6646
Biochemistry 1992,31, 6646-6659
Hydroxyethylene Isostere Inhibitors of Human Immunodeficiency Virus- 1 Protease: Structure-Activity Analysis Using Enzyme Kinetics, X-ray Crystallography, and Infected T-cell Assays+,$ Geoffrey B. Dreyer,'.s Dennis M. Lambert,ll Thomas D. Meek,$ Thomas J. Carr,$ Thaddeus A. Tomaszek, Jr,,s Annabellee V. Fernandez,ll Henry Bartus,ll Emilio Cacciavillani,* Anne M. Hassell,l,# Michael Minnich,o Stephen R. Petteway, Jr.) and Brian W. MetcalP Departments of Medicinal Chemistry, Antiinfectives, Macromolecular Sciences, and Protein Biochemistry, SmithKIine Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, Pennsylvania I9406
Mitchell Lewis The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania I91 04 Received October 21, 1991; Revised Manuscript Received March 24, I992
ABSTRACT: Analogues of peptides ranging in size from three to six amino acids and containing the hy-
droxyethylene dipeptide isosteres PheSGly, PhePAla, PheSNorVal, PheSLeu, and PheSPhe, where S denotes replacement of C O N H by (S)-CH(OH)CH*, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (Ki)with purified HIV-1 protease depend strongly on the isostere in the order PheSGly > PheSAla > PheSNorVal > PheSLeu > PheSPhe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. Ki values are progressively less dependent on inhibitor length as the size of the P1' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is PheSGly, PheSAla, PheSNorVal, and PheSPhe, have been determined by X-ray crystallography (resolution 2.3-3.2 A). The crystals exhibit symmetry consistent with space group P61 with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The PheSGly inhibitor adopts an altered conformation that places its P3'valine side chain partially in the hydrophobic S1' pocket, thus suggesting an explanation for the greater dependence of the Ki value on inhibitor length in the PheSGly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S 2 S 2 ' are optimally occupied. The Ki values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of Ki and those of MIC or ICSO.ICs0 values can approach those of Ki but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.
The aspartic protease of the human immunodeficiencyvirus1 (H1V-l)l carries out the posttranslational processing of the viral gag and gag-pol proteins into functional viral components (Ratner et al., 1985; Kramer et al., 1986; Debouck et al., 1987; Petteway et al., 1991). The initial translation product of the gag gene, Pr55, is cleaved to yield the structural proteins of the virion core, namely, p17, p24, p7, and p6 (Veronese et al., 1987). Processing of the gag-pol translation product releases the viral replicative enzymes (protease, reverse trant Supported in part by Grant GM-39526 from the National Institutes of Health. A preliminary account of this work was presented at the 22nd National Medicinal Chemistry Symposium, Austin, TX, July 30, 1990. t The crystallographic coordinates have been deposited in the Brookhaven Protein Data Bank under the file name laaq. * Author to whom correspondence should be addressed. i Department of Medicinal Chemistry, L420, SmithKline Beecham Pharmaceuticals. 11 Department of Antiinfectives, SmithKlineBeecham Pharmaceuticals. 1 Department of Macromolecular Sciences, SmithKline Beecham Pharmaceuticals. i+ Present address: Glaxo Inc., 5 Moore Drive, Research Triangle Park, N C 27709. O Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals.
0006-2960/92/043 1-6646$03.OO/O
scriptase/ribonuclease H, and integrase). The protease is essential to the retroviral life cycle, since disrupting the proteolytic activity of HIV- 1 through either site-directed mutagenesis (Kohl et al., 1988; Gottlinger et al., 1989; Peng et al., 1989) or addition of specific protease inhibitors (Meek et al., 1990; Roberts et al., 1990; Ashorn et al., 1990; Lambert et al., 1992) results in formation of noninfectious virions of aberrant morphology and prevents the spread of infection. These observations have led to extensive efforts to develop effective drugs targeting HIV- 1 protease. Analogues of peptide substrates in which the scissile dipeptide is replaced with mimetics of the proteolysis transition Abbreviations: AIDS, acquired immunodeficiencysyndrome;HIV1, human immunodeficiency virus type 1; Boc, left-butyloxycarbonyl;
Cbz, benzyloxycarbonyl; HPLC, high-performance liquid chromatography; 'HNMR, proton nuclear magneticresonance;NOE, nuclear Overhauser effect; TBDMS, terr-butyldimethylsilyl;THF, tetrahydrofuran; IC50, 50% inhibitory concentration by RT analysis; MIC, minimum inhibitory concentration by Western blot analysis; RT, reverse transcriptase;TCIDso, 50%infectious dose by viral titration in tissue culture; DMSO, dimethyl sulfoxide;EDTA, ethylenediaminetetraaceticacid; FBS, fetal bovine serum; Mes, 2-(N-morpholino)ethanesulfonic acid; PAGE. polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
0 1992 American Chemical Society
SAR Analysis of HIV-1 Protease Inhibition state are effective inhibitorsof purified HIV-1 protease (Dreyer et al., 1989; Tomasselli et al., 1990; Rich et al., 1990; Roberts et al., 1990; Ashorn et al., 1990; Vacca et al., 1991; Lyle et al., 1991). In a direct comparison of several such transitionstate mimetics, hydroxyethyleneisosteres afforded particularly potent HIV-1 protease inhibitors in a purified enzyme assay (Dreyer et al., 1989). Some but not all of these inhibitors blocked viral replication in infected T-cell assays, suggesting that their antiviral activity depends not only on inhibition of the purified enzyme but also on molecular properties relating to their cell permeability and stability (Meek et al., 1990). Realization of the antiviral potential of protease inhibitors willdependon the ability to proceed frominitial lead structures to molecules which possess the spectrum of antiviral potency, low toxicity, and bioavailability required in a therapeutic agent. Low oral bioavailability and rapid biliary excretion of peptidelike inhibitors of human renin (also an aspartic protease) have impeded the development of such agents for hypertension (Humphrey & Ringrose, 1986;Greenlee, 1990). While factors affecting oral absorption and clearance mechanismsare poorly understood, efforts to overcome these problems include reduction of molecular weight and number of amide bonds and adjustment of lipophilicity. To provide a foundation for these efforts, in the present study we have used synthetic chemistry, enzymology,and X-ray crystallography to develop a detailed understanding of the relationships of inhibitor structureto enzyme inhibition. We have examined the effects of changes within a series of hydroxyethylene isosteres on their potency in the inhibition of purified HIV-1 protease, and from this we propose a minimum inhibitor structure necessary for tight-binding inhibition. We have also determined the three-dimensional structures of four complexes of HIV-1 protease with hydroxyethylene isostere inhibitors. The binding modes within these complexes help explain observed structure-activity relationships, point out the probable enzymeinhibitor interactions involved in tight-binding inhibition, and suggest new inhibitor designs. An essential component in guiding inhibitor modifications is the identification of relevant cell-based antiviral assays. A range of cell types is involved in HIV infection and AIDS pathogenesis (Fauci, 1988), each of which could respond differently to protease inhibitors. In this study, we have evaluated the antiviral activities of hydroxyethylene isosteres in two different infected cell systems and compared them with their enzyme inhibition constants. In one assay, within chronically infected T-cells, we have examined the ability of these compounds to inhibit processing of Pr55 into its functional components, thus interrupting the assembly of infectious virions. In a second, acute infection assay, we have examined their ability to inhibit infection of uninfected Tcells.
MATERIALS AND METHODS Synthesis of Inhibitors General. Tetrahydrofuran (THF) and toluene were distilled from sodium/benzophenone ketyl. Ether refers to commercial anhydrous diethyl ether. All other reagents and solventswere from commercial sources and were used without further purification. Flash chromatography was performed with E. Merck Kieselgel60. Analytical and preparative HPLC were performed with Dynamax silica ( 5 pm) (Rainin). Proton nuclear magnetic resonance (‘H NMR) spectra were recorded at 250 MHz on a Bruker AM 250 spectrometer. Spectral and analytical data for all compounds are available as supplementary material.
Biochemistry, Vol. 31, No. 29, I992 6647
(tert-Butyloxycarbonyl)phenylalanal(2). To a fine suspensionof LiAlH4 (0.912 g) in ether (80mL) at-45 OC under Ar was added amide 1 (6.16 g) [prepared by the procedure of Goel et al. (1989): clear oil, [a11365 = 31’ (ethanol)] in ether (20 mL) with stirring. After 5 min the mixture was allowed to warm to 10 OC over 30 min and then was recooled to 4 5 OC, and a solution of 5.30 g of KHS04 in 15 mL of water was added cautiously. The cooling bath was removed and the thick mixture was stirred for 30 min. Ether (100 mL) and CH2Cl2 (100 mL) were added, the mixture was filtered through Celite, and the filter cake was rinsed with 100 mL each of ether and CH2Cl2. The filtrate was extracted with ice-cold 1 N HCl(3 X 50 mL), ice-cold 5% NazCOs (2 X 50 mL), and 100mL of brine and then dried over MgS04, filtered, and concentrated by rotary evaporation at 30 OC to provide 2 (4.18 g, 84% yield) as a white solid: mp 82-83.5 OC; [a]D = +35.3’ (C = 1-00, CH2C12). (5SR,6S)-7-Phenyl-6- [(tert-butyloxycarbonyl)amino]-5hydroxyhept-I-ene (3a,b). 4-Bromo-1-butene (7.61 mL) was added over 25 min to a stirred suspension of Mg turnings (2.16 g) in ether (35 mL) under Ar, causing vigorous reflux. The mixture was heated to gentle reflux for an additional 20 min and then cooled to 0 OC (yield of Grignard reagent determined by titration: 65-70%). A solution of aldehyde 2 (5.26 g) in toluene (40 mL) was added over 10 min. After 1.5 h at 0 OC the clear reaction mixture was quenched cautiously with 3 N HCl. Extraction with ethyl acetate and concentration provided a yellow solid, which was dissolved in 50 mL of CH3OH and stirred with NaBH4 (200 mg) for 10 min. The mixture was diluted with 1 N HCl, extracted with CH2C12, and concentrated. Flash chromatography of the residue (gradient: 20%40% ethyl acetate in hexanes) provided 5.03 g of 3a,b as a white solid (78% yield). Analytical HPLC (15% ethyl acetate in hexanes) indicated a diastereomeric ratio (5S):(5R)of 7.20:l.OO. Analytical samples of 3a and 3b were obtained by preparative HPLC. For 3a (5S), mp 71-72 OC. Anal. C, H. N. For 3b (5R), mp 148-149 OC. Anal. C, H, N. (5S)-5- [ ( I ’S)-l’-[ (tert-Butyloxycarbonyl)amino]-2’phenylethyl] tetrahydrofuran-2-one (4a). To a solution of alcohols 3a,b (5.00 g; 3a:3b = 7.2:l.O) in ethyl acetate (50 mL) were added triethylamine (4.56 mL), acetic anhydride (3.09 mL), and 4-(dimethy1amino)pyridine (50 mg). After 3 h excess ethanol was added; the mixture was washed with 3 N HCl, 5% NaHC04, and water and then concentrated. The residue was stirred rapidly in 75 mL of 5 5 : 1 benzene/water/ acetic acid with (n-Bu)dNBr (160 mg) and KMn04 (7.9 g) for 2 h with intermittent cooling. The thick black mixture was cooled to 0 OC and 75 mL of saturated aqueous NaHSO3 was added. After 15 min, the resulting white mixture was filtered through Celite, and the filtrate was extracted with ether. The ether extract was washed with water, dried (MgS04), filtered, and concentrated to a foam (6.9 g), which was then taken up in methanol (100 mL) and stirred at 60 O C with NaOCH3 (ca. 10 g) for 18 h. The mixture was concentrated to ca. 25 mL, diluted with 10% HCl, and extracted with CH2C12. The organic layer was dried over MgS04, filtered, and stirred with p-toluenesulfonic acid (80 mg) and 3-A molecular sieves ( 5 g) for 24 h. Filtration, concentration, and flash chromatography (gradient: 20% 30% ethyl acetate in hexanes) provided lactone 4a (2.59 g, 52% yield), followed by lactone 4b. For 4a, mp 95-96 OC. Anal. C, H, N. Alkylation of Lactone 4a (Zllustrated for Compound 5). (JR,5S)-5-[(I S)-I’-[(tert-Butyloxycarbonyl)amino]-2’-
6648 Biochemistry, Vol. 31, No. 29, 1992
Dreyer et al.
phenylethyll-3-methyltetrahydrofuran-2-one (5). A solution anoyl]ualyl]ualineMethylEster (19). Compound 18 (99 mg, 0.129 mmol) was stirred in 3 mL of methanol with 30 mg of of lactone 4a (1.07 g, 3.50 mmol) in THF (3.0 mL) was added 10% Pd on carbon under hydrogen (1 atm) for 2.5 h. Filtration over 5 min to a solution of lithium diisopropylamide(monotetand concentration provided the unblocked peptide 19a (75.3 rahydrofuran) (7.70 mmol) in cyclohexane(5.1 mL) and T H F mg, 92%yield). Isobutyl chloroformate (1 6.9 pL, 0.13 mmol) (5.0 mL) under Ar at -78 OC. After 20 min, hexamethwas added to a -40 OC solution of Cbz-Ala (29 mg, 0.13 ylphosphoric triamide (7.0 mmol) was added, followed by mmol) andN-methylmorpholine (16.5 pL, 0.15 mmol) inTHF methyl iodide (7.0 mmol). After an additional 45 min at -78 "C, the mixture was diluted with 1 N HCl and extracted with (0.8 mL) under Ar with stirring. After 5 min compound 19a (75.3 mg) was added as a solution in THF (1.0 mL). The CHzC12. The organic layer was concentrated, and the residue cold bath was removed, and after 4 h extractive workup was purified by flash chromatography (30%ethyl acetate in followed by flash chromatography (50: 1 methanol/CHCl3) hexanes) and then by HPLC (20%ethyl acetate in hexanes) provided compound 19 (93.3 mg, 94%yield). The lH NMR to provide lactone 5 (883 mg, 79%yield): mp 134.5-135.0 spectrum of this compound reveals conformation isomerism. OC. Anal. C, H , N . (2R,4S,5S)-[[2-Methyl-4-hydroxy-5-[[ [(benzyloxycarLactones 6,7, and 10 were prepared by procedures similar 6onyl)alanyI]aIanyl]amino]-6-phenylhexanoyl]ualyl]uato that described for lactone 5, using allyl bromide, methallyl line Methyl Ester (Zm). Compound 19 (93.3 mg) was bromide, or benzyl bromide as the alkylating agent (yields: dissolved in trifluoroacetic acid (1 mL). After 10 min the 70%,72%, and 77%, respectively). solution was diluted with CHCl3 and shaken with 5% NaOH. Lactones 8 and 9 were prepared in quantitative yield by The resulting emulsion was extracted with 90:lO:l CHCls/ catalytic hydrogenation of lactones 6 and 7 (1 atm of H2,10% methanol/water, and then the organic layers were washed Pd/C, ethyl acetate). with water and concentrated. The residue was slurried in Preparation of Silyl-Protected Isosteres (Illustrated for CHzCl2 and filtered through a Celite plug, after which the Compound 12). (2R,4S,5S)-2-Methyl-4-(tert-butyldimeth80:20:2 CHCl3/methanol/ clean product was eluted with ylsiloxy)-5-[(tert-butyloxycarbonyl)umino]-6-phenylhexwater. Concentration of the eluant provided compound IVb anoic Acid (12). The procedure of Evans et al. (1985) was (60 mg, 74% yield) as a white solid: mp 238-240 OC. Anal. used, beginning with 830 mg of lactone 5. The crude product C, H, N. was purified by flash chromatography (gradient: 2 7 4 % Enzymology methanol in CHCl3) to provide 0.10 g of lactone 5 (12%yield) followed by 1.02 g of compound 12 (87%yield): mp 152-1 54 Source of Enzyme. Recombinant HIV-1 protease (DeOC. Anal. C, H, N. The lH NMR spectrum of compound bouck et al., 1987) was purified to apparent homogeneity 12 displays evidence of conformational isomerism (approxfrom bacterial cell lysates as described by Grant et al. (1991) imate 2:5 ratio of two conformers),a phenomenonalso observed and its protein content determined chromatographically as with other silyl-protected isosteres. described by Strickler et al. (1989). The purified protease Synthesis of Peptide Analogues (Illustratedfor Compound was routinely stored in 50 mM sodium acetate (pH 5.0), 0.35 ZVb). (2R,4S,5S)-[[2-Methyl-4-(tert-butyldimethylsiloxy)-M NaC1,l mM EDTA, 1 mM dithiothreitol, and 40%glycerol 5-[(tert-butyloxycarbony I)amino]-6-phenylhexanoyll ualyl]at -80 OC. d i n e Methyl Ester (16). Isobutyl chloroformate (34 pL, Enzyme Inhibition Studies. Protease inhibition was de0.26 mmol) was added dropwise to a stirring solution of 4termined with the substrate Ac-Arg-Ala-Ser-Gln-Asn-Tyrsiloxy acid 12 (1 12 mg, 0.250 mmol) and N-methylmorphine Pro-Val-Val-NH2 (Km= 5.5 mM, k, = 29 s-l) as described (33 pL, 0.30 mmol) in THF (1.5 mL) under Ar at -40 OC. (Moore et al., 1989; Dreyer et al., 1989). Inhibitors were After 15 min, more N-methylmorpholine (50 pL, 0.45 mmol) dissolved in DMSO and diluted into reaction mixtures to a was added, followed by a solution of valylvaline methyl ester final DMSO content of 10%. For inhibitors with inhibition hydrochloride (100 mg, 0.375 mmol) in THF (1 .O mL). The constants (Ki) greater than 40 nM, assay mixtures contained mixture was allowed to warm to room temperature and after the substrate (1 mM), inhibitor (10-500 nM or 1-50 pM), 12 h was diluted with 5% HCl, extracted with ethyl acetate, and 10% DMSO in 50 pL of 50 mM Mes (pH 6.0), 1 mM concentrated, and purified by flash chromatography (25:1 EDTA, 200 mM NaCl, 1 mM dithiothreitol, and 0.1%Triton CHCl3/methanol) to provide 16 (154 mg, 93% yield) as a X-100 (MENDT buffer). Reaction was initiated by addition white solid. of purified protease (30 nM). Reaction mixtures were (2R,4S,5S)-[ [2-Methyl-4-(tert-butyldimethylsiloxy)-5- incubated at 37 OC for 20 min, then quenched with 1% trifluoroacetic acid (50 pL), and analyzed by reversed-phase [ [(benzyloxycarbonyl)alany~ amino]-6-phenylhexanoyl]uaHPLC. Inhibition constants were obtained by the method of lyl]ualineMethyl Ester (18). Compound 16 (154 mg, 0.233 Dixon (1953) assuming competitive inhibition. mmol) was dissolved in trifluoroacetic acid (1 mL). After 5 For more potent inhibitors (KiC 40 nM), the substrate (1 min the solution was diluted with 5% Na2CO3 and extracted mM) was preincubated at 37 OC with inhibitor (0-100 nM) three times with CH2C12. The combined organic layers were in 50 p L of MENDT buffer (pH 6.0) containing 10%DMSO. washed with water and concentrated to provide 17 (124 mg, Reaction was initiated by addition of protease (1-40 nM). 95%). Isobutyl chloroformate (25 pL, 0.19 mmol) was added Reactions were quenched after 60 min (
