Ferrocene-based inhibitors of hepatitis C virus replication that target NS5A with low picomolar in vitro antiviral activity.

An unprecedented series of organometallic HCV (hepatitis C virus) NS5A (nonstructural 5A protein) replication complex inhibitors that incorporates a 1,1'-ferrocenediyl scaffold was explored. This scaffold introduces the elements of linear flexibility and non-planar topology that are unconventional for this class of inhibitors. Data from 2-D NMR spectroscopic analyses of these complexes in solution support an anti (unstacked) arrangement of the pharmacophoric groups. Several complexes demonstrate single-digit picomolar in vitro activity in an HCV genotype-1b replicon system. One complex to arise from this investigation (10a) exhibits exceptional picomolar activity against HCV genotype 1a and 1b replicons, low hepatocellular cytotoxicity, and good pharmacokinetic properties in rat.

ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

Selenophene-containing inhibitors of type IIA bacterial topoisomerases.

We investigated compounds related to the previously reported antistaphyloccocal agent AVE6971 in an effort to attenuate inhibition of hERG potassium channel current that has been noted for this and related antibacterial drug classes. While most modifications of the original thiophene group compromised antibacterial activity, one selenophene analogue displayed (i) improved activity against the primary target enzyme DNA gyrase, (ii) similar activities against a panel of MRSA clinical isolates, and (iii) reduced hERG channel inhibition.

A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery.

Real-time RT-PCR and Northern blot are employed for the measurement of HCV RNA but suffer from multiple purification steps, high cost, and relatively large variability. In this study, a hybridization method for HCV RNA detection is described. This method does not need RNA purification, and is sensitive enough to detect HCV RNA present in replicon cellular lysates harvested from a single well of a 96-well plate. Fixation of RNA by UV cross-linking is crucial for this sensitivity. A linear relationship exists between hybridization signal and cell density ranging from 10(5) to as few as 300 cells per well. The signal-to-background ratio is greater than 40 and the Z factor is above 0.7. Using several known anti-HCV agents, dose-response curves and EC(50) values generated from hybridization were similar to those obtained from a luciferase assay. This method has been successfully applied to replicons of different HCV subtypes and hepatitis B virus in our laboratory. In summary, this hybridization assay is sensitive, highly reproducible, easy to handle, and a valuable tool for antiviral drug discovery.

Selection of replicon variants resistant to ACH-806, a novel hepatitis C virus inhibitor with no cross-resistance to NS3 protease and NS5B polymerase inhibitors.

We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.

Discovery of novel dialkyl substituted thiophene inhibitors of HCV by in silico screening of the NS5B RdRp.

A novel 5,4-dialkyl substituted thiophene was discovered by in silico screening of the 3D polymerase crystal structure (1GX6) that demonstrated single digit micromolar HCV inhibition activity in the replicon assay and dose-dependent inhibition in the replicase complex assay. Subsequently, SAR was explored with a small set of dialkyl and tetrahydro-benzo thiophenes. Since these thiophenes inhibit synthesis of both, single- and double-stranded RNAs, their mechanism of action is distinct from other known HCV inhibitors.

Small molecule inhibitors of E. coli primase, a novel bacterial target.

Bacterial primase is essential for DNA replication in Gram-positive and Gram-negative bacteria. It is also structurally distinct from eukaryotic primases, and therefore an attractive, but under-explored, target for therapeutic intervention. We applied virtual screening to discover primase inhibitors, and subsequently several commercially available analogs of these initial hits showed potent primase inhibition and in vitro antibacterial activity. This work provides a 3D pharmacophore for primase ligands, SAR trends, and leads that can be further optimized.

Hepatitis C virus (HCV) NS5B nonnucleoside inhibitors specifically block single-stranded viral RNA synthesis catalyzed by HCV replication complexes in vitro.

Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

Synthesis of well-defined tower-shaped 1,3,5-trisubstituted adamantanes incorporating a macrocyclic trilactam ring system.

We describe the synthesis of two novel well-defined tower-shaped 1,3,5-trisubstituted adamantanes 30 and 33 that incorporate a macrocyclic trilactam ring system. Each nanoscale molecule has a broad tripodal base consisting of three identical sulfur-containing termini as the tripod feet, 4-acetylsulfanylmethylphenyl units in the case of 30 and 3,5-bis(acetylsulfanylmethyl)phenyl units in the case of 33. The sulfur atoms are designed to bind the molecules trivalently to the apex of a gold-coated commercial AFM tip through formation of three S-Au bonds. The rigid adamantane-derived head unit with a single hydrogen atom at the apex is designed to scan the sample. Molecules 30 and 33 are synthesized from 1,3,5-triethynyladamantane by a series of Sonogashira coupling reactions involving terminal alkynes and aryl iodides. A macrocyclic trilactam unit is included for added rigidity. We demonstrate that molecule 30 is sufficiently large and rigid to be visualized by a conventional AFM tip. These nanoscale molecules may also find application as chemically well-defined nanoscale objects for calibration of AFM tips.