Mimotopes of the hyper variable region 1 of the hepatitis C virus induce cross-reactive antibodies directed against discontinuous epitopes.

Hepatitis C virus (HCV) is a major cause worldwide of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and the development of an effective vaccine represents a high priority goal. The hyper variable region 1 (HVR1) of the second envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. To be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1, selected from a specialized phage library using HCV patients' sera. Some of the cross-reacting anti-mimotope antibodies elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

ADAM-HCV, a new-concept diagnostic assay for antibodies to hepatitis C virus in serum.

We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides. Peptides that mimic several immunodominant epitopes of the virus were used to develop a novel type of diagnostic assay which efficiently detects antibodies to HCV in serum. This type of analysis provides a conclusive diagnosis for many patients identified as indeterminate according to presently available serological assays.

A new method for chemoselective conjugation of unprotected peptides to dauno- and doxorubicin.

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.

Measurement of homonuclear three-bond J(H(N)Halpha) coupling constants in unlabeled peptides complexed with labeled proteins: application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV).

A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H(N)Halpha) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the 'quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV).

A personal account of the role of peptide research in drug discovery: the case of hepatitis C.

Although peptides themselves are not usually the end products of a drug discovery effort, peptide research often plays a key role in many aspects of this process. This will be illustrated by reviewing the experience of peptide research carried out at IRBM in the course of our study of hepatitis C virus (HCV). The target of our work is the NS3/4A protease, which is essential for maturation of the viral polyprotein. After a thorough examination of its substrate specificity we fine-tuned several substrate-derived peptides for enzymology studies, high-throughput screening and as fluorescent probes for secondary binding assays. In the course of these studies we made the key observation: that the protease is inhibited by its own cleavage products. Single analog and combinatorial optimization then derived potent peptide inhibitors. The crucial role of the NS4A cofactor was also addressed. NS4A is a small transmembrane protein, whose central domain is the minimal region sufficient for enzyme activation. Structural studies were performed with a peptide corresponding to the minimal activation domain, with a series of product inhibitors and with both. We found that NS3/4A is an induced fit enzyme, requiring both the cofactor and the substrate to acquire its bioactive conformation; this explained some puzzling results of 'serine-trap' type inhibitors. A more complete study on NS3 activation, however, requires the availability of the full-length NS4A protein. This was prepared by native chemical ligation, after sequence engineering to enhance its solubility; structural studies are in progress. Current work is focused on the P' region of the substrate, which, at variance with the P region, is not used for ground state binding to the enzyme and might give rise to inhibitors showing novel interactions with the enzyme.

Induction of cross-reactive humoral immune response by immunization with mimotopes of the hypervariable region 1 of the hepatitis C virus.

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.

Optimization of the P'-region of peptide inhibitors of hepatitis C virus NS3/4A protease.

Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A have been described, all taking advantage of binding to the S subsite of the enzyme. Inspection of the S' subsite of NS3/4A shows binding pockets which might be exploited for inhibitor binding, but due to the fact that ground-state binding to the S' subsite is not used by the substrate, this does not represent a suitable starting point. We have now optimized S'-binding in the context of noncleavable decapeptides spanning P6-P4'. Binding was sequentially increased by introduction of the previously optimized P-region [Ingallinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' residue, and combinatorial optimization of positions P2'-P3'. The overall process led to an increase in binding of more than 3 orders of magnitude, with the best decapeptide showing IC(50) < 200 pM. The binding mode of the decapeptides described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subsite. Therefore, these peptides may represent an entry point for a novel class of NS3 inhibitors.

Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer.

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody.

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies.

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.

A high-throughput radiometric assay for hepatitis C virus NS3 protease.

A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease.

Mechanisms of hepatitis C virus NS3 proteinase inhibitors.

The NS3 serine proteinase is regarded as one of the preferred targets for the development of therapeutic agents against hepatitis C virus (HCV). Possible mechanisms of NS3 inhibitors include: (i) interference with the activation of the enzyme by its NS4A cofactor; (ii) binding to the structural zinc site; and (iii) binding to the active site. These mechanisms have been explored in detail by structural analysis of the enzyme. (i) The NS4A cofactor binds to the amino-terminal beta-barrel domain of the NS3 proteinase bringing about several conformational changes that result in enzyme activation. The interaction between NS3 and NS4A involves a very large surface area and therefore it is not a likely target for the development of inhibitors. (ii) The NS3 proteinase contains a structural zinc binding site. Spectroscopic studies have shown that changes in the conformation of this metal-binding site correlate with changes in the specific activity of the enzyme, and the NS3 proteinase is inhibited by compounds capable of extracting zinc from its native coordination sphere. (iii) Based on the observation that the NS3 proteinase undergoes inhibition by its cleavage products, potent, active site-directed inhibitors have been generated. Kinetic studies, site-directed mutagenesis, and molecular modelling have been used to characterize the interactions between the NS3 proteinase and its product inhibitors.

Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments.

Alpha-synuclein (alpha-syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We have shown that human wild-type alpha-syn, mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. Here we report that aggregates of NAC and alpha-syn proteins induced apoptotic cell death in human neuroblastoma SH-SY5Y cells. These findings indicate that accumulation of alpha-syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Product inhibition of the hepatitis C virus NS3 protease.

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products.

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase.

The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral membrane protein that acts as an activator of the NS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A beta-strand contributed by NS4A is clamped between two beta-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4'), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottom by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P1 position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the K(m) values of the respective substrates and follow the order NS4A < NS5A < NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.

Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.