Bacterial Transcription as a Target for Antibacterial Drug Development

Abstract

Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

FIG 1

RNAP structure and functional motifs. In all panels the structure of the Thermus thermophilus RNAP elongation complex was used. (A and B) Side and front views, respectively. α subunit, dark gray; β subunit, light gray; β′ subunit, medium gray; ω subunit, black. The DNA primary channel and secondary channel for NTP entry are highlighted with yellow circles. Template-strand DNA, green; nontemplate-strand DNA, orange; RNA, red. The active-site magnesium ion is shown as a cyan sphere. (C and D) Enlarged views from panels A and B, respectively, with functional regions colored and labeled. CH region, pale green; rudder, cyan; switch, red; active-site residues (NADFDGD), purple; β link, orange; F loop, magenta, fork loop II, yellow; bridge helix (BH), blue; trigger loop (TL), green; lid, bronze; zipper, brown; β flap tip, salmon. The approximate path of RNA through the RNA exit channel is indicated by the arrow in panel D. Structure images were prepared using PDB files 1IW7 and 2O5I in PyMol v1.7.4 (Schrödinger, LLC).

FIG 2

Compounds that inhibit bacterial transcription. The compounds featured in this review are shown as chemical structures, illustrating the diversity in size and complexity of RNAP inhibitors. The compounds are arranged with respect to their targets. The naphthyl group of rifampin in the primary-channel inhibitors is circled, with the equivalent region of sorangicin also circled. Numbered circles: streptolol (1) and monosaccharide (2) moieties of streptolydigin, squarate ring (3), benzylamine ring (4) and piperidine (5) groups of squaramides, and steroid-like carboxylic acid (6) and indolone (7) groups of DSHS0057. See the text for further details.

FIG 3

Antibiotics that bind close to the active site. (A) Space-filled structure of T. thermophilus RNAP holoenzyme with core subunits (α, β, β′, and ω) in gray and initiation factor σ shown as a slate blue cartoon. The catalytic Mg²⁺ ion is shown as a cyan sphere, and its location is indicated by the red arrow and star. The rifamycin/sorangicin binding site is shown in green, and the area used in panels B and C is boxed. The approximate rotations for regions shown in panels B and C are indicated with arrows. In all panels the catalytic Mg²⁺ ion is shown as a cyan sphere, and in panels B and C an initiating RNA dinucleotide (orange) is shown adjacent to the active site. (B) Rifampin (RIF) (red) and sorangicin (SOR) (yellow) have overlapping binding sites. (C) Rifabutin (RBT) (magenta) has improved binding to holoenzyme through interaction with D513 (red stick) located on a region of the σ factor called the σ₃ loop. When benzoxazinorifamycin 2b (BZR) (blue) is bound, the C-3′ tail causes a distortion in the σ₃ loop (teal), preventing it interacting with the template strand of DNA. (D) GE23077 (orange) binds in the i and i+1 sites, inhibiting RNA synthesis. The binding site is shown in pale green. ATP entering RNAP via the secondary channel is shown in red. Rifamycin SV (RifSV) (orange) binds near to GE23077, and the two molecules can be covalently linked to form a compound with activity against rifampin-resistant RNAP. Structure images were prepared using PDB files 1IW7, 1YNJ, 1YNN, 2A68, 2O5J, 4G7O, 4KN4, and 4OIR in PyMol v1.7.4 (Schrödinger, LLC).

FIG 4

Inhibitors that target the bridge helix and trigger loop. (A and B) Bridge helix (BH) (blue) and trigger loop (TL) (green) in alternative conformations. The BH and TL colors are conserved in all panels in this figure, and active-site Mg²⁺ ions are shown as cyan spheres. Bending of the BH in panel A, indicated by the dashed straight arrow, aids translocation of the transcript to help incorporation of the incoming NTP, shown in red behind the BH. The TL is in the “open” conformation, allowing NTP entry into the active site. In panel B, the BH is straight and the TL has formed extended α helices behind the BH, forming a “closed” conformation that helps position the incoming NTP (red) in the active site. The movement of the TL from an open to a closed conformation is indicated with the curved dashed arrow in panel A. (C) Streptolydigin (STL) bound in a space-filled model of RNAP. Amino acids involved in interaction with STL are colored yellow. The STL pocket formed by the BH and β fork loop II is circled. The TL in the open conformation (shown as a cartoon) clashes with the tetramic moiety of STL, providing an understanding of how its deletion can stabilize STL binding. (D) Salinamide (SAL) bound within the secondary channel adjacent to the TL and CBR703 bound adjacent to the F loop (magenta). The binding site for SAL is shown in orange and that for CBR703 in yellow. RNAP surface and cartoon elements are shown in semitransparent form so that CBR703 within its binding site can be seen. Conformational change of the TL from the open to the closed form (transparent cartoons) will be sterically blocked by SAL. Structure images were prepared using PDB files 1IW7, 1ZYR, 2O5J, 4G7O, 4MEX, 4OIP, 4XSX, and 4ZH2 in PyMol v1.7.4 (Schrödinger, LLC).

FIG 5

Secondary-channel binding compounds. (A) Tagetitoxin bound inside the secondary channel. The binding site is shown in yellow with the TL (green) in the closed conformation as a cartoon to aid visualization of tagetitoxin, and the BH is shown in blue. (B) Binding site for microcin J25 in yellow, with the TL surface rendered in the open conformation. No structure of microcin J25 in complex with RNAP is available, but modeling indicates that it would block the secondary channel, preventing NTP entry (rendered as a semitransparent red surface). Structure images were prepared using PDB files 1PP5 and 2BE5 in PyMol v1.7.4 (Schrödinger, LLC).

FIG 6

Transcription factor interaction network. The space-filled RNAP elongation complex is shown in gray, with template-strand DNA in green and nontemplate-strand DNA in orange. Arrows indicate known interactions, although the binding sites for Rho and NusE on RNAP are not known. Subscript N and C indicate N- and C-terminal protein domains. NusG_N binds to the CH region of RNAP and across the active channel and has an overlapping binding site with region 2.2 of initiation factor σ. NusG_C can partner switch between Rho and NusE as appropriate. NusE forms a heterodimer with NusB and binds RNAP. NusA_N and region 4 of σ have overlapping binding sites on the β flap tip helix at the top of the RNA exit channel. All of these interactions could potentially be exploited in the development of new antimicrobial compounds. Structure images were prepared using PDB file 2O5I in PyMol v1.7.4, Schrödinger, LLC.

FIG 7

Inhibition of transcription termination factor Rho activity. (A) The Rho hexamer in its open-ring conformation. Four subunits are shown in slate blue with the remaining subunits in gray and white, sandwiching the Rho inhibitor bicyclomycin (red sticks). The bicyclomycin binding site is boxed. (B) Expanded view of bicyclomycin binding site area, with protein shown as ribbons. The bicyclomycin binding site is highlighted in yellow, with contributing amino acids shown as sticks. ATP is shown in blue in stick format and Mg²⁺ as a cyan sphere. Structure images were prepared using PDB file 1XPO in PyMol v1.7.4 (Schrödinger, LLC).

FIG 8

NusE is an “interaction hub” that links transcription and translation. (A) Nonoverlapping interactions that NusE makes with NusB (red), NusG_C (yellow), boxA RNA from an rRNA operon leader (green), and double-stranded RNA (dsRNA) (cyan), representative of the boxB hairpin from an rRNA operon leader. (B) Location of NusE (gray) (also known as ribosomal protein S10) in the context of a translating ribosome. The 30S subunit is shown in green and the 50S subunit in cyan. NusG_C (yellow) would not be sterically inhibited from interacting with NusE integrated into the 30S ribosomal subunit and so could be important in linking transcription with translation. Structure images were prepared using PDB files 2JVV, 2KVQ, 3CXC, 3D3B, 3R2C, and 3R2D in PyMol v1.7.4 (Schrödinger, LLC).