SummarySince 1967, Rifamaycin (RIF) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of RIF-resistant (RIF R ) TB, ~41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIF R mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon Rifampin (RMP) binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RIF affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIF. Analysis of interactions of RMP with three common RIF R mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIF R TB.
is a critical threat to human health due to the increased prevalence of rifampin resistance (RMP). Fitness defects have been observed in RMP mutants with amino acid substitutions in the β subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β' subunit of RNAP. To identify factors contributing to the fitness defects observed , several RNA transcription assays were utilized to probe initiation, elongation, termination, and 3'-RNA hydrolysis with the wild-type and RMP RNAPs. We found that the less prevalent RMP mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMP mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β' subunit.
A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC50 values below 10 μM, while candesartan cilexetil had an IC50 value of approximately 67 µM against Mpro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC50) value was the only one greater than the limit of the assay (>100 μM).
The respiratory virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly every aspect of life worldwide, claiming the lives of over 3.9 million people globally, at the time of this publication. Neutralizing humanized nanobody (V
H
H)-based antibodies (V
H
H-huFc) represent a promising therapeutic intervention strategy to address the current SARS-CoV-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity V
H
H bacteriophage library, several potent neutralizing V
H
H-huFc antibodies were identified and evaluated for their capacity to tightly bind to the SARS-CoV-2 receptor-binding domain, to prevent binding of SARS-CoV-2 spike (S) to the cellular receptor angiotensin-converting enzyme 2, and to neutralize viral infection. Preliminary preclinical evaluation of multiple V
H
H-huFc antibody candidates demonstrate that they are prophylactically and therapeutically effective
in vivo
against
wildtype
SARS-CoV-2. The identified and characterized V
H
H-huFc antibodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.
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