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.

The structural and dynamic behavior of the ionic liquid 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide ([C(6)mim][NTf(2)]) in chloroform has been studied by experimental measurements of (1)H and (19)F self-diffusion coefficients, viscosity, and excess molar volume in the concentration range of 0.001-1.0 mol·kg(-1) and temperatures ranging from 15 to 45 °C. Within measurement uncertainty, the (1)H and (19)F self-diffusion coefficients are identical at the same experimental conditions of concentration and temperature, indicating that even to the lowest measured concentrations the cation and anion are not completely dissociated. The combined experimental data indicates a progression from ion pairing to aggregate formation as concentration increases where at concentrations near 0.1 mol·kg(-1) aggregate formation becomes dominant. Concurrently with the formation of the IL aggregates at higher concentrations, we also observe an apparent breakdown of the validity of the Stokes-Einstein equation, which we explain by translational motion to become dominated by individual ion pairs moving rapidly between IL aggregates.

Rifampin, a semi-synthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semi-synthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases, and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the β subunit, blocking the path of the RNA transcript. The C3′-tail of benzoxazinorifamycin fits a cavity between the β subunit and σ factor. We propose that, in addition to blocking RNA exit, the benzoxazinorifamycin C3′-tail changes the σ region3.2 loop position, which influences the template DNA at the active site thereby reducing the efficiency of transcription initiation. This study supports expansion of structure–activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.

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Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP), comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis. To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that utilizes malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (βS531L) containing a mutation (β’V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of WT and RifR E. coli and M. tuberculosis RNAPs. Four compounds identified from the screen are active against M. tuberculosis in culture at concentrations below 200 μM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan assay interference (PAINS).