Anton Poliakov

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.

Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus.

Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

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.

Refolding of a recombinant full-length non-structural (NS3) protein from hepatitis C virus by chromatographic procedures

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) was expressed as inactive aggregates in Escherichia coli. The protein was refolded by chromatographic techniques of which ion exchange chromatography was best for crude samples and gel filtration best for partially purified samples. Immobilized metal ion affinity chromatography showed intermediate performance. Gradient procedures enhanced the recovery of active NS3 protein.

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.

Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors

From L-alpha-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of substrate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised and coupled to a model pentapeptide furnishing a set of hexapeptide inhibitors. Promising inhibitory activities with K(i) values of 0.18 microM (11b, P1 electrophilic alpha,beta-unsaturated ketone), 0.46 microM (12e, P1 electrophilic alkyl ketone) and 0.98 microM (10e, P1 non-electrophilic alkenyl alcohol as diastereomeric mixture). The reference hexapeptide product inhibitor had a K(i) value of 1.54 microM (14, P1 Abu-OH). The electrophilic inhibitors exhibit increased potency as compared with the corresponding product inhibitor, and notably also the non-electrophilic P1 alkenyl alcohol 10e. This represents the first example of non-electrophilic inhibitors that are not P1 amides or product inhibitors.

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.