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• W2085304196 abstract "The key to unlocking the secret? The nonstructural protein NS5A has no known enzymatic function, but it is essential for hepatitis C virus (HCV) replication and has been proposed to function as a regulator for key replication events. The first clinically validated NS5A inhibitor, developed via chemical genetics screens with HCV replicons, was recently described and this work is highlighted here. The hepatitis C virus (HCV) infects approximately 200 million people worldwide (∼3 % of the population), including 4–5 million in the United States. Most are unaware of HCV infections that have not advanced enough to produce liver disease (cirrhosis or liver cancer). Approximately 12 000 Americans are estimated to die from this virus each year. Current treatment, consisting of pegylated interferon-α (PEG-IFN-α) plus the nucleoside antiviral drug ribavirin, is ineffective in more than 50 % of patients infected with genotype 1 HCV. This is the most aggressive and common strain in the Americas, Europe, Japan and China. Unfortunately, the conventional therapy is accompanied by poorly tolerated side effects. These facts have led to aggressive competition between pharmaceutical companies striving to develop new, more effective anti-HCV drugs.1–3 Hepatitus C is an enveloped virus with a 9.6 kb RNA genome that encodes a polyprotein consisting of about 3 000 amino acids. Cellular and viral proteases process this polyprotein into several structural proteins, plus several nonstructural proteins. The latter are designated NS2, NS3, NS4A, NS4B, NS5A and NS5B. NS2 and NS3 exhibit protease activity, and NS4A acts as a cofactor for NS3. NS4B induces changes in cellular membranes that are important for viral replication, while NS5B catalyses RNA synthesis. NS5A has no known enzymatic function, but it is essential for HCV replication and has been proposed to function as a regulator for key replication events.3 Over the last several years, drug companies have mainly focused on inhibiting the protease function of NS3 or the RNA polymerase activity of NS5B. Several such drugs are now in clinical trials, with two (Vertex's telaprevir and Merck's boceprevir) in phase III clinical trials at the time of this writing. According to a May 2010 press release,4 telaprevir produced a sustained viral response in 75 % of patients, reducing the patient's viral load to below detectible levels. This is excellent news, but it does not greatly affect the need for new anti-HCV drugs operating by other mechanisms. Like many other viruses, HCV mutates rapidly, so it is expected that combinations of drugs will be needed to prevent and manage drug resistance, with the eventual aim of eradicating the virus. Traditionally, medicines were discovered by empirical testing, first of natural products derived from plants, later of dyes derived from coal tar or synthesized in the laboratory. Ehrlich's “magic bullet” hypothesis, stating that every drug has a specific “receptor” (e.g., enzyme or receptor protein) that is responsible for its effect, eventually changed the focus of drug research. Investigators began to look for small organic molecules that could selectively interact with specific proteins that are important in disease processes. By the late twentieth century, genetic screens were used to identify protein function in many organisms. This involves randomly producing mutations in cells, screening the resulting mutants to generate a characteristic of interest, and identifying the corresponding mutations in specific genes. By the end of the twentieth century, an alternate approach called “chemical genetics” was developed and applied to the discovery of new drugs.5 Chemical genetics, in the same “forward” sense as the classical genetic screen just described, involves screening libraries of small organic molecules in cells, selecting a compound that produces the phenotype of interest, and identifying the corresponding protein target. The process of discovery and development of antiviral drugs faces unique challenges.6 Viruses do not replicate outside the host cell, so drugs that inhibit viral replication must exploit subtle biological differences between virus-infected cells and normal, uninfected cells. Certain proteins and processes native to the host cell can enable virion entry, replication of viral constituents, or budding of nascent virus particles; hence, they are included as potential targets for antiviral drugs. In order to avoid toxicity and various side effects, the drug industry has traditionally tried to achieve high potency and selectivity against proteins that are characteristic of the virus, not of the host cell. New drug leads are typically discovered by screening compounds for inhibition of specific viral proteins in vitro, or by screening compounds for inhibition of viral replication in cells. The latter method, which resembles the chemical genetics approach, is broader than specifically targeted screens because compounds that inhibit replication by any mechanism can be discovered, including inhibitors of viral entry or budding of nascent virions (for screens conducted in cell cultures). The dream of in silico drug design has only been realized for one class of drugs: HIV protease inhibitors.7, 8 Historically, many drug companies have followed a conservative approach, developing new drugs that operate by the same mechanism as previously developed drugs. A drug target is fully validated by the existence of profitable drugs operating by this mechanism of action; such drugs are often potent, nontoxic, and produce minimal side effects. While more interesting scientifically, validation of novel drug targets is risky and very expensive, though also potentially more lucrative in the long run. Validation of new targets for antiviral drugs is particularly important because drugs operating by different mechanisms are usually synergistic and combination therapy delays the onset of resistant strains, which must simultaneously develop multisite mutations conferring resistance to more than one drug. A recent report of the first clinical validation of a novel NS5A inhibitor can accordingly be considered a breakthrough in anti-HCV drug research.9 Early in 2010, a team from Bristol–Myers Squibb reported the novel HCV NS5A inhibitors shown in Figure 1.10 These compounds were identified by means of a high-throughput screen that simultaneously measures inhibition of HCV replication, selectivity relative to a related virus (bovine viral diarrhea, BVDV), and cellular toxicity in a 96-well format.11 The HCV replication assay utilized human liver cells (Huh-7) transfected with HCV RNA comprising the portion of the viral genome encoding proteins including NS3 through NS5B.12 Intracellular replication of this HCV “replicon” was detected by measuring NS3 protease activity with a FRET assay employing a peptide substrate bearing two fluorescent labels.13 BVDV replication was also detected by fluorescence, measuring luciferase produced in Huh-7 cells transfected with RNA to produce a BVDV replicon containing the luciferase gene. Cytotoxicity was measured by a conventional method involving transformation of Alamar blue dye by cellular enzymes. By means of this screen, BMS-858 was identified as a novel anti-HCV lead. Initially, the concentration of BMS-858 producing 50 % inhibition of wild-type HCV replicon replication (EC50) was measured as 0.6–1.0 μM, with low cytotoxicity (CC50 >50 μM) and no detectible activity toward the BVDV replicon. A series of related compounds were then synthesized and screened, revealing BMS-824 with an EC50 value of 5 nM against the wild-type HCV replicon. Remarkably, removal of just one atom from the structure of BMS-858 results in a more than 100-fold increase in potency. Structures of previously identified anti-HCV NS5A inhibitors.10 The HCV replicon system12 provided the first reliable, cell-based model for HCV replication and enabled the development of high-throughput screens for anti-HCV drugs. Detecting intracellular replication of a portion of the HCV genome has powerful advantages for drug discovery, relative to assays involving specific HCV proteins. By this means, drugs operating by a broader range of mechanisms can be found, including drugs that interact with viral proteins that do not have (or have unknown) enzymatic activity. While the method has been described as “mechanistically unbiased”,9 in actuality, it is restricted to processes related to intracellular replication and cannot detect potential drugs that might be able to inhibit binding/entry of the virus to host cells, or formation/budding of infectious virus particles. In the case at hand, the target of the BMS-858/824 series was identified as the HCV protein NS5A by characterizing mutations in drug-resistant replicons.10 The replicon cell system then became a useful tool for investigating interactions between NS5A and the drugs, enabling testing with single-site mutants and various HCV genotypes. Because of differences in the amino acid composition of the NS5A protein, BMS-824 was found to be far less potent against the genotype 1a HCV replicon than against genotype 1b, as shown in Table 1. Further analogue studies unveiled the symmetrical compound BMS-665, which has similar potency against genotype 1b as BMS-824, but also has significant ability to inhibit genotype 1a replication (Table 1). EC50 [nM] genotype 1b (strain=Con1) genotype 1a (strain=H77) BMS-824[a] 18 >10 000 BMS-665[a] 11 393 BMS-790052[b] 0.009 0.050 In May 2010, a more potent inhibitor of HCV NS5A was reported, namely BMS-790052.9 The structure of this drug is shown in Figure 2 along with those of two analogues (1 and 2), which were used to confirm the mechanism of action. As can be seen in Table 1, BMS-790052 inhibits replication of both HCV replicon genotypes with EC50 values in the range of 9–50 pM. This drug is also active against several other HCV replicon genotypes and has a therapeutic index (CC50/EC50) of at least 100 000 in vitro. Despite having a molecular weight of more than 700 g mol−1, BMS-790052 is orally bioavailable and distributes effectively into the liver. It displays additive-to-synergistic effects with other anti-HCV drugs, and it has advanced into clinical trials. Biotin-conjugated drugs 1 and 2, which bear a much closer structural relationship to BMS-665, were reported in the same publication as BMS-790052.9 Compound 1, with the same S,S-proline configuration as BMS-665, inhibited replication of the genotype 1b HCV replicon with an EC50 value of 33 nM, while the R,R-enantiomer (2) was inactive. These two biotinylated compounds were added to separate flasks of HCV 1b replicon cells and their lysates were treated with streptavidin-agarose beads to isolate bound proteins, which were analyzed by gel electophoresis and immunoblotting. Compound 1 was found to bind HCV protein NS5A in this experiment, while inactive control compound 2 did not. Structures of an anti-HCV NS5A inhibitor in clinical trials (BMS-790052) and biotin conjugates 1 and 2.9 NS5A is a 447 amino acid protein that apparently dimerizes by self-association of domain I at the N terminus. The series of drugs described here clearly bind to domain I of NS5A, according to observed mutations in drug-resistant HCV replicon strains. The symmetrical (pallindromic) structures of many of these drugs suggest that they bind across the dimer interface at the site of symmetry of the protein aggregate. This is reminiscent of the interaction of inhibitors with HIV protease, which is smaller (only 99 amino acids) but also dimerizes, with its active site pierced by the axis of symmetry of the assembly.7, 8 Chemists designed a number of C2-symmetric molecules as HIV protease inhibitors, reasoning that affinity could be maximized by matching the symmetry of a drug with that of its receptor. Early observations of rapid onset of drug resistance led to the concern that this approach had a serious weakness: single-site mutation in a homodimer would weaken drug–receptor interaction at two sites, rather than one. Only non-C2-symmetric compounds eventually became viable anti-HIV protease inhibitors, but it is not clear whether rapid onset of resistance was really a practical limitation of the symmetrical candidates. On the other hand, low aqueous solubility and poor bioavailability have been associated with symmetry in some HIV protease inhibitors.14 It would be very interesting to know in the case of the BMS NS5A inhibitors what is the influence of molecular symmetry on solubility, bioavailability, and onset of drug resistance. This drug discovery program aimed at inhibitors of HCV NS5A is certainly an excellent example of increased willingness of drug companies to invest in higher risk efforts to validate new targets. Other HCV NS5A inhibitors have been reported,15, 16 but BMS-790052 is apparently the most advanced in the clinic. The method of its development—employing high-throughput screening of drug libraries in cell-based replicon assays—is also significant as a model for others to follow in the discovery of drugs operating by unconventional mechanisms. This is a powerful demonstration of the utility of chemical genetics in drug discovery, though some important questions remain unanswered. For example, the exact manner in which the BMS compounds interact with HCV NS5A has not been described. One can hope that this question will be answered soon, perhaps through the X-ray structure of NS5A domain I bound to one of these drugs. Also, NS5A is known to be essential in HCV replication, but its exact role remains undiscovered. Again, a potent NS5A inhibitor, such as BMS-790052, may be the key to unlocking this secret." @default.
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• W2085304196 date "2010-09-06" @default.
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• W2085304196 title "Drugs for Hepatitis C: Unlocking a New Mechanism of Action" @default.
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