Highlights d REGEN-COV retains neutralization potency against current variants of concern/interest d In vitro escape studies can predict emergence of viral variants in animals and humans d 3 noncompeting mAb in combination reduce variant risk compared to a combination of 2 d Treatment with REGEN-COV in humans does not lead to emergence of viral variants

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.Mycobacterium tuberculosis | differentially detectable | phenotypic tolerance | rifampin | thioridazine

The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Identification of compounds that target metabolically diverse subpopulations of Mycobacterium tuberculosis (Mtb) may contribute to shortening the course of treatment for tuberculosis. This study screened 270,000 compounds from GlaxoSmithKline's collection against Mtb in a nonreplicating (NR) state imposed in vitro by a combination of four host-relevant stresses. Evaluation of 166 confirmed hits led to detailed characterization of 19 compounds for potency, specificity, cytotoxicity, and stability. Compounds representing five scaffolds depended on reactive nitrogen species for selective activity against NR Mtb, and two were stable in the assay conditions. Four novel scaffolds with activity against replicating (R) Mtb were also identified. However, none of the 19 compounds was active against Mtb in both NR and R states. There was minimal overlap between compounds found active against NR Mtb and those previously identified as active against R Mtb, supporting the hypothesis that NR Mtb depends on distinct metabolic pathways for survival.