Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti,Via Pontina Km 30,600-00040, Pomezia (Roma), Italy
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Abstract |
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Keywords: HCV/loop mimetic/NS3 protease/minibody/protease inhibitor
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Introduction |
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HCV is the major agent of transfusion-associated and community acquired non-A, non-B hepatitis worldwide (Choo et al., 1989; Houghton et al., 1991
) and is estimated to infect about 0.21% of the world's population. Chronic HCV infection, which develops in about half of the patients (Alter, 1995
; Bisceglie, 1995
), leads to liver cirrhosis and hepatocellular carcinoma (Chien et al., 1992
). The current treatment for HCV infected patients is
-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., 1993a
,b
). 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., 1993
). 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., 1996
; Love et al., 1996
; Yan et al., 1998
). 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., 1997; Martin et al., 1997
) and a first principle design approach (Martin et al., 1998
) 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., 1997
). 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., 1994
), 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., 1998
; Martin et al., 1998
). It has been demonstrated that the main interactions between the enzyme and peptide inhibitors based on the substrate sequence (spanning P1P6) are provided by P6, P5 and P1 residues (Ingallinella et al., 1998
; Martin et al., 1998
; Steinkühler et al., 1998
). Furthermore, the deletion of negatively charged amino acids P6 and P5 correlate with a dramatic decrease of the inhibitor potency by a factor of 150600. 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., 1998; Martin et al., 1998
). In some favorable cases, these can be converted into small molecular weight compounds amenable to optimization through traditional medicinal or combinatorial chemistry strategies.
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Materials and methods |
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Microbiological and recombinant DNA techniques were carried out according to standard protocols (Ausubel et al., 1994) 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., 1994
). 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 NdeINruI 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., 1997
) using inverse PCR (Hemsley et al., 1989
). 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, 1995
). 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., 1994) and hcVH (Davies and Riechmann, 1995
; Martin et al., 1997
) have been described.
Purification of the NS3 protease domain
Expression and purification were performed as previously described (De Francesco et al., 1996). Protein concentration was estimated by quantitative amino acid analysis. Purity of the enzyme was checked on silver-stained SDSPAGE and by reversed-phase (RP) high-pressure liquid chromatography (HPLC).
NS3 protease assays
Recombinant protein assays were performed in 50 mM TrisHCl 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., 1996). Samples were analyzed as described before (Martin et al., 1997
). The IC50 values were calculated by fitting inhibition data to the following equation using Kaleidagraph software
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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 MichaelisMenten kinetics. The Ki values were determined as described elsewhere (Martin et al., 1997). 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., 1997
).
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., 1997).
Peptide synthesis
The linear peptide (Pep-L) was prepared by standard Fmoc/ t-Bu solid-phase synthesis (Atherton and Sheppard, 1989) 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., 1993
). 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., 1994
). Completion of the reaction was estimated by the usual colour tests (Kaiser et al., 1970
; Hanckock and Battersby, 1976
). Similar treatment with HOBt/DIEA gave substantially lower yields. The peptide was cleaved from the resin by treatment with Reagent B (Sole and Barany, 1992
), 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).
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Results |
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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 4. 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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Discussion |
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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., 1996; Martin et al., 1998
), makes it difficult to design a small molecular weight competitive inhibitor. As a complementary approach to combinatorial synthetic chemistry (Ingallinella et al., 1998
), 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., 1997
) and human pancreatic soluble trypsin inhibitor, yet no competitive inhibitors were obtained from the affinity selection of a minibody library (Dimasi et al., 1997
). 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., 1997
). 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., 1994, 1996
) 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 minibodypIII 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., 1997
).
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., 1998; Martin et al., 1998
; Steinkühler et al., 1998
). 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., 1998
). 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 150600-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., 1998). This observation confirms the results obtained in previous work (Martin et al., 1998
) 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., 1998). 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., 1998
). 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
-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., 1999
). Consequently, its binding mode is probably different from that of the substrate or `canonical' inhibitors (Martin et al., 1998
). 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., 1998). 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 104) of the experimentally available minibody repertoire (Martin et al., 1994
) 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., 1988; Saragovi et al., 1991
; Chen et al., 1992
; Satoh et al., 1997
; Zhang et al., 1997
). 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., 1996
), which has shown its potential for the optimization of the unconstrained NS3 peptide inhibitors.
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Acknowledgments |
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Notes |
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Received October 8, 1998; revised May 5, 1999; accepted May 11, 1999.