Identification of hepatitis C virus NS5A inhibitors.

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was approximately 5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.

Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical Stilbenes.

The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity. Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an EC50 = 86 pM.

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.

N-(5-chloro-2-(hydroxymethyl)-N-alkyl-arylsulfonamides as gamma-secretase inhibitors.

A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity.

Inhibitors of Abeta production: solid-phase synthesis and SAR of alpha-hydroxycarbonyl derivatives.

Inhibitors of amyloid-beta (Abeta) protein production have been widely pursued as a potential treatment for Alzheimer's disease. Following the identification of a 5 microM screening hit, SAR was initiated using solid-phase synthetic techniques. Two series of alpha-hydroxy esters and ketones which are sub-micromolar inhibitors of Abeta production were identified. The most potent alpha-hydroxyketone identified is approximately 30-fold more potent than the initial lead.

Differentially functionalized diamines as novel ligands for the NPY2 receptor.

The synthesis of novel ligands for the NPY(2) receptor using solid phase split pool methodology is described. One of the analogues, diamine 16, was found to be a potent NPY(2) binder.