CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

Abstract

RNA polymerase inhibitors like the CBR class that target the enzyme's complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg 2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/ Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3′-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.CBR inhibitors | RNA polymerase | transcription inhibition | X-ray crystallography B acteria harbor a single RNA polymerase (RNAP) enzyme (subunit composition α 2 ββ′ω, ∼400 kDa) that performs all transcription in the cell. The bacterial RNAP is a proven target for antimicrobials. The rifamycins (Rifs), which bind to and inhibit the bacterial RNAP (1-3), are potent, broad-spectrum antimicrobials and are the lynchpin of current tuberculosis therapy (4). Nevertheless, the emergence of multidrug-resistant pathogens highlights the importance of discovering novel antimicrobials (5). New RNAP inhibitors can also be used as tools to probe transcriptional mechanisms.Artsimovitch et al. (6) described a new class of bacterial RNAP inhibitors, the CBR compounds. Single amino acid substitutions in the RNAP β and β′ subunits that conferred resistance to CBR compounds (CBR R ) defined a pocket near the RNAP surface bounded by β′ residues of the F loop and the N-terminal part of the bridge helix (BH), and β residues neighboring fork-loop 2 (FL2) and the βDII loop, all RNAP structural elements shown to play important roles in the enzyme's nucleotide addition cycle (NAC) (7-20). The genetically defined CBR site is ∼31 Å from the RNAP active site Mg 2+ ion and is also distinct from the binding sites of other well-characterized bacterial RNAP inhibitors (SI Appendix, Table S1). Inhibition of RNAP by the CBR compounds appears to be mechanistically distinct as well; the CBR compounds allosterically inhibit known catalytic activities of the RNAP active site preferentially at pause sites but have lesser to no effects on translocation (6, 21).The prototype CBR compound [3-(trifluoromethyl)-N-phenylbenzamidoxime, designated CBR703] (Fig. 1 A and B), identified by screening a library of compounds for inhibition of Escherichia coli RNAP, yielded 50% inhibitory co...

Bacterial Transcription as a Target for Antibacterial Drug Development

“…Despite resistance arising at 14 separate loci, the rate of resistance due to spontaneous mutation in one tested strain was extremely low at Ͻ1 ϫ 10 Ϫ12 , which is 10 2 -to 10 4 -fold lower than those for other antibiotics, suggesting that the CBR binding site may have potential as a useful resistancerefractory binding site (110). Interestingly, two mutations (␤= P750L and ␤= F773V) make cells dependent on CBR compounds for growth as well as conferring resistance, although the mechanism for this is currently unknown (108). The predicted structure of CBR703 bound to RNAP determined by in silico docking was very similar to what was found in the X-ray crystal structures, except it was inserted into the two hydrophobic pockets the wrong way around (107,108,110).…”

“…CBR703, together with CBR9379 and CBR9393, demonstrated the ability to inhibit transcription elongation by stabilizing elongation complex isomerization and slowing translocation (107). The inhibitory activity of these compounds is proposed to be due to an allosteric effect that prevents TL folding, mediated via the F-loop, and the inhibition of BH movement at its N-terminal hinge (108). An interesting aspect of their antibacterial activity is their ability to inhibit biofilm formation, which is especially important in a clinical setting as biofilms are a major source of nosocomial infection (109).…”

“…4D) (108,110). Specifically, CBR703 forms interactions with ␤ residues V550, H551, P552, Y555, R637, G640, E641, and S642 and ␤= residues K749, P750, I755 (part of the F-loop), P770, F773, I744, and H777 (BH N terminus) (108).…”

“…4D) (108,110). Specifically, CBR703 forms interactions with ␤ residues V550, H551, P552, Y555, R637, G640, E641, and S642 and ␤= residues K749, P750, I755 (part of the F-loop), P770, F773, I744, and H777 (BH N terminus) (108). Most of these interactions are hydrophobic, with only ␤ S642 making a polar interaction with the amidoxime moiety of CBR7903 (108,110).…”

“…Specifically, CBR703 forms interactions with ␤ residues V550, H551, P552, Y555, R637, G640, E641, and S642 and ␤= residues K749, P750, I755 (part of the F-loop), P770, F773, I744, and H777 (BH N terminus) (108). Most of these interactions are hydrophobic, with only ␤ S642 making a polar interaction with the amidoxime moiety of CBR7903 (108,110). The structural information agrees well with data inferring the binding site from analysis of resistance mutants (at ␤ P560, R637 and S642) (106), and this was further elaborated by saturation mutagenesis experiments that identified a further 11 resistance alleles (110).…”