Development and Standardization of an Immuno-Quantified Solid

solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for ...
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Anal. Chem. 1997, 69, 1746-1752

Development and Standardization of an Immuno-Quantified Solid Phase Assay for HIV-1 Aspartyl Protease Activity and Its Application to the Evaluation of Inhibitors S. Fournout,†,| F. Roquet,†,| S. L. Salhi,‡ R. Seyer,§ V. Valverde,⊥ J. M. Masson,⊥ P. Jouin,§ B. Pau,*,† M. Nicolas,† and V. Hanin†

Laboratoire d’Immunoanalyse et Innovation en Biologie Clinique, CNRS UMR 9921, and Laboratoire d’Immunologie et Biotechnologie, Faculte´ de Pharmacie, 34060 Montpellier Cedex 2, France, Laboratoire de Synthe` se Peptidique et Reconnaissance Mole´ culaire, CNRS UPR 9023, CCIPE, 34094 Montpellier Cedex 5, France, and INSA and Institut de Pharmacologie et de Biologie Structurale, 31000 Toulouse, France

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this Cterminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor. HIV-1, the agent responsible for the acquired immunodeficiency syndrome (AIDS), encodes a protease that is required for the production of infective progeny virions.1 The protease permits the posttranslational processing of the gag and gag-pol gene products into the functional core proteins and viral enzymes. Given the key role of the protease in the retroviral life cycle, the use of protease inhibitors in combination with nucleoside analogues has the potential of being a potent regimen for the treatment of AIDS and related diseases.2 The HIV-1 protease belongs to the class of aspartyl proteases.3-6 There are seven known cleavage sites for the HIV-1 protease among the gag and gag-pol gene products. Although there is no absolute sequence homology at the cleavage sites, three of these †

Laboratoire d’Immunoanalyse et Innovation en Biologie Clinique. Laboratoire d’Immunologie et Biotechnologie. Laboratorie de Synthe`se Peptidique et Reconnaissance Mole´culaire. ⊥ INSA and Institut de Pharmacologie et de Biologie Structurale. | To be considered as equal co-authors. (1) Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach, J. C.; Dixon, R. A. F.; Scolnick, E. M.; Sigal, I. S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4686-4690. (2) Moyle, G.; Gazzard, B. Drugs 1996, 51, 701-712. (3) Toh, H.; Ono, M.; Saigo, K.; Miyata, T. Nature 1985, 315, 691. (4) Pearl, L. H.; Taylor, W. R. Nature 1987, 329, 351-354. (5) Seelmeier, S.; Schmidt, H.; Turk, V.; Von der Helm, K. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6612-6616. ‡ §

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sites are highly conserved in retroviral polyproteins; it consists of a pentapeptide with the consensus sequence (Ser/Thr)-X-X′(Tyr/Phe)-Pro. Cleavage occurs between the aromatic residue Tyr or Phe and the Pro residue. Darke et al.7 established a minimum sequence of seven amino acid residues containing this pentapeptide sequence, which serves as a substrate. Based on these studies and that of Dreyer et al.,8 we have chosen the heptapeptide Ac-Ser-Ala-Ala-Phe-Pro-Val-Val as substrate to measure aspartyl protease activity. A large number of crystal structures of the HIV-1 protease, free or complexed with inhibitors, have been determined9 and used to design substrate analogues with specific inhibitory activity.10-12 Many of these HIV-1 protease inhibitors are peptidomimetics: at the substrate cleavage site, their scissile peptide bond has been replaced by non-hydrolyzable transitionstate analogue moieties. In our study, various synthetic pseudopeptide inhibitors, corresponding to analogues of previously described peptidomimetics,8,13-16 were assayed. Several different HIV-1 protease assays have been developed to test a variety of compounds for their inhibitory activity.17 These assays are based on product separation by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Another type of assay relies on the use of a chromogenic, (6) Meek, T. D.; Dayton, B. D.; Metcalf, B. W.; Dreyer, G. B.; Strickler, J. E.; Gorniak, J. G.; Rosenberg, M.; Moore, M. L.; Magaard, V. W.; Debouck, C. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1841-1845. (7) Darke, P. L.; Nutt, R. F.; Brady, S. F.; Garsky, V. M.; Ciccarone, T. M.; Leu, C. T.; Lumma, P. K.; Freidinger, R. M.; Veber, D. F.; Sigal, I. S. Biochem. Biophys. Res. Commun. 1988, 156, 297-303. (8) Dreyer, G. B.; Metcalf, B. W.; Tomaszek, T. A., Jr.; Carr, T. J.; Chandler, A. C., III; Hyland, L.; Fakhoury, S. A.; Magaard, V. W.; Moore, M. L.; Strickler, J. E.; Debouck, C.; Meek, T. D. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9752-9756. (9) Wlodawer, A.; Erickson, J. W. Annu. Rev. Biochem. 1993, 62, 543-585. (10) Moore, M. L.; Dreyer, G. B. Perspect. Drug Discovery Des. 1993, 1, 85108. (11) Vacca, J. P. Methods Enzymol. 1994, 241, 311-333. (12) Kempf, D. J. Methods Enzymol. 1994, 241, 334-353. (13) Rich, D. H.; Green, J.; Toth, M. V.; Marshall, G. R.; Kent, S. B. H. J. Med. Chem. 1990, 33, 1285-1288. (14) Camp, N. P.; Hawkins, P. C. D.; Hitchcock, P. B.; Gani, D. Bioorg. Med. Chem. Lett. 1992, 2, 1047-1052. (15) Darke, P. L.; Leu, C. T.; Davis, L. J.; Heimbach, J. C.; Diehl, R. E.; Hill, W. S.; Dixon, R. A. F.; Siga, I. S. J. Biol. Chem. 1989, 264, 2307-2312. (16) Krau ¨ sslich, H. G.; Ingraham, R. H.; Skoog, M. T.; Wimmer, E.; Pallai, P. V.; Carter, C. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 807-811. (17) Hellen, C. U. T. Methods Enzymol. 1994, 241, 46-57. S0003-2700(96)01075-X CCC: $14.00

© 1997 American Chemical Society

Table 1. Formulas of the Substrate and Its Different Fragments Synthetized for This Study and Reactivity of the Anti-C2-Terminal Product Antiserum hapten name

formula

IC50 value (µM)

S1 C1 C2 C3 C4 C5

Ac-S-A-A-F-P-V-VAhx-C Ac-S-A-A-F P-V-VAhx-C A-F-P-V-VAhx-C F-P-V-VAhx-C V-VAhx-C

nia nia 0.02 209 28 nia

a

No detectable inhibition at the highest concentration tested: 312

µM.

fluorogenic, or radiolabeled synthetic peptide substrate. Solid phase immunoassays have also been published to monitor the activity of the HIV-1 protease;18-20 they use either an anti-substrate antibody18 or a protein recombinant substrate and an anti-product antiserum.19,20 Here, we have developed and standardized a solid phase assay for the detection of HIV-1 protease activity as an alternative to previous methods. Our assay is an adaptation of the previously described catELISA.21 It is based on the immobilization of the biotinylated synthetic peptide substrate, Ac-Ser-Ala-Ala-Phe-ProVal-Val-Ahx-Cys-biot, on a microtiter plate coated with streptavidin. To measure the enzymatic activity on this coated substrate, a polyclonal rabbit antiserum was produced for specific detection and quantification of the C-terminal fragment product, which remains linked to the plate after substrate hydrolysis. This assay was standardized by reference to HPLC and applied to the evaluation of the potency of synthetic pseudopeptide inhibitors. EXPERIMENTAL SECTION Solid Phase Synthesis of Substrates and Haptens. Synthesis of the substrate (S1, Table 1) has been described by Hanin et al.22 Synthesis of C1, C2, C3, C4, and C5, which are fragments of S1 (Table 1), followed the same protocol. Inhibitors (pepstatin, E64, and aprotinin) were obtained from Boehringer (Mannheim, Germany). The synthesis of pseudopeptide haptens H1,23 H2,24 H3, H4, H5,22 and H925 has already been described. H10 and H11 were prepared according to the following procedure: phosphino dipeptide analogues BocPheΨ[P(O)OH]GlyOH and BocPheΨ[P(O)OHCH]ProOH were obtained after NaOH saponification of the corresponding ethyl esters, which were prepared by hexamethyldisilazane (HMDS) activation of Bocamino-2-phenethylphosphonous acid and reaction with ethyl bromoacetate26 or methyl cyclopentene-2-carboxylate.27 (18) Sarubbi, E.; Nolli, M. L.; Andronico, F.; Stella, S.; Saddler, G.; Selva, E.; Siccardi, A.; Denaro, M. FEBS Lett. 1991, 279, 265-269. (19) Mansfeld, H. W.; Schulz, S.; Gru ¨tz, G.; Von Baehr, R.; Ansorge, S. J. Immunol. Methods 1993, 161, 151-155. (20) Yu, S. L.; Wang, N.; Liou, C. Y.; Syu, W. J. J. Virol. Methods 1995, 53, 6373. (21) Tawfik, D. S.; Green, B. S.; Chap, R.; Sela, M.; Eshhar, Z. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 373-377. (22) Hanin, V.; Campagne, J. M.; Dominice, C.; Mani, J. C.; Dufour, M. N.; Jouin, P.; Pau, B. J. Immunol. Methods 1994, 173, 139-147. (23) Maffre-Lafon, D.; Escale, R.; Dumy, P.; Vidal, J. P.; Girard, J. P. Tetrahedron Lett. 1994, 35, 4097-4098. (24) Maffre-Lafon, D.; Escale, R.; Dumy, P.; Vidal, J. P.; Girard, J. P. Peptides 1994; Escom: Leiden, The Netherlands, 1995; pp 686-687. (25) Campagne, J.-M.; Coste, J.; Guillou, L.; Heitz, A.; Jouin P. Tetrahedron Lett. 1993, 34, 4181-4184.

BocPheΨ[P(O)OH]GlyOH: HPLC [SFCC Ultrabase column C8, 5 µm, 4.6 mm × 150 mm, linear gradient of water (0.1% TFA) and acetonitrile (0.1% TFA) with acetonitrile from 20 to 80% over 20 min at 1.5 mL/min] tR ) 9.33 min; 31P NMR (DMSO-d6, 81 MHz) δ 37.9; MS FAB+ m/z 344 (MH+). Anal. Calcd for C15H22NO6P: C, 52.48; H, 6.41; N, 4.08. Found: C, 52.22; H, 6.66; N, 4.19. BocPheΨ[P(O)OHCH]ProOH: HPLC (as above) tR ) 10.7 min; 31P NMR (DMSO-d6, 81 MHz) δ (relative intensity of four diastereoisomers) 49.0, 48.0, 48.0, 47.5 (40/6/47/13); MS FAB+ m/z 398 (MH+). Anal. Calcd for C19H28NO6P: C, 57.43; H, 7.05; N, 3.53. Found: C, 57.28; H, 7.38; N, 3.42. Solid phase synthesis of inhibitors H10 and H11 proceeded according to the work of Campagne et al.25 The peptides were purified by preparative HPLC and used as diastereoisomeric mixtures. H10: HPLC (as above except that the acetonitrile gradient was from 10 to 60%) tR ) 7.05, 7.35, 7.61, 7.87 min; HRMS calcd 981.452, found 981.465. H11: HPLC (as above except acetonitrile gradient from 10 to 100%) tR ) 6.94, 7.11 min; HRMS calcd 1026.473, found 1026.507. C-Terminal Biotinylation of Peptides. Ten microliters of dimethyl sulfide and 100 µL of dimethyl sulfoxide (DMSO) were used to solubilize 5 mg of peptide (S1 or C2) and 5.4 mg of iodoacetyl-LC-biotin (Pierce Europe B.V., Oud Beijerland, Holland); 5 µL of diisopropylethylamine was then added. This solution was allowed to stand for 1 h in the dark at room temperature, and then the biotinylated peptide was purified by RP-HPLC using a 22 mm × 250 mm Vydac C18 preparative column with a linear gradient of water (0.1% TFA) and acetonitrile (0.1% TFA) with acetonitrile from 0 to 60% over 60 min at 10 mL/ min. The absorbance was monitored at 214 and 254 nm. Purified S1-biot and C2-biot were identified by FAB mass spectrometry: S1-biot, m/z 1330 (MH+); C2-biot, m/z 912 (MH+). Production of the HIV-1 Protease. The plasmid pAra 14 containing the β-gal-protease (PR107) fusion gene was described by Valverde et al.28 For induction of gene expression in Escherichia coli strain MC1061, overnight cultures were diluted 20-fold in LB broth containing 100 µg/mL of ampicillin and incubated at 37 °C until they reached an optical density of 0.5-0.7 at 600 nm. The expression of the protease was induced by adding 0.2% arabinose. After induction for 5 h at 30 °C, the cell culture was then harvested by centrifugation and the pellet washed with a neutralization buffer (20 mM Tris-HCl, 0.1 M NaCl, pH 7.5). Aliquots of 1 mL of bacterial solution were centrifuged for 10 min at full speed in a microfuge: the supernatant was discarded and the pellet frozen at -80 °C. For the experiment, each pellet was diluted with 400 µL of a lysing buffer [50 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 5 mM EDTA, 0.1% dithiotreitol (DTT), 0.01% lysozyme, 0.1% NP 40, 1 mM phenylmethylsulfonyl fluoride (PMSF) in dimethyl formamide] and gently mixed during 2 h at 4 °C. The solution was centrifuged and the supernatant (called protease extract) recovered and conserved at -80 °C in aliquots of 50 µL. An extract from noninduced E. coli cells, as well as an extract from E. coli cells lacking the HIV-1 protease expression vector, was used as control. (26) Allen, M. C.; Fuhrer, W.; Tuck, B.; Wade, R.; Wood, J. M. J. Med. Chem. 1989, 32, 1652-1661. (27) Boyd, E. A.; Regan, A. C.; James, K. Tetrahedron Lett. 1992, 33, 813-816. (28) Valverde, V.; Lemay, P.; Masson, J. M.; Gay, B.; Boulanger, P. J. Gen. Virol. 1992, 73, 639-651.

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Assay of HIV-1 Protease Activity by HPLC. Proteolysis was performed at 37 °C in a tube containing 25 µL of S1-biot (94 µM) as substrate, 20 µL of protease extract (1:10 dilution), and protease buffer (50 mM CH3COONa, 1 mM EDTA, 1 mM DTT, 0.3 M NaCl, pH 5.5), to a final volume of 200 µL. At different times, samples of the reaction mixture were removed, quenched (0.1% TFA in water), and frozen at -80 °C. Reaction products were analyzed by HPLC using a C8 analytical column (Kromasil C8RP, 4.5 mm × 135 mm, 5 µm) with a solution of 10% acetonitrile in 0.1% TFA. Elution was carried out at room temperature with a linear gradient of acetonitrile from 10 to 70% in 0.1% TFA during 15 min at a flow rate of 1 mL/min. The volume of the injected sample was 20 µL. Absorbance was monitored at 220 nm. The cleavage products (C1 and C2-biot) and the substrate (S1-biot) were used as internal standards for peak identification, standard curve construction, and proteolysis estimation. Anti-C-Terminal Product Antiserum Production and Purification. Peptide C2 was conjugated to the carrier protein thyroglobulin (Tg) as previously described.22 Rabbits were injected subcutaneously with 10 µg of this complex in phosphatebuffered saline (PBS) emulsified with Freund’s complete adjuvant in a 1:1 volume. Three boost injections were given in Freund’s incomplete adjuvant at 4-week intervals. Serum bleedings were carried out 2 weeks after each boost and tested for their reactivity against C1, C2, and S1 in an indirect ELISA described hereafter. Peptide S1 was coupled to thiopropyl-Sepharose 6B, via its cysteine residue, in 0.1 M ammonium acetate buffer, pH 5, containing 1 mM EDTA according to the manufacturer’s instructions (Pharmacia, Uppsala, Sweden). To remove antibodies crossreacting with S1, 500 µL of anti-C-terminal product antiserum was incubated with the gel (333 mg) overnight at 4 °C under shaking. The supernatant containing the anti-C-terminal product antiserum (called anti-C2 antiserum) elicited against the C-terminal product was recovered and checked by indirect ELISA and competitive ELISA (described below) for absence of reactivity against S1, S1biot, and the smaller fragments listed in Table 1. Competitive ELISA. Microtiter plates (Nunc, Maxisorp, Roskilde, Denmark) were directly coated with 100 µL/well of a 5 µg/mL solution of streptavidin (Boehringer, Mannheim, Germany) in PBS overnight at 4 °C. The plates were washed three times with PBS containing 0.1% (v/v) Tween 20 (PBS-T) and blocked with 200 µL of a solution of 1% (w/v) gelatin (Sigma, Saint Quentin Fallavier, France) in PBS (PBS-G) for 1 h at 37 °C. The plates were then washed three times, and 100 µL/well of a 1 nM solution of C2-biot was added to the wells and incubated for 1 h at 37 °C. After three washings, the plates were incubated for 90 min at 37 °C with a solution containing the anti-C2 antiserum and various concentrations of the competitor haptens listed in Table 1. The anti-C2 antiserum was diluted to give an absorbance of 1.0-1.5 at 490 nm in the absence of competitor to ensure a linear doseresponse curve. After three washings, horseradish peroxidaseconjugated AffiniPure goat anti-rabbit IgG, Fc fragment specific (Jackson,West Grove, PA), diluted 1:5000 in PBS-T containing 1% gelatin (PBS-T-G), was added to the wells. After 1 h at 37 °C, 100 µL of o-phenylenediamine (4 mg/mL) containing 0.03% (v/v) hydrogen peroxide in 0.1 M citrate buffer, pH 5, was added, and the microtiter plates were incubated for 30 min in the dark, at room temperature. Finally, the reaction was stopped by the addition of 50 µL of 4 N H2SO4, and the resulting absorbance was measured at 490 nm with an automated microtiter plate reader 1748

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(Dynatech MR 5000). This assay was alternatively used as an indirect ELISA when no competitor was introduced. Construction of Standard Curves of the C-Terminal Product (C2-Biot) by Indirect ELISA and by HPLC. To construct the ELISA standard curve, known concentrations (between 0.03 and 0.51 nM) of the biotinylated product C2 (C2-biot) were added to streptavidin-coated wells, incubated for 1 h at 37 °C, and detected by adding anti-C2 antiserum at a dilution of 1:10 000. The next steps, i.e., addition and detection of the anti-rabbit antibodies, were the same as those described above for the competitive ELISA. For HPLC analysis, we constructed a standard curve by injecting known concentrations of C2-biot (between 18 and 138 µM) onto a C8 analytical column (Kromasil C8-RP, 4.5 mm × 135 mm, 5 µm) as described above and plotting a curve of the peak area versus C2-biot concentration. Determination of HIV-1 Protease Activity and Assay of Enzyme Inhibitor Potency by catELISA. Streptavidin-coated plates were blocked with a solution of PBS-G and incubated with 100 µL of various concentrations of biotinylated substrate (S1biot) in PBS-T-G for 1 h at 37 °C. After being washed three times with PBS-T, plates were incubated with 100 µL of various dilutions of the protease extract at 37 °C in protease buffer for 90 min. In inhibition experiments, the test inhibitors (synthetic haptens designed in our laboratory or commercially available products) were added at various concentrations in protease buffer to the protease extract (final dilution 1:300), and the mixture was incubated in the microtiter plates for 90 min. The plates were then washed and incubated for 90 min at 37 °C with the anti-C2 antiserum diluted at 1:10 000 in PBS-T-G. After washings, binding of anti-C2 antibodies to the plate was detected as in indirect ELISA. Comparison of the HPLC and ELISA Methods for CTerminal Product Quantification. Enzymatic hydrolysis was performed in solution as described in the HPLC assay. At different times, aliquots of the reaction mixture were removed. The quantity of C2-biot appearing in these samples was measured by HPLC and by indirect ELISA. For ELISA, the reaction mixture was diluted (from 1:4000 to 1:12 000) in PBS-T-G containing 50 µM pepstatin and incubated for 1 h at 37 °C in streptavidin-coated wells. The indirect ELISA was then performed as described above. RESULTS Cleavage of the Soluble Biotinylated Substrate S1 (S1Biot) by the HIV-1 Protease Extract. To demonstrate that the HIV-1 protease was able to cleave S1-biot, HPLC analysis was used to check for appearance of reaction products C1- and C2-biot (Table 1). The reference chromatographic profile of a mixture containing synthetic S1-biot (tR ) 7.91 min) and the two synthetic products, C1 (tR ) 6.07 min) and C2-biot (tR ) 7.04 min), is given in panel a (Figure 1). Panels b and c show the chromatographic profiles of samples corresponding to the enzymatic cleavage of S1-biot at t ) 5 (b) and 60 min (c). As compared with the HPLC profile at 5 min, the HPLC profile at 60 min showed a decrease of the peak corresponding to the substrate (tR ) 8.13 min) and an increase of two species with retention times of 5.97 and 7.02 min, corresponding to C1 and C2-biot, respectively. We noticed a difference in the retention times of the substrate S1-biot in the profiles shown in Figure 1. To verify that the peaks at 8.13 and 8.45 min corresponded to S1-biot, we coinjected the internal standard S1-biot and the reaction mixture. Under these condi-

Figure 1. HPLC analysis of the biotinylated substrate and the reaction products. (a) 20 µL of a solution containing 2.5 µg of S1biot, 2.5 µg of C1, and 2.5 µg of C2-biot was injected. (b, c) Protease activity at t ) 5 min (b) and at t ) 60 min (c). Assays were performed under the conditions described in the Experimental Section.

tions, the retention time for S1-biot was, indeed, prolonged. An extract of cell culture which did not contain the pAra vector as well as an extract of a noninduced cell culture showed no protease activity (data not shown). This demonstrates that the assay was selective for the recombinant retroviral protease and that host proteases did not significantly hydrolyze the substrate under these conditions. Characterization of the Anti-C2-Terminal Product Antiserum. The nonbiotinylated peptide C2 was coupled to thyroglobulin and used as an immunogen to produce an antiproduct antiserum. The cross-reacting antibodies able to bind the substrate S1 were completely removed by adsorption on S1-Sepharose gel as assessed by indirect ELISA on S1-biot (data not shown). The specificity of this affinity-purified antiserum was then determined by competitive ELISA. The IC50 values are shown in Table 1. At 1 µg/mL (about 2 µM), only soluble C2 inhibited (100% inhibition) the binding of polyclonal antibodies to the adsorbed C2-biot. The antiproduct antiserum also exhibited some reactivity for C3 and C4, but this was very weak, as demonstrated by the high IC50 values. These results illustrate the high specificity of anti-C2 antiserum for the C-terminal product C2. Standardization of the catELISA for Monitoring Proteolytic Activity. S1-biot was immobilized by binding to streptavidincoated microtiter plates and incubated with HIV-1 protease extract, and the C2-biot reaction product was detected by indirect ELISA using anti-C2 antiserum. Figure 2a shows that the signal (absorbance at 490 nm) which is directly related to the amount of product formed during the reaction, at a fixed enzyme concentration, increased as the concentration of the immobilized substrate increased. The working range of S1-biot was between 3 and 22 nM. The effect of protease concentration on the signal was assessed (Figure 2b). As expected, the quantity of product generated by the enzymatic reaction decreased as the dilution of protease extract increased. The working range of protease dilution was between 1:250 and 1:750. As controls, we used an extract of cell culture which did not contain the pAra vector and an extract of noninduced cell culture. As expected, no signal was detected when S1-biot was incubated with these supernatants (data not shown).

Figure 2. Solid phase enzymatic cleavage of S1-biot by the protease extract. (a) A fixed amount of protease extract, diluted 1:100, was added to wells coated with increasing concentrations of streptavidin-bound S1-biot. The microtiter plates were then incubated for 90 min at 37 °C. (b) Increasing dilutions of protease extract were incubated in wells coated with S1-biot (22 nM). The C-terminal product of the enzymatic cleavage was detected as described in the Experimental Section.

To construct the standard curve of C2-biot, serial dilutions of C-terminal product C2 were assayed by indirect ELISA. To verify that all the C2-biot was linked to the coated streptavidin, C2-biot at known concentrations was incubated in a first streptavidincoated well; the supernatants were then removed and transferred to a second streptavidin-coated well. The presence of C2-biot was assessed by using the anti-C2 antiserum. At the highest concentration of C2-biot, 51 nM, no signal was detectable in the second streptavidin-coated well (data not shown), indicating that all the C2-biot was captured by the first streptavidin-coated well. Similarly, by using an anti-substrate antibody,22 we verified that, at the concentration (22 nM) used in the test, all the S1-biot was captured by the streptavidin-coated wells. Using the C2-biot standard curve (Figure 3) of the indirect ELISA, we calculated the amount of C-terminal product appearing during proteolysis of a known amount of S1-biot substrate. From these results, a relationship between the signal from ELISA and the percentage of S1-biot hydrolyzed was established. Quantification of the Solid Phase Proteolysis of S1-Biot by HIV-1 Protease: Comparison with Proteolysis in Solution Determined by HPLC and Indirect ELISA. To compare the ELISA and HPLC analyses, a standard curve for the detection of C2-biot by HPLC was constructed by injecting known amounts of Analytical Chemistry, Vol. 69, No. 9, May 1, 1997

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Figure 3. Standard curve for the biotinylated C-terminal product (C2-biot) assayed by indirect ELISA. Errors bars indicate standard deviation, and symbols without error bars indicate that this bar is within the symbol.

Figure 4. (a) HPLC standard curve for the product C2-biot. (b) Correlation between the percentage of S1-biot hydrolyzed in solution as determined by indirect ELISA and by HPLC after incubation with the aspartyl HIV-1 protease extract for 5, 10, 20, 40, and 60 min.

C2-biot and plotting the peak area against the concentration of C2-biot (Figure 4a). In this assay, the lower detection limit of C2-biot is about 20 µM. In a comparative ELISA versus HPLC study, a proteolytic reaction in solution was performed as described in the Experimental Section. Samples were removed at different times and divided into two parts: one part was used in an HPLC experiment, whereas the other part was determined by indirect ELISA. Due to the low sensitivity of the HPLC 1750

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detection, a higher concentration of S1-biot (400 times) and HIV-1 protease extract (10 times) was needed for the comparison with ELISA. For the ELISA experiment, the samples were thus diluted so that the absorbance limit of the plate reader was not reached. The dilution buffer was supplemented with pepstatin to inhibit the protease. The correlation coefficient of 0.99 and the linear regression curve (y ) -0.69 + 1.37x, P < 0.001) show that there is a strong correlation between our assay and the HPLC assay (Figure 4b). Assay of Protease Inhibitors. To check the usefulness of our method for the screening of protease inhibitors, assays using pepstatin, a HIV-1 protease inhibitor,8,15,16 were carried out. The pepstatin concentration required to reduce S1-biot hydrolysis by 50% (IC50) was estimated at 0.3 µM. E64, a typical inhibitor of thiol proteases, and aprotinin, a known inhibitor of serine proteases, were not able to inhibit the HIV-1 aspartyl protease activity in our ELISA at concentrations of 2.8 and 0.15 µM, respectively (data not shown). These results showed that our assay was reliable and specific. The IC50 values of a set of pseudopeptides designed in our laboratory were determined (Table 2). H4 was found to be the most potent compound and could be considered as a good inhibitor of HIV-1 aspartyl protease, whereas H3, H5, H11, H9, and H10 were weak inhibitors; H1 and H2 did not inhibit the protease. DISCUSSION A rapid and easily immuno-quantified solid phase enzyme assay for studying the proteolytic activity of the HIV-1 protease was developed by using a crude preparation of the protease, a streptavidin-bound biotinylated synthetic substrate, and a highly specific anti-C-terminal product antiserum introduced in an indirect ELISA. The improvements of this enzyme assay by comparison with the catELISA of Tawfik et al.21 rely on both the use of a biotinylated substrate and the overall standardization of the test, therefore permitting determination of the amount of substrate hydrolyzed. The use of a crude preparation of the HIV-1 protease preserves the stability of the protease. This preparation was found to be active on the biotinylated substrate either in solution as shown by HPLC or immobilized on streptavidin-coated well as determined by indirect ELISA. Our method uses a chemically defined synthetic substrate and thus eliminates the need to extract and refold a recombinant substrate like in Sarubbi’s18 and Mansfeld’s19 immunoenzymatic assays. The use of a biotinylated substrate or products for coating the microtiter plates presents several advantages since biotin is a small molecule easily conjugated to various chemical functions, and the coupling yields an extremely stable complex with streptavidin which is not affected by extreme conditions of pH or by the presence of denaturating agents.29 We chose to introduce a thioether linkage between the C-terminal cysteine of the peptide and the N-iodoacetyl-N-biotinylhexylenediamine because these conjugates are chemically stable and can be purified and unambiguously characterized. Furthermore, in comparison with a disulfide linkage, thioethers are not susceptible to exchange reactions with cysteine residues present in proteins in the bioassays. Also, the presence of the biotin moiety on the C-terminal end of the substrate allows the cleavage product (C2(29) Wilchek, M.; Bayer, E. A. Trends Biochem. Sci. 1989, 14, 408-412.

Table 2. Structure and IC50 Values of the Synthetic Pseudopeptides Tested as Potential Inhibitors of HIV-1 Aspartyl Protease

a

No detectable inhibition at the highest concentration tested: 357

µM.

biot) to be retained on the streptavidin-coated wells, whereas the C1 fragment is released into solution and removed by washing. The use of purified biotinylated peptides (substrate and C-terminal product) permitted us to determine the percentage of substrate hydrolyzed. We constructed ELISA and HPLC standard curves to determine the relationship between the concentration of C2-biot and the absorbance at 490 nm or peak area. Thus, knowing the concentration of S1-biot added to the streptavidincoated wells or injected in HPLC and the quantity of the enzymatically generated C2-biot by reference to the standard curves, we were able to calculate the percentage of hydrolysis of the biotinylated substrate, S1-biot. This quantification constitutes a clear advantage over the enzymatic method developed by Yu et al.20 Indeed, they used a crude preparation of substrate, and as such they could not quantify it. We validated our method by comparing the percentage of S1-biot hydrolyzed in solution by HPLC and ELISA. The percentage of S1-biot hydrolyzed in solution as determined by these two methods was found to be highly correlated. An important point in this catELISA for HIV-1 aspartyl protease is the high specificity of the anti-C2-terminal product antiserum.

It specifically recognized C2 and C2-biot. At the concentrations used in the assay, the substrate S1, its N-terminal fragment resulting from the proteolytic cleavage (C1), and the fragment C5 which corresponds to the proline deletion in C2 were not recognized by the affinity purified anti-C2-terminal product antiserum. On the other hand, the anti-C2-antiserum reactedsalthough to a minor extentswith fragments C3 and C4 (Table 1). The solid phase hydrolysis of S1-biot in the presence of various dilutions of protease extract was maximum for a dilution of 1:200. This sensitivity can be attributed to the high potency of the antiserum and to the very small amount of substrate captured by the streptavidin-coated solid phase (∼22 nM). The HPLC standard curve for C2-biot showed that we could not detect less than 18 µg/mL (20 µΜ) of C2-biot by this method; in contrast, by catELISA, as little as 9 pg/mL (0.1 nM) of product could be measured. This method is thus extremely sensitive. This performance is likely to be due to the high-affinity anti-product antibodies we used and to the signal amplification conferred by peroxidase labeling of anti-rabbit IgG antibodies. There is growing interest in developing specific inhibitors of HIV-1 aspartyl protease as possible therapeutic agents in the treatment of AIDS. Since TLC and HPLC are not adapted to largescale screening and the characterization of inhibitors, we analyzed the possibility of using our standardized catELISA for the simultaneous screening of several inhibitors of this enzyme. We first tested aprotinin and E64 as negative controls and pepstatin as a positive control. The weak inhibitory effect measured for pepstatin is comparable with previously published values.8,15,16 We next characterized inhibitors which were synthesized by application of a general design strategy of replacing the P1-P1′ (Phe-Pro) cleavage point in the substrate (S1) with transition-state analogues (Table 2, in which X represents the Phe-Pro substitute). This method was shown to produce tight-binding inhibitors of HIV-1 protease.10,11 The hydroxymethylene transition state analogue H5 (X ) AHPPA, (3S,4S)Pheψ[CHOH]Gly) did not show better inhibitory activity than pepstatin. This confirmed the results of Rich et al.13 on Boc-Ser-Leu-Asn-AHPPA-Ile-Val-OMe, Ki ) 2.6 µM, and those of Dreyer et al.8 on Boc-Ser-Ala-Ala-AHPPA-ValVal-OMe, Ki ) 1.2 µM, obtained by HPLC-monitored peptidolytic assay. The reduced peptide isostere H3 (IC50 ) 1.5 µM) was also a weak inhibitor, and this is consistent with potencies of similar compounds (Ac-Ser-Ala-Ala-Pheψ[CH2N]Pro-Val-Val-NH2, Ki ) 38 µM;8 Ac-Ser-Gln-Asn-Pheψ[CH2N]Pro-Val-Val-NH2, IC50 ) 13 µM13). A better inhibitory potency was obtained with the phosphinate H9 and the phosphinate H10. H9 is comparable to the phosphinate analogue (Ser-Ala-Ala-Pheψ[P(O)OHCH2]Gly-ValVal-OMe, Ki ) 4.4 µM) described by Dreyer et al.8 Moreover, we observed that the phosphinate H11, with the added proline residue, is a weak inhibitor. We noticed that introduction of the phosphonamidate function into inhibitors H1 and H2 was detrimental to the activity, as previously observed for Boc-Asn-Pheψ[P(O)OMeN]Pro-Ile-NHiBu, IC50 ) 100 µM.14 Maximum inhibition was observed for the hydroxyethylamine isosteric replacement of the Phe-Pro peptide bond (H4). This hydroxyethylamine peptide mimic was previously exploited by Rich et al.13 and later by Alewood et al.30 (Ac-Ser-Leu-Asn-Pheψ[CHOHCH2N]Pro-Ile-Val-OMe, Ki ) 0.6 nM,13 IC50 ) 5 nM30) to design potent inhibitors. These results demonstrate that our assay is effective (30) Alewood, P. F.; Brinkworth, R. I.; Dancer, R. J.; Garnham, B.; Jones, A.; Kent, S. B. H. Tetrahedron Lett. 1992, 33, 977-980.

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for the rapid and convenient screening of synthetic HIV-1 aspartyl protease inhibitors. The catELISA was further validated by assaying two commercially available HIV-1 protease inhibitors, Saquinavir (Roche) and Ritonavir (Abbott); they were both found to exhibit potent inhibitory activity. We have developed a fast, specific, and quantitative assay for measuring HIV-1 aspartyl protease activity by immunological detection of the reaction product. Moreover, this test allows the specific detection of the Phe-Pro bond cleavage of the enzyme substrate. The system can be automated and should be useful for screening large numbers of HIV-1 protease inhibitors. Finally, this quantified catELISA can be used for all biological or chemical reactions, provided that the reaction product is immunologically detectable.

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ACKNOWLEDGMENT This work was supported in part by a grant from Elf Aquitaine and by the European Network on Antibody Catalysis (ENABC). We kindly acknowledge Roche Products Ltd. and Abbott Laboratories for the generous gifts of Saquinavir and Ritonavir, respectively. The authors thank J. M. Campagne and D. M. Maffre for synthesizing the pseudopeptides.

Received for review October 18, 1996. Accepted February 21, 1997.X AC961075H X

Abstract published in Advance ACS Abstracts, April 1, 1997.