Virus NS3 Protease Research Articles

Genetic diversity of NS3 protease from Brazilian HCV isolates and possible implications for therapy with direct-acting antiviral drugs

The hepatitis C virus (HCV) NS3 protease has been one of the molecular targets of new therapeutic approaches. Its genomic sequence variability in Brazilian HCV isolates is poorly documented. To obtain more information on the magnitude of its genetic diversity, 114 Brazilian HCV samples were sequenced and analysed together with global reference sequences. Genetic distance (d) analyses revealed that subtype 1b had a higher degree of heterogeneity (d = 0.098) than subtypes 1a (d = 0.060) and 3a (d = 0.062). Brazilian isolates of subtype 1b were distributed in the phylogenetic tree among sequences from other countries, whereas most subtype 1a and 3a sequences clustered into a single branch. Additional characterisation of subtype 1a in clades 1 and 2 revealed that all but two Brazilian subtype 1a sequences formed a distinct and strongly supported (approximate likelihood-ratio test = 93) group of sequences inside clade 1. Moreover, this subcluster inside clade 1 presented an unusual phenotypic characteristic in relation to the presence of resistance mutations for macrocyclic inhibitors. In particular, the mutation Q80K was found in the majority of clade 1 sequences, but not in the Brazilian isolates. These data demonstrate that Brazilian HCV subtypes display a distinct pattern of genetic diversity and reinforce the importance of sequence information in future therapeutic approaches.

Synthesis of [14C]boceprevir, [13C3]boceprevir, and [D9]boceprevir, a hepatitis C virus protease inhibitor

Boceprevir is a hepatitis C virus (HCV) NS3 protease inhibitor for HCV treatment. [14C]Boceprevir (SCH 503034, trade name Victrelis) was synthesized from K14CN in 11 steps with an overall yield of 16.4%. [13C3]Boceprevir was synthesized in 16 steps with a 2.5% overall yield. The carbon‐13 in the molecule was distributed along the peptide chain. [D9]Boceprevir was synthesized from [D9]‐t‐butylamine in four steps with an overall yield of 69%. Copyright © 2012 John Wiley & Sons, Ltd.

New developments in the management of hepatitis C virus infection: focus on boceprevir

Chronic hepatitis C virus infection is an important public health problem, and the standard treatment (combination of pegylated interferon-α and ribavirin) has an effectiveness rate of only 40%–50%. Novel virus-specific drugs have recently been designed, and multiple compounds are under development. The approval for the clinical use of direct-acting antivirals in 2011 (boceprevir [BOC] and telaprevir, viral NS3 protease inhibitors) has increased recovery rates by up to 70%. Therefore, a highly effective treatment has been envisioned for the first time. This paper focuses on BOC and the implementation of new BOC-based treatment regimes.

Novel, potent and orally bioavailable indolizidinone-derived inhibitors of the hepatitis C virus NS3 protease

A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature. The synthesis, preliminary SAR and pharmacokinetic profiling of this compact, indolizidinone-derived scaffold are described.

Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir

BackgroundTelaprevir and boceprevir are highly selective hepatitis C virus (HCV) NS3/4A proteaseinhibitors in phase 3 development. Viral breakthrough during mono- and triple-therapies with PEG-interferon and ribavirin and relapse is associated with resistance. ObjectivesPotential persistence of resistance mutations during long-term follow-up should be analyzed. Study designClonal sequence analysis of the NS3-protease gene was performed at long-term follow-up in HCV genotyp-1 infected patients who received telaprevir or boceprevir within phase-1b studies for comparison with resistant variants present directly after the end-of-treatment. ResultsAfter a median follow-up of 4.2years in 28 of 82 patients HCV-RNA was still detectable. Resistance variants were detected in two of 14 telaprevir- and in four of 14 boceprevir-treated patients. For telaprevir patients two low-level (V36M, V36A) and one high-level (A156T) mutation associated with resistance were detected at low frequencies (4–9% of the clones). In five boceprevir-treated patients four low level mutations (V36A, T54A/S, V55A) were observed at low frequencies (1–10%) while in one patient additionally a combined variant (T54S+R155K) was detected at 94%. Presence of resistant variants at long-term follow-up was not predictable by variants detected at the end-of-treatment. In one patient a V55A variant which was dominant already at baseline was still detectable at long-term follow-up. ConclusionsIn the majority of patients after short-term treatment with telaprevir or boceprevir wild-type NS3-protease isolates are detectable by clonal sequencing at long-term follow-up. Detectable resistance mutations in single patients are not predictable by initial frequencies of variants.

Screening and rank ordering of reversible mechanism‐based inhibitors of Hepatitis C virus NS3 protease using electrospray ionization mass spectrometry

An affinity-selection study using size exclusion chromatography (SEC) combined with off-line electrospray ionization mass spectrometry (ESI-MS) was performed on libraries of peptidic α-ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 µM) was incubated with equimolar amounts (100 µM) of 49 reversible mechanism-based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme-inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease-inhibitor complexes by SEC spin columns. The eluate of the SEC was immediately analyzed by direct-infusion ESI-MS. An enzyme-inhibitor complex, with a molecular mass corresponding to the NS3 protease binding to the preferred inhibitor, SCH212986, was the only molecular species detected. By increasing the molar ratio of HCV NS3 protease to inhibitors to 1:2 while keeping the inhibitors' concentration constant, the complex of the second most tightly bound inhibitor, SCH215426, was also identified. Although the potencies of these inhibitors were virtually un-measurable by kinetic assays, a rank order of CVS4441 > SCH212986 > SCH215426 was deduced for their inhibition potencies by direct competition experiment with CVS4441 (K(i)*>80 µM). As discussed in the article, through judicious application of this strategy, even large libraries of fairly weak, reversible and slow-binding inhibitors could be rapidly screened and rank ordered to provide critical initial structure-activity insights.

Tripeptide inhibitors of dengue and West Nile virus NS2B–NS3 protease

A series of tripeptide aldehyde inhibitors were synthesized and their inhibitory effect against dengue virus type 2 (DENV2) and West Nile virus (WNV) NS3 protease was evaluated side by side with the aim to discover potent flaviviral protease inhibitors and to examine differences in specificity of the two proteases. The synthesized inhibitors feature a varied N-terminal cap group and side chain modifications of a P2-lysine residue. In general a much stronger inhibitory effect of the tripeptide inhibitors was observed toward WNV protease. The inhibitory concentrations against DENV2 protease were in the micromolar range while they were submicromolar against WNV. The data suggest that a P2-arginine shifts the specificity toward DENV2 protease while WNV protease favors a lysine in the P2 position. Peptides with an extended P2-lysine failed to inhibit DENV2 protease suggesting a size-constrained S2 pocket. Our results generally encourage the investigation of di- and tripeptide aldehydes as inhibitors of DENV and WNV protease.

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.

Novel, potent, and orally bioavailable phosphinic acid inhibitors of the hepatitis C virus NS3 protease

A potent and novel class of product-like inhibitors of the HCV NS3 protease was discovered by employing a phosphinic acid as a carboxylate isostere. The replicon activity and pharmacokinetic profile of this series of compounds was optimized by exploring the substitution of the phosphinic acid, as well as conformationally constraining these compounds through macrocyclization. The syntheses and preliminary biological evaluation of these phosphinic acids is described.

Complexity and Catalytic Efficiency of Hepatitis C Virus (HCV) NS3 and NS4A Protease Quasispecies Influence Responsiveness to Treatment with Pegylated Interferon plus Ribavirin in HCV/HIV-Coinfected Patients

The role of the hepatitis C virus (HCV) NS3/4A protease in ablating the signaling pathway involved in the production of alpha/beta interferon (IFN-α/β) suggests a relationship between NS3/4A proteolytic activity and a patient's response to IFN-based therapy. To identify viral factors associated with the HCV treatment response, we analyzed the pretreatment NS3/4A protease gene quasispecies composition of 56 HCV genotype 1-HIV-1-coinfected patients treated in our clinic with pegylated IFN (pegIFN) plus ribavirin (RBV). The catalytic efficiency of the dominant (i.e., the most abundant) quasispecies was also assayed for Cardif cleavage and correlated with treatment outcome. A total of 1,745 clones were isolated and sequenced. Significantly less nucleotide quasispecies heterogeneity and lower Shannon entropy values were detected within the responder group (P < 0.05). A correlation was also found between the efficiency of NS3/4A protease Cardif cleavage and therapy outcome. Proteases from sustained responder patients were more efficient at processing Cardif (mean ± standard error of the mean [SEM], 0.8960 ± 0.05568; n = 19) than proteases from nonresponders (mean ± SEM, 0.7269 ± 0.05306; n = 37; P < 0.05). Finally, the amino acid p distance (the proportion [p] of nucleotide sites at which two sequences being compared are different) was significantly shorter in patients with an interleukin-28B (IL-28B) risk allele (P < 0.01), suggesting that IL-28B risk allele carriers exert a lower positive selection pressure on the NS3/4A protease. NS3/4A protease efficiency in cleaving Cardif may be associated with the pegIFN-RBV treatment response, as shown in our cohort of HIV-HCV-coinfected patients. Greater NS3/4A nucleotide heterogeneity and higher Shannon entropy values in nonresponders suggest that less HCV quasispecies complexity may favor a better response to pegIFN-RBV.

Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with direct-acting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a single-step binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK(a) changes within the catalytic triad associated with binding of the bromo-quinoline moiety.

Inhibition of hepatitis C virus replication by herbal extract: Phyllanthus amarus as potent natural source

Hepatitis C virus infection is a major health problem worldwide. Developing effective antiviral therapy for HCV is the need of the hour. The viral enzymes NS3 protease and NS5B RNA dependent RNA polymerase are essential enzymes for polyprotein processing and viral RNA replication and thus can be potential targets for screening anti-HCV compounds. A large number of phytochemicals are present in plants, which are found to be promising antiviral agents. In this study, we have screened inhibitory effect of different plant extracts against the NS3 and NS5B enzymes of hepatitis C virus. Methanolic extracts were prepared from various plant materials and their inhibitory effects on the viral enzymes were determined by in vitro enzyme assays. Effect on viral RNA replication was investigated by using TaqMan Real time RT-PCR. Interestingly, Phyllanthus amarus root (PAR) extract showed significant inhibition of HCV-NS3 protease enzyme; whereas P. amarus leaf (PAL) extract showed considerable inhibition of NS5B in the in vitro assays. Further, the PAR and PAL extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. However, both PAR and PAL extracts did not show cytotoxicity in Huh7 cells in the MTT assay. Furthermore, addition of PAR together with IFN-α showed additive effect in the inhibition of HCV RNA replication. Results suggest the possible molecular basis of the inhibitory activity of PA extract against HCV which would help in optimization and subsequent development of specific antiviral agent using P. amarus as potent natural source.

Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa‐2b

Treatment with hepatitis C virus (HCV)-NS3-protease inhibitors lead to the selection of resistant variants. Viral kinetics and resistance profiles in patients who are re-treated with the same protease inhibitor are unknown. Viral kinetics and NS3-resistance mutations obtained by clonal sequencing of the NS3-protease were analyzed in nine HCV-genotype-1-infected nonresponder patients who were sequentially treated with boceprevir (400 mg t.i.d.) for 1 week, peginterferon-alfa-2b for 2 weeks and combination of the two for 2 weeks in varying order. In addition to predominant wild-type isolates, previously described boceprevir-resistant mutations (V36, T54, R155, A156, V170) were observed. Furthermore, two resistant mutations (Q41, F43) were detected for the first time in vivo. In three patients, mutations selected after initial treatment with boceprevir were re-selected during subsequent boceprevir exposure. However, mutational patterns after the first and second exposure to boceprevir were different in five patients. In one patient, a viral variant (V55A) known to reduce susceptibility to boceprevir was the predominant variant observed at baseline and throughout treatment and was associated with a shallow viral decline. Different resistance mutations were selected during treatment with boceprevir ± peginterferon. Sequential short-term dosing of boceprevir was not associated with accumulation of resistant variants but pre-existing variants may impair virologic response.

NS5A inhibitors: A new breakthrough for the treatment of chronic hepatitis C

COMMENTARY ON: Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O'Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG. Nature. 2010;465(7294):96–100. Copyright (2010). Abstract reprinted with permission from Macmillan Publishers Ltd . http://www.ncbi.nlm.nih.gov/pubmed/20410884 Abstract: The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24h post-dose that was sustained for an additional 120h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.

Conformational Stability of Hepatitis C Virus NS3 Protease

The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states.

Virtual Ligand Screening of the National Cancer Institute (NCI) Compound Library Leads to the Allosteric Inhibitory Scaffolds of the West Nile Virus NS3 Proteinase

Viruses of the genus Flavivirus are responsible for significant human disease and mortality. The N-terminal domain of the flaviviral nonstructural (NS)3 protein codes for the serine, chymotrypsin-fold proteinase (NS3pro). The presence of the nonstructural (NS)2B cofactor, which is encoded by the upstream gene in the flaviviral genome, is necessary for NS3pro to exhibit its proteolytic activity. The two-component NS2B-NS3pro functional activity is essential for the viral polyprotein processing and replication. Both the structure and the function of NS2B-NS3pro are conserved in the Flavivirus family. Because of its essential function in the posttranslational processing of the viral polyprotein precursor, NS2B-NS3pro is a promising target for anti-flavivirus drugs. To identify selective inhibitors with the reduced cross-reactivity and off-target effects, we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3pro interface rather than the NS3pro active site. Using virtual ligand screening of the diverse, ∼275,000-compound library and the catalytic domain of the two-component West Nile virus (WNV) NS2B-NS3pro as a receptor, we identified a limited subset of the novel inhibitory scaffolds. Several of the discovered compounds performed as allosteric inhibitors and exhibited a nanomolar range potency in the in vitro cleavage assays. The inhibitors were also potent in cell-based assays employing the sub-genomic, luciferase-tagged WNV and Dengue viral replicons. The selectivity of the inhibitors was confirmed using the in vitro cleavage assays with furin, a human serine proteinase, the substrate preferences of which are similar to those of WNV NS2B-NS3pro. Conceptually, the similar in silico drug discovery strategy may be readily employed for the identification of inhibitors of other flaviviruses.

Susceptibility of Treatment-Naive Hepatitis C Virus (HCV) Clinical Isolates to HCV Protease Inhibitors

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.

Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

BackgroundThe dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.MethodsAlanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined.ResultsKinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole.ConclusionsKinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: The basis of the genotype 1b vs. 3a inhibitor potency shift

The first structural characterization of the genotype 3a Hepatitis C Virus NS3 protease is reported, providing insight into the differential susceptibility of 1b and 3a proteases to certain inhibitors. Interaction of the 3a NS3 protease with a P2–P4 macrocyclic and a linear phenethylamide inhibitor was investigated. In addition, the effect of the NS4A cofactor binding on the conformation of the protease was analyzed. Complexation of NS3 with the phenethylamide inhibitor significantly stabilizes the protease but binding does not involve residues 168 and 123, two key amino acids underlying the different inhibition of genotype 1b vs. 3a proteases by P2–P4 macrocycles. Therefore, we studied the dynamic behavior of these two residues in the phenethylamide complex, serving as a model of the situation in the apo 3a protein, in order to explore the structural basis of the inhibition potency shift between the proteases of the genotypes 1b and 3a.

Discovery of achiral inhibitors of the hepatitis C virus NS3 protease based on 2(1 H)-pyrazinones

Herein, the design, synthesis and inhibitory potency of a series of novel hepatitis C virus (HCV) NS3 protease inhibitors are presented. These inhibitors are based on a 2(1 H)-pyrazinone P3 scaffold in combination with either a P2 phenylglycine or a glycine, and they were evaluated on the wild type as well as on two resistant variants of the enzyme, A156T and D168V. Molecular modelling suggested that the aromatic side-chain of the P2 phenylglycine occupies the same space as the substituent in position 6 on the pyrazinone core. The versatile synthetic route applied for the pyrazinone synthesis made a switch between the two positions easily feasible, resulting in phenyl- or benzyl substituted pyrazinones and leaving glycine as the P2 residue. Of several P1–P1′ residues evaluated, an aromatic P1–P1′ scaffold was found superior in combination with the new P3–P2 building block. As a result, an entirely new type of achiral and rigidified inhibitors was discovered, with the best of the novel inhibitors having fourfold improved potency compared to the corresponding tripeptide lead. We consider these achiral inhibitors highly suitable as starting points for further optimization.