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

F. Martin, C. Steinkühler, M. Brunetti, A. Pessi, R. Cortese, R. De Francesco and M. Sollazzo1

Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti,Via Pontina Km 30,600-00040, Pomezia (Roma), Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.

Keywords: HCV/loop mimetic/NS3 protease/minibody/protease inhibitor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of small protein scaffolds (Bianchi et al., 1995Go; McConnell and Hoess, 1995Go; Nord et al., 1995Go; Vita et al., 1995Go; Zhao et al., 1995Go; Smith et al., 1998Go) and minimized versions of natural proteins (Pessi et al., 1993Go; Li et al., 1995Go; Braisted and Wells, 1996Go) has been proposed as a means of generating conformationally defined structures with a potential as pharmacophores (Sollazzo et al., 1995Go; Zhao et al., 1995Go). Minimized antibody-like ß-proteins that recognize target molecules with high specificity and affinity have been developed (Pessi et al., 1993Go; Martin et al., 1994Go, 1996Go, 1997Go; Davies and Riechmann, 1995Go; Dimasi et al., 1997Go; Lauwereys et al., 1998Go). In order to test if these ligands can serve as a source of potential pharmacophores convertible into lead compounds we targeted the NS3 serine protease of HCV.

HCV is the major agent of transfusion-associated and community acquired non-A, non-B hepatitis worldwide (Choo et al., 1989Go; Houghton et al., 1991Go) and is estimated to infect about 0.2–1% of the world's population. Chronic HCV infection, which develops in about half of the patients (Alter, 1995Go; Bisceglie, 1995Go), leads to liver cirrhosis and hepatocellular carcinoma (Chien et al., 1992Go). The current treatment for HCV infected patients is {alpha}-interferon alone or in combination with ribavirin, which however is only partially effective (reviewed by Poynard and Opolon, 1998). In addition, as a vaccine against HCV is not yet available, there is undoubtedly a need for more effective anti-HCV agents. The structure of the HCV RNA genome and the processing mechanism of its polyprotein have been characterized in considerable detail. The HCV polyprotein is cleaved into 10 mature proteins by host signal peptidases, and by virus encoded proteases (Grakoui et al., 1993aGo,bGo). A serine protease located in the N-terminal region (180 residues) of the multidomain NS3 protein is responsible for the four cleavage events that occur downstream of the NS3 protein itself (Tomei et al., 1993Go). The NS3 protease requires as a cofactor the 54 residue NS4A viral protein that binds to the N-terminal region of the protease domain (Kim et al., 1996Go; Love et al., 1996Go; Yan et al., 1998Go). Inhibition of the NS3 protease is expected to produce non-infective viral particles and the search for specific NS3 protease inhibitors is thus the subject of major efforts towards developing effective HCV treatments.

As a first step toward our main objective, we have used phage display (Dimasi et al., 1997Go; Martin et al., 1997Go) and a first principle design approach (Martin et al., 1998Go) to identify macromolecules that inhibit the NS3 protease. Both approaches yielded competitive inhibitors using several scaffolds, except for the minibody that resulted in the isolation of a noncompetitive inhibitor (Dimasi et al., 1997Go). It can be argued that, from a drug discovery point of view, it is desirable to have a competitive rather than a noncompetitive inhibitor of the protease as such a compound will be less prone to inducing the emergence of escape mutants in vivo. In addition, a noncompetitive inhibitor may target a site of the enzyme surface which may not be available at the level of the polyprotein. The failure to identify a competitive inhibitor using the minibody could be attributed either to the affinity-selection scheme adopted or to the properties of this particular scaffold. Because we wished to pursue this system further and to avoid limitations intrinsic to the affinity-selection schemes, we adopted the following approach. Out of a collection of about 103 sequenced minibody mutants, from an original repertoire of more than 107 (Martin et al., 1994Go), a panel of candidate ligands were chosen and tested individually using an in vitro protease assay. Criteria for selecting potential ligands were based upon characteristics of natural NS3 substrates and known specific inhibitors (Ingallinella et al., 1998Go; Martin et al., 1998Go). It has been demonstrated that the main interactions between the enzyme and peptide inhibitors based on the substrate sequence (spanning P1–P6) are provided by P6, P5 and P1 residues (Ingallinella et al., 1998Go; Martin et al., 1998Go; Steinkühler et al., 1998Go). Furthermore, the deletion of negatively charged amino acids P6 and P5 correlate with a dramatic decrease of the inhibitor potency by a factor of 150–600. In the same way, the P1 cysteine residue has been demonstrated to be the most crucial element for substrate recognition (Martin et al., 1998 and references therein). We thus chose to test minibodies as candidate inhibitors that displayed either two contiguous anionic residues (acid anchor), to mimic the minimal substrate N-terminal portion, or cysteine residues (P1 anchor) in their variable regions.

From the testing of such a biased panel we report the identification and characterization of a competitive minibody inhibitor (Mbic) of HCV NS3 protease. This macromolecule provided the basis for the synthesis of a cyclic peptide mimicking the Mbic active site-binding loop which maintains significant activity against the target enzyme. This result validates the approach of using macromolecule ligands for a given target for identifying pharmacophores and confirms the requirements for NS3 inhibitor recognition identified earlier (Ingallinella et al., 1998Go; Martin et al., 1998Go). In some favorable cases, these can be converted into small molecular weight compounds amenable to optimization through traditional medicinal or combinatorial chemistry strategies.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microbiological and recombinant DNA techniques

Microbiological and recombinant DNA techniques were carried out according to standard protocols (Ausubel et al., 1994Go) or as recommended by suppliers. Phage DNA encoding minibodies were amplified by PCR and cloned in the pT7 expression vector as previously described (Martin et al., 1994Go). Mbic-H1 was constructed by PCR amplification of Mb70 phage DNA, a previously isolated f1 clone displaying a minibody bearing the sequence KMSFRPK in its H1 loop. The NdeI–NruI DNA fragment of this minibody, encompassing the MB70 H1 loop and its flanking framework residues, was exchanged with that of the Mbic by subcloning thus generating the double loop recombinant minibody Mbic-H1. For the construction of hcVH the sequence encoding the Mbic loop was grafted into the hcVH-N5 clone (Martin et al., 1997Go) using inverse PCR (Hemsley et al., 1989Go). The oligonucleotides were designed as follows: VHup 5'-AGCTGGACTTTGACTATTGGGGC-3', VHdwn 5'-TTCGATACCTCTAGCACAGTAATACAC-3', and the oligonucleotide 5'-GGAAACAA- AGCTATGACCATG-3' was used as primer to sequence the constructs. Expression and purification of the hcVH recombinant protein was performed as previously described (Davies and Riechmann, 1995Go). Alanine scanning variants were generated by inverse PCR using Mbic DNA as a template and the following set of primers: Up1, 5'-G1GTTAG1CACCACCGAGTACT-3'; Up2, 5'-G1GGCAG1CACCACCGAGTACT-3'; Dwn1, 5'-T2CTATACCTCGCGAAGCAGC-3'; Dwn2, 5'-T2CTGCACCTCGCGAAGCAGC-3', with degenerated positions 1 and 2 being mixtures of (C/A) and (T/G) respectively, with 66% of first base. Twenty-five PCR cycles were performed at 92°C for 2 min, 37°C for 2 min and 72°C for 12 min.

Expression and purification of the minibody and hcVH variants

Expression and purification of minibodies (Bianchi et al., 1994Go) and hcVH (Davies and Riechmann, 1995Go; Martin et al., 1997Go) have been described.

Purification of the NS3 protease domain

Expression and purification were performed as previously described (De Francesco et al., 1996Go). Protein concentration was estimated by quantitative amino acid analysis. Purity of the enzyme was checked on silver-stained SDS–PAGE and by reversed-phase (RP) high-pressure liquid chromatography (HPLC).

NS3 protease assays

Recombinant protein assays were performed in 50 mM Tris–HCl pH 7.5, 2% CHAPS, 50% glycerol, 30 mM DTT using 150 nM recombinant NS3 protease purified from Escherichia coli as previously described (Steinkühler et al., 1996Go). Samples were analyzed as described before (Martin et al., 1997Go). The IC50 values were calculated by fitting inhibition data to the following equation using Kaleidagraph software

where [I] is the inhibitor concentration, maximum activity is that of the enzyme in the absence of inhibitor and S is the slope factor of the curve. Inhibition mechanisms were determined by performing substrate titration experiments using concentrations of substrate peptide between 3.5 and 750 µM in the absence and presence of three different inhibitor concentrations. Initial rates of cleavage were determined on samples with <10% conversion. Kinetic parameters were calculated by determining the equations of the fitted curves corresponding to the four inhibitor concentrations assuming Michaelis–Menten kinetics. The Ki values were determined as described elsewhere (Martin et al., 1997Go). Peptide assays were performed in 50 mM HEPES pH 7.5, 1% CHAPS, 15% glycerol, 30 mM DTT, 15 µM pep4A of sequence KKKGSVVIVGRIILSGR, using 20 nM purified recombinant NS3. Reversibility of Mbic binding was assessed as described previously (Martin et al., 1997Go).

HLE and PPK protease assays

HLE was purchased from Worthington Biochemical Corporation, and its substrate (Me-o-Suc-Ala-Ala-Pro-Val-pNA) from Calbiochem®. PPK was purchased from Calbiochem® and its substrate Chromozym (D-Pro-Phe-Arg-pNA) from Boehringer Mannheim. Assays were carried out as described previously (Martin et al., 1997Go).

Peptide synthesis

The linear peptide (Pep-L) was prepared by standard Fmoc/ t-Bu solid-phase synthesis (Atherton and Sheppard, 1989Go) using PyBOP/HOBt/DIEA activation (3-fold excess of acylant, 30 min coupling time). The cyclic peptide (Pep-C) was prepared with the same method as protected, resin bound precursor with the free N-terminus and the Glu side-chain protected as allyl ester (Kates et al., 1993Go). The allyl group was removed by treatment with tetrakis(phenylphosphine)palladium (0) in chloroform containing 5% acetic acid and 2.5% N-methylmorpholine (2 h, room temperature), and the resin was then extensively washed with DMF, 0.5% DIEA/0.5% sodium diethyldithiocarbamate in DMF, and DMF. The purity of the peptide at this stage was >90% as assessed after deprotection by mass spectrometry. Cyclization was accomplished by 16 h treatment at room temperature of the peptide-resin with a 3-fold excess of HATU/DIEA (Carpino et al., 1994Go). Completion of the reaction was estimated by the usual colour tests (Kaiser et al., 1970Go; Hanckock and Battersby, 1976Go). Similar treatment with HOBt/DIEA gave substantially lower yields. The peptide was cleaved from the resin by treatment with Reagent B (Sole and Barany, 1992Go), and purified (>95%) by preparative HPLC. The combined yield (synthesis/cyclization/purification) was 32%. The identity of the peptide was established by mass spectrometry (calculated: 769.5; found: 769.4).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo shows the amino acid sequence of the panel selected for testing among approximately 103 minibody mutants. The molecules display cysteine (preferred P1 residue) or negatively charged (acid anchor) residues in various positions of their variable loops. Figure 1Go shows the presumed fold of the engineered minibody molecule as described in detail previously (Pessi et al., 1993Go; Tramontano et al., 1994Go). The genes encoding these proteins were amplified by PCR using the phage DNA as a template and subcloned into the pT7 vector for expression in E.coli as described previously (Martin et al., 1994Go). To improve their solubility, a lysine tail was genetically fused to the C-terminus of these polypeptides and the expressed minibodies were purified and refolded before testing. The purified proteins were tested in the NS3 protease assay at a single concentration in triplicate. In this experiment the 20 kDa protease domain was incubated with the peptide corresponding to the central part (residues 21–34) of the NS4A cofactor (pep 4A) and with the purified minibody at 8 µM concentration. After 15 min preincubation, a 13mer substrate corresponding to the NS4A/NS4B cleavage site was included in the reaction and the product released was separated and quantified by HPLC chromatography. Using this assay, 13 of the 14 minibodies tested showed marginal or no inhibition (0–10%), values considered within the range of non-specific interactions as shown by the control wild-type minibody. The exception was clone No. 5 (Mbic), which almost completely abolished protease activity and was thus characterized further.


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Table I. Amino acid sequences corresponding to the loops of the sorted minibody panel
 


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Fig. 1. Schematic ribbon representation of the minibody structural model. Hypervariable H1 and H2 loops are represented in red and green. The amino (N) and carboxy (C) termini are indicated. The model was drawn using InsightII.

 
Mbic is a single H2 loop recombinant with respect to the wild type minibody and bears the sequence GIEELD. To accurately determine its potency we titrated the NS3 protease residual activity as a function of inhibitor concentration. The IC50 of Mbic was deduced from the inhibition plot shown in Figure 2Go and estimated to be 0.97 ± 0.06 µM. Next, we tested the specificity of Mbic by using two commercially available serine protease assays, human leukocyte elastase (HLE) and porcine kallikrein (PPK). Neither of these two enzymes was inhibited by the highest testable concentration of Mbic (12 µM). In addition, the NS3–Mbic association was shown to be reversible (data not shown). In order to determine the Mbic inhibitory mechanism, substrate titration experiments were carried out in the absence and presence of increasing amounts of inhibitor. Experimental data represented in a Lineweaver–Burke plot (Figure 3Go) show the pattern of a typical competitive inhibitor with a Ki of 0.40 µM.



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Fig. 2. Determination of IC50 of the protein inhibitors. The recombinant NS3 20 kDa protease fragment (20 nM) was incubated in the presence of increasing amounts of purified inhibitors (see legends, inset), 10 µM substrate and 15 µM NS4A cofactor. The cleaved peptide product concentration was determined by HPLC (average of duplicate samples). The percentage of NS3 residual activity (y-axis) was determined as function of the inhibitor concentration (x-axis), and the IC50 was derived.

 


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Fig. 3. Mechanism of inhibition of NS3 protease activity by Mbic. Recombinant NS3 protease (20 nM) activity was determined at 25°C in 50 mM HEPES pH 7.5, 30 mM DTT, 15 µM NS4A peptide, 1% CHAPS and 15% glycerol. Double-reciprocal of NS3 inhibition by Mbic were determined in the absence (squares) and in the presence of 1.6 µM (triangles), 3.3 µM (circles) and 6.6 µM (diamonds) purified Mbic. Each value is the average of duplicate experiments.

 
The role played by individual Mbic residues in the recognition of the NS3 protease was analyzed by three sets of experiments. First, the role of the H1 loop was evaluated by drastically altering its amino acid composition from wild type to a random sequence (KMSFRPK). The mutant, Mbic-H1, was tested in the protease assay and its IC50 was calculated to be 1.86 ± 0.16 µM, in the same range as that of the Mbic one (Figure 2Go). It can be concluded that the H1 loop does not contribute to the formation of the ternary complex Mbic-NS3-NS4A. A second experiment was designed to investigate if some amino acids belonging to the minibody scaffold were interacting with the 20 kDa NS3 protease. To this end, we grafted the Mbic H2 loop onto another presentation scaffold molecule, by inserting the sequence GIEELD in a hypervariable loop of a human `camelized' VH domain, hcVH (Davies and Riechmann, 1995Go). Because the minibody belongs to the immunoglobulin family (Pessi et al., 1993Go; Tramontano et al., 1994Go) the loop grafting results in a similar `presentation' in both molecules yet within a different environment. Unfortunately, the homologous grafting into the H2 loop of hcVH did not yield a soluble protein. On the contrary, the grafting into the H3 loop of hcVH produced an inhibitor with an IC50 of 1.5 ± 0.12 µM (Figure 2Go), virtually equivalent to that of Mbic.

Having established that all the information necessary for binding was included within the Mbic H2 loop, a third experiment was devised to identify which residues were involved in the recognition process. We thus performed an alanine scanning of the Mbic H2 loop by replacing one or two adjacent positions by alanine residues except for the Gly residue as reported in Figure 4Go. The Mbic alanine mutants were purified to homogeneity and tested at 1 µM concentration (corresponding to the Mbic IC50) in triplicate samples. The results clearly show that the two negative charges present at the centre of the loop are the main players in the interaction between Mbic and the NS3 protease active site. The double mutant bearing two alanine residues at these positions (Mbic.34) is virtually inactive. The aspartic acid residue at position six of the loop also contributes to binding, albeit to a lesser extent. The side chains of residues at positions 2 and 5 probably do not make contacts with the protease since their replacement by alanine does not affect the potency of these inhibitors.



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Fig. 4. Alanine scanning of the Mbic H2 loop. One or two adjacent residues in the Mbic bioactive loop were replaced by alanine residues. The minibodies variants were tested in triplicate samples at 1 µM concentration in the protease assay. The percentage of NS3 activity inhibition (x-axis) is reported as function of the loop sequence (y-axis).

 
Since all the information necessary for binding was contained within the Mbic H2 loop, we decided to excise the active sequence (Ac-GIEELD-CONH2) out of the scaffold and test its activity. With the aim of maintaining some of the constraints that are provided by the minibody scaffold in the loop-mimetic, we prepared an end-to-tail cyclic peptide with the same sequence. As the NS3 protease is very sensitive to oxidation and requires reducing agents in its assay buffer, it was not possible to close the hexapeptide by a disulfide bridge. Therefore cyclization was performed by adding an extra glutamic acid at the C-terminus of the peptide and its side chain carboxylic acid was engaged in an intra molecular peptide bond with the N-terminus of the peptide. By adopting such a strategy we obtained a cyclic conformation insensitive to DTT (30 mM) required in the protease assay. The constrained peptide (pep-C) was synthesized on solid phase, cyclized and HPLC purified before testing. To quantify the contribution of the constraint, a linear peptide bearing the same sequence (pep-L) was also synthesized. As shown in Figure 5Go, pep-L inhibits only 40% of protease activity at 2 mM (the highest concentration testable) whereas in the same conditions, pep-C fully inhibits enzyme activity. The IC50 of the minibody loop mimetic (pep-C) was deduced from the titration curve and was estimated to be 534 ± 24 µM. This experiment demonstrates that the sequence in itself is an important determinant since pep-L shows a dose dependent inhibition, but that a suitable presentation of the pharmacophore is crucial. The same sequence is about one order of magnitude more active when constrained. The IC50 value of Pep-C albeit modest, was nevertheless sufficient to test the mechanism of action. Substrate titration experiments were performed in the presence of increasing concentrations of hexapeptide. The plot shown in Figure 6Go demonstrates that the competitive nature of the pep-C inhibitor and a Ki value of 125 µM was determined.



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Fig. 5. Determination of the NS3 residual activity as a function of pep-L (circles) and pep-C (squares) concentration. Each point is the average of duplicate samples.

 


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Fig. 6. Mechanism of inhibition of pep-C. Lineweaver–Burke representation of NS3 inhibition by the minibody loop mimetic in the absence (open squares) and in the presence of 295 (plain squares), 590 (circles) and 1180 µM (triangles) inhibitor. Each value is the average of duplicate experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been proposed that conformationally defined peptide structures may have a potential as pharmacophores (reviewed by Sollazzo et al., 1995). On the basis of this notion minimized protein scaffolds have been developed (Pessi et al., 1993Go; Li et al., 1995Go; Braisted and Wells, 1996Go). In particular, minimized antibody-like ß-proteins that can recognize targets with high specificity and affinity (Martin et al., 1994Go, 1996Go, 1997Go; Davies and Riechmann, 1995Go; Lauwereys et al., 1998Go) are believed to be promising tools.

The X-ray structure determination of the NS3 protease has revealed that this viral protein is smaller than the average cellular serine proteases. The nature of the NS3 substrate binding site (Love et al., 1996Go; Martin et al., 1998Go), makes it difficult to design a small molecular weight competitive inhibitor. As a complementary approach to combinatorial synthetic chemistry (Ingallinella et al., 1998Go), we attempted to tackle this problem also by using phage display of macromolecule repertoires to affinity select competitive inhibitors of NS3 that can provide useful information on potential pharmacophores. This strategy led to the isolation of competitive inhibitors of NS3 protease using human hcVH (Martin et al., 1997Go) and human pancreatic soluble trypsin inhibitor, yet no competitive inhibitors were obtained from the affinity selection of a minibody library (Dimasi et al., 1997Go). The failure to isolate a minibody competitive inhibitor was rather unexpected. It was unlikely that this failure was due to the intrinsic nature of the minibody scaffold as it is derived from that of an immunoglobulin heavy chain variable domain. This latter approach has proven adequate in yielding competitive inhibitors of NS3 protease (Martin et al., 1997Go). It is more probable that the failure to isolate a competitive inhibitor could be attributed to the inadequate affinity selection conditions. With this in mind, in order to sort candidate competitive inhibitors, we explored the approach described here based on known sequence requirements that drive NS3 protease substrate recognition.

Previous experiments (Martin et al., 1994Go, 1996Go) indicated that the result of an affinity selection using a repertoire of minibody variants is always the sum of two parameters. Namely, the KD value of the ligand for the target molecule and the phage ability to infect E.coli. The displayed minibody may alter the pIII phage protein function, which in turn is responsible for recognition of the bacterial pilus. Each minibody–pIII fusion will probably reduce the phage infectivity. On the other hand, the interaction between the pIII fusion and the bacterial pilus is also influenced by the nature of the minibody loop sequences. These uncontrolled parameters introduce another variable to the selection process, favouring the most infective phage as the number of cycles increase. During the selection cycles the pool is also enriched for phage bearing higher affinity binders. Thus because of these effects it is likely that the weaker binders may be lost. Considering the above, it is unlikely that Mbic would have been identified from the minibody repertoire by applying a classical panning strategy, especially considering its moderate affinity (Dimasi et al., 1997Go).

We thus pre-sorted among about 1000 randomly sequenced minibody variants, a panel of molecules bearing sequence features that are known to be important for the recognition of substrates by NS3 protease as previously established (Ingallinella et al., 1998Go; Martin et al., 1998Go; Steinkühler et al., 1998Go). It has also been shown that HCV-NS3 protease activity is susceptible to product inhibition by the P region of the NS4A/B substrate Ac-DEMEEC-COOH (Steinkühler et al., 1998Go). The presence of negatively charged residues in the best substrates cleaved by NS3 in trans (P6 and P5 positions), appears to be an absolute requirement for efficient recognition. These observations show that the substrate binding is mainly driven by electrostatic interactions. We therefore tested minibodies displaying two contiguous anionic residues including clones displaying them either at the centre or at the edge of each loop to test the maximum number of possible configurations. In addition, minibodies having a single negative charge in each loop were also tested. For the display of the cysteine residues, our choice was more limited; except for one clone this amino acid was only found in the H1 loop among the clones of our collection.

It can be concluded from the experiments described here that the Mbic H2 loop is responsible for the inhibitory activity, and within such a loop three residues specifically play the most important role. The amino acid sequence of this loop (GIEELD) would seem to mimic part of the P region of the NS4A/B cleavage site (DEMEEC), particularly the P6, P5 and P3 positions. This finding is well in agreement with the alanine scanning experiment which demonstrates that by replacing the two central negative charges of the minibody loop the inhibitory activity is virtually lost. This should be correlated with the decrease in potency by 150–600-fold that occurs when the inhibitor size is reduced from a hexa- to a tetra-peptide by deleting the acidic P6, P5 couple (Ingallinella et al., 1998Go). This observation confirms the results obtained in previous work (Martin et al., 1998Go) also emphasizing the importance of negative charges (acid anchor) present in the inhibitor corresponding to the P6, P5 region of the substrate.

The potency of the Mbic inhibitor is in the micromolar range. Albeit modest, this is similar to that of the product inhibitor derived from the natural cleavage site which is 1 µM (Ingallinella et al., 1998Go). Even if these two inhibitors interact with the same portion of the protease and with the same affinity, their binding modes are quite different. During characterization of the NS3 cleavage product inhibition, two important binding determinants were identified on the DEMEEC peptide, namely the P6, P5 and P1 residues. The latter is crucial for both its side chain and for the C-terminal carboxylate moiety. Substituting the C-terminal carboxylate with an amide decreases the potency from 1 to 160 µM, while eliminating the P1 side chain by substitution of Cys with a Gly residue decreases potency by 80-fold (Ingallinella et al., 1998Go). The Mbic H2 loop has no amino acid residue capable of fitting efficiently into the S1 pocket of the enzyme (Cys) and also lacks a free C{alpha}-carboxylate. In addition, its residues are likely to be in ß-turn conformation whereas the product inhibitor is known to bind to the protease in an extended conformation (Cicero et al., 1999Go). Consequently, its binding mode is probably different from that of the substrate or `canonical' inhibitors (Martin et al., 1998Go). In the light of the alanine scanning results, where only three of the six amino acids were shown to contribute to binding, it is reasonable to think that there is a partial occupancy of the substrate binding site, probably in the S6-5 and possibly in the S3 pocket. From the X-ray structure it can be observed that the NS3 protease exhibits large areas of charge concentration, especially around the substrate binding region, where several patches of cationic amino acids, without any neutralizing anionic side chains nearby, are present. These cationic amino acids give rise to an extended area of strong positive electrostatic potential along the P-site of the substrate binding region. The electrostatic potential of the substrate binding region of NS3 protease is believed to be mainly characterized by amino acids Arg161 and Lys165 which are likely to favour interactions around the active site with substrate negative charges close to P5 and P6. The difference in potency between Mbic and the corresponding linear peptide is to be attributed to the constraints imposed by the scaffold. By forcing the binding loop to adopt a pre-established active conformation, the minibody inhibitor has an entropic advantage over the linear peptide which instead has many more degrees of freedom. Introducing conformational constraints into the excised active sequence improves potency, though the difference with Mbic is about two orders of magnitude. This result is not surprising and similar results have been described elsewhere (Smythe and von Itsein, 1994), as the cyclic hexapeptide is still a rather flexible molecule that in the unbound state is likely to adopt thousands of different conformations.

The result of this approach led us to the synthesis of a cyclic peptide that is a weak but specific inhibitor of the NS3 protease (Ki, 125 µM), yet its potency is considerably higher than that of linear unoptimized hexapeptide inhibitors lacking the P1 side chain and the carboxylate elements. The IC50 of the P1 truncated Ac-DEMEE-OH was not measurable under the experimental conditions used. In the optimized inhibitor Ac-DEDifICha-Cys-OH (IC50, 40 nM), truncation of P1 to Ac-DEDifI-Cha-OH led to 3000-fold decrease in potency (IC50, 117 µM). Deletion of the P1 side chain (Cys) also resulted in a considerable loss of potency (IC50, 35 µM) of the optimized unconstrained hexapeptide inhibitor (Ingallinella et al., 1998Go). Our finding confirms that the paradigm of turning a protein ligand into a low molecular weight active compound is a valid one and in some cases can be a viable strategy for lead discovery (Sollazzo et al., 1995 and references therein). It is worth noting that Mbic was identified using only a very small fraction (approximately 10–4) of the experimentally available minibody repertoire (Martin et al., 1994Go) and thus it can be extrapolated that, in principle, by sampling a larger fraction more potent inhibitors could be found.

In the past 10 years, synthetic chemistry strategies have been developed to enable the conversion of immunoglobulin complementarity-determining regions (CDRs) in particular, and ß-turns in general, into chemical leads (Kahn et al., 1988Go; Saragovi et al., 1991Go; Chen et al., 1992Go; Satoh et al., 1997Go; Zhang et al., 1997Go). Converting peptides into small chemical molecules may overcome the problems of poor bioavailability, rapid degradation, antigenicity and high cost which often limits the use of proteinaceous biopharmaceuticals. By reducing Mbic into a small functional conformationally-constrained peptide we have achieved another step towards the design of non-peptide low molecular weight inhibitors of the HCV NS3 protease. The strategy described here should not be considered an alternative but rather complementary to lead evolution through synthetic combinatorial chemistry (Mc Bride et al., 1996Go), which has shown its potential for the optimization of the unconstrained NS3 peptide inhibitors.


    Acknowledgments
 
We would like to thank S.Altamura and U.Koch for useful discussions, M.Cerretani and S.Serafini for HLE and PPK assays, S.Alcali for peptide synthesis, F.Bonelli and F.Naimo for mass spectrometry, G.Biasiol for providing NS3 protease, C.Volpari for valuable technical assistance, J.Clench and B.McManus for helping with bibliographical searches and for reviewing the manuscript.


    Notes
 
1 To whom correspondence should be addressed; email: Sollazzo{at}IRBM.IT Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alter,M.J. (1995) Blood, 85, 1681–1695.[Free Full Text]

Atherton,E. and Sheppard,R.C. (1989) Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, Oxford.

Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1994) Current Protocols in Molecular Biology. Greene Publishing Associates, New York.

Bianchi,E., Venturini,S., Pessi,A., Tramontano,A. and Sollazzo,M. (1994) J. Mol. Biol., 236, 649–659.[ISI][Medline]

Bianchi,E., Folgori,A., Wallace,A., Nicotra,M., Acali,S., Phalipon,A., Barbato,G., Bazzo,R., Cortese,R., Felici,F. and Pessi,A. (1995) J. Mol. Biol., 247, 154–160.[ISI][Medline]

Bisceglie,A.M. (1995) Semin. Liver Dis., 15, 64–69.[ISI][Medline]

Braisted,A.C. and Wells,J.A. (1996) Proc. Natl Acad. Sci. USA, 93, 5688–5692.[Abstract/Free Full Text]

Carpino,L.A., El-Faham,A., Minor,C. and Albericio,F. (1994) J. Chem. Soc. Chem. Commun., 201–203.

Chen,S. et al. (1992) Proc. Natl Acad. Sci. USA, 89, 5872–5876.[Abstract]

Chien,D.Y. et al. (1992) Proc. Natl Acad. Sci. USA, 89, 10011–10015.[Abstract]

Choo,Q.L., Kuo,G., Weiner,A.J., Overby,L.R., Bradley,D.W. and Houghton,M. (1989) Science, 244, 359–362.[ISI][Medline]

Cicero,D.O., Barbato,G., Koch,U., Ingallinella,P., Bianchi,E., Nardi,M.C., Steinkühler,C., Cortese,R., Matassa,V., De Francesco,R., Pessi,A. and Bazzo,R. (1999) J. Mol. Biol., 289, 385–396.[ISI][Medline]

Davies,J. and Riechmann,L. (1995) Biotechnology, 13, 475–479.[ISI][Medline]

De Francesco,R., Urbani,A., Nardi,M.C., Tomei,L., Steinkühler,C. and Tramontano,A. (1996) Biochemistry, 35, 13282–13287.[ISI][Medline]

Dimasi,N., Martin,F., Volpari,C., Brunetti,M., Biasiol,G., Altamura,S., Cortese,R., De Francesco,R., Steinkühler,C. and Sollazzo,M. (1997) J. Virol., 71, 7461–7469.[Abstract]

Grakoui,A., McCourt,D.W., Wychowski,C., Feinstone,S.M. and Rice,C.M (1993a) J. Virol., 67, 2832–2843.[Abstract]

Grakoui,A., Wychowski,C., Lin,C., Feinstone,S.M. and Rice,C.M. (1993b) J. Virol., 67, 1385–1395.[Abstract]

Hanckock,W.S. and Battersby,J.E. (1976) Anal. Biochem., 71, 260–264.[ISI][Medline]

Hemsley,A., Arnheim,N., Toney,M.D., Cortopassi,G. and Galas,D.J. (1989) Nucleic Acids Res., 17, 6545–6551.[Abstract]

Houghton,M., Weiner,A., Han,J., Kuo,G. and Choo,Q.L. (1991) Hepatology, 14, 381–388.[ISI][Medline]

Ingallinella,P., Altamura,S., Bianchi,E., Taliani,M., Ingenito,R., Cortese,R., De Francesco,R., Steinkühler,C. and Pessi,A. (1998) Biochemistry, 37, 8906–8914.[ISI][Medline]

Kahn,M.I., Wilke,S., Chen,B., Fujita,K., Lee,Y.H. and Johnson,M.E. (1988) J. Mol. Recogn., 1, 75–79.[Medline]

Kaiser,E., Colescott,R.L., Bossinger,C.D. and Cook,P.I. (1970) Anal. Biochem., 34, 595–598.[ISI][Medline]

Kates,S.A., Sole,N.A., Johnson,C.R., Hudson,D., Barany,G. and Albericio,F. (1993) Tetrahedron Lett., 34, 1549–1552.[ISI]

Kim,J.L. et al. (1996) Cell, 87, 343–355.[ISI][Medline]

Lauwereys,M., Arbabi Ghahroudi,M., Desmyter,A., Kinne,J., Holzer,W., De Genst,E., Wyns,L. and Muyldermans,S. (1998) EMBO J., 17, 3512–3520.[Abstract/Free Full Text]

Li,B., Tom,J.Y., Oare,D., Yen,R., Fairbrother,W.J., Wells,J.A. and Cunningham,B.C. (1995) Science, 270, 1657–1660.[Abstract]

Love,R.A., Parge,H.E., Wickersham,J.A., Hostomsky,Z., Habuka,N., Moomaw,E.W., Adachi,T. and Hostomska,Z. (1996) Cell, 87, 331–342.[ISI][Medline]

Martin,F., Toniatti,C., Salvati,A.L., Venturini,S., Ciliberto,G., Cortese,R. and Sollazzo,M. (1994). EMBO J., 13, 5303–5309.[Abstract]

Martin,F., Toniatti,C., Salvati,A.L., Ciliberto,G., Cortese,R. and Sollazzo,M. (1996) J. Mol. Biol., 255, 86–97.[ISI][Medline]

Martin,F., Volpari,C., Steinkühler,C., Dimasi,N., Brunetti,M., Biasiol,G., Altamura,S., Cortese,R., De Francesco,R. and Sollazzo,M. (1997) Protein Engng, 10, 607–614.[Abstract]

Martin,F., Dimasi,N., Volpari,C., Perrera,C., Di Marco,S., Brunetti,M., Steinkühler,C., De Francesco,R. and Sollazzo,M. (1998) Biochemistry, 37, 11459–11468.[ISI][Medline]

Mc Bride,J.D., Freeman,N., Domingo,G.J. and Leatherbarrow,R.J. (1996) J. Mol. Biol., 259, 819–827.[ISI][Medline]

McConnell,S.J. and Hoess,R.H. (1995) J. Mol. Biol., 250, 460–470.[ISI][Medline]

Nord,K., Nilsson,J., Nilsson,B., Uhlen,M. and Nygren,P.A. (1995) Protein Engng, 8, 601–608.[Abstract]

Pessi,A., Bianchi,E., Crameri,A., Venturini,S., Tramontano,A. and Sollazzo,M. (1993) Nature, 362, 367–369.[ISI][Medline]

Poynard,T. and Opolon,P. (1998) Hepatology, 27, 1443–1444.[ISI][Medline]

Saragovi,H.U., Fitzpatrick,D., Raktabutr,A., Nakanishi,H., Kahn,M.I. and Green,M.I. (1991) Science, 253, 792–795.[ISI][Medline]

Satoh,T., Aramini,J.M., Li,S., Friedman,T.M., Gao,J., Edling,A.E., Townsend,R., Koch,U., Choksi,S., Germann,M.W., Korngold,R. and Huang,Z. (1997) J. Biol. Chem., 272, 12175–12180.[Abstract/Free Full Text]

Sole,N.A. and Barany,G. (1992) J. Org. Chem., 57, 5399–5403.[ISI]

Sollazzo,M., Bianchi,E., Felici,F., Cortese,R. and Pessi,A. (1995) In Cortese,R. (ed.) Combinatorial Libraries. de Gruyter, Berlin, pp. 127–143.

Steinkühler,C., Urbani,A., Tomei,L., Biasiol,G., Sardana,M., Bianchi,E., Pessi,A. and De Francesco,R. (1996) J. Virol., 70, 6694–6700.[Abstract]

Steinkühler,C., Biasiol,G., Brunetti,M., Urbani,A., Koch,U., Cortese,R., Pessi,A. and De Francesco,R. (1998) Biochemistry, 37, 8899–8905.[ISI][Medline]

Smith,G.P., Patel,S.U., Windass,J.D., Thornton,J.M., Winter,G. and Griffiths,A.D. (1998) J. Mol. Biol., 277, 317–332.[ISI][Medline]

Smythe,M.L. and von Itzsein,M. (1994) J. Am. Chem. Soc., 116, 2725–2733.[ISI]

Tomei,L., FaillaC., Santolini,E., De Francesco,R. and LaMonica,N. (1993) J. Virol., 67, 4017–4026.[Abstract]

Tramontano,A., Bianchi,E., Venturini,S., Martin,F., Pessi,A. and Sollazzo,M. (1994) J. Mol. Recogn., 7, 9–24.[Medline]

Vita,C., Roumestand,C., Toma,F. and Menez,A. (1995) Proc. Natl Acad. Sci. USA, 92, 6404–6408.[Abstract]

Yan,Y. et al. (1998) Protein Sci., 7, 837–847.[Abstract/Free Full Text]

Zhang,X. et al. (1997) Nature Biotechnol., 15, 150–154.[ISI][Medline]

Zhao,B.M., Helms,L.R., DesJarlais,R.,L., Abdel-Meguid,S.S. and Wetzel,R. (1995) Nature Struct. Biol., 2, 1131–1137.[ISI][Medline]

Received October 8, 1998; revised May 5, 1999; accepted May 11, 1999.