Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-β-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs.

Synthesis and evaluation of 2'-dihalo ribonucleotide prodrugs with activity against hepatitis C virus.

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver.

Highlights of the Structure-Activity Relationships of Benzimidazole Linked Pyrrolidines Leading to the Discovery of the Hepatitis C Virus NS5A Inhibitor Pibrentasvir (ABT-530).

Curative interferon and ribavirin sparing treatments for hepatitis C virus (HCV)-infected patients require a combination of mechanistically orthogonal direct acting antivirals. A shared component of these treatments is usually an HCV NS5A inhibitor. First generation FDA approved treatments, including the component NS5A inhibitors, do not exhibit equivalent efficacy against HCV virus genotypes 1-6.

Synthesis and Biological Characterization of Aryl Uracil Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of ABT-072, a trans-Stilbene Analog with Good Oral Bioavailability.

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species.

Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir.

Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6.

Discovery of fluorobenzimidazole HCV NS5A inhibitors.

Research toward a next-generation HCV NS5A inhibitor has identified fluorobenzimidazole analogs that demonstrate potent, broad-genotype in vitro activity against HCV genotypes 1-6 replicons as well as HCV NS5A variants that are orders of magnitude less susceptible to inhibition by first-generation NS5A inhibitors in comparison to wild-type replicons. The fluorobenzimidazole inhibitors have improved pharmacokinetic properties in comparison to non-fluorinated benzimidazole analogs. Discovery of these inhibitors was facilitated by exploring SAR in a structurally simplified inhibitor series.

In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.

Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.

Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system.

The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer.

Regulation of transcription by 6S RNAs: insights from the Escherichia coli and Bacillus subtilis model systems.

Whereas, the majority of bacterial non-coding RNAs and functional RNA elements regulate post-transcriptional processes, either by interacting with other RNAs via base-pairing or through binding of small ligands (riboswitches), 6S RNAs affect transcription itself by binding to the housekeeping holoenzyme of RNA polymerase (RNAP). Remarkably, 6S RNAs serve as RNA templates for bacterial RNAP, giving rise to the de novo synthesis of short transcripts, termed pRNAs (product RNAs). Hence, 6S RNAs prompt the enzyme to act as an RNA-dependent RNA polymerase (RdRP).

Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A.

We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5R analogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency.

6S RNA: recent answers--future questions.

6S RNA is a non-coding RNA, found in almost all phylogenetic branches of bacteria. Through its conserved secondary structure, resembling open DNA promoters, it binds to RNA polymerase and interferes with transcription at many promoters. That way, it functions as transcriptional regulator facilitating adaptation to stationary phase conditions.

Mapping the spatial neighborhood of the regulatory 6S RNA bound to Escherichia coli RNA polymerase holoenzyme.

Bacterial 6S RNA interacts specifically with RNA polymerase acting as transcriptional regulator. Until now, no detailed characterization of the spatial arrangement of the non-coding RNA within the three-dimensional structure of RNA polymerase has been performed. Here we present results obtained with the chemical nuclease FeBABE tethered to distinct positions of RNA polymerase σ(70) subunit.

High potency improvements to weak aryl uracil HCV polymerase inhibitor leads.

Described herein is the development of a potent non-nucleoside, small molecule inhibitor of genotype 1 HCV NS5B Polymerase. A 23 μM inhibitor that was active against HCV polymerase was further elaborated into a potent single-digit nanomolar inhibitor of HCV NS5B polymerase by additional manipulation of the R and R1 substituents. Subsequent modifications to improve physical properties were made in an attempt to achieve an acceptable pharmacokinetic profile.

A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli.

6S RNA is a bacterial transcriptional regulator,which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ 70 -containing RNA polymerase. This inhibitory RNA polymerase ∼ 6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs.

Aryl uracil inhibitors of hepatitis C virus NS5B polymerase: synthesis and characterization of analogs with a fused 5,6-bicyclic ring motif.

The synthesis and structure-activity relationships of a novel aryl uracil series which contains a fused 5,6-bicyclic ring unit for HCV NS5B inhibition is described. Several analogs display replicon cell culture potencies in the low nanomolar range along with excellent rat pharmacokinetic values.

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2.

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations.

RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12.

Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E.

6S RNA - an old issue became blue-green.

6S RNA from Escherichia coli acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with E. coli.

Identification of aryl dihydrouracil derivatives as palm initiation site inhibitors of HCV NS5B polymerase.

Aryl dihydrouracil derivatives were identified from high throughput screening as potent inhibitors of HCV NS5B polymerase. The aryl dihydrouracil derivatives were shown to be non-competitive with respect to template RNA and elongation nucleotide substrates. They demonstrated genotype 1 specific activity towards HCV NS5B polymerases.

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae.

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure.

RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants.

The LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci.

Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides.

Transcription of rRNAs in Escherichia coli is directed from seven redundant rRNA operons, which are mainly regulated by their P1 promoters. Here we demonstrate by in vivo measurements that the amounts of individual rRNAs transcribed from the different operons under normal growth vary noticeably although the structures of all the P1 promoters are very similar. Moreover, we show that starvation for amino acids does not affect the seven P1 promoters in the same way.

The E. coli anti-sigma factor Rsd: studies on the specificity and regulation of its expression.

Background: Among the seven different sigma factors in E. coli σ(70) has the highest concentration and affinity for the core RNA polymerase. The E.

Structural basis for CRISPR RNA-guided DNA recognition by Cascade.

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner.

Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety.

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.