Eva Åkerblom

Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): A comparative study of different C-terminals

Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3 (protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentafluoroethyl ketones, alpha-keto acids, and alpha-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides). Bioisosteric replacement with the tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfonamide group yielded more potent inhibitors. The hexapeptide inhibitors Suc-Asp-D-Glu-Leu-Ile-Cha-Nva-NHSO(2)Ph and Suc-Asp-D-Glu-Leu-Ile-Cha-ACPC-NHSO(2)Ph with K(i) values of 13.6 and 3.8 nM, respectively, were approximately 20 times more potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor containing the native cysteine, Suc-Asp-D-Glu-Leu-Ile-Cha-Cys-OH (K(i)=28 nM). The acyl sulfonamide group constitutes a very promising C-terminal functionality that allows for prime site optimization.

Tetrapeptides as Potent Protease Inhibitors of Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase)

A library of tetrapeptides was evaluated for Hepatitis C Virus NS3 protease inhibitor activity in an in vitro assay system comprising the native bifunctional full-length NS3 (protease-helicase/NTPase) protein. Tetrapeptides with K(i) values in the high nanomolar range were identified, for example Suc-Chg-Glu-2-Nal-Cys (K(i)=0.27+/-0.03 microM) and Suc-Dif-Glu-Glu-Cys (K(i)=0.40+/-0.10 microM). Furthermore, it was shown that the inhibitory potencies are not affected significantly by assay ionic strength. As suggested by molecular modelling, potential binding interactions of the tetrapeptide inhibitors with the helicase domain might explain the data and structure-activity relationships thus obtained. Hence, we postulate that the full-length NS3 assay is a relevant system for inhibitor identification, offering new opportunities for inhibitor design.

Inhibition of hepatitis C virus NS3 protease activity by product-based peptides is dependent on helicase domain.

Structure activity relationships (SARs) of product-based inhibitors of hepatitis C virus NS3 protease were evaluated using an in vitro assay system comprising the native bifunctional full-length NS3 (protease-helicase/NTPase). The results were compared to previously reported data derived from the corresponding NS3 protease domain assay. Shortening the length of the protease inhibitors from hexapeptides to tripeptides revealed that the decrease in potency was much less when determined in the assay system with the full-length NS3 protein. Disagreements in SARs at different positions (P5 P2) were also discovered. Taken together, the results suggest that the impact of the helicase domain upon protease inhibitor binding is substantial.

Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3

This study of the full‐length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild‐type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10‐fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full‐length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.

Hepatitis C virus NS3 protease inhibitors comprising a novel aromatic P1 moiety.

Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hepatitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P(1) moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.

Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents

Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel pi-pi-interaction between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized that the introduction of a vinyl on the phenylglycine might strengthen this pi-pi-interaction. Thus, herein is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both D- and L-phenylglycine were found to be effective inhibitors, with a slight preference for the d-epimers. Furthermore, prime-side alkenylic extension of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the nanomolar range (approximately 35 nM), potencies which were retained on mutant variants of the protease.

Mechanistic studies of electrophilic protease inhibitors of full length hepatic C virus (HCV) NS3

The inhibition mechanism of electrophilic peptide-based protease inhibitors of full-length hepatitis C virus (HCV) NS3 has been investigated by determining the Ki-values for a series of compounds differing in the electrophilicity and acidity of the C-terminal residue at pH-values above and below the pKa of the catalytic histidine (6.85) and at two different ionic strengths. Electrophilic compounds with a pentafluoroethyl ketone group showed stronger inhibition at pH 8 than pH 6, as expected for a mechanism requiring an unprotonated catalytic histidine. However, the difference was only significant at high ionic strength. In contrast, electrophilic compounds with an acidic C-terminal group or a cyclic P1 residue showed a lower inhibitory effect at pH 8 than at pH 6, inconsistent with a mechanism-based inhibition. Moreover, all electrophilic compounds had an unexpectedly strong inhibition at pH 6, when mechanism-based inhibition is unlikely. The results suggest that for some of the electrophilic compounds the reactive group may not be properly positioned in the active site and that binding of these inhibitors is a result of non-covalent interactions. The nature of these interactions is discussed.

Discovery of pyrazinone based compounds that potently inhibit the drug-resistant enzyme variant R155K of the hepatitis C virus NS3 protease

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on β-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the β-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype 1a, both wild-type (Ki = 30 nM) and R155K (Ki = 2 nM), and genotype 3a (Ki = 5 nM).

Different Types of P1 Residues in Peptide-Based Inhibitors of Hepatitis C Virus Full-Length NS3 Protease

In a project aimed at the design and synthesis of inhibitors to the hepatitis C virus (HCV) NS3 serine protease, peptides containing different types of P1 residues were compared: 1) pentafluoroethyl ketones, since substrate-based peptides containing electrophilic groups in the P1 position are classical inhibitors of serine proteases; 2) tetrazoles, since HCV NS3 protease is uniquely inhibited by its N-terminal cleavage products [1] and tetrazoles are known metabolically stable bioisosteres for carboxylic acids; 3) ketotetrazoles, since peptide α-ketoacids work as electrophilic inhibitors of the HCV NS3 protease [2] and oc-ketotetrazoles should serve as α-ketoacid bioisosteres and; 4) carboxylic acids, as reference compounds.