

Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes.

DNA damage in oocytes can cause infertility and birth defects. DNA double-strand breaks (DSBs) are highly deleterious and can substantially impair genome integrity. Homologous recombination (HR)-mediated DNA DSB repair plays dominant roles in safeguarding oocyte quantity and quality. However, little is known regarding the key players of the HR repair pathway in oocytes. Here, we identified oocyte-specific gene Ooep as a novel key component of the HR repair pathway in mouse oocytes. OOEP was required for efficient ataxia telangiectasia mutated (ATM) kinase activation and Rad51 recombinase(RAD51)focal accumulation at DNA DSBs. Ooep null oocytes were defective in DNA DSB repair and prone to apoptosis upon exogenous DNA damage insults. Moreover, Ooep null oocytes exhibited delayed meiotic maturation. Therefore, OOEP played roles in preserving oocyte quantity and quality by maintaining genome stability. Ooep expression decreased with the advance of maternal age, suggesting its involvement in maternal aging.

Insights into the molecular mechanism underlying CD4-dependency and neutralization sensitivity of HIV-1: a comparative molecular dynamics study on gp120s from isolates with different phenotypes.

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion. Although structures of prefusion Env have been determined and phenotypes relevant to the CD4 dependency and the neutralization sensitivity for various HIV-1 isolates have been identified, the detailed structural dynamics and energetics underlying these two phenotypes have remained elusive. In this study, two unliganded structural models of gp120, one from the CD4-dependent, neutralization-resistant isolate H061.14 and the other from the CD4-independent, neutralization-sensitive R2 strain, were constructed, and subsequently were subjected to multiple-replica molecular dynamics (MD) simulations followed by free energy landscape (FEL) construction. Comparative analyses of MD trajectories reveal that during simulations R2-gp120 demonstrated larger structural fluctuations/deviations and higher global conformational flexibility than H061.14-gp120. Close comparison of local conformational flexibility shows that some of the structural regions involving direct interactions with gp41 and adjacent gp120 subunits in the context of the closed trimeric Env exhibit significantly higher flexibility in R2-gp120 than in H061.14-gp120, thus likely increasing the probability for R2-Env to open the trimer crown and prime gp41 fusogenic properties without induction by CD4. Collective motions derived from principal component analysis (PCA) reveal that R2-gp120 is prone to spontaneous transition to the neutralization-sensitive CD4-bound state while H061.14-gp120 tends to maintain the neutralization-resistant unliganded state. Finally, comparison between FELs reveals that R2-gp120 has larger conformational entropy, richer conformational diversity, and lower thermostability than H061.14-gp120, thus explaining why R2-gp120 is more structurally unstable and conformationally flexible, and has a higher propensity to transition to the CD4-bound state than H061.14-gp120. The present results reveal that the differences in dynamics and energetics between R2-gp120 and H061.14-gp120 impart Env trimers with distinct capacities to sample different states (i.e., R2-Env samples more readily the open state while H061.14-Env is more inclined to maintain the closed state), thus shedding light on the molecular mechanism underlying the HIV-1 phenotype associated with CD4 dependency/neutralization sensitivity.

Effects of co-administration of ketamine and ethanol on the dopamine system via the cortex-striatum circuitry.

AIM: Ketamine and ethanol are increasingly being used together as recreational drugs in rave parties. Their effects on the dopamine (DA) system remain largely unknown. This study aimed to investigate the effects of consuming two different concentrations of ketamine with and without alcohol on the DA system. MATERIALS AND METHODS: We employed the conditioned place preference (CPP) paradigm to evaluate the rewarding effects of the combined administration of two different doses of ketamine (30mg/kg and 60mg/kg) with ethanol (0.3156g/kg). We evaluated the effects of the combined drug treatment on the transcriptional output of tyrosine hydroxylase (TH), dopa decarboxylase (DDC), synaptosomal-associated protein 25 (SNAP25), and vesicular monoamine transporter 2 (VMAT2) as well as protein expression level of brain-derived neurotrophic factor (BDNF) in rat prefrontal cortex (PFC) and striatum. KEY FINDINGS: We found that rats exhibited a dose-dependent, drug-paired, place preference to ketamine and ethanol associated with an elevated DA level in the striatum but not in the PFC. Moreover, treatment involving low- or high-dose ketamine with or without ethanol caused a differential regulatory response in the mRNA levels of the four DA metabolism genes and the cellular protein abundance of BDNF via the cortex-striatum circuitry. SIGNIFICANCE: This study investigated the molecular mechanisms that occur following the combined administration of ketamine and ethanol in the DA system, which could potentially lead to alterations in the mental status and behavior of ketamine/ethanol users. Our findings may aid the development of therapeutic strategies for substance abuse patients.