HCV Protease Research Articles

Design of Hepatitis C Protease Inhibitors Based on Knottin Fold

AbstractKnottins are very stable peptides (also called miniproteins) containing approximately 30 amino acid residues. Their structures are stabilized by three disulfide bonds forming a characteristic ‘pseudo‐knotted’ structure. This research was devoted to the design of potential inhibitors of human hepatitis C (HCV) NS3 protease by means of knottin fold using loop grafting, amino acid residues replacements and L/D amino acid epimerization. The initial structure used for design was a trypsin inhibitor from Momordica cochinchinensis seeds, MCOTI‐II. Modifications of the knottin template for optimization of the NS3 protease‐peptide interaction included deletion of several residues from the N‐terminus, replacement of residues in‐ and outside the inhibitor loop and replacement of certain L‐amino acids by their D‐stereoisomers. Stability of modified knottins and their complexes with HCV protease were evaluated by molecular dynamics simulation. Binding energy values for protein‐knottin complexes were calculated by MM‐GBSA method. The designed modified knottin characterized by the highest stability of the complex with HCV NS3 protease, was proposed for further experimental testing.

Novel Pan-Coronavirus 3CL Protease Inhibitor MK-7845: Biological and Pharmacological Profiling.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

Description and In silico ADME Studies of US-FDA Approved Drugs or Drugs under Clinical Trial which Violate the Lipinski’s Rule of 5

Background & Objective: Christopher A. Lipinski, in 1997, formulated Lipinski’s rule of five for drug-likeness prediction of potent molecules. It states that molecular weight (less than 500 Daltons), octanol/water partition coefficient (not exceeding more than 5), hydrogen bond acceptor (no more than 10), and hydrogen bond donor (no more than 5) are important for good oral bioavailability. Many drugs among various important classes such as antibiotics, anti-cancer, HIV and HCV protease inhibitors, immunosuppressants, cardiovascular, antifungal, and other miscellaneous classes are approved by FDA or other drug regulatory authorities as clinical use lie beyond the rule of five. In this review, beyond the rule of 5 drugs belonging to these classes (which are either currently approved or under clinical study) are explored and their ADME properties are analyzed. Methods: Data of 73 beyond the rule of 5 drugs, belonging to various classes, were collected and their ADME properties were calculated using the Qikprop prediction program of maestro 12.9 module of Schrodinger software. Result: Out of 73 drugs, 4 had at least 1 Rule of 5 (Ro5) violation, 16 had at least 2, 31 had at least 3 out of which 22 drugs had 4, Ro5 violations. Conclusion: Drugs not obeying the rule of five may also serve as good clinical candidates and potential candidates should not be discarded only on the basis of this rule.

Structure-based development and preclinical evaluation of the SARS-CoV-2 3C-like protease inhibitor simnotrelvir

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CLpro) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CLpro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CLpro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

Repurposing of HIV/HCV protease inhibitors against SARS-CoV-2 3CLpro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that caused the global COVID-19 outbreak. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a key role in virus replication and has become an ideal target for antiviral drug design. In this work, we have employed bioluminescence resonance energy transfer (BRET) technology to establish a cell-based assay for screening inhibitors against SARS-CoV-2 3CLpro, and then applied the assay to screen a collection of known HIV/HCV protease inhibitors. Our results showed that the assay is capable of quantification of the cleavage efficiency of 3CLpro with good reproducibility (Z′ factor is 0.59). Using the assay, we found that 9 of 26 protease inhibitors effectively inhibited the activity of SARS-CoV-2 3CLpro in a dose-dependent manner. Among them, four compounds exhibited the ability to bind to 3CLproin vitro. HCV protease inhibitor simeprevir showed the most potency against 3CLpro with an EC50 vale of 2.6 μM, bound to the active site pocket of 3CLpro in a predicted model, and importantly, exhibited a similar activity against the protease containing the mutations P132H in Omicron variants. Taken together, this work demonstrates the feasibility of using the cell-based BRET assay for screening 3CLpro inhibitors and supports the potential of simeprevir for the development of 3CLpro inhibitors.

Uridine-Derived 4-Aminophenyl 1-Thioglucosides: DFT Optimized FMO, ADME, and Antiviral Activities Study

The biological roles of carbohydrates, especially their synthetic glycoconjugates, are at the forefront of research, and their applications and research are in the development of an impressive area of carbohydrate-derived vaccines. Considering this, several thioglucosides 2-6 conjugated with para-aminophenyl and uridine moieties are investigated for their possible application as hepatitis antiviral applications. Geometry optimization of these uridine thioglucosides by density functional theory (DFT) indicated that their glucopyranose rings are in 4C1 chair conformation with β-glucosidic linkage. Frontier molecular orbital (FMO), molecular electrostatic potential (MEP), lower hardness, and softness calculations indicated their higher electrophilic nature. Solubility and ADMET studies indicated that the uridine thioglucosides might be safer drugs to use. With these encouraging results, molecular docking of 2-6 was conducted with hepatitis C virus (HCV, 3su4) and hepatitis B virus (HBV, 5e0i) proteases where the binding affinities for 2-6 were found higher than the standard anti-hepatitis drug sofosbuvir. Overall, the thioglucosides showed a better binding affinity with HBV protease (5e0i) than the HCV protease (3su4), indicating their prospect against these viruses.

Determination of Binding Potential of HCV Protease Inhibitors Against to SARS-CoV-2 Papain-like Protease wtih Computational Docking

Background: SARS-CoV-2, a novel coronavirus that caused a pandemic respiratory disease, has recently emerged from China. Since it is a life-threatening virus, investigation of curative medications along with protective vaccines still maintains its importance. Drug repurposing is a reasonable and immediate approach to combat SARS-CoV-2 infection by identifying inhibitory molecules from marketed drugs. PL protease (PLpro) is one of the essential enzymes for the progression of SARS-CoV-2 replication and life cycle. Objective: We aimed to investigate the potential of 4 HCV protease inhibitors as probable repurposing drugs in Covid-19 treatment. Methods: In order to understand possible binding affinity of HCV protease inhibitors, Boceprevir, Grazoprevir, Simeprevir, and Telaprevir, against PLpro, we performed docking analysis in silico. Docking study was accomplished using AutoDock 4.2 Software. Potential druggable pockets on PLpro were predicted by DoGSiteScorer tool in order to explore any overlapping with binding regions and these pockets. Results: This analysis demonstrated Boceprevir, Grazoprevir, Simeprevir and Telaprevir interacted by PLpro with binding energies (kcal/mol) of -4.97, -4.24, -6.98, -1.08, respectively. Asn109, one of the interacted residues with both Boceprevir and Simeprevir, is a neighbouring residue to catalytic Cys111. Additionally, Telaprevir notably interacted with catalytic His272 in the active site. Conclusion: Present study explains the binding efficiency and repurposing potential of certain HCV protease inhibitors against to SARS-CoV-2 PLpro enzyme. Docking sites and potential druggability of ligands were also crosschecked by the estimation of druggable pockets. Thereby our results can promote promising preliminary data for research on drug development in the fight of Covid-19.

Адекватность применения прогностических систем при оценке тяжести цирроза печени

Assessment of the severity of liver cirrhosis as a mandatory parameter includes a characteristic of the degree of its compensation. Detailed historical research has shown that this characteristic of liver disease has no authorship, no precise definition, and seems to be a matter of course for the medical community. Moreover, the term “decompensation” (the adjustment of certain functions due to the adaptation of other organs and systems) does not reflect the pathophysiology of changes developing in patients with terminal liver cirrhosis. When determining the degree of compensation for cirrhosis, physicians are forced to focus on the classification of Child and Turcotte in Pugh's modification. This system was developed to assess the outcome of surgical treatment of portal hypertension and validated to predict the survival of patients with liver cirrhosis (expectation of an event in the future) in the short and medium term, but cannot be orrectly applied to assess the severity of liver cirrhosis (at the time of assessment). The inadequacy of the Child-Pugh prognostic system for assessing the severity of liver cirrhosis was shown by the example of determining the possibility of using HCV protease inhibitors in patients with complicated (“decompensated”) liver cirrhosis. It is necessary to develop new principles for assessing the severity of liver cirrhosis, in particular, in relation to the disorders of drug metabolism and the potential toxicity of drugs and their metabolites.

Amino Acid and Peptide-Based Antiviral Agents.

A significant number of antiviral agents used in clinical practice are amino acids, short peptides, or peptidomimetics. Among them, several HIV protease inhibitors (e. g. lopinavir, atazanavir), HCV protease inhibitors (e. g. grazoprevir, glecaprevir), and HCV NS5A protein inhibitors have contributed to a significant decrease in mortality from AIDS and hepatitis. However, there is an ongoing need for the discovery of new antiviral agents and the development of existing drugs; amino acids, both proteinogenic and non-proteinogenic in nature, serve as convenient building blocks for this purpose. The synthesis of non-proteinogenic amino acid components of antiviral agents could be challenging due to the need for enantiomerically or diastereomerically pure products. Herein, we present a concise review of antiviral agents whose structures are based on amino acids of both natural and unnatural origin. Special attention is paid to the synthetic aspects of non-proteinogenic amino acid components of those agents.

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors.

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics.

The rapid growth of COVID-19 cases is causing an increasing death toll and also paralyzing the world economy. De novo drug discovery takes years to move from idea and/or pre-clinic to market, and it is not a short-term solution for the current SARS-CoV-2 pandemic. Drug repurposing is perhaps the only short-term solution, while vaccination is a middle-term solution. Here, we describe the discovery path of the HCV NS3–4A protease inhibitors boceprevir and telaprevir as SARS-CoV-2 main protease (3CLpro) inhibitors. Based on our hypothesis that α-ketoamide drugs can covalently bind to the active site cysteine of the SARS-CoV-2 3CLpro, we performed docking studies, enzyme inhibition and co-crystal structure analyses and finally established that boceprevir, but not telaprevir, inhibits replication of SARS-CoV-2 and mouse hepatitis virus (MHV), another coronavirus, in cell culture. Based on our studies, the HCV drug boceprevir deserves further attention as a repurposed drug for COVID-19 and potentially other coronaviral infections as well.

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

Development of a Large-Scale Route to Glecaprevir: Synthesis of the Side Chain and Final Assembly

The preceding article described the development of the large-scale synthetic route to macrocycle 3 of glecaprevir (1), a potent HCV protease inhibitor. This article describes the development of the...

A Note on the Potential Clinical Use of Sofosbuvir to Treat COVID-19: The Importance of Protease Inhibitors

In this manuscript we argue that the use of Sofosbuvir as a possible medication in the clinical treatment of COVID-19 can be complemented with HCV protease inhibitors given the homology between the HCV NS3/4A protease and the SARS-CoV-2 Mpro protease.

In Silico Evaluation of the Effectivity of Approved Protease Inhibitors against the Main Protease of the Novel SARS-CoV-2 Virus.

The coronavirus disease, COVID-19, caused by the novel coronavirus SARS-CoV-2, which first emerged in Wuhan, China and was made known to the World in December 2019 turned into a pandemic causing more than 126,124 deaths worldwide up to April 16th, 2020. It has 79.5% sequence identity with SARS-CoV-1 and the same strategy for host cell invasion through the ACE-2 surface protein. Since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. The 3D structure of the SARS-CoV-2 main protease was compared with the 3D structures of seven proteases, which are drug targets, and docking analysis to the SARS-CoV-2 protease structure of thirty four approved and on-trial protease inhibitors was performed. Increased 3D structural similarity between the SARS-CoV-2 main protease, the HCV protease and α-thrombin was found. According to docking analysis the most promising results were found for HCV protease, DPP-4, α-thrombin and coagulation Factor Xa known inhibitors, with several of them exhibiting estimated free binding energy lower than −8.00 kcal/mol and better prediction results than reference compounds. Since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. DPP-4 inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe SARS-CoV-2 induced pneumonia.

Hepatitis C Virus Drugs Simeprevir And Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV2 Replication

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold.Funding: This research was supported by grants from the National Institutes of Health (R01-GM120574 to GTM) and RPI Center for Computational Innovations (to KB and GTM). This research was also partly funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract #,HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grant U19AI142733 U19AI135972 and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to AG-S.Conflict of Interest: A provisional patent application related to these, studies has been filed. GTM is a founder of Nexomics Biosciences, Inc. This, relationship has no conflict of interest with respect to this study. GTM and RMK are inventors in patents owned jointly by Rutgers University and the University of Texas at Austin concerning the use of specific compounds as antivirals against influenza virus. These patents have no conflict of interest for this study. AG-S is inventor in patents and patent application owned by the Icahn School of Medicine concerning the use of specific antiviral compounds. This inventorship has no conflict of interest with respect to this study.

A novel molecular mechanism to explain mutations of the HCV protease associated with resistance against covalently bound inhibitors

NS3 is an important therapeutic target for direct-acting antiviral (DAA) drugs. However, many patients treated with DAAs have unsustained virologic response (UVR) due to the high mutation rate of HCV. The aim of this work was to shed some light on the puzzling molecular mechanisms of the virus’s of patients who showed high viral loads even under treatment with DAA. Bioinformatics tools, molecular modelling analyses were employed to identify mutations associated with HCV resistance to boceprevir and possible structural features related to this phenomenon. We identified two mutations of NS3 that may be associated with HCV resistance: D168N and L153I. The substitution D168N was previously reported in the literature as related with drug failure. Additionally, we identified that its molecular resistance mechanism can be explained by the destabilization of receptor-ligand hydrogen bonds. For the L153I mutation, the resistance mechanism is different from previous models reported in the literature. The L153I substitution decreases the S139 deprotonation susceptibility, and consequently, this mutation impairs the covalent binding between the residue S139 from NS3 and the electrophilic trap on boceprevir, which can induce drug failure. These results were supported by the time course analysis of the mutations of the NS3 protease, which showed that boceprevir was designed for enzymes with an L residue at position 153; however, the sequences with I153 are predominant nowadays. The results presented here could be used to infer about resistance in others DAA, mainly protease inhibitors.

Anti-HCV protease potential of endophytic fungi and cytotoxic activity

Abstract Forty-eight endophytic fungal strains were isolated and purified from ten Egyptian medicinal plants and their culture broth extracts were explored for HCV protease inhibitory activity and cytotoxicity. The ethyl acetate extracts of Alternaria alternata PGL-3, Cochlibolus lunatus PML-17, Nigrospora sphaerica EPS-38, followed by Emerecilla nidulans RPL-21 showed the most potent inhibition of HCV NS3/4A protease with IC50 17.0, 20.5, 33.6, and 54.6 μg/ml, respectively, with low cytotoxicity except for the later. The extracts of Emericella nidulans RSL-24, Fusarium oxysporum SML-41, Emericella nidulans RPL-21, and Penicillium sp. RSL-43 exhibited strong cytotoxic activity against human breast cancer cell lines (MCF-7) with IC50 10.8, 11.0, 12.5, and 13.7 μg/ml, respectively. Emericella nidulans RSS-22, Emericella nidulans RSL-24, and Fusarium oxysporum SML-41 displayed a potent cytotoxic effect on human liver cancer cell lines (HEP-G2) with IC5014.8, 20.3 and 24.0 μg/ml, respectively. Alternariol and alternariol -9-methyl ether were isolated from the ethyl acetate extract of Alternaria alternata PGL-3 whereas, emericellin, shamixanthone, arugosin C were isolated from the ethyl acetate extract of Emericella nidulans RPL-21. The results suggest Alternaria alternata PGL-3 endophyte from Punica granatum peel as a source of antiviral lead.

Anticancer and Antiviral Diketopiperazine Produced by the Red Sea Endophytic Fungus Penicillium chrysogenum

Scale-up fermentation of the endophytic fungus Penicillium chrysogenum in biomaltpeptone media followed by cytotoxicity-guided fractionation led to the isolation of haenamindole, an unusual diketopiperazine (DKP) alkaloid, along with other five known DKPs. Haenamindole was elucidated on the basis of comprehensive 1D and 2D NMR spectroscopic including 15N-HSQC and 15NHMBC and mass spectrometric analyses. The compound possesses the secondary hydroxamic acid functionality of N-piperazindione ring system confirmed by methylation in sodium dimethyl sulfate and dry dimethylformamide to yield haenamindole-22-N-methyl ether. However, haenamindole demonstrated weak HCV protease activity with an IC50 value of 76.3µM, its cytotoxicity profiling in a panel of up to 12 cell lines indicated significant cytotoxicity of the compound with pronounced selectivity for colon-38 cancer cells compared to the human normal cells.

Controlling protein function with HCV protease.

Two methods for controlling protein activities with small molecules provide a general solution to a long-standing challenge in mammalian synthetic biology.