Inhibitors of human immunodeficiency virus-1 protease SURESHC.
TYAGI'
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Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A. Received October 29, 1991 TYAGI,S. C. 1992. Inhibitors of human immunodeficiency virus-1 protease. Biochem. Cell Biol. 70: 309-315. We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) 27 nM), resulting in pure competitive inhibition of the can be characterized by a high-affinity mode (Ki = 478 hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 pM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 + 1 pM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 pM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease. Key words: HIV-1 protease, inhibitors, pepstatin, haloperidol, tryptophan fluorescence. TYAGI,S. C. 1992. Inhibitors of human immunodeficiency virus-1 protease. Biochem. Cell Biol. 70 : 309-315. Nous avons montrt que la rtaction de la pepstatine A avec la prottase du virus-1 de l'immunodtficience humaine (HIV-1 prottase) peut Etre caractbisk par un mode d'affinitt tlevt (Ki = 478 27 nM) rtsultant en une pure inhibition compttitive de l'activitt hydrolytique de la HIV-1 prottase B l'tgard d'un substrat fluorogkne. La liaison de la pepstatine dans ce mode induit un dtplacement vers le bleu dans la fluorescence endogkne provenant des rtsidus de tryptophane dans la HIV-1 prottase. Ce dtplacement est maximal en prCsence de pepstatine 10 pM. En revanche, l'halopbidol rtagit avec la HIV-1 prottase avec une affinitt plus faible (Ki = 19 1 pM) selon un mode qui rtsulte en une pure inhibition non compttitive de l'activitt hydrolytique de la HIV-1 prottase. La liaison de l'haloptridol dans ce mode induit un dtplacement vers le rouge dans la fluorescence endogkne provenant des rtsidus de tryptophane dans la HIV-1 prottase. Ce dtplacement est maximal en prbence de I'haloptridol200 pM. L'addition B la fois de pepstatine et d'haloptridol en des concentrations dans la garnme des valeurs de leur Ki entraine une inhibition additive de l'activitt hydrolytique de la HIV-1 prottase de mEme qu'un effet additif sur la fluorescence du tryptophane de la prottase. Cependant, B des concentrations saturantes de pepstatine et d'haloptridol, l'effet de l'halopbidol, tel que mesurt par les changements dans la fluorescence intrinskque de la HIV-1 prottase, est prtdominant. Mots cl4s : HIV-1 prottase, inhibiteurs, pepstatine, halopbidol, fluorescence de tryptophane. [Traduit par la rtdaction]
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Introduction Human immunodeficiency virus, a member of the retroviral group, is the causative agent of AIDS (Barre-Sinoussi et al. 1983; Gallo et al. 1984). Proteolytic processing of structural components and replicative enzymes by a virally encoded protease is an essential step in the maturation of infectious viral particles. This protease also has the capacity to degrade some of the cytoskeletal proteins of the host cell (Wallin et al. 1990; Shoeman et al. 1991; Tomasselli et al. 1991), and thus, is an important target for development of antiviral drugs. The protease is a member of the family of aspartic proteases, like pepsin and renin, but unlike these enzymes, exists as a dimer of the identical subunits, with each monomer contributing an Asp-Thr-Gly sequence to the active site (Toh et al. 1985; Navia et al. 1989; Miller et al. 1989). ABBREVIATIONS: HIV-1 protease, human immunodeficiency virus-1 protease; AIDS, acquired immunodeficiency syndrome; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; Bz-, benzoyl-; MeO-, methoxy-; kDa, kilodalton(s). 'present address: Division of Cardiology, Department of Medicine, Medical Sciences Building, Room No. 431A, University of Missouri, Columbia, MO 65212. U.S.A. Printed in Canada / Imprim6 au Canada
Rational design of drugs directed against HIV-1 protease requires an understanding of the mechanism by which it interacts with and catalyzes the hydrolysis of substrate. Substrate peptides, with cleavage sites at Phe-Pro or TyrPro, have been used to measure protease activity (Darke et al. 1988; Billich et al. 1988; Krausslich et al. 1989; Graves et al. 1988). It is difficult, however, to perform rigorous mechanistic analysis in these assays because there is no detectable signal during catalysis. Recently, several studies including our own (Tyagi and Carter 1992) have described the use of fluorescent substrates and energy transfer techniques (Matayoshi et al. 1990a, 1990b; Tamburini et al. 1990), as well as chromogenic substrates (Nashed et al. 1989; Dilanni et al. 1990), which should help in this regard. The transition-state analogue concept (Wolfenden 1976; Dreyer et al. 1989; Ashorn et al. 1990; Meek et al. 1990; Roberts et al. 1990) suggests a general strategy for the design of inhibitors of the aspartic proteases in which the scissile carbonyl of an oligopeptide substrate is replaced by a nonhydrolyzable analogue with tetrahedral geometry. This strategy has proven fruitful in the search for renin inhibitors (Fischer 1988). The natural product pepstatin, containing the unusual amino acid statine, binds with dissociation constants in the subnanomolar range (Dreyer et al. 1989) and is a potent inhibitor specific for most aspartic proteases.
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Exceptions are the mammalian aspartic protease renin (Boger et al. 1983) and the retroviral proteases (Giam and Boros 1988; Darke et al. 1989; Seelmeier et al. 1988; Katoh et al. 1987), for which pepstatin is much less inhibitory. Peptide-based inhibitors with submicromolar inhibitory activity towards protease in vivo have been shown t o be effective in reducing viral infectivity in cultured T4 cells and in inhibition of gag polyprotein processing in vitro (Meek et al. 1990; McQuade et al. 1990). Recently, Zhang et al. (1991) have reported that a C-terminal sequence of HIV-1 protease, tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, is an excellent inhibitor of this enzyme. They have shown that this tetrapeptide binds t o the inactive protomers and prevents their association into the active dimer and cause a dissociative inhibition of HIV-1 protease. They have reported a dissociation constant (Kd) for the dimer of 3.5 nM and an inhibition constant (Ki) for the tetrapeptide of 45 kM. However, peptide-based inhibitors are often therapeutically ineffective when orally administered, stimulating interest in a search for nonpeptidic inhibitors (Rich et al. 1990; Dreyer et al. 1989). Structure-based design of nonpeptide inhibitors for HIV-1 protease was described by DesJarlais et al. (1990), who discovered that the drug haloperidol (Fig. 1) has some inhibitory activity against HIV-1 protease. Based on X-ray crystallographic data, DesJarlais et al. (1990), Miller et al. (1989), Swain et al. (1990), Erickson et al. (1990), Fitzgerald et al. (1990), and Rao et al. (1991) postulated that haloperidol may induce conformational changes in HIV-1 protease by binding at the dimer interface (DesJarlais et al. 1990). Precise experimental support for this prediction is limited. The present work is aimed at further characterizing the nature of the inhibition of HIV-1 protease by pepstatin and haloperidol and the relationship of this inhibition t o alterations in protease structure. We have examined inhibition of HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate peptide by pepstatin and haloperidol. In addition, we have studied the effect of pepstatin and haloperidol o n the endogenous fluorescence of the two tryptophan residues in HIV-1 protease t o see if these inhibitors induce perturbations in the structure of the HIV-1 protease.
Materials and methods Materials HIV-1 protease was expressed and isolated from Escherichia coli according to the method of Graves et al. (1989) and was a generous gift from Dr. M. C. Graves (Department of Molecular Genetics, Hoffmann-La Roche Inc. Nutley, N.J.). Protease was 98% pure as judged by SDS-PAGE and silver staining. Pepstatin A, haloperidol, cytochrome c, EDTA. DMSO, urea, DTT, Bz-ArgGly-Phe-Pro-MeO-Na (substrate), and Pro-MeO-Na (product) were from Sigma (St. Louis, Mo.). All other reagents were of standard grade. Enzymatic activity Hydrolytic activity of HIV-1 protease was assayed with Bz-ArgGly-Phe-Pro-MeO-Na. The release of Pro-MeO-Na was monitored by recording fluorescence at 420 nm with excitation at 346 nm in a 3 x 3 rnm (120-pL capacity) cuvette. The substrate concentration was varied from 0.5 to 6 mM. Based on weight measurements of lyophilized sample of this 22-kDa protein, HIV-1 protease concentration ranged from approximately 0.1-1 pM in the different studies reported here. All measurements were carried out in 100 mM sodium acetate (pH 6) containing 1 M NaCl, 1 mM
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FIG. 1. Chemical structure of haloperidol. EDTA, 1 mM DTT, and 10% DMSO. The final HIV-1 protease concentration was set at 200 nM for determination of the dissociation constant Kiof pepstatin or haloperidol. Kinetic data were collected for 5 min. The fluorescence increase was recorded at each second beginning just after mixing. The initial velocity was calculated from fluorescence changes over the fist 20-30 s. During this time the increases in fluorescencewere linear with no lag period. The data were converted into the concentration of released product (Pro-MeO-Na) according to the following: Moles Pro-MeO-Na released = F x [Pro-MeO-Na],/F, where F, [Pro-MeO-Na], and F, are the initial fluorescence reading as the function of time, and concentration and fluorescence of Pro-MeO-Na of the standard, respectively. The fluorescence of standard Pro-MeO-Na was recorded at the same time and the same instrument settings as for the experimental reaction. The initial velocities (mol Pro-MeO-Na released/s) in the presence of inhibitor (V)were all normalized to the velocity in the absence of the inhibitor (V,).
Enzymatic inhibition Various concentrations of pepstatin and haloperidol ranging from 0.5 to 10 pM for pepstatin and 5 to 100 pM final concentration for haloperidol were mixed with 0.2 p M HIV-1 protease at 25°C prior to addition of Bz-Arg-Gly-Phe-Pro-MeO-Na, to give a final substrate concentration ranging from 2 to 4 mM. Comparable results were obtained when the order of addition of inhibitor and substrate was reversed, showing that a steady state was established within the manual mixing time. All reactions were carried out in 100 mM sodium acetate, 1 M NaCl, 1 mM EDTA, 1 mM DTT, and 10% DMSO. Each data point was done in triplicate and enzymatic hydrolysis rates were fit to the Michaelis-Menten equation using a nonlinear regression program ("Enzfitter" from Biosoft). Spectroscopic methods Absorption spectra were recorded with a Hewlett Packard 8452A diode array spectrophotometer. The concentration of HIV-1 protease was determined by employing the Bradford (1976) dye-binding assay. The concentration of protease was also checked by absorbance at 280 nm, in experiments where relative comparisons were made. The concentration of Bz-Arg-Gly-Phe-Pro-MeO-Na (substrate) was based on weight measurements and compared with the concentrationdetermined by extinction coefficient for naphthyl derivatives at about 340 nm ( E = 6000 M - .cm - ') (Turner and Brand 1968). The concentrations of Pro-MeO-Na (product), pepstatin, and haloperidol were based on weight measurements. Fluorescence spectra were recorded on a computer-controlled Spex Datamate spectrofluorometer connected to a circulating water bath to control temperature. The excitation and emission slits were adjusted for 5 nm band-pass width. Spectra were recorded at 1-nm intervals and corrected for base line and instrument response. Samples were prepared and incubated for appropriate times prior to measurements at 25°C. Tryptophan fluorescence was determined with )h, at 295 nm. Protein solutions used for these measurements had an absorbance of less than 0.05 to avoid inner filter effects (Lakowicz 1983). Pepstatin and haloperidol do not absorb in the wavelength range where tryptophan absorbs or emits from approximately 290 to 350 nm.
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Analytical gel filtration A 20-pL sample (20 pg) of HIV-1 protease was loaded on to a 1 x 18 cm Sephacryl S-200 column equilibrated with buffer A
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[Pepstatinl/[HIV- I Protease] FIG. 2. Inhibition of HIV-1 protease (200 nM) by various concentrations of pepstatin A. After addition of pepstatin to the enzyme, the residual hydrolytic activity was assayed by addition of various concentrations of Bz-Arg-Gly-Phe- Pro-MeO-Na as described in Materials and methods. The hydrolytic rates (V) were all normalized to the rate observed in the absence of pepstatin (V,). The curve is a nonlinear least-squares fit to the data points, according to the tight-binding inhibition equation (Williams and Morrison 1979). Data were obtained at 2 mM Bz-Arg-Gly-Phe-Pro-MeO-Na. Insert: replots of the data at 2 (m) and 4 mM (a) substrate concentrations. The lines are the linear least-squares fit to the data.
(100 mM sodium acetate (pH 6.0), 1 M NaC1, 1 mM EDTA, 1 mM DTT, and 10% DMSO) at 4°C. The fractions of 0.38 mL were collected and measured for their protein fluorescence at 340 nm when excited at 295 nm. The band-pass slits were 5 nm for both the excitation and emission, respectively. The hydrolytic activity of these fractions was measured by employing Bz-Arg-Gly-PhePro-MeO-Na as a fluorogenic substrate. Cytochrome c (12.5 kDa) was employed as a marker protein on this column.
Results and discussion Inhibition of hydrolytic activity of HIV-1 protease In order to conform to the criteria outlined by Silverman (1988) for analysis of inhibition by the partition ratio method, we varied the ratios of pepstatin to HIV-1 protease from 2.5 to 50 and the ratios of haloperidol to enzyme from 25 to 500 in the hydrolytic assays. Pepstatin is a tightly bound inhibitor of HIV-1 protease. The level of inhibition of protease by pepstatin was independent of the order of addition of substrate and inhibitor within the limits of manual mixing times, justifying the assumption of equilibrium binding. Under conditions in which a significant fraction of the inhibitor is bound, the inhibition can be analyzed by the partition ratio method (Silverman 1988). The inhibition is sensitive to the substrate concentration. As can be seen from Fig. 2, the slope of this plot, Ki,,,, is dependent on substrate concentration and reflects some competitive character. The data can be fit according to Henderson plots to conform to a model of simple competitive inhibition (Fig. 2) (Green and Work 1953; Morrison 1969; Henderson 1972; Bieth 1974; Empie and Laskowski 1982; Bieth 1984):
where [pepstatinlo and [HIV-1 Proteaselo are the initial total concentrations of pepstatin and HIV-1 protease, respectively. [S], K,, and Ki are the substrate concentration, binding constant of substrate to enzyme, and the dissociation constant between enzyme and inhibitor, respectively. Vand Vo are the initial rates of substrate hydrolysis in the presence and absence of inhibitor, respectively. From plots at several different substrate concentrations, we computed a value of Ki for pepstatin of 478 & 27 nM. The hydrolysis of Bz-Arg-Gly-Phe-Pro-MeO-Na by HIV-1 protease in the absence of inhibitors conformed to simple Michaelis-Menten kinetics. The apparent value of K, for this fluorogenic substrate under the conditions we employed is 2.0 + 0.2 mM (Tyagi and Carter 1992). Pepstatin has been recognized as a peptide analog inhibitor of HIV-1 protease, which binds to the enzyme with an affinity in the low micromolar range (Darke et al. 1989). The mode of inhibition by pepstatin has been described as mixed by Darke et al. (1989) and as linear competitive by Dreyer et al. (1989). Under our assay conditions, we observed that pepstatin appeared to have a purely competitive mode of inhibition with a Ki in the submicromolar range. This transition-state analog appeared to possess a structure that was optimal for interactions with active site residues in HIV-1 protease. Pepstatin, once bound, appeared to prevent substrate binding to the enzyme; we were hindered from obtaining any evidence for effects of pepstatin on catalytic activity under these conditions, since the competitive mode of inhibition was complete. Inhibition of HIV-1 protease by haloperidol was found to be associated with binding of relatively low affinity: at a ratio of haloperidol to HIV-1 protease of 10 pM to 0.2 pM, the hydrolytic activity was reduced only 10-15%. The extent
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[Haloperidol]/[HIV- 1 Proteasel FIG.3. Inhibition of HIV-1 protease (200 nM) by various concentrations of haloperidol. After addition of haloperidol to the enzyme, the residual hydrolytic activity was assayed by addition of various concentrations of Bz-Arg-Gly-Phe-Pro-MeO-Na as described in Materials and methods. The hydrolytic rates (V) were all normalized to the rate observed in the absence of haloperidol (Vo).The curve is a nonlinear least-squares fit to the data points, according to the modification of Dixon (1953) equation. Data were obtained at 2 mM Bz-Arg-Gly-Phe-Pro-MeO-Na.Insert: replots of the data at 2 (w) and 4 mM ( 0 )substrate concentrations. The line is the linear leastsquares fit to the data. of inhibition was independent of the incubation period, indicating rapid (within manual mixing time) binding between haloperidol and protease, justifying the assumption of a steady state model. Accordingly, we analyzed this inhibition (Fig. 3) by a modification of the Dixon plot (1953): 1 -
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TABLE1. Additive effect of pepstatin A and haloperidol as inhibitors of hydrolytic activity of Bz-Arg-Gly-Phe-Pro-MeO-Na by HIV-1 protease: hydrolysis of 2 m M Bz-Arg-Gly-Phe-Pro-MeO-Na by 200 nM HIV-1 protease in 100 mM sodium acetate (pH 6.0) - 1 M NaCl, 1 mM EDTA - 1 mM DTT 10% DMSO at 2S°C, in the presence or absence of inhibitors
1- V V [haloperidol] Reactants V/V , In this expression, the inhibitor concentration [haloperidol] is rigorously defined as the concentration of free inhibitor, HIV-1 protease 1.00 HIV-1 protease + pepstatin 0.52 but since the total inhibitor concentration is about two HIV-1 protease + haloperidol 0.54 orders of magnitude greater than that of the total enzyme HIV-1 protease + pepstatin + haloperidol 0.23 concentration, the amount of bound inhibitor can be disregarded. The value of KisaPp for this mode of inhibition NOTB:Five min prior to addition of substrate, 200 nM emzyme was added to the 1 pM pepstatin and 20 pM haloperidol, respectively, and is independent of substrate concentration and therefore the subsequent hydrolytic rate V was compared with the rate observed reflects noncompetitive character. The general expression in the absence of any added inhibitor. Yo. gives a true value of Ki in this case. The value of Ki for pounds at their apparent Ki values to 200 nM HIV-1 prohaloperidol inhibition computed from the data in Fig. 3 is tease to see if they were additive with each other for inhibi19 + 1 pM. tion of hydrolytic activity of the enzyme (Table 1). In the DesJarlais et al. (1990) found that inhibition of hydrolysis presence of 1 pM pepstatin alone, the hydrolysis of 2 mM of a decapeptide (Ala-Thr-Leu-Asn-Phe-Pro-Ile-Ser-ProBz-Arg- Gly-Phe-Pro-MeO-Na was inhibited 48%, whereas Trp) by haloperidol was of the mixed type with both parin the presence of 20 @ haloperidol, I the hydrolysis was tial competitive and noncompetitive components. Under our assay conditions, we observed that inhibition of Bz-Arg-Glyinhibited 46%. Hydrolysis was inhibited 77% in the presence of a mixture of 1 pM pepstatin + 20 pM haloperidol, Phe-Pro-MeO-Na, a shorter peptide, was purely nonsuggesting that both inhibitors do not compete for the same competitive with an affinity to HIV-1 protease in the low site on the enzyme, but rather are approximately additive micromolar range (Fig. 3). in their inhibition of hydrolytic activity of HIV-1 protease. Pepstatin and haloperidol are noncompetitive with each Effect of pepstatin and haloperidol on HIV-1 protease other tryptophan fluorescence Since pepstatin appears to bind tightly to HIV-1 protease as a competitive inhibitor, whereas haloperidol binds as a The two tryptophan residues in HIV-1 protease give rise noncompetitive inhibitor, we added both of these comto a characteristic emission spectrum with a maximum
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Fraction no.
Wavelength FIG. 4. Endogenous tryptophan fluorescence of HIV-1 protease: HIV-1 protease (- . -,0.2 pM), HIV-1 protease plus 10 pM pepstatin A (- - -), and HIV-1 protease plus 200 pM haloperidol (-). The samples were incubated prior to recording the spectra in pH 6 buffer. The spectra were corrected for buffer base line, instrument response, and contributions due to free pepstatin and haloperidol, respectively. The excitation was 295 nm.The slits were 5 nm for both the excitation and emission wavelengths.
around 340 nm with excitation at 295 nm. The effect of pepstatin and haloperidol on this endogenous fluorescence was studied over a range of pepstatin and haloperidol concentrations to identify effects which might be ascribed to binding. In the presence of 10 pM pepstatin, the tryptophan fluorescence of 0.2 pM HIV-1 protease was shifted about 5 nm to shorter wavelengths. Similarly, when 200 pM haloperidol was added to 0.2 pM HIV-1 protease, the endogenous fluorescence from Trp residues in HIV-1 protease was shifted about 7 nrn to longer wavelengths (Fig. 4). These changes were reversed upon dilution of the pepstatin and haloperidol, respectively, indicating that neither haloperidol nor pepstatin irreversibly denatures the protease. The red shift upon haloperidol binding implies more Trp exposure to solution and therefore a more open conformation and the blue shift upon pepstatin binding implies more Trp surrounded by the hydrophobic residue@)and therefore induces compact conformation in the protease structure. Since pepstatin, a transition-state analog, appears to bind tightly to the active site in the enzyme and to induce a compact conformation in protease structure, whereas haloperidol binds less tightly and induces an open conformation in the protease structure, we added both of these inhibitors at their apparent Kiand at 10 times their apparent Kivalues, respectively, to 200 nM HIV-1 protease to see if they were also additive for induction of conformation changes in the enzyme. In the presence of l pM pepstatin the changes in Trp fluorescence were about half the magnitude of the changes observed at saturating con-
FIG. 5. Analytical gel filtration of HIV-1 protease on Sephacryl S-200. Twenty microlitre (20 pg) of HIV-1 protease was loaded on the column. Fractions of 0.38 mL each were collected and their fluorescence at 340 nm was measured when excited at 295 nm. The band-pass slits were 5 nm for both the excitation and emission, respectively. The protein was eluted with buffer A (100 rnM sodium acetate (pH 6.0). 1 M NaCI, 1 mM EDTA, 1 mM DTT, and 10% DMSO) (m), with buffer A plus 100 pM pepstatin (a), and with buffer A plus 2 mM haloperidol (A). All buffer solutions were at 4OC.
centration of pepstatin. Similarly 20 pM haloperidol induced changes in Trp fluorescence, about half the magnitude of the changes observed in the presence of saturating amount of haloperidol. This indicates also that the binding events detected by fluorescence are probably equivalent to the inhibitory binding sites on the protease. However, in the presence of mixture of 10 pM pepstatin and 200 pM haloperidol, tryptophan fluorescence was red shifted as has been seen for haloperidol alone (Fig. 4) after correcting the emission spectra for free pepstatin and haloperidol in the buffer. These data suggest that haloperidol alters the conformation of the protease in a way that cannot be reversed by pepstatin binding. However, changes in tryptophan fluorescence induced by pepstatin alone appeared to be reversed by haloperidol binding in the HIV-1 protease. This may suggest that haloperidol binds to the dissociated subunit (protomer) more tightly than the dimer of the protease. Since pepstatin and haloperidol are additive in their inhibition, it may be possible that the effect of pepstatin on fluorescence is too small to be reliably detected on top of a bigger signal, e.g., red shift from haloperidol binding. X-ray crystallographicexamination indicates that transitionstate analogs (inhibitor or substrate) bound at the active site can induce movement around the flap region of the enzyme, resulting in a more compact conformation in the protease structure (Miller et al. 1989; Erickson et al. 1990). This is supported by our observation that binding of pepstatin to HIV-1 protease induces a blue shift in the intrinsic Trp fluorescence of the protease. This blue shift indicates compactness in the structure of protease which produces a more hydrophobic environment or steric coverage around Trp residue@)by pepstatin. The possible Trp residue for steric coverage by pepstatin may be the ~ r p which ~ , is closer to the active site than ~r~~~(Miller et al. 1989; Erickson et al. 1990; Pearl and Taylor 1987). The binding of haloperidol to the enzyme induced a reduction in the catalytic activity and no alteration in substrate
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binding. This mode of binding is supported not only by our kinetic analysis of substrate hydrolysis but also by the red shift in Trp fluorescence. This red shift suggests a decrease in hydrophobicity in the environment of at least one of the two indole side chains in HIV-1 protease. Changes in the intrinsic Trp fluorescence of HIV-1 protease suggested that the high-affinity competitive mode of binding of pepstatin to the protease can be altered by the low-affinity noncompetitive binding of haloperidol to the enzyme, when both inhibitors are present at concentrations sufficient to saturate their binding sites if they were added separately. These studies suggested that binding of a transition-state analog (pepstatin or substrate analog) at the active site may be disrupted by the binding of a nonpeptidic compound at the noncompetitive site in the protease. This may suggest that the binding of haloperidol to the monomer is stronger than to the dimer of HIV-1 protease, so that it prevents dimer formation.
Effect of inhibitors on the apparent elution profile on a gel filtration column of protease As suggested by structural modeling, the retroviral proteases would most resemble the structure of the other aspartic proteases if they were assembled as dimers (Pearl and Taylor 1987). This prediction was confirmed by crystallography (Miller et al. 1989; Erickson et al. 1990). The molecular mass of HIV-1 protease obtained under denaturing conditions was in accord with the value of 11 kDa found upon SDS-PAGE analysis of the purified protease. Analytical gel filtration of purified protease on Sephacryl S-200 demonstrated that HIV-1 protease fluorescence and activity eluted in a sharp peak at 8.2 mL (fraction no. 21-22) (Fig. 5). The position of this peak in the elution profile was shifted to 10 mL (fraction no. 26) when a saturating amount (2 mM) haloperidol was present in the elution buffer A. This peak contained only fluorescence, but no activity. However, the presence of 100 FM pepstatin in buffer A did not change the elution profile of HIV-1 protease on this column under the conditions we applied. The peak in the presence of pepstatin also contained fluorescence, but no activity. Cytochrome c (12.5 kDa) was employed as the protein calibration standard under the same identical conditions as used above in buffer A and was eluted at 9.75 mL. HIV-I protease migrates as a dimer on gel filtration column (Meek et al. 1989). We have shown, based on the elution profile on an analytical gel filtration column, that the presence of haloperidol appears to induce conformational changes in the protease structure in a way that the protease-haloperidol migrates as a monomer complex. This result is consistent with our observation, based on the intrinsic fluorescence of protease in the presence of haloperidol and pepstatin, that haloperidol induces an open conformation whereas pepstatin induces a compact conformation in the protease structure. The possibility that the effect of pepstatin on fluorescence is too small to be reliably detected on top of a bigger signal towards the longer wavelength induced by binding of haloperidol may not be consistent with the elution pattern of protease on gel filtration in the presence of a saturating concentration of haloperidol, which appears to induce dissociation of the protease dimer. These observations support a rational basis for design of synthetic HIV-1 protease inhibitors with high specificity and
affinity. Evidence of interactions of the transition-state inhibitor with the enzyme through hydrophobic domains is seen in the characteristic changes in the emission spectra of bound pepstatin. In addition, nonhydrophobic interactions are apparently involved in binding of haloperidol. These two modes of interaction between the HIV-1 protease and the inhibitors are structurallyvery different and suggest that they may be combined to obtain inhibitors that are both potent and stable in chemical therapy. These studies may serve as a useful lead for the development of a new class of antiretroviral agents. Acknowledgements The author thanks Drs. Carol Carter and Mary Graves for providing HIV-1 purified protease, and Drs. Sanford R. Simon, Carl Moos, and Rodney A. Bednar for their critical review of the manuscript. This work was supported in part by grants AI-25993 and HL-14262 from the National Institutes of Health. Ashorn, P., McQuade, T., Thaisrivongs, S., et al. 1990. An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proc Natl. Acad. Sci. U.S.A. 87: 7472-7476. Barre-Sinoussi, F., Cherman, G.C., Rey, G., et al. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science (Washington, D.C.), 220: 868-870. Bieth, J. 1974. Some kinetic consequences of the tight binding of protein-protease-inhibitors to proteolytic enzymes and their application to the determination of dissociation constants. BayerSymp. 5: 463-469. Bieth, J. 1984. In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem. Med. 32: 387-397. Billich, S., Knoop, M.-T., Hansen, J., et al. 1988. Synthetic peptides as substrates and inhibitors of HIV-1 protease. J. Biol. Chem. 263: 17 905 - 17 908. Boger, J., Lohr, N.S., Ulm, E.H., et al. 1983. Novel renin inhibitors containing the amino acid statine. Nature (London), 303: 81-84. Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Darke, P.L., Nutt, R.F., Brady, S.F., et al. 1988. HIV-1 protease specificity of peptide cleavage is sufficient. Biochem. Biophys. Res. Commun. 156: 297-303. Darke, P L., Leu, C.-T., Davis, L.J., et al. 1989. HIV-1 protease. J Biol. Chem. 264: 2307-2312. DesJarlais, R.L., Seibel, G.L., Kuntz, I.D., et al. 1990. Structurebased design of nonpeptide inhibitors specific for the HIV-1 protease. Proc. Natl. Acad. Sci. U.S.A. 87: 6644-6648. Dilanni, C.L., Davis, L.J., Holloway, M.K., et al. 1990. Characterization of an active single polypeptide form of the HIV-1 protease. J. Biol. Chem. 265: 17 348 - 17 354. Dixon, M. 1953. The determination of enzyme inhibition constants. Biochem. J. 55: 170-171. Dreyer, G.B., Metcalf, B.W., Tomaszek, T.A., et al. 1989. Inhibition of HIV-1 protease in vitro: rational design of substrate analogue inhibitors. Proc. Natl. Acad Sci. U.S.A. 86: 9752-9756. Empie, M.W., and Laskowski, M., Jr. 1982. Thermodynamics and kinetics of single residue replacements in avian ouomucoid third domain: effect of inhibitor interactions with serine proteases. Biochemistry, 21: 2274-2284. Erickson, J.W., Neidhart, D.J., VanDrie, J., et al. 1990. Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science (Washington, D.C.), 249: 527-533.
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Fischer, G. 1988. Trends in protease inhibition. Nat. Prod. Rep. 5: 465-495. Fitzgerald, P.M.D., McKeever, B.M., VanMiddlesworth, J.F., et al. 1990. Crystallographic analysis of a complex between HIV-1 protease and acetyl-pepstatin at 2.0 A resolution. J. Biol Chem. 265: 14 209 - 14 219. Gallo, R.C., Salahuddin, S.Z., Popovic, M., et al. 1984. Frequent detection and isolation of cytopathic retrovirus (HTLV-111) from patients with AIDS and at risk for AIDS. Science (Washington, D.C.), 224: 500-502. Giam, C.-Z., and Boros, I. 1988. In vivo and in vitro autoprocessing of HIV protease expressed in Escherichia coli. J. Biol. Chem. 263: 14 617 - 14 620. Graves, M.C., Lim, J.J., Helmer, E.P., and Kramer, RA. 1988. An 11-kDa form of HIV-1 protease expressed in Escherichia coli is sufficient for enzymatic activity. Proc. Natl. Acad Sci. U.S.A. 85: 2449-2453. Graves, M.C., Lim, J.J., Zicopoulos, M.A. et al. 1989. Expression and characterization of human immunodeficiency virus-1 protease. In Proteases of retroviruses. Proceedings of the Colloquium C 52, 14th International Congress of Biochemistry, Prague, Czechoslovakia, July 10-15, 1988. Edited by V. Kostka. Walter de Gruyer, Berlin. pp. 83-92. Green, N.M., and Work, E. 1953. Pancreatic trypsin inhibitor. Biochem. J. 54: 347-352. Henderson, P.J.F. 1972. A linear equation that describes the steady-state kinetics of enzymes and subcellular particles indicating with tightly bound inhibitors. Biochem. J. 127: 321-333. Kotoh, I., Yasunaga, T., Ikawa, Y., and Yoshinaka, Y. 1987. Inhibition of retroviral protease activity by an aspartyl protease inhibitor. Nature (London), 329: 654-656. Krausslich, H.-G., Ingraham, R.H., Skoog, M.T., et al. 1989. Activity of purified biosynthetic proteinase of HIV on natural substrates and synthetic peptides. Proc. Natl. Acad. Sci. U.S.A. 86: 807-811. Lakowicz, J.R. 1983. Principles of fluorescence spectroscopy. Plenum, New York. Matayoshi, E.D., Wang, G.T., Krafft, G.A., and Erickson, J. 1990a. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science (Washington, D.C.), 247: 954-958. Matayoshi, E.D., Wang, G.T., Krafft, G.A., and Erickson, J. 1990b. Novel fluorogenic substrates for assaying retroviral proteolytic activity by resonance energy transfer. Biophys. J. 57: 37a. McQuade, T.J., Tomasselli, A.G., Liu, L., et al. 1990. A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science (Washington, D.C.), 247: 454-456. Meek, T.D., Dayton, B.D., Metcalf, B.W., et al. 1989. HIV-1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease. Proc. Natl. Acad. Sci. U.S.A. 86: 1841-1845. Meek, T.D., Lambert, D.M., Dreyer, G.B., etal. 1990. Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. Nature (London), 343: 90-92. Miller, M., Schneider, J., Sathyanarayana, B.K., et al. 1989. Structure of complex of sypthetic HIV-1 protease with a substratebased inhibitor at 2.3 A resolution. Science (Washington, D.C.), 246: 1149-1 152. Morrison, J.F. 1969. Kinetics of the reversible inhibition of enzyme-
catalyzed reactions by tight-binding inhibitors. Biochim. Biophys. Acta, 185: 269-286. Nashed, N.T., Louis, J.M., Sayer, J.M., et al. 1989. Continuous spectroscopic assay for retroviral proteases of HIV-1 and AMV. Biochem. Biophys. Res. Commun. 163: 1079-1085. Navia, M.A., Fitzgerald, P.M.D., McKeever, B.M., et al. 1989. Three-dimensional structure of aspartyl protease from HIV-1. Nature (London), 337: 615-620. Pearl, L., and Taylor, W. 1987. A structural model for the retroviral proteases. Nature (London), 329: 351-354. Rao, J.K.M., Erickson, J.W., and Wlodawer, A. 1991. Structural and evolutionary relationship between retroviral and eucaryotic aspartic proteinases. Biochemistry, 30: 4663-467 1. Rich, D.H., Green, J., Mihaly, V.T., et al. 1990. Hydroethylamine analogues of the p17/p24 substrate cleavage site are tight-binding inhibitors of HIV protease. J. Med. Chem. 33: 1285-1288. Roberts, N., Martin, J.A., Kinchington, D., et al. 1990. Rational design of peptide-based HIV proteinase inhibitors. Science (Washington, D.C.), 248: 358-361. Seelmeier, S., Schmidt, H., Turk, V., and von der Helm, K. 1988. HIV has an aspartic-type protease that can be inhibited by pepstatin A. Proc. Natl. Acad. Sci. U.S.A. 85: 6612-6616. Shoeman, R.L., Kesselmeier, C., Mothes, E., et al. 1991. Nonviral cellular substrates for HIV-1 protease. FEBS Lett. 278: 199-203. Silverman, R. 1988. Mechanism-based enzyme inactivators: chemistry and enzymology. CRC Press, Boca Raton, Fla. p. 22 Swain, A.L., Miller, M.M., Green, J., et al. 1990. X-ray crystallographic structure of a complex between a synthetic protease of HIV-1 and a substrate-based hydroxyethylamine inhibitor. Proc. Natl. Acad. Sci. U.S.A. 87: 8805-8809. Tamburini, P.P., Dreyer, R.N., Hansen, J., et al. 1990. A fluorometric assay for HIV protease activity using highperformance liquid chromatography. Anal. Biochem. 186: 363-368. Toh, H., Ono, M., Saigo, K., and Miyata, T. 1985. Retroviral protease-like sequence in the yeast transposon TYl. Nature (London), 315: 691-692. Tomasselli, A.G., Hui, J.O., Adams, L., et al. 1991. Actin, troponin C, Alzheimer amyloid precursor protein and prointerleukin 1,8 as substrates of the protease from HIV. J. Biol. Chem. 266: 14 548 - 14 553. Turner, D.C., and Brand, L. 1968. Quantitation estimation of protein binding site polarity: fluorescence of N-arylaminonaphthalenesulfonates. Biochemistry, 7: 3381-3390. Tyagi, S.C., and Carter, C.A. 1992. Continuous assay of the hydrolytic activity of HIV-1 protease. Anal. Biochem. 200: 143-148. Wallin, M., Deinum, J., Goobar, L., and Danielson, U.H. 1990. Proteolytic cleavage of microtubule-associated proteins by retroviral proteinases. J. Gen. Virol. 71: 1985-1991. Williams, J.W., and Morrison, J.F. 1979. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 63: 437-467. Wolfenden, R. 1976. Transition state analog inhibitors and enzyme catalysis. Annu. Rev. Biophys. Bioeng. 5: 271-306. Zhang, Z.-Y., Poorman, R.A., Maggiora, L.L., et al. 1991. Dissociative inhibition of dimeric enzymes. J. Biol. Chem. 266: 15 591 - 15 594.
TYAGI'
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Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A. Received October 29, 1991 TYAGI,S. C. 1992. Inhibitors of human immunodeficiency virus-1 protease. Biochem. Cell Biol. 70: 309-315. We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) 27 nM), resulting in pure competitive inhibition of the can be characterized by a high-affinity mode (Ki = 478 hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 pM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 + 1 pM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 pM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease. Key words: HIV-1 protease, inhibitors, pepstatin, haloperidol, tryptophan fluorescence. TYAGI,S. C. 1992. Inhibitors of human immunodeficiency virus-1 protease. Biochem. Cell Biol. 70 : 309-315. Nous avons montrt que la rtaction de la pepstatine A avec la prottase du virus-1 de l'immunodtficience humaine (HIV-1 prottase) peut Etre caractbisk par un mode d'affinitt tlevt (Ki = 478 27 nM) rtsultant en une pure inhibition compttitive de l'activitt hydrolytique de la HIV-1 prottase B l'tgard d'un substrat fluorogkne. La liaison de la pepstatine dans ce mode induit un dtplacement vers le bleu dans la fluorescence endogkne provenant des rtsidus de tryptophane dans la HIV-1 prottase. Ce dtplacement est maximal en prCsence de pepstatine 10 pM. En revanche, l'halopbidol rtagit avec la HIV-1 prottase avec une affinitt plus faible (Ki = 19 1 pM) selon un mode qui rtsulte en une pure inhibition non compttitive de l'activitt hydrolytique de la HIV-1 prottase. La liaison de l'haloptridol dans ce mode induit un dtplacement vers le rouge dans la fluorescence endogkne provenant des rtsidus de tryptophane dans la HIV-1 prottase. Ce dtplacement est maximal en prbence de I'haloptridol200 pM. L'addition B la fois de pepstatine et d'haloptridol en des concentrations dans la garnme des valeurs de leur Ki entraine une inhibition additive de l'activitt hydrolytique de la HIV-1 prottase de mEme qu'un effet additif sur la fluorescence du tryptophane de la prottase. Cependant, B des concentrations saturantes de pepstatine et d'haloptridol, l'effet de l'halopbidol, tel que mesurt par les changements dans la fluorescence intrinskque de la HIV-1 prottase, est prtdominant. Mots cl4s : HIV-1 prottase, inhibiteurs, pepstatine, halopbidol, fluorescence de tryptophane. [Traduit par la rtdaction]
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Introduction Human immunodeficiency virus, a member of the retroviral group, is the causative agent of AIDS (Barre-Sinoussi et al. 1983; Gallo et al. 1984). Proteolytic processing of structural components and replicative enzymes by a virally encoded protease is an essential step in the maturation of infectious viral particles. This protease also has the capacity to degrade some of the cytoskeletal proteins of the host cell (Wallin et al. 1990; Shoeman et al. 1991; Tomasselli et al. 1991), and thus, is an important target for development of antiviral drugs. The protease is a member of the family of aspartic proteases, like pepsin and renin, but unlike these enzymes, exists as a dimer of the identical subunits, with each monomer contributing an Asp-Thr-Gly sequence to the active site (Toh et al. 1985; Navia et al. 1989; Miller et al. 1989). ABBREVIATIONS: HIV-1 protease, human immunodeficiency virus-1 protease; AIDS, acquired immunodeficiency syndrome; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; Bz-, benzoyl-; MeO-, methoxy-; kDa, kilodalton(s). 'present address: Division of Cardiology, Department of Medicine, Medical Sciences Building, Room No. 431A, University of Missouri, Columbia, MO 65212. U.S.A. Printed in Canada / Imprim6 au Canada
Rational design of drugs directed against HIV-1 protease requires an understanding of the mechanism by which it interacts with and catalyzes the hydrolysis of substrate. Substrate peptides, with cleavage sites at Phe-Pro or TyrPro, have been used to measure protease activity (Darke et al. 1988; Billich et al. 1988; Krausslich et al. 1989; Graves et al. 1988). It is difficult, however, to perform rigorous mechanistic analysis in these assays because there is no detectable signal during catalysis. Recently, several studies including our own (Tyagi and Carter 1992) have described the use of fluorescent substrates and energy transfer techniques (Matayoshi et al. 1990a, 1990b; Tamburini et al. 1990), as well as chromogenic substrates (Nashed et al. 1989; Dilanni et al. 1990), which should help in this regard. The transition-state analogue concept (Wolfenden 1976; Dreyer et al. 1989; Ashorn et al. 1990; Meek et al. 1990; Roberts et al. 1990) suggests a general strategy for the design of inhibitors of the aspartic proteases in which the scissile carbonyl of an oligopeptide substrate is replaced by a nonhydrolyzable analogue with tetrahedral geometry. This strategy has proven fruitful in the search for renin inhibitors (Fischer 1988). The natural product pepstatin, containing the unusual amino acid statine, binds with dissociation constants in the subnanomolar range (Dreyer et al. 1989) and is a potent inhibitor specific for most aspartic proteases.
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Exceptions are the mammalian aspartic protease renin (Boger et al. 1983) and the retroviral proteases (Giam and Boros 1988; Darke et al. 1989; Seelmeier et al. 1988; Katoh et al. 1987), for which pepstatin is much less inhibitory. Peptide-based inhibitors with submicromolar inhibitory activity towards protease in vivo have been shown t o be effective in reducing viral infectivity in cultured T4 cells and in inhibition of gag polyprotein processing in vitro (Meek et al. 1990; McQuade et al. 1990). Recently, Zhang et al. (1991) have reported that a C-terminal sequence of HIV-1 protease, tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, is an excellent inhibitor of this enzyme. They have shown that this tetrapeptide binds t o the inactive protomers and prevents their association into the active dimer and cause a dissociative inhibition of HIV-1 protease. They have reported a dissociation constant (Kd) for the dimer of 3.5 nM and an inhibition constant (Ki) for the tetrapeptide of 45 kM. However, peptide-based inhibitors are often therapeutically ineffective when orally administered, stimulating interest in a search for nonpeptidic inhibitors (Rich et al. 1990; Dreyer et al. 1989). Structure-based design of nonpeptide inhibitors for HIV-1 protease was described by DesJarlais et al. (1990), who discovered that the drug haloperidol (Fig. 1) has some inhibitory activity against HIV-1 protease. Based on X-ray crystallographic data, DesJarlais et al. (1990), Miller et al. (1989), Swain et al. (1990), Erickson et al. (1990), Fitzgerald et al. (1990), and Rao et al. (1991) postulated that haloperidol may induce conformational changes in HIV-1 protease by binding at the dimer interface (DesJarlais et al. 1990). Precise experimental support for this prediction is limited. The present work is aimed at further characterizing the nature of the inhibition of HIV-1 protease by pepstatin and haloperidol and the relationship of this inhibition t o alterations in protease structure. We have examined inhibition of HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate peptide by pepstatin and haloperidol. In addition, we have studied the effect of pepstatin and haloperidol o n the endogenous fluorescence of the two tryptophan residues in HIV-1 protease t o see if these inhibitors induce perturbations in the structure of the HIV-1 protease.
Materials and methods Materials HIV-1 protease was expressed and isolated from Escherichia coli according to the method of Graves et al. (1989) and was a generous gift from Dr. M. C. Graves (Department of Molecular Genetics, Hoffmann-La Roche Inc. Nutley, N.J.). Protease was 98% pure as judged by SDS-PAGE and silver staining. Pepstatin A, haloperidol, cytochrome c, EDTA. DMSO, urea, DTT, Bz-ArgGly-Phe-Pro-MeO-Na (substrate), and Pro-MeO-Na (product) were from Sigma (St. Louis, Mo.). All other reagents were of standard grade. Enzymatic activity Hydrolytic activity of HIV-1 protease was assayed with Bz-ArgGly-Phe-Pro-MeO-Na. The release of Pro-MeO-Na was monitored by recording fluorescence at 420 nm with excitation at 346 nm in a 3 x 3 rnm (120-pL capacity) cuvette. The substrate concentration was varied from 0.5 to 6 mM. Based on weight measurements of lyophilized sample of this 22-kDa protein, HIV-1 protease concentration ranged from approximately 0.1-1 pM in the different studies reported here. All measurements were carried out in 100 mM sodium acetate (pH 6) containing 1 M NaCl, 1 mM
1992
FIG. 1. Chemical structure of haloperidol. EDTA, 1 mM DTT, and 10% DMSO. The final HIV-1 protease concentration was set at 200 nM for determination of the dissociation constant Kiof pepstatin or haloperidol. Kinetic data were collected for 5 min. The fluorescence increase was recorded at each second beginning just after mixing. The initial velocity was calculated from fluorescence changes over the fist 20-30 s. During this time the increases in fluorescencewere linear with no lag period. The data were converted into the concentration of released product (Pro-MeO-Na) according to the following: Moles Pro-MeO-Na released = F x [Pro-MeO-Na],/F, where F, [Pro-MeO-Na], and F, are the initial fluorescence reading as the function of time, and concentration and fluorescence of Pro-MeO-Na of the standard, respectively. The fluorescence of standard Pro-MeO-Na was recorded at the same time and the same instrument settings as for the experimental reaction. The initial velocities (mol Pro-MeO-Na released/s) in the presence of inhibitor (V)were all normalized to the velocity in the absence of the inhibitor (V,).
Enzymatic inhibition Various concentrations of pepstatin and haloperidol ranging from 0.5 to 10 pM for pepstatin and 5 to 100 pM final concentration for haloperidol were mixed with 0.2 p M HIV-1 protease at 25°C prior to addition of Bz-Arg-Gly-Phe-Pro-MeO-Na, to give a final substrate concentration ranging from 2 to 4 mM. Comparable results were obtained when the order of addition of inhibitor and substrate was reversed, showing that a steady state was established within the manual mixing time. All reactions were carried out in 100 mM sodium acetate, 1 M NaCl, 1 mM EDTA, 1 mM DTT, and 10% DMSO. Each data point was done in triplicate and enzymatic hydrolysis rates were fit to the Michaelis-Menten equation using a nonlinear regression program ("Enzfitter" from Biosoft). Spectroscopic methods Absorption spectra were recorded with a Hewlett Packard 8452A diode array spectrophotometer. The concentration of HIV-1 protease was determined by employing the Bradford (1976) dye-binding assay. The concentration of protease was also checked by absorbance at 280 nm, in experiments where relative comparisons were made. The concentration of Bz-Arg-Gly-Phe-Pro-MeO-Na (substrate) was based on weight measurements and compared with the concentrationdetermined by extinction coefficient for naphthyl derivatives at about 340 nm ( E = 6000 M - .cm - ') (Turner and Brand 1968). The concentrations of Pro-MeO-Na (product), pepstatin, and haloperidol were based on weight measurements. Fluorescence spectra were recorded on a computer-controlled Spex Datamate spectrofluorometer connected to a circulating water bath to control temperature. The excitation and emission slits were adjusted for 5 nm band-pass width. Spectra were recorded at 1-nm intervals and corrected for base line and instrument response. Samples were prepared and incubated for appropriate times prior to measurements at 25°C. Tryptophan fluorescence was determined with )h, at 295 nm. Protein solutions used for these measurements had an absorbance of less than 0.05 to avoid inner filter effects (Lakowicz 1983). Pepstatin and haloperidol do not absorb in the wavelength range where tryptophan absorbs or emits from approximately 290 to 350 nm.
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Analytical gel filtration A 20-pL sample (20 pg) of HIV-1 protease was loaded on to a 1 x 18 cm Sephacryl S-200 column equilibrated with buffer A
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[Pepstatinl/[HIV- I Protease] FIG. 2. Inhibition of HIV-1 protease (200 nM) by various concentrations of pepstatin A. After addition of pepstatin to the enzyme, the residual hydrolytic activity was assayed by addition of various concentrations of Bz-Arg-Gly-Phe- Pro-MeO-Na as described in Materials and methods. The hydrolytic rates (V) were all normalized to the rate observed in the absence of pepstatin (V,). The curve is a nonlinear least-squares fit to the data points, according to the tight-binding inhibition equation (Williams and Morrison 1979). Data were obtained at 2 mM Bz-Arg-Gly-Phe-Pro-MeO-Na. Insert: replots of the data at 2 (m) and 4 mM (a) substrate concentrations. The lines are the linear least-squares fit to the data.
(100 mM sodium acetate (pH 6.0), 1 M NaC1, 1 mM EDTA, 1 mM DTT, and 10% DMSO) at 4°C. The fractions of 0.38 mL were collected and measured for their protein fluorescence at 340 nm when excited at 295 nm. The band-pass slits were 5 nm for both the excitation and emission, respectively. The hydrolytic activity of these fractions was measured by employing Bz-Arg-Gly-PhePro-MeO-Na as a fluorogenic substrate. Cytochrome c (12.5 kDa) was employed as a marker protein on this column.
Results and discussion Inhibition of hydrolytic activity of HIV-1 protease In order to conform to the criteria outlined by Silverman (1988) for analysis of inhibition by the partition ratio method, we varied the ratios of pepstatin to HIV-1 protease from 2.5 to 50 and the ratios of haloperidol to enzyme from 25 to 500 in the hydrolytic assays. Pepstatin is a tightly bound inhibitor of HIV-1 protease. The level of inhibition of protease by pepstatin was independent of the order of addition of substrate and inhibitor within the limits of manual mixing times, justifying the assumption of equilibrium binding. Under conditions in which a significant fraction of the inhibitor is bound, the inhibition can be analyzed by the partition ratio method (Silverman 1988). The inhibition is sensitive to the substrate concentration. As can be seen from Fig. 2, the slope of this plot, Ki,,,, is dependent on substrate concentration and reflects some competitive character. The data can be fit according to Henderson plots to conform to a model of simple competitive inhibition (Fig. 2) (Green and Work 1953; Morrison 1969; Henderson 1972; Bieth 1974; Empie and Laskowski 1982; Bieth 1984):
where [pepstatinlo and [HIV-1 Proteaselo are the initial total concentrations of pepstatin and HIV-1 protease, respectively. [S], K,, and Ki are the substrate concentration, binding constant of substrate to enzyme, and the dissociation constant between enzyme and inhibitor, respectively. Vand Vo are the initial rates of substrate hydrolysis in the presence and absence of inhibitor, respectively. From plots at several different substrate concentrations, we computed a value of Ki for pepstatin of 478 & 27 nM. The hydrolysis of Bz-Arg-Gly-Phe-Pro-MeO-Na by HIV-1 protease in the absence of inhibitors conformed to simple Michaelis-Menten kinetics. The apparent value of K, for this fluorogenic substrate under the conditions we employed is 2.0 + 0.2 mM (Tyagi and Carter 1992). Pepstatin has been recognized as a peptide analog inhibitor of HIV-1 protease, which binds to the enzyme with an affinity in the low micromolar range (Darke et al. 1989). The mode of inhibition by pepstatin has been described as mixed by Darke et al. (1989) and as linear competitive by Dreyer et al. (1989). Under our assay conditions, we observed that pepstatin appeared to have a purely competitive mode of inhibition with a Ki in the submicromolar range. This transition-state analog appeared to possess a structure that was optimal for interactions with active site residues in HIV-1 protease. Pepstatin, once bound, appeared to prevent substrate binding to the enzyme; we were hindered from obtaining any evidence for effects of pepstatin on catalytic activity under these conditions, since the competitive mode of inhibition was complete. Inhibition of HIV-1 protease by haloperidol was found to be associated with binding of relatively low affinity: at a ratio of haloperidol to HIV-1 protease of 10 pM to 0.2 pM, the hydrolytic activity was reduced only 10-15%. The extent
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[Haloperidol]/[HIV- 1 Proteasel FIG.3. Inhibition of HIV-1 protease (200 nM) by various concentrations of haloperidol. After addition of haloperidol to the enzyme, the residual hydrolytic activity was assayed by addition of various concentrations of Bz-Arg-Gly-Phe-Pro-MeO-Na as described in Materials and methods. The hydrolytic rates (V) were all normalized to the rate observed in the absence of haloperidol (Vo).The curve is a nonlinear least-squares fit to the data points, according to the modification of Dixon (1953) equation. Data were obtained at 2 mM Bz-Arg-Gly-Phe-Pro-MeO-Na.Insert: replots of the data at 2 (w) and 4 mM ( 0 )substrate concentrations. The line is the linear leastsquares fit to the data. of inhibition was independent of the incubation period, indicating rapid (within manual mixing time) binding between haloperidol and protease, justifying the assumption of a steady state model. Accordingly, we analyzed this inhibition (Fig. 3) by a modification of the Dixon plot (1953): 1 -
-
Ki.app
+
TABLE1. Additive effect of pepstatin A and haloperidol as inhibitors of hydrolytic activity of Bz-Arg-Gly-Phe-Pro-MeO-Na by HIV-1 protease: hydrolysis of 2 m M Bz-Arg-Gly-Phe-Pro-MeO-Na by 200 nM HIV-1 protease in 100 mM sodium acetate (pH 6.0) - 1 M NaCl, 1 mM EDTA - 1 mM DTT 10% DMSO at 2S°C, in the presence or absence of inhibitors
1- V V [haloperidol] Reactants V/V , In this expression, the inhibitor concentration [haloperidol] is rigorously defined as the concentration of free inhibitor, HIV-1 protease 1.00 HIV-1 protease + pepstatin 0.52 but since the total inhibitor concentration is about two HIV-1 protease + haloperidol 0.54 orders of magnitude greater than that of the total enzyme HIV-1 protease + pepstatin + haloperidol 0.23 concentration, the amount of bound inhibitor can be disregarded. The value of KisaPp for this mode of inhibition NOTB:Five min prior to addition of substrate, 200 nM emzyme was added to the 1 pM pepstatin and 20 pM haloperidol, respectively, and is independent of substrate concentration and therefore the subsequent hydrolytic rate V was compared with the rate observed reflects noncompetitive character. The general expression in the absence of any added inhibitor. Yo. gives a true value of Ki in this case. The value of Ki for pounds at their apparent Ki values to 200 nM HIV-1 prohaloperidol inhibition computed from the data in Fig. 3 is tease to see if they were additive with each other for inhibi19 + 1 pM. tion of hydrolytic activity of the enzyme (Table 1). In the DesJarlais et al. (1990) found that inhibition of hydrolysis presence of 1 pM pepstatin alone, the hydrolysis of 2 mM of a decapeptide (Ala-Thr-Leu-Asn-Phe-Pro-Ile-Ser-ProBz-Arg- Gly-Phe-Pro-MeO-Na was inhibited 48%, whereas Trp) by haloperidol was of the mixed type with both parin the presence of 20 @ haloperidol, I the hydrolysis was tial competitive and noncompetitive components. Under our assay conditions, we observed that inhibition of Bz-Arg-Glyinhibited 46%. Hydrolysis was inhibited 77% in the presence of a mixture of 1 pM pepstatin + 20 pM haloperidol, Phe-Pro-MeO-Na, a shorter peptide, was purely nonsuggesting that both inhibitors do not compete for the same competitive with an affinity to HIV-1 protease in the low site on the enzyme, but rather are approximately additive micromolar range (Fig. 3). in their inhibition of hydrolytic activity of HIV-1 protease. Pepstatin and haloperidol are noncompetitive with each Effect of pepstatin and haloperidol on HIV-1 protease other tryptophan fluorescence Since pepstatin appears to bind tightly to HIV-1 protease as a competitive inhibitor, whereas haloperidol binds as a The two tryptophan residues in HIV-1 protease give rise noncompetitive inhibitor, we added both of these comto a characteristic emission spectrum with a maximum
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Fraction no.
Wavelength FIG. 4. Endogenous tryptophan fluorescence of HIV-1 protease: HIV-1 protease (- . -,0.2 pM), HIV-1 protease plus 10 pM pepstatin A (- - -), and HIV-1 protease plus 200 pM haloperidol (-). The samples were incubated prior to recording the spectra in pH 6 buffer. The spectra were corrected for buffer base line, instrument response, and contributions due to free pepstatin and haloperidol, respectively. The excitation was 295 nm.The slits were 5 nm for both the excitation and emission wavelengths.
around 340 nm with excitation at 295 nm. The effect of pepstatin and haloperidol on this endogenous fluorescence was studied over a range of pepstatin and haloperidol concentrations to identify effects which might be ascribed to binding. In the presence of 10 pM pepstatin, the tryptophan fluorescence of 0.2 pM HIV-1 protease was shifted about 5 nm to shorter wavelengths. Similarly, when 200 pM haloperidol was added to 0.2 pM HIV-1 protease, the endogenous fluorescence from Trp residues in HIV-1 protease was shifted about 7 nrn to longer wavelengths (Fig. 4). These changes were reversed upon dilution of the pepstatin and haloperidol, respectively, indicating that neither haloperidol nor pepstatin irreversibly denatures the protease. The red shift upon haloperidol binding implies more Trp exposure to solution and therefore a more open conformation and the blue shift upon pepstatin binding implies more Trp surrounded by the hydrophobic residue@)and therefore induces compact conformation in the protease structure. Since pepstatin, a transition-state analog, appears to bind tightly to the active site in the enzyme and to induce a compact conformation in protease structure, whereas haloperidol binds less tightly and induces an open conformation in the protease structure, we added both of these inhibitors at their apparent Kiand at 10 times their apparent Kivalues, respectively, to 200 nM HIV-1 protease to see if they were also additive for induction of conformation changes in the enzyme. In the presence of l pM pepstatin the changes in Trp fluorescence were about half the magnitude of the changes observed at saturating con-
FIG. 5. Analytical gel filtration of HIV-1 protease on Sephacryl S-200. Twenty microlitre (20 pg) of HIV-1 protease was loaded on the column. Fractions of 0.38 mL each were collected and their fluorescence at 340 nm was measured when excited at 295 nm. The band-pass slits were 5 nm for both the excitation and emission, respectively. The protein was eluted with buffer A (100 rnM sodium acetate (pH 6.0). 1 M NaCI, 1 mM EDTA, 1 mM DTT, and 10% DMSO) (m), with buffer A plus 100 pM pepstatin (a), and with buffer A plus 2 mM haloperidol (A). All buffer solutions were at 4OC.
centration of pepstatin. Similarly 20 pM haloperidol induced changes in Trp fluorescence, about half the magnitude of the changes observed in the presence of saturating amount of haloperidol. This indicates also that the binding events detected by fluorescence are probably equivalent to the inhibitory binding sites on the protease. However, in the presence of mixture of 10 pM pepstatin and 200 pM haloperidol, tryptophan fluorescence was red shifted as has been seen for haloperidol alone (Fig. 4) after correcting the emission spectra for free pepstatin and haloperidol in the buffer. These data suggest that haloperidol alters the conformation of the protease in a way that cannot be reversed by pepstatin binding. However, changes in tryptophan fluorescence induced by pepstatin alone appeared to be reversed by haloperidol binding in the HIV-1 protease. This may suggest that haloperidol binds to the dissociated subunit (protomer) more tightly than the dimer of the protease. Since pepstatin and haloperidol are additive in their inhibition, it may be possible that the effect of pepstatin on fluorescence is too small to be reliably detected on top of a bigger signal, e.g., red shift from haloperidol binding. X-ray crystallographicexamination indicates that transitionstate analogs (inhibitor or substrate) bound at the active site can induce movement around the flap region of the enzyme, resulting in a more compact conformation in the protease structure (Miller et al. 1989; Erickson et al. 1990). This is supported by our observation that binding of pepstatin to HIV-1 protease induces a blue shift in the intrinsic Trp fluorescence of the protease. This blue shift indicates compactness in the structure of protease which produces a more hydrophobic environment or steric coverage around Trp residue@)by pepstatin. The possible Trp residue for steric coverage by pepstatin may be the ~ r p which ~ , is closer to the active site than ~r~~~(Miller et al. 1989; Erickson et al. 1990; Pearl and Taylor 1987). The binding of haloperidol to the enzyme induced a reduction in the catalytic activity and no alteration in substrate
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binding. This mode of binding is supported not only by our kinetic analysis of substrate hydrolysis but also by the red shift in Trp fluorescence. This red shift suggests a decrease in hydrophobicity in the environment of at least one of the two indole side chains in HIV-1 protease. Changes in the intrinsic Trp fluorescence of HIV-1 protease suggested that the high-affinity competitive mode of binding of pepstatin to the protease can be altered by the low-affinity noncompetitive binding of haloperidol to the enzyme, when both inhibitors are present at concentrations sufficient to saturate their binding sites if they were added separately. These studies suggested that binding of a transition-state analog (pepstatin or substrate analog) at the active site may be disrupted by the binding of a nonpeptidic compound at the noncompetitive site in the protease. This may suggest that the binding of haloperidol to the monomer is stronger than to the dimer of HIV-1 protease, so that it prevents dimer formation.
Effect of inhibitors on the apparent elution profile on a gel filtration column of protease As suggested by structural modeling, the retroviral proteases would most resemble the structure of the other aspartic proteases if they were assembled as dimers (Pearl and Taylor 1987). This prediction was confirmed by crystallography (Miller et al. 1989; Erickson et al. 1990). The molecular mass of HIV-1 protease obtained under denaturing conditions was in accord with the value of 11 kDa found upon SDS-PAGE analysis of the purified protease. Analytical gel filtration of purified protease on Sephacryl S-200 demonstrated that HIV-1 protease fluorescence and activity eluted in a sharp peak at 8.2 mL (fraction no. 21-22) (Fig. 5). The position of this peak in the elution profile was shifted to 10 mL (fraction no. 26) when a saturating amount (2 mM) haloperidol was present in the elution buffer A. This peak contained only fluorescence, but no activity. However, the presence of 100 FM pepstatin in buffer A did not change the elution profile of HIV-1 protease on this column under the conditions we applied. The peak in the presence of pepstatin also contained fluorescence, but no activity. Cytochrome c (12.5 kDa) was employed as the protein calibration standard under the same identical conditions as used above in buffer A and was eluted at 9.75 mL. HIV-I protease migrates as a dimer on gel filtration column (Meek et al. 1989). We have shown, based on the elution profile on an analytical gel filtration column, that the presence of haloperidol appears to induce conformational changes in the protease structure in a way that the protease-haloperidol migrates as a monomer complex. This result is consistent with our observation, based on the intrinsic fluorescence of protease in the presence of haloperidol and pepstatin, that haloperidol induces an open conformation whereas pepstatin induces a compact conformation in the protease structure. The possibility that the effect of pepstatin on fluorescence is too small to be reliably detected on top of a bigger signal towards the longer wavelength induced by binding of haloperidol may not be consistent with the elution pattern of protease on gel filtration in the presence of a saturating concentration of haloperidol, which appears to induce dissociation of the protease dimer. These observations support a rational basis for design of synthetic HIV-1 protease inhibitors with high specificity and
affinity. Evidence of interactions of the transition-state inhibitor with the enzyme through hydrophobic domains is seen in the characteristic changes in the emission spectra of bound pepstatin. In addition, nonhydrophobic interactions are apparently involved in binding of haloperidol. These two modes of interaction between the HIV-1 protease and the inhibitors are structurallyvery different and suggest that they may be combined to obtain inhibitors that are both potent and stable in chemical therapy. These studies may serve as a useful lead for the development of a new class of antiretroviral agents. Acknowledgements The author thanks Drs. Carol Carter and Mary Graves for providing HIV-1 purified protease, and Drs. Sanford R. Simon, Carl Moos, and Rodney A. Bednar for their critical review of the manuscript. This work was supported in part by grants AI-25993 and HL-14262 from the National Institutes of Health. Ashorn, P., McQuade, T., Thaisrivongs, S., et al. 1990. An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proc Natl. Acad. Sci. U.S.A. 87: 7472-7476. Barre-Sinoussi, F., Cherman, G.C., Rey, G., et al. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science (Washington, D.C.), 220: 868-870. Bieth, J. 1974. Some kinetic consequences of the tight binding of protein-protease-inhibitors to proteolytic enzymes and their application to the determination of dissociation constants. BayerSymp. 5: 463-469. Bieth, J. 1984. In vivo significance of kinetic constants of protein proteinase inhibitors. Biochem. Med. 32: 387-397. Billich, S., Knoop, M.-T., Hansen, J., et al. 1988. Synthetic peptides as substrates and inhibitors of HIV-1 protease. J. Biol. Chem. 263: 17 905 - 17 908. Boger, J., Lohr, N.S., Ulm, E.H., et al. 1983. Novel renin inhibitors containing the amino acid statine. Nature (London), 303: 81-84. Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Darke, P.L., Nutt, R.F., Brady, S.F., et al. 1988. HIV-1 protease specificity of peptide cleavage is sufficient. Biochem. Biophys. Res. Commun. 156: 297-303. Darke, P L., Leu, C.-T., Davis, L.J., et al. 1989. HIV-1 protease. J Biol. Chem. 264: 2307-2312. DesJarlais, R.L., Seibel, G.L., Kuntz, I.D., et al. 1990. Structurebased design of nonpeptide inhibitors specific for the HIV-1 protease. Proc. Natl. Acad. Sci. U.S.A. 87: 6644-6648. Dilanni, C.L., Davis, L.J., Holloway, M.K., et al. 1990. Characterization of an active single polypeptide form of the HIV-1 protease. J. Biol. Chem. 265: 17 348 - 17 354. Dixon, M. 1953. The determination of enzyme inhibition constants. Biochem. J. 55: 170-171. Dreyer, G.B., Metcalf, B.W., Tomaszek, T.A., et al. 1989. Inhibition of HIV-1 protease in vitro: rational design of substrate analogue inhibitors. Proc. Natl. Acad Sci. U.S.A. 86: 9752-9756. Empie, M.W., and Laskowski, M., Jr. 1982. Thermodynamics and kinetics of single residue replacements in avian ouomucoid third domain: effect of inhibitor interactions with serine proteases. Biochemistry, 21: 2274-2284. Erickson, J.W., Neidhart, D.J., VanDrie, J., et al. 1990. Design, activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science (Washington, D.C.), 249: 527-533.
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