Scott D. Seiwert

Antifibrotic activities of pirfenidone in animal models

Pirfenidone is an orally active small molecule that has recently been evaluated in large clinical trials for the treatment of idiopathic pulmonary fibrosis, a fatal disease in which the uncontrolled deposition of extracellular matrix leads to progressive loss of lung function. This review describes the activity of pirfenidone in several well-characterised animal models of fibrosis in the lung, liver, heart and kidney. In these studies, treatment-related reductions in fibrosis are associated with modulation of cytokines and growth factors, with the most commonly reported effect being reduction of transforming growth factor-&bgr;. The consistent antifibrotic activity of pirfenidone in a broad array of animal models provides a strong preclinical rationale for the clinical characterisation of pirfenidone in pulmonary fibrosis and, potentially, other conditions with a significant fibrotic component.

Preclinical Characteristics of the Hepatitis C Virus NS3/4A Protease Inhibitor ITMN-191 (R7227)

ABSTRACT Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.

RNA aptamers as pathway-specific MAP kinase inhibitors

BACKGROUND In eukaryotic cells, many intracellular signaling pathways have closely related mitogen activated protein kinase (MAPK) paralogs as central components. Although MAPKs are therefore obvious targets to control the cellular responses resulting from the activation of these signaling pathways, the development of inhibitors which target specific cell signaling pathways involving MAPKs has proven difficult. RESULTS We used an RNA combinatorial approach to isolate RNAs that inhibit the in vitro phosphorylation activity of extracellular regulated kinase 2 (ERK2). These inhibitors block phosphorylation by ERK1 and ERK2, but do not inhibit Jun N-terminal kinase or p38 MAPKs. Kinetic analysis indicates these inhibitors function at high picomolar concentrations through the steric exclusion of substrate and ATP binding. In one case, we identified a compact RNA structural domain responsible for inhibition. CONCLUSIONS RNA reagents can selectively recognize and inhibit MAPKs involved in a single signal transduction pathway. The methodology described here is readily generalizable, and can be used to develop inhibitors of MAPKs involved in other signal transduction pathways. Such reagents may be valuable tools to analyze and distinguish homologous effectors which regulate distinct signaling responses.

Inhibition and Binding Kinetics of the Hepatitis C Virus NS3 Protease Inhibitor ITMN-191 Reveals Tight Binding and Slow Dissociative Behavior

The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.

Monitoring post-translational modification of proteins with allosteric ribozymes.

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.

Treatment of chronic hepatitis C patients with the NS3/4A protease inhibitor danoprevir (ITMN-191/RG7227) leads to robust reductions in viral RNA: A phase 1b multiple ascending dose study

BACKGROUND & AIMS Danoprevir is a potent and selective inhibitor of the hepatitis C virus (HCV) NS3/4A serine protease. The present study assessed the safety, pharmacokinetics, and antiviral activity of danoprevir in a randomized, placebo-controlled, 14-day multiple ascending dose study in patients with chronic HCV genotype 1 infection. METHODS Four cohorts of treatment-naïve (TN) patients (100 mg q12 h, 100 mg q8 h, 200 mg q12 h, 200 mg q8 h) and one cohort of non-responders (NR) to prior pegylated interferon alfa-ribavirin treatment (300 mg q12 h) were investigated. RESULTS Danoprevir was safe and well tolerated; adverse events were generally mild, transient and were not associated with treatment group or dose level. Danoprevir displayed a slightly more than proportional increase in exposure with increasing daily dose and was rapidly eliminated from the plasma compartment. Maximal decreases in HCV RNA were: -3.9 log(10)IU/ml and -3.2 log(10)IU/ml in TN receiving 200 mg q8 h and 200 mg q12 h, respectively. End of treatment viral decline in these two cohorts was within 0.1 log(10)IU/ml of the viral load nadir. HCV RNA reduction in NR was more modest than that observed in upper dose TN cohorts. The overall incidence of viral rebound was low (10/37) and was associated with the R155K substitution in NS3 regardless of the HCV subtype. CONCLUSIONS Danoprevir was safe and well tolerated when administered for 14 days in patients with chronic HCV genotype 1 infection. Treatment resulted in sustained, multi-log(10) IU/ml reductions in HCV RNA in upper dose cohorts. These results support further clinical evaluation of danoprevir in patients with chronic HCV.

Virologic Escape during Danoprevir (ITMN-191/RG7227) Monotherapy Is Hepatitis C Virus Subtype Dependent and Associated with R155K Substitution

ABSTRACT Danoprevir is a hepatitis C virus (HCV) NS3/4A protease inhibitor that promotes multi-log10 reductions in HCV RNA when administered as a 14-day monotherapy to patients with genotype 1 chronic HCV. Of these patients, 14/37 experienced a continuous decline in HCV RNA, 13/37 a plateau, and 10/37 a rebound. The rebound and continuous-decline groups experienced similar median declines in HCV RNA through day 7, but their results diverged notably at day 14. Plateau group patients experienced a lesser, but sustained, median HCV RNA decline. Baseline danoprevir susceptibility was similar across response groups but was reduced significantly at day 14 in the rebound group. Viral rebound in genotype 1b was uncommon (found in 2/23 patients). Population-based sequence analysis of NS3 and NS4A identified treatment-emergent substitutions at four amino acid positions in the protease domain of NS3 (positions 71, 155, 168, and 170), but only two (155 and 168) were in close proximity to the danoprevir binding site and carried substitutions that impacted danoprevir potency. R155K was the predominant route to reduced danoprevir susceptibility and was observed in virus isolated from all 10 rebound, 2/13 plateau, and 1/14 continuous-decline patients. Virus in one rebound patient additionally carried partial R155Q and D168E substitutions. Treatment-emergent substitutions in plateau patients were less frequently observed and more variable. Single-rebound patients carried virus with R155Q, D168V, or D168T. Clonal sequence analysis and drug susceptibility testing indicated that only a single patient displayed multiple resistance pathways. These data indicate the ascendant importance of R155K for viral escape during danoprevir treatment and may have implications for the clinical use of this agent.

Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors

Hepatitis C virus (HCV) is a major health burden, with an estimated 170 million chronically infected individuals worldwide, and a leading cause of liver transplantation. Patients are at increased risk of developing liver cirrhosis, hepatocellular carcinoma and even liver failure. In the past two decades, several approaches have been adopted to inhibit non-structural viral proteins. The RNA-dependent RNA polymerase (NS5B) of HCV is one of the attractive validated targets for development of new drugs to block HCV infection. In this review, we report the recent progress made towards identifying and developing benzothiadiazines as HCV NS5B polymerase inhibitors. The substituted benzothiadiazine class was identified by HTS in 2002 as an NS5B inhibitor. Further optimization and modification of the core has improved the potency and pharmacokinetic properties of substituted benzothiadiazines. Research on palm site-binding benzothiadiazine analogs and related derivatives and analogs is discussed in this article.

HCV NS5B polymerase inhibitors 1: Synthesis and in vitro activity of 2-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives

2-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition and replicon activity are discussed.

HCV NS5B polymerase inhibitors 3: Synthesis and in vitro activity of 3-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives

3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.