Valproate-coenzyme A conjugate blocks opening of receptor binding domains in the spike trimer of SARS-CoV-2 through an allosteric mechanism.

The receptor-binding domains (RBDs) of the SARS-CoV-2 spike trimer exhibit "up" and "down" conformations often targeted by neutralizing antibodies. Only in the "up" configuration can RBDs bind to the ACE2 receptor of the host cell and initiate the process of viral multiplication. Here, we identify a lead compound (3-oxo-valproate-coenzyme A conjugate or Val-CoA) that stabilizes the spike trimer with RBDs in the down conformation. Val-CoA interacts with three R408 residues, one from each RBD, which significantly reduces the inter-subunit R408-R408 distance by ∼ 13 Å and closes the central pore formed by the three RBDs. Experimental evidence is presented that R408 is part of a triggering mechanism that controls the prefusion to postfusion state transition of the spike trimer. By stabilizing the RBDs in the down configuration, this and other related compounds can likely attenuate viral transmission. The reported findings for binding of Val-CoA to the spike trimer suggest a new approach for the design of allosteric antiviral drugs that do not have to compete for specific virus-receptor interactions but instead hinder the conformational motion of viral membrane proteins essential for interaction with the host cell. Here, we introduce an approach to target the spike protein by identifying lead compounds that stabilize the RBDs in the trimeric "down" configuration. When these compounds trimerize monomeric RBD immunogens as co-immunogens, they could also induce new types of non-ACE2 blocking antibodies that prevent local cell-to-cell transmission of the virus, providing a novel approach for inhibition of SARS-CoV-2.

Translocation pause of remdesivir-containing primer/template RNA duplex within SARS-CoV-2's RNA polymerase complexes.

The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg2+-pyrophosphate (PPi) binds more tightly to the polymerase when the added RMP is at the third primer position than in the AMP added complex. The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1'-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.

Structural Insights into Binding of Remdesivir Triphosphate within the Replication-Transcription Complex of SARS-CoV-2.

Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication-transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5'-end to the last 3'-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3'-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5'-UUUAU-3' next to stem-loop (SL) 1'. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

Function of m6A and its regulation of domesticated animals' complex traits.

N6-methyladenosine (m6A) is the most functionally important epigenetic modification in RNA. The m6A modification widely exists in mRNA and noncoding RNA, influences the mRNA processing, and regulates the secondary structure and maturation of noncoding RNA. Studies showed the important regulatory roles of m6A modification in animal's complex traits, such as development, immunity, and reproduction-related traits. As an important intermediate stage from animal genome to phenotype, the function of m6A in the complex trait formation of domestic animals cannot be neglected. This review discusses recent research advances on m6A modification in well-studied organisms, such as human and model organisms, and introduces m6A detection technologies, small-molecule inhibitors of m6A-related enzymes, interaction between m6A and other biological progresses, and the regulation mechanisms of m6A in domesticated animals' complex traits.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes. Current studies showed that the m6A modification widely regulates a series of life processes, such as biological metabolism, growth and development, inflammation, and cancer. Understanding the m6A process of domestic animals can provide a new breakthrough for further promoting animal production performance and improving reproduction and disease resistance. Thus, this review briefly introduces m6A-related enzymes, m6A detection technologies, small-molecule inhibitors of m6A-related enzymes, and interaction between m6A and other biological progresses. In addition, the regulation mechanisms of m6A in domesticated animals' complex traits are elaborated and discussed.

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis.

BACKGROUND: Staphylococcus aureus (S. aureus) mastitis is one of the most difficult diseases to treat in lactating dairy cows worldwide. S. aureus with different lineages leads to different host immune responses. Long non-coding RNAs (lncRNAs) are reported to be widely involved in the progress of inflammation. However, no research has identified stable lncRNAs among different S. aureus strain infections. In addition, folic acid (FA) can effectively reduce inflammation, and whether the inflammatory response caused by S. aureus can be reduced by FA remains to be explored. METHODS: lncRNA transcripts were identified from Holstein mammary gland tissues infected with different concentrations of S. aureus (in vivo) and mammary alveolar cells (Mac-T cells, in vitro) challenged with different S. aureus strains. Differentially expressed (DE) lncRNAs were evaluated, and stable DE lncRNAs were identified in vivo and in vitro. On the basis of the gene sequence conservation and function conservation across species, key lncRNAs with the function of potentially immune regulation were retained for further analysis. The function of FA on inflammation induced by S. aureus challenge was also investigated. Then, the association analysis between these keys lncRNA transcripts and hematological parameters (HPs) was carried out. Lastly, the knockdown and overexpression of the important lncRNA were performed to validate the gene function on the regulation of cell immune response. RESULTS: Linear regression analysis showed a significant correlation between the expression levels of lncRNA shared by mammary tissue and Mac-T cells (P < 0.001, R2 = 0.3517). lncRNAs PRANCR and TNK2-AS1 could be regarded as stable markers associated with bovine S. aureus mastitis. Several HPs could be influenced by SNPs around lncRNAs PRANCR and TNK2-AS1. The results of gene function validation showed PRANCR regulates the mRNA expression of SELPLG and ITGB2 within the S. aureus infection pathway and the Mac-T cells apoptosis. In addition, FA regulated the expression change of DE lncRNA involved in toxin metabolism and inflammation to fight against S. aureus infection. CONCLUSIONS: The remarkable association between SNPs around these two lncRNAs and partial HP indicates the potentially important role of PRANCR and TNK2-AS1 in immune regulation. Stable DE lncRNAs PRANCR and TNK2-AS1 can be regarded as potential targets for the prevention of bovine S. aureus mastitis. FA supplementation can reduce the negative effect of S. aureus challenge by regulating the expression of lncRNAs.

Mechanism of Inhibition of the Reproduction of SARS-CoV-2 and Ebola Viruses by Remdesivir.

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.

AMOEBA binding free energies for the SAMPL7 TrimerTrip host-guest challenge.

As part of the SAMPL7 host-guest binding challenge, the AMOEBA force field was applied to calculate the absolute binding free energy for 16 charged organic ammonium guests to the TrimerTrip host, a recently reported acyclic cucurbituril-derived clip host structure with triptycene moieties at its termini. Here we report binding free energy calculations for this system using the AMOEBA polarizable atomic multipole force field and double annihilation free energy methodology. Conformational analysis of the host suggests three families of conformations that do not interconvert in solution on a time scale available to nanosecond molecular dynamics (MD) simulations. Two of these host conformers, referred to as the "indent" and "overlap" structures, are capable of binding guest molecules. As a result, the free energies of all 16 guests binding to both conformations were computed separately, and combined to produce values for comparison with experiment. Initial ranked results submitted as part of the SAMPL7 exercise had a mean unsigned error (MUE) from experimental binding data of 2.14 kcal/mol. Subsequently, a rigorous umbrella sampling reference calculation was used to better determine the free energy difference between unligated "indent" and "overlap" host conformations. Revised binding values for the 16 guests pegged to this umbrella sampling reference reduced the MUE to 1.41 kcal/mol, with a correlation coefficient (Pearson R) between calculated and experimental binding values of 0.832 and a rank correlation (Kendall τ) of 0.65. Overall, the AMOEBA results demonstrate no significant systematic error, suggesting the force field provides an accurate energetic description of the TrimerTrip host, and an appropriate balance of solvation and desolvation effects associated with guest binding.

Phase I/II trial of orteronel (TAK-700)--an investigational 17,20-lyase inhibitor--in patients with metastatic castration-resistant prostate cancer.

PURPOSE: The androgen receptor pathway remains active in men with prostate cancer whose disease has progressed following surgical or medical castration. Orteronel (TAK-700) is an investigational, oral, nonsteroidal, selective, reversible inhibitor of 17,20-lyase, a key enzyme in the production of androgenic hormones. EXPERIMENTAL DESIGN: We conducted a phase I/II study in men with progressive, chemotherapy-naïve, metastatic castration-resistant prostate cancer, and serum testosterone <50 ng/dL. In the phase I part, patients received orteronel 100 to 600 mg twice daily or 400 mg twice a day plus prednisone 5 mg twice a day. In phase II, patients received orteronel 300 mg twice a day, 400 mg twice a day plus prednisone, 600 mg twice a day plus prednisone, or 600 mg once a day without prednisone. RESULTS: In phase I (n = 26), no dose-limiting toxicities were observed and 13 of 20 evaluable patients (65%) achieved ≥50% prostate-specific antigen (PSA) decline from baseline at 12 weeks. In phase II (n = 97), 45 of 84 evaluable patients (54%) achieved a ≥50% decline in PSA and at 12 weeks, substantial mean reductions from baseline in testosterone (-7.5 ng/dL) and dehydroepiandrosterone-sulfate (-45.3 µg/dL) were observed. Unconfirmed partial responses were reported in 10 of 51 evaluable phase II patients (20%). Decreases in circulating tumor cells were documented. Fifty-three percent of phase II patients experienced grade ≥3 adverse events irrespective of causality; most common were fatigue, hypokalemia, hyperglycemia, and diarrhea. CONCLUSIONS: 17,20-Lyase inhibition by orteronel was tolerable and results in declines in PSA and testosterone, with evidence of radiographic responses.

A 6-week randomized, placebo-controlled trial of CP-316,311 (a selective CRH1 antagonist) in the treatment of major depression.

OBJECTIVE: The corticotropin-releasing hormone (CRH) system is implicated in the pathogenesis of several psychiatric disorders, including major depressive disorder. This study was designed to evaluate the safety and efficacy of CP-316,311, a selective nonpeptide antagonist of corticotropin-releasing hormone type 1 (CRH(1)) receptors, in the treatment of recurrent major depressive disorder. METHOD: Of a total of 167 patients with recurrent major depression who were screened, 123 were randomly assigned to receive 400 mg of CP-316,311 twice daily, or 100 mg of sertraline daily, or placebo in a 6-week fixed-dose, double-blind, double-dummy, parallel-group, placebo- and sertraline-controlled trial. The primary efficacy analysis compared the change in score from baseline to endpoint on the 17-item Hamilton Depression Rating Scale (HAM-D) between the CP-316,311 and placebo groups. A group sequential design was used to support early trial termination based on efficacy or futility at a planned interim analysis. RESULTS: The evaluable data set for the interim analysis included 28 patients in the CP-316,311 group, 31 patients in the placebo group, and 30 patients in the sertraline group. In the interim analysis, the change from baseline in the HAM-D score at the final visit was not significantly different between the CP-316,311 and placebo groups, while change from baseline between the sertraline and placebo groups was significantly different. Given these results, futility was declared for CP-316,311 and the trial was terminated. CONCLUSIONS: Although CP-316,311 was safe and well tolerated in this study population, it failed to demonstrate efficacy in the treatment of major depression.