Structure–activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC 50 ) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC 99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

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“…TMC435350 was prepared by a method described previously (36). Human recombinant IFN-␣ was purchased from Calbiochem (La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

Lin¹,

Lenz²,

Fanning³

et al. 2009

Antimicrob Agents Chemother

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182

142

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“…Over the past decade, pharmaceutical companies have further developed these lead compounds. Many structure-activity-relationship (SAR) studies have been performed to evaluate the effect of different functional moieties on protease inhibition at positions P4-P1′ (11)(12)(13)(14)(15)(16)(17). Crystal structures have been determined of the NS3/4A protease domain bound to a variety of inhibitors as well as of several drug resistant protease variants, such as R155K and V36M (18,19).…”

mentioning

confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

181

245

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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“…However, the historical difficulties in producing high yields of pure, soluble, and active hPOL (or domains thereof) have prevented the full power of structural and biophysical methods in this research. These approaches typically help catalyze successful drug discovery programs (22)(23)(24)(25). Our work, however, could help change this situation for HBV, as TP and RT-RH 303-778 can be rapidly and easily purified and were soluble over a wide pH range (and at high protein concentrations).…”

Section: Discussionmentioning

confidence: 99%

“…Despite being such an important target, our understanding of hPOL structure-function and the ability to generate novel antivirals targeting its diverse functions currently lags behind that of important enzymes from other pathogenic viruses (21)(22)(23)(24)(25). This situation arises from the extreme difficulties in obtaining sufficient yields of soluble, active recombinant hPOL that are needed to drive forward structural biology and high-throughput drug discovery programs (26)(27)(28)(29)(30)(31).…”

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confidence: 99%

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Vörös

Urbanek

Rautureau

et al. 2014

J Virol

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Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies-broad-spectrum antivirals-are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly ␣-helical structures with small amounts of ␤-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCEThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of muchneeded new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV. Hepatitis B virus (HBV) is a highly infectious, species-specific pathogen that causes serious liver diseases, including cancer, and causes 600,000 deaths annually (1). While excellent prophylactic HBV vaccines exist, these are ineffective against extant chronic HBV infections which affect 350 million people worldwide. Approved therapies for chronic HBV infections include immuno-modulators (e.g., interferon therapies) and nucleoside analogues that inhibit the reverse transcriptase domain of hPOL, the multifunctional viral polymerase of human HBV. These reverse transcriptase inhibitors, currently our best weapons against HBV, are not complete cures and have unwanted side effects (2, 3). While reverse transcriptase inhibitors are initially very effective at...

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Structure–activity relationship study on a novel series of cyclopentane-containing macrocyclic inhibitors of the hepatitis C virus NS3/4A protease leading to the discovery of TMC435350

Cited by 128 publications

References 20 publications

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

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