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- EMDB-25570: Cryo-EM structure of Rifamycin bound to E. coli RNAP and rrnBP1 p... -
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Open data
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Basic information
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Title | Cryo-EM structure of Rifamycin bound to E. coli RNAP and rrnBP1 promoter complex | |||||||||
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Function / homology | ![]() sigma factor antagonist complex / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Shin Y / Murakami KS | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Optimization of Benzoxazinorifamycins to Improve RNA Polymerase Inhibition and Treatment of Tuberculosis. Authors: Walajapet Rajeswaran / Shireen R Ashkar / Pil H Lee / Larisa Yeomans / Yeonoh Shin / Scott G Franzblau / Katsuhiko S Murakami / Hollis D Showalter / George A Garcia / ![]() Abstract: Rifampin (RMP), a very potent inhibitor of the (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from ...Rifampin (RMP), a very potent inhibitor of the (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIF) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIF) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue () that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (∼30-fold > RMP) activity against RIF MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, has a 7 day MBC that is ∼10-fold more potent than RMP. These results suggest that may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of ∼2 g of at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 168.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.3 KB 22.3 KB | Display Display | ![]() |
Images | ![]() | 36.9 KB | ||
Filedesc metadata | ![]() | 8.5 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7szjMC ![]() 7szkC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.12 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
+Entire : Rifamycin bound to E. coli RNAP and rrnBP1 promoter complex
+Supramolecule #1: Rifamycin bound to E. coli RNAP and rrnBP1 promoter complex
+Macromolecule #1: DNA-directed RNA polymerase subunit alpha
+Macromolecule #2: DNA-directed RNA polymerase subunit beta
+Macromolecule #3: DNA-directed RNA polymerase subunit beta'
+Macromolecule #4: DNA-directed RNA polymerase subunit omega
+Macromolecule #5: RNA polymerase sigma factor RpoD
+Macromolecule #6: DNA (57-MER)
+Macromolecule #7: DNA (49-MER)
+Macromolecule #8: RIFAMPICIN
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ZINC ION
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 7.5 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: C-flat-2/1 / Mesh: 400 | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: OTHER |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3301 / Average electron dose: 45.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: OTHER |
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Initial angle assignment | Type: OTHER |
Final angle assignment | Type: OTHER |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.0) / Number images used: 251427 |