scholarly journals Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase: pseudouridimycin

2017 ◽  
Author(s):  
Sonia I. Maffioli ◽  
Yu Zhang ◽  
David Degen ◽  
Thomas Carzaniga ◽  
Giancarlo Del Gatto ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Drug Development ◽  
Rna Polymerase ◽  
Binding Site ◽  
Bacterial Pathogens ◽  
Nucleoside Analog ◽  
Cross Resistance ◽  
Nucleoside Triphosphate ◽  
Antibacterial Drug ◽  
Bacterial Rna

There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs1–2. Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV3and HCV4. NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites4-5could limit resistance emergence4-5. Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model ofStreptococcus pyogenesperitonitis. PUM inhibits RNAP through a binding site on RNAP (the "i+1" NTP binding site) and mechanism (competition with UTP for occupancy of the "i+1" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.

scholarly journals Activity of and Development of Resistance to Corallopyronin A, an Inhibitor of RNA Polymerase

2011 ◽  
Vol 55 (5) ◽  
pp. 2413-2416 ◽  
Author(s):  
Katherine Mariner ◽  
Martin McPhillie ◽  
Rachel Trowbridge ◽  
Catriona Smith ◽  
Alex J. O'Neill ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Rna Polymerase ◽  
Inhibitory Concentration ◽  
Drug Candidate ◽  
Antibacterial Drug ◽  
Content Type ◽  
Development Of Resistance ◽  
Bacterial Rna ◽  
Resistant Mutants

ABSTRACTWe explored the properties of corallopyronin A (CorA), a poorly characterized inhibitor of bacterial RNA polymerase (RNAP). It displayed a 50% inhibitory concentration of 0.73 μM against RNAP, compared with 11.5 nM for rifampin. The antibacterial activity of CorA was also inferior to rifampin, and resistant mutants ofStaphylococcus aureuswere easily selected. The mutations conferring resistance resided in therpoBandrpoCsubunits of RNAP. We conclude that CorA is not a promising antibacterial drug candidate.

scholarly journals GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yu Zhang ◽  
David Degen ◽  
Mary X Ho ◽  
Elena Sineva ◽  
Katherine Y Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Active Center ◽  
Transcription Initiation ◽  
Cyclic Peptide ◽  
Cross Resistance ◽  
Peptide Antibiotic ◽  
Nucleotide Binding Sites ◽  
Bacterial Rna ◽  
Polymerase I

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.

scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

scholarly journals Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

2020 ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Nucleoside Analogue ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1–4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25–7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3’-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3’-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3’-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

scholarly journals Furanyl-Rhodanines Are Unattractive Drug Candidates for Development as Inhibitors of Bacterial RNA Polymerase

2010 ◽  
Vol 54 (10) ◽  
pp. 4506-4509 ◽  
Author(s):  
Katherine R. Mariner ◽  
Rachel Trowbridge ◽  
Anil K. Agarwal ◽  
Keith Miller ◽  
Alex J. O'Neill ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Rna Polymerase ◽  
Dna Synthesis ◽  
Malate Dehydrogenase ◽  
Membrane Damage ◽  
Macromolecular Synthesis ◽  
Drug Candidates ◽  
The Third ◽  
Bacterial Rna ◽  
Inhibitor Drug

ABSTRACT Previous studies suggest that furanyl-rhodanines might specifically inhibit bacterial RNA polymerase (RNAP). We further explored three compounds from this class. Although they inhibited RNAP, each compound also inhibited malate dehydrogenase and chymotrypsin. Using biosensors responsive to inhibition of macromolecular synthesis and membrane damaging assays, we concluded that in bacteria, one compound inhibited DNA synthesis and another caused membrane damage. The third rhodanine lacked antibacterial activity. We consider furanyl-rhodanines to be unattractive RNAP inhibitor drug candidates.

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