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• W2604824245 abstract "•Crystal structures of Mycobacterium tuberculosis RNA polymerase (Mtb RNAP)•Crystal structures of Mtb RNAP in complex with antituberculosis drug rifampin•Crystal structures of Mtb RNAP in complex with new antituberculosis compound D-AAP1•Different binding sites and simultaneous binding of rifampin and D-AAP1 Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds—Nα-aroyl-N-aryl-phenylalaninamides (AAPs)—that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds—Nα-aroyl-N-aryl-phenylalaninamides (AAPs)—that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif. Rifampin (Rif) is the cornerstone of current antituberculosis therapy (World Health Organization, 2016World Health OrganizationGlobal Tuberculosis Report 2016. WHO, 2016Google Scholar, Rothstein, 2016Rothstein D.M. Rifamycins, alone and in combination.Cold Spring Harb. Perspect. Med. 2016; 6: a027011Crossref Scopus (58) Google Scholar, Aristoff et al., 2010Aristoff P.A. Garcia G.A. Kirchhoff P.D. Showalter H.D. Rifamycins--obstacles and opportunities.Tuberculosis (Edinb.). 2010; 90: 94-118Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The emergence and spread of Rif-resistant Mycobacterium tuberculosis (Mtb) is an urgent public health crisis (0.6 million new cases annually; World Health Organization, 2016World Health OrganizationGlobal Tuberculosis Report 2016. WHO, 2016Google Scholar). Rif resistance in Mtb arises from substitution of residues of the binding site for Rif on its molecular target, Mtb RNA polymerase (RNAP) (Rothstein, 2016Rothstein D.M. Rifamycins, alone and in combination.Cold Spring Harb. Perspect. Med. 2016; 6: a027011Crossref Scopus (58) Google Scholar, Aristoff et al., 2010Aristoff P.A. Garcia G.A. Kirchhoff P.D. Showalter H.D. Rifamycins--obstacles and opportunities.Tuberculosis (Edinb.). 2010; 90: 94-118Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Intensive efforts are underway to identify Rif derivatives that are unaffected by substitutions in the Rif binding site and to identify novel, non-Rif-related RNAP inhibitors that function through binding sites on RNAP that do not overlap the Rif binding site and thus are unaffected by substitutions in the Rif binding site (Aristoff et al., 2010Aristoff P.A. Garcia G.A. Kirchhoff P.D. Showalter H.D. Rifamycins--obstacles and opportunities.Tuberculosis (Edinb.). 2010; 90: 94-118Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, Lee and Borukhov, 2016Lee J. Borukhov S. Bacterial RNA polymerase-DNA interaction–the driving force of gene expression and the target for drug action.Front. Mol. Biosci. 2016; 3: 73PubMed Google Scholar). However, these efforts have been hampered by the unavailability of structural information for Mtb RNAP or a closely related bacterial RNAP. Crystal structures of bacterial RNAP hitherto have been available only for Thermus aquaticus (Taq), Thermus thermophilus (Tth), and Escherichia coli (Eco) RNAP, which have ≤40% sequence identity with Mtb RNAP (Murakami, 2015Murakami K.S. Structural biology of bacterial RNA polymerase.Biomolecules. 2015; 5: 848-864Crossref PubMed Scopus (77) Google Scholar), although a paper reporting a structure of Mycobacterium smegmatis RNAP recently has appeared (Hubin et al., 2017Hubin E.A. Fay A. Xu C. Bean J.M. Saecker R.M. Glickman M.S. Darst S.A. Campbell E.A. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA.eLife. 2017; 6: e22520Crossref PubMed Scopus (73) Google Scholar). Here, we report a series of crystal structures of Mtb RNAP transcription initiation complexes at 3.8–4.4 Å resolution. We report structures of Mtb RNAP transcription initiation complexes alone, in complex with Rif, in complex with a non-Rif-related RNAP inhibitor, and in complex with both Rif and a non-Rif-related RNAP inhibitor. To determine crystal structures of Mtb RNAP transcription initiation complexes, we used a strategy analogous to the strategy used previously to determine structures of Tth RNAP transcription initiation complexes (Zhang et al., 2012Zhang Y. Feng Y. Chatterjee S. Tuske S. Ho M.X. Arnold E. Ebright R.H. Structural basis of transcription initiation.Science. 2012; 338: 1076-1080Crossref PubMed Scopus (249) Google Scholar), i.e., crystallization of Mtb RNAP σA holoenzyme in complex with a synthetic nucleic-acid scaffold that mimics the single-stranded DNA (ssDNA) transcription bubble and downstream double-stranded DNA (dsDNA) of a catalytically competent RNAP promoter open complex (RPo) or with the same synthetic nucleic-acid scaffold in the presence of synthetic RNA oligomers corresponding to 2-, 3-, and 4-nt RNA products (RNAP-promoter initial transcribing complexes, RPitc2, RPitc3, and RPitc4) (Table 1; Figure 1).Table 1Structure Data Collection and Refinement StatisticsComplexMtb RPoMtb RPo + 2-nt RNAMtb RPo + 3-nt RNAMtb RPo + 4-nt RNAMtb RPo + RifMtb RPo + Rif + 2-nt RNAMtb RPo + Rif + 3-nt RNAMtb RPo + Rif + 4-nt RNAMtb RPo + D-AAP1Mtb RPo + D-IX336Mtb RPo + Rif + D-AAP1Mtb Se-β′MtbSIPDB codePDB: 5UHAPDB: 5UH9PDB: 5UH5PDB: 5UH8PDB: 5UHBPDB: 5UH6PDB: 5UHCPDB: 5UHDPDB: 5UHEPDB: 5UHFPDB: 5UHGPDB: 5UH7Data collection sourceAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDAPS 19-IDData CollectionSpace groupP212121P212121P212121P212121P212121P212121P212121P212121P212121P212121P212121P21Cell Dimensionsa, b, c150.2,163.5,195.6 Å154.5,164.6,201.9.0 Å150.3,163.6,195.5 Å152.0,163.7,197.8 Å154.4,164.2,200.2 Å153.2,164.9,199.9 Å150.1,167.4,195.2 Å152.1,163.1,197.9 Å151.5,162.1,195.7 Å152.4,163.5,197.4 Å151.4,162.1,194.3 Å40.8, 97.5, 53.7 Åα, β, γ90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 90.0°, 90.0°90.0°, 93.3°, 90.0°Resolution50.0–3.9 Å (4.0–3.9 Å)50.0–4.4 Å (4.6–4.4 Å)50.0–3.8 Å (3.9–3.8 Å)50.0–4.2 Å (4.3–4.2 Å)50.0–4.2 Å (4.4–4.2 Å)50.0–3.8 Å (3.9–3.8 Å)50.0–3.8 Å (3.9–3.8 Å)50.0–4.0 Å (4.1–4.0 Å)50.0–4.0 Å (4.2–4.0 Å)50.0–4.3 Å (4.5–4.3 Å)50.0–4.0 Å (4.1–4.0 Å)50.0–2.2 Å (2.3–2.2 Å)Number of unique reflections44,08031,06150,20735,87433,86947,44048,27541,86439,80532,77641,65321,229Rmerge0.219 (0.837)0.191 (0.542)0.175 (0.710)0.172 (0.669)0.066 (0.543)0.134 (0.800)0.169 (0.903)0.096 (0.875)0.216 (0.716)0.203 (0.913)0.243 (0.801)0.075 (0.508)Rmeas0.226 (0.877)0.202 (0.624)0.184 (0.764)0.183 (0.740)0.072 (0.609)0.142 (0.901)0.178 (0.965)0.101 (0.931)0.230 (0.781)0.212 (0.956)0.258 (0.862)0.086 (0.593)Rpim0.057 (0.253)0.062 (0.300)0.055 (0.273)0.060 (0.299)0.028 (0.270)0.046 (0.401)0.053 (0.331)0.030 (0.312)0.076 (0.301)0.065 (0.428)0.087 (0.313)0.040 (0.302)CC1/2 (highest resolution shell)0.4220.5390.4710.4890.6970.4320.3430.4130.5240.6320.4900.791I/σI10.1 (1.5)7.6 (1.8)13 (2.0)8.8 (1.4)20.2 (2.0)14.2 (1.6)11.0 (2.0)23.5 (1.9)8.0 (1.9)10.3 (1.3)8.1(2.1)16.5 (5.4)Completeness99.2% (99.4%)93.3% (81.7%)99.7% (99.3%)96.1% (82.6%)97.0% (89.0%)97.7% (92.6%)97.4% (94.3%)99.8% (98.6%)99.2% (96.7%)99.0% (92.8%)99.8% (99.9%)99.4% (96.8%)Redundancy15.0 (8.2)7.6 (3.4)10.4 (7.4)7.8 (4.4)5.9 (4.5)7.5 (4.3)9.9 (7.5)11.1 (8.2)7.4 (6.0)12.1 (8.5)8.3 (7.4)4.3 (3.5)RefinementResolution48.9–3.9 Å49.9–4.4 Å49.6–3.8 Å48.6–4.2 Å49.8–4.3 Å50.0–3.8 Å48.8–3.8 Å49.5–4.0 Å49.3–4.0 Å48.5–4.3 Å49.2–4.0 Å36.1–2.2 ÅNumber of unique reflections34,56428,53446,46429,90829,56239,78343,85438,08934,62228,58139,39918,943Number of test reflections1,9881,9931,9971,9872,0051,9962,0001,9902,0073,1352,0141,835Rwork/Rfree0.22/0.27 (0.25/0.33)0.28/0.33 (0.30/0.34)0.19/0.24 (0.24/0.30)0.20/0.25 (0.21/0.25)0.21/0.26 (0.23/0.26)0.21/0.26 (0.25/0.31)0.20/0.26 (0.27/0.32)0.21/0.26 (0.26/0.32)0.21/0.26 (0.25/0.28)0.20/0.26 (0.24/0.30)0.22/0.26 (0.28/0.34)0.22/0.25 (0.30/0.34)Number of AtomsProtein25,99526,03326,07826,09825,99426,03326,05625,97325,95225,97325,9642,153Ligand/ion333362626262302989N/AWaterN/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/A45Root-Mean-Square (RMS) DeviationsBond lengths0.005 Å0.004 Å0.004 Å0.004 Å0.003 Å0.004 Å0.004 Å0.004 Å0.005 Å0.003 Å0.003 Å0.010 ÅBond angles0.793°0.681°0.763°0.692°0.709°0.730°0.733°0.737°0.804°0.701°0.702°1.170°MolProbity StatisticsClash score8.58.08.97.67.07.17.17.59.48.07.45.4Rotamer outliers1.8%1.4%2.2%1.4%1.3%1.6%1.7%1.6%2.1%2.2%1.7%0.0%Cβ outliers0%0%0%0%0%0%0%0%0%0%0%0%Ramachandran PlotFavored93.3%94.2%93.5%94.2%94.5%94.0%94.0%94.1%93.9%93.2%94.5%98.8%Outliers0.7%0.5%0.7%0.6%0.5%0.5%0.5%0.5%0.5%0.8%0.5%0.0%The highest resolution shells are in parentheses. Open table in a new tab The highest resolution shells are in parentheses. The resulting structures of Mtb RPo and RPitc are similar to previously reported structures of Tth RPo and RPitc (Zhang et al., 2012Zhang Y. Feng Y. Chatterjee S. Tuske S. Ho M.X. Arnold E. Ebright R.H. Structural basis of transcription initiation.Science. 2012; 338: 1076-1080Crossref PubMed Scopus (249) Google Scholar) in overall structural organization; in sequence-specific interactions between RNAP holoenzyme and promoter −10, discriminator, and core recognition elements; and in sequence-independent interactions between RNAP holoenzyme and template-strand ssDNA and downstream dsDNA (Figures 1B, 2, S1, and S2). The structures reveal two distinctive features. First, the structures reveal the conformation and interactions of an ∼100-residue taxon-specific sequence insertion present in the β′ subunit of RNAP from mycobacterial and closely related acinetobacterial taxa (β′MtbSI; β′In1 in Lane and Darst, 2010Lane W.J. Darst S.A. Molecular evolution of multisubunit RNA polymerases: sequence analysis.J. Mol. Biol. 2010; 395: 671-685Crossref PubMed Scopus (120) Google Scholar; β′ residues 140–229, Figures 3A–3C). The structures of Mtb RPo and RPitc show that β′MtbSI folds as an extraordinarily long (∼70-Å) α-helical coiled-coil (Figures 3A and 3B), and a structure at 2.2-Å resolution of an isolated protein fragment corresponding to β′MtbSI shows a superimposable conformation and thus shows that β′MtbSI folds independently (Figure 3B; Table 1). In the structures of Mtb RPo and RPitc, the β′MtbSI coiled-coil emerges from the RNAP clamp module and extends across the RNAP active-center cleft (Figures 3A and 3C). We infer that the β′MtbSI coiled-coil serves as a “gate” that helps trap and secure DNA within the active-center cleft and that presumably must open, rotating about a “hinge” formed by the short unstructured segments connecting β′MtbSI to the remainder of β′, to allow DNA to enter the active-center cleft (Figure 3C). Consistent with the inference from the structures that β′MtbSI helps trap and secure DNA within the active-center cleft, deletion of β′MtbSI strongly reduces the stability of Mtb RPo (10-fold defect; Figure 3D). We suggest that the taxon-specific sequence insertion β′MtbSI, like the taxon-specific stabilizing general transcription factors CarD and RbpA (Davis et al., 2015Davis E. Chen J. Leon K. Darst S.A. Campbell E.A. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD.Nucleic Acids Res. 2015; 43: 433-445Crossref PubMed Scopus (47) Google Scholar, Hubin et al., 2017Hubin E.A. Fay A. Xu C. Bean J.M. Saecker R.M. Glickman M.S. Darst S.A. Campbell E.A. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA.eLife. 2017; 6: e22520Crossref PubMed Scopus (73) Google Scholar), is a mycobacterial adaptation that helps overcome the unusual intrinsic instability of the mycobacterial RPo (Davis et al., 2015Davis E. Chen J. Leon K. Darst S.A. Campbell E.A. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD.Nucleic Acids Res. 2015; 43: 433-445Crossref PubMed Scopus (47) Google Scholar). Second, the structures provide information about the position of σR1.1, a σ module that, based on fluorescence resonance energy transfer (FRET) data, occupies the dsDNA binding site within the RNAP active-center cleft in RNAP holoenzyme but is displaced from the dsDNA binding site upon formation of RPo (Mekler et al., 2002Mekler V. Kortkhonjia E. Mukhopadhyay J. Knight J. Revyakin A. Kapanidis A.N. Niu W. Ebright Y.W. Levy R. Ebright R.H. Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.Cell. 2002; 108: 599-614Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar; Figures 3A and 3C). A structure of Eco RNAP holoenzyme shows that σR1.1 occupies the dsDNA binding site in RNAP holoenzyme (Bae et al., 2013Bae B. Davis E. Brown D. Campbell E.A. Wigneshweraraj S. Darst S.A. Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ70 domain 1.1.Proc. Natl. Acad. Sci. USA. 2013; 110: 19772-19777Crossref PubMed Scopus (106) Google Scholar), consistent with the FRET data (Mekler et al., 2002Mekler V. Kortkhonjia E. Mukhopadhyay J. Knight J. Revyakin A. Kapanidis A.N. Niu W. Ebright Y.W. Levy R. Ebright R.H. Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.Cell. 2002; 108: 599-614Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), but previous structures of bacterial RPo and RPitc have not resolved σR1.1 (Zhang et al., 2012Zhang Y. Feng Y. Chatterjee S. Tuske S. Ho M.X. Arnold E. Ebright R.H. Structural basis of transcription initiation.Science. 2012; 338: 1076-1080Crossref PubMed Scopus (249) Google Scholar). The structures of Mtb RPo and RPitc show clear, unambiguous electron density for one α helix of σR1.1, the α helix that connects σR1.1 to the remainder of σ (H4; numbered as in Zhang et al., 2012Zhang Y. Feng Y. Chatterjee S. Tuske S. Ho M.X. Arnold E. Ebright R.H. Structural basis of transcription initiation.Science. 2012; 338: 1076-1080Crossref PubMed Scopus (249) Google Scholar; Figures 3A and 3C). Strikingly, whereas in Eco RNAP holoenzyme, H4 is oriented perpendicular to the floor of the dsDNA binding site and occupies the dsDNA binding site (green in Figures 1B and S3A), in Mtb RPo and RPitc, H4 is rotated by ∼100°, about a “pivot” formed by the short unstructured segment between σR1.1 and the remainder of σ, and is oriented essentially parallel to the floor of the dsDNA binding site, outside the dsDNA binding site in Mtb RPo and RPitc (orange in Figures 3C and S3A). The rotation of H4 in RPo and RPitc displaces the center of H4 by ∼30 Å from its position in RNAP holoenzyme, consistent with the FRET data (Mekler et al., 2002Mekler V. Kortkhonjia E. Mukhopadhyay J. Knight J. Revyakin A. Kapanidis A.N. Niu W. Ebright Y.W. Levy R. Ebright R.H. Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.Cell. 2002; 108: 599-614Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), and positions H4 between β′MtbSI and dsDNA (Figures 3C and S3A). Assuming Mtb σR1.1 has approximately the same molecular volume as Eco σR1.1, σR1.1 in Mtb RPo and RPitc occupies essentially the entire space between β′MtbSI and dsDNA, potentially making a continuous chain of β′MtbSI-σR1.1-dsDNA interactions that close the active center and help trap and secure dsDNA in the active center (Figures 3C and S3B). Consistent with this inference, deletion of Mtb σR1.1 strongly reduces the stability of Mtb RPo (5-fold defect), and deletion of both Mtb σR1.1 and β′MtbSI very strongly reduces the stability of RPo (>70-fold defect; Figure 3D). We suggest that the stabilizing effect of Mtb σR1.1, functioning together with β′MtbSI, is a taxon-specific mycobacterial adaptation that further helps overcome the unusual intrinsic instability of mycobacterial RPo (Davis et al., 2015Davis E. Chen J. Leon K. Darst S.A. Campbell E.A. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD.Nucleic Acids Res. 2015; 43: 433-445Crossref PubMed Scopus (47) Google Scholar). We note that, in Eco RNAP, in contrast to Mtb RNAP, deletion of σR1.1 does not reduce the stability of RPo (Vuthoori et al., 2001Vuthoori S. Bowers C.W. McCracken A. Dombroski A.J. Hinton D.M. Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.J. Mol. Biol. 2001; 309: 561-572Crossref PubMed Scopus (57) Google Scholar). We determined a crystal structure of Mtb RPo in complex with Rif using analogous procedures (Figures 4A–4C; Table 1). The experimental electron density for Rif could be fitted in a manner that placed the ansa ring of Rif in a ring-shaped density feature, placed the (4-methyl-1-piperazinyl)iminomethyl side chain of Rif in an adjoining tab-shaped density feature, and positioned Rif H-bond-forming atoms adjacent to RNAP complementary H-bond-forming atoms (Figures 4B and 4C). The resulting structure shows RNAP-Rif interactions similar to those in previously reported structures of Taq RNAP, Tth RNAP, and Eco RNAP in complex with rifamycins (Campbell et al., 2001Campbell E.A. Korzheva N. Mustaev A. Murakami K. Nair S. Goldfarb A. Darst S.A. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase.Cell. 2001; 104: 901-912Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar, Artsimovitch et al., 2005Artsimovitch I. Vassylyeva M.N. Svetlov D. Svetlov V. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Tahirov T.H. Vassylyev D.G. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.Cell. 2005; 122: 351-363Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Molodtsov et al., 2013Molodtsov V. Nawarathne I.N. Scharf N.T. Kirchhoff P.D. Showalter H.D. Garcia G.A. Murakami K.S. X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins.J. Med. Chem. 2013; 56: 4758-4763Crossref PubMed Scopus (46) Google Scholar) but, in this case, with a Rif binding-site sequence from a bacterial species for which Rif is a clinically relevant antibacterial drug (Figures 4B and 4C). The structure shows direct H-bonded contacts between Rif and two of the three residues most frequently substituted in Rif-resistant Mtb clinical isolates (β H526 and S531) and direct van der Waals interactions between Rif and the third (β D516; Rothstein, 2016Rothstein D.M. Rifamycins, alone and in combination.Cold Spring Harb. Perspect. Med. 2016; 6: a027011Crossref Scopus (58) Google Scholar, Aristoff et al., 2010Aristoff P.A. Garcia G.A. Kirchhoff P.D. Showalter H.D. Rifamycins--obstacles and opportunities.Tuberculosis (Edinb.). 2010; 90: 94-118Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Figures 4B and 4C). A series of crystal structures of Mtb RPitc in complex with Rif were obtained by crystallizing a pre-formed Mtb RNAP-Rif complex with nucleic-acid scaffolds containing RNA oligomers corresponding to 2-, 3-, and 4-nt RNA products (Table 1). The results graphically demonstrate that Rif inhibits transcription through a “steric occlusion” mechanism that prevents extension of 2- to 3-nt RNA products to yield longer RNA products—a mechanism that previously had been proposed (Campbell et al., 2001Campbell E.A. Korzheva N. Mustaev A. Murakami K. Nair S. Goldfarb A. Darst S.A. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase.Cell. 2001; 104: 901-912Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar, Mustaev et al., 1994Mustaev A. Zaychikov E. Severinov K. Kashlev M. Polyakov A. Nikiforov V. Goldfarb A. Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.Proc. Natl. Acad. Sci. USA. 1994; 91: 12036-12040Crossref PubMed Scopus (48) Google Scholar, Feklistov et al., 2008Feklistov A. Mekler V. Jiang Q. Westblade L.F. Irschik H. Jansen R. Mustaev A. Darst S.A. Ebright R.H. Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center.Proc. Natl. Acad. Sci. USA. 2008; 105: 14820-14825Crossref PubMed Scopus (68) Google Scholar) but had not been directly demonstrated and had been controversial (Artsimovitch et al., 2005Artsimovitch I. Vassylyeva M.N. Svetlov D. Svetlov V. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Tahirov T.H. Vassylyev D.G. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.Cell. 2005; 122: 351-363Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Feklistov et al., 2008Feklistov A. Mekler V. Jiang Q. Westblade L.F. Irschik H. Jansen R. Mustaev A. Darst S.A. Ebright R.H. Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center.Proc. Natl. Acad. Sci. USA. 2008; 105: 14820-14825Crossref PubMed Scopus (68) Google Scholar) (Figures 4D and 4E; Table 1). Thus, whereas in the absence of Rif, 2-, 3-, and 4-nt RNA products fully base pair to the DNA template strand (Figure 4D), in the presence of Rif, the 5′ nucleotide of a 3-nt RNA is unpaired, unstacked, and rotated by ∼40°, due to steric clash with Rif (Figure 4E, second and fourth panels), and a 4-nt RNA appears to be unable to interact stably with the complex (i.e., shows no density; Figure 4E, third panel). All the Mtb RPo-Rif and RPitc-Rif structures show clear, unambiguous electron density for the RNAP active-center catalytic Mg2+, unlike the structure of Tth RNAP-Rif (Artsimovitch et al., 2005Artsimovitch I. Vassylyeva M.N. Svetlov D. Svetlov V. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Tahirov T.H. Vassylyev D.G. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.Cell. 2005; 122: 351-363Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and inconsistent with the alternative, “allosteric” mechanism for Rif function proposed in Artsimovitch et al., 2005Artsimovitch I. Vassylyeva M.N. Svetlov D. Svetlov V. Perederina A. Igarashi N. Matsugaki N. Wakatsuki S. Tahirov T.H. Vassylyev D.G. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.Cell. 2005; 122: 351-363Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar (Figures 4D and 4E). By high-throughput screening of a library of 114,000 synthetic compounds using a 384-well microplate-based fluorescence-detected assay of promoter-dependent transcription by Mtb RNAP σA holoenzyme (details to be provided elsewhere), we have identified a class of small-molecule inhibitors of Mtb RNAP: Nα-aroyl-N-aryl-phenylalaninamides (AAPs) (Figure 5A). The prototype of the class, D-AAP1, exhibits potent, selective, stereospecific activity against mycobacterial RNAP (potent inhibition of Mtb RNAP but poor inhibition of other bacterial RNAP and human RNAP I, II, and III; Figures 5A and S4A) and exhibits potent, selective, stereospecific activity against mycobacteria (potent activity against Mtb, M. avium, and M. smegmatis but poor activity against other bacterial and mammalian cells; Figures 5A and S4A). A crystal structure of Mtb RPo in complex with D-AAP1, determined by soaking pre-formed crystals of Mtb RPo with D-AAP1, defines the binding site, orientation, and interactions of D-AAP1 (Figure 5B; Table 1). Synthesis of a D-AAP1 analog containing a carbon-to-selenium substitution, D-IX336, followed by crystal soaking, X-ray diffraction analysis, and selenium-anomalous-dispersion analysis confirms the identified binding site, orientations, and interactions (Figures 5A–5C and S4B; Table 1). The structures reveal that AAPs bind to Mtb RNAP at a binding site centered on the N terminus of the RNAP bridge helix (an α helix that bridges the RNAP active-center cleft and forms one wall of the RNAP active center; Figures 5B and 5C). The three aromatic rings of the AAP bind in three pocket-like subsites (Figures 5B–5D). Alanine substitution of a residue of the observed binding site results in AAP resistance, both for RNAP-inhibitory activity and for antibacterial activity, confirming the functional importance of the observed interactions (Figure 5E). The structures enable rational, structure-based optimization of AAPs to improve potencies and properties. In particular, the structures show that the methyl group of ring “C” of the AAP projects into an unoccupied area with volume sufficient to accommodate at least six non-H atoms (Figures 5B–5D), allowing substitution of this position with diverse chemical functionality, including both linear and cyclic substituents. Lead-optimization efforts (to be described elsewhere) confirm the utility of the structures for structure-based lead optimization and, in particular, confirm the ability to improve potency and properties by substitution of the methyl group of ring C. The structures in Figure 5 show that the binding site on RNAP for AAPs differs from, and does not overlap, the binding site on RNAP for Rif (Figures 4A, 5B, and 5C). The binding site on RNAP for AAPs is similar in location to the binding site for CBRs, a class of compounds that inhibit Gram-negative bacterial RNAP but do not inhibit mycobacterial RNAP (Artsimovitch et al., 2003Artsimovitch I. Chu C. Lynch A.S. Landick R. A new class of bacterial RNA polymerase inhibitor affects nucleotide addition.Science. 2003; 302: 650-654Crossref PubMed Scopus (87) Google Scholar, Feng et al., 2015Feng Y. Degen D. Wang X. Gigliotti M. Liu S. Zhang Y. Das D. Michalchuk T. Ebright Y.W. Talaue M. et al.Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles.Structure. 2015; 23: 1470-1481Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, Bae et al., 2015Bae B. Nayak D. Ray A. Mustaev A. Landick R. Darst S.A. CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition.Proc. Natl. Acad. Sci. USA. 2015; 112: E4178-E4187Crossref PubMed Scopus (26) Google Scholar; Figure S5). Thus, both AAPs (in Mtb RNAP) and CBRs (in Gram-negative RNAP) interact with the RNAP bridge-helix N terminus (Figures 5B–5D and S5). We infer that AAPs are mycobacteria-selective inhibitors that function through the bridge-helix N terminus target, and CBRs are Gram-negative-selective inhibitors that function through the bridge-helix N terminus. Comparison of structures of Mtb RNAP-AAP complexes and Eco RNAP-CBR complexes reveals the basis for the difference in selectivity, i.e., because of sequence differences, Mtb RNAP has a three-pocket site complementary to an AAP, with three rings, but Eco RNAP has a two-pocket site complementary to a CBR, with two rings (Figures S5 and S6). The crucial sequence differences apparently include β residue 642 and β′ residues 757 and 771, which line or approach the pocket that is present, and accommodates AAP ring “A,” in Mtb RNAP, but is absent in Eco RNAP (residues numbered as in Eco RNAP; Figures S5 and S6). The binding-site residues for AAPs and CBRs are not conserved in human RNAP I, RNAP II, and RNAP III (Figure S6), consistent with, and accounting for, the observations that AAPs and CBRs do not inhibit human RNAP I, RNAP II, and RNAP III (Figure 5A; Feng et al., 2015Feng Y. Degen D. Wang X. Gigliotti M. Liu S. Zhang Y. Das D. Michalchuk T. Ebright Y.W. Talaue M. et al.Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles.Structure. 2015; 23: 1470-1481Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Based on the similarity in binding sites of AAPs and CBRs, AAPs most likely inhibit RNAP through a mechanism similar to that of CBRs, i.e., interference with bridge-helix conformational dynamics required for nucleotide addition (Feng et al., 2015Feng Y. Degen D. Wang X. Gigliotti M. Liu S. Zhang Y. Das D. Michalchuk T. Ebright Y.W. Talaue M. et al.Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles.Structure. 2015; 23: 1470-1481Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, Bae et al., 2015Bae B. Nayak D. Ray A. Mustaev A. Landick R. Darst S.A. CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition.Proc" @default.
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