Specificity and Inhibition of Proteases from Human ...

THE JOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 265, No. 24, Issue of August 25, pp. 14675-14683,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Specificity and Inhibition of Proteases from Human Immunodeficiency Viruses 1 and 2*

(Received for publication, March 26, 1990)

Alfred0 G. Tomasselli, John 0. Hui, Tomi K. Sawyer, Douglas J. Staples, Carol Bannow, Ilene M. Reardon, W. Jeffrey Howe, Dianne L. DeCamp$$, Charles S. CraikSll, and Robert L. Heinriksonll From the Biopolymer Chemistry and Computational Chemistry Units, Discovery Research Division, The Upjohn Company, Kalamazoo, Michigan 49001 and the SDepartment of Pharmaceutical Chemistry, The University of California, San Francisco, California 94143-0446

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several PI-PI’ pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys- PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-l enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265,408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-l and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-l gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-l protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-l enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and PI’, but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Rela- tive to PI-PI’ statine or Phe*[CHzN]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa\k[CH(OH)CHz]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-l protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-l protease suggest that most of the nonconservative amino acid replacements occur in regions well outside

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health Grant GM39552. ll Supported by National Institutes of Health Grant GM39552. 11 To whom correspondence should be addressed: The Upiohn Com-

pany, 7240-267-119,.Kalamazoo, MI 49001.

the catalytic cleft, while only subtle structural differences exist within the active site. In addition, energy- based modeling of a peptide fragment patterned after one of the observed substrates indicated that class 3 substrates may be interacting with charged protease side chains clustered at the ends of the binding cleft.

The aspartyl protease encoded in the pol gene of retrovi- ruses is essential for viral maturation (l), and the protease from human immunodeficiency virus (HIV)’ has been targeted as a possible therapeutic intervention point in the treatment of acquired immunodeficiency syndrome (AIDS). Numerous reports have appeared in the recent literature describing the purification and characterization of HIV protease produced by recombinant or synthetic means (2-ll), the tertiary structural analysis of this (12-14), and a related retroviral enzyme (15), and studies of enzyme chemistry (6, 16), specificity, and inhibition (6, 7, 17-23). In addition, an x-ray crystallographic model of the HIV-l protease bound to a synthetic pseudopeptide inhibitor has been reported recently by Wlodawer and co-workers (24). This level of structural detail in our understanding of enzyme-inhibitor interactions has been complemented on the functional side by recent descriptions of HIV protease inhibitors that block viral maturation and polyprotein processing, respectively, in HIV-l- infected cell culture assays (25,26) and in a cell model system which mimics HIV-l infectivity (25). Therefore, both from a structural and functional point of view, the protease has been characterized at a high level of sophistication, and this serves as a basis for drug design and implementation in AIDS therapy.

Most of the findings published thus far with respect to the protease have been obtained from studies of the enzyme from HIV-l, that form of the virus that is associated with AIDS in most of the Western world. However, it is now clear that HIV-2 is a distinct etiologic factor that is also of importance in human AIDS. Since the first description of its genomic sequence (27), HIV-2 was recognized as a separate virus that closely resembles simian immunodeficiency virus (SIV). Evi- dence for the possible origins of HIV-l and HIV-2 from a reservoir of African lentiviruses, collectively termed SIV, was presented in a recent paper by Hirsch et al. (28), and the implications of these findings were discussed further by Doo- little (29). Whatever the evolutionary history of these viruses,

’ The abbreviations used are: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; SIV, simian immunodeficiency virus; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate.

14675

Compared Specificities of HIV-l and HIV-2 Proteases

therapeutic approaches to AIDS must take into account in- fections derived from HIV-2, and if the protease is the target, it is crucial to establish similarities and differences between enzymes from HIV-l and HIV-2. Moreover, at the present time, the concept of a protease inhibitor as a drug against AIDS has yet to be validated in an animal model. One reasonable choice here is the monkey (30), and the close similarity between the sequences both of the HIV-2 and SIV proteases (28) and of their polyprotein substrates (31) would imply that a study of the HIV-2 enzyme should provide important information for design of inhibitors for testing in non-human primate models.

assay except for the protease. Another set of controls included the

The present paper describes a comparison of the proteases from HIV-l and HIV-2 relative to their substrate specificities and with respect to their inhibition by a variety of compounds. Furthermore, the recent availability of x-ray crystal structures of HIV-l protease has enabled modeling studies to be undertaken in conjunction with the experimental work. The com- bined results suggest structural factors that may contribute to the observed substrate preferences.

EXPERIMENTAL PROCEDURES

Materials

Recombinant HIV-1 protease was prepared from Escherichia coli inclusion bodies as described bv Tomasselli et al. (6). The recombinant enzyme from HIV-Z, expr&sed in a soluble f&m in yeast, was purified 2,3 according to similar methods; the lyophilized, homogene- ous protein was eluted from the reverse-phase HPLC column and refolded by the same methods employed for HIV-1 protease (6). Concentrations of the enzymes were established both by amino acid analysis and by titration with the substrate-based inhibitor Val-Ser- Gln-Asn-Leu\k[CH(OH)CHz]Val-Ile-Val (6). Pseudomonas exotoxin, PE66, and derivatives thereof in which domain I is either missing (LysPE40), or replaced by the first two domains of soluble CD4 [CD4(178)PE40],were the same as described in detail in an earlier

Dublication (19). and were the generous gift of Dr. David J. FitzGerald, National Cancer Institute, N;?tional Institutes of Health. Peptides were synthesized by solid-phase technology (32) employing a model 430-A Peptide Synthesizer from Applied Biosystems Inc.; purity was assessed by HPLC analysis. Chemical authenticity was established for all peptide substrates and inhibitors by amino acid analysis and FAB-MS. Solvents for protein sequence analysis, HPLC, and other routine laboratory chemicals were of the highest grade commercially available.

Methods

HIV Protease Assay-both HIV-l and HIV-2 proteases were as- sayed against the octapeptide, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, corresponding to the sequence of the natural HIV-l gag polyprotein sequence from residue 128 to 135 (33). Conditions for the assays and for monitoring the course of hydrolysis of the Tyr-Pro bond by HPLC were detailed in earlier publications (6, 19).

Protein substrates, PE66, LysPE40, and CD4(178)PE40 were cleaved by the two proteases under essentially the same conditions, but hydrolysis was monitored in these cases by quantitative SDS- polyacrylamide gel electrophoresis (19). CD4( 178)PE40 (100 pg) was incubated for 7 h at 30 “C with 0.40 pg of HIV-1 protease or 0.55 pg of the HIV-2 enzyme in 200 ~1 of 0.2 M sodium acetate, pH 5.5, containing 10% glycerol and 5% ethylene glycol. Samples were with- drawn at various times and subjected to reducing SDS-polyacrylamide gel electrophoresis in order to follow the progress of reaction. Se- quence analysis was performed on samples from the last time points of each hydrolysis mixture so that sites of cleavage could be docu- mented. LysPE40 was cleaved under similar conditions, except that reactions were carried out for 4 h in a volume of 100 ~1 with 0.7 pg of protease.

With all of the studies involving protein substrates, control reactions were included which contained all of the ingredients of the

* S. Pichuantes, L. Bab6, P. J. Barr, and C. S. Craik, manuscript submitted.

” L. Bab&, S. Pichuantes, and C. S. Craik, manuscript submitted.

tight binding HIi protease inhibitor Val-Ser-Gln-Asn- Leu+ICH(OH)CHplVal-Ile-Val (U-85548E) in order to Drove that any observed prot&ysis was due, exclusively, to the retrbviral protease action (6).

Amirzo Acid Analysis-Compositional analysis of proteins and peptides were obtained by conventional automated ion-exchange chromatography on a Beckman model 6300 analyzer. Samples were hydrolyzed in 6 N HCI for 24 h in vacua at 110 ‘C. Hydrolyzates were dried in a Speed Vat Concentrator (Savant), and the residues were dissolved in buffer at pH 2.2 (Na-S; Beckman) for application to the analyzer.

Sequence Analysis-Protein and peptide sequencing was performed by automated Edman degradation in an Applied Biosystems Inc. model 470 sequencer fitted with an on-line HPLC analyzer (model 120-A) for phenylthiohydantoin amino acids. Peaks from the latter were integrated by a Nelson Analytical 3000 Series chromatography data system connected in parallel with the recorder to the output of the HPLC system.

Quantitative SDS-Polyacrylamide Gel Electrophoresis-Laemmli (34) gels (12%) were run under reducing conditions in either mercap- toethanol or dithiothreitol and stained with Coomassie Blue for detection of proteins. Stained gels were analyzed in a Visage 110 scanner from BioImage, Ann Arbor, MI, which integrates the optical density of the gel bands and thus provides a means of quantitation for assaying the time course of cleavage of protein substrates by the HIV proteases.

Molecular Modeling-Molecular modeling studies made use of various components of the Mosaic software package for model building, graphical visualization, and energy refinements.4 Two x-ray crystal structures were used in the modeling work: 1) complexed HIV-l protease/MVTlOl reduced-bond inhibitor (Ac-Thr-Ile-Nleq[CH*. NH]Nle-Gln-Arg-NHa (24); coordinates supplied by Dr. A. Wlodawer, National Cancer Institute, Frederick Cancer Research Facility), and 2) uncomplexed HIV-1 protease monomer (Brookhaven Protein Data Bank entry 3HVP (12)). A model of the protease dimer was constructed from the coordinates of the monomer by applying the spec- ified (Y, X, Z) transformation.

One of the cleavage sites observed for both HIV-1 and HIV-2 proteases had a Glu-Glu pair in the substrate P2’-P3’ positions. To examine the extent to which these residues could interact with charged side chains that are clustered at the end of the HIV-l protease active site cleft (24), the following approach was used. A Leu-Ala- Glu-Glu fragment was constructed, using Mosaic, with all backbone angles set to 180 degrees. The tetrapeptide was then manually docked into the uncomplexed HIV-l protease dimer model, parallel to the lengthwise axis of the cleft, so as to position the Leu and Ala residues at approximately the P, and P1’ locations relative to the catalytic Asp side chains. The geometry of the tetrapeptide and all protease atoms within 10 A of it was then relaxed by 700 iterations of energy minimization using the AMBER force field (35), as implemented in Mosaic/Macromodel2.5, and the PRCG minimizer. Strong positional constraints placed on the catalytic aspartyl residues and the “scissile” CONH atoms held the tetrapeptide fragment in position during the procedure. Protein atoms up to 5 A beyond the freely moving atoms were increasingly constrained, but beyond that 5 8, shell all protein atoms were frozen. Prior to the minimization, hydrogen atoms were added to the appropriate heteroatoms and to one of the catalytic aspartyl residues. The qualitative model that resulted was then used in an examination of charged “substrate” side chain interactions with protease residues.

RESULTS

Hydrolysis of Pseudomonas Exotoxin Derivatives--In an earlier paper, we demonstrated that two Pseudomonas exotoxin derivatives, LysPE40 and CD4(178)PE40, were hydrolyzed by the HIV-l protease at bonds within interdomain regions (19). Interestingly, cleavage occurred at two sites in LysPE40, a Leu-Leu and a Leu-Ala bond representative of class 3 substrates (31). The expected Tyr-Pro bond in the interdomain region was not hydrolyzed (19). Since we had

4 W. J. Howe, J. R. Blinn, J. B. Moon, T. R. Hagadone, G. J. White, M. W. Schulz, manuscript submitted. Mosaic is based, in part, on MacroModel, developed by W. C. Still and co-workers, Columbia University.

Compared Specificities of HIV-l and HIV-2 Proteases 14677

already characterized these novel, non-viral proteins as substrates for the HIV-l protease, we were interested in deter- mining the course of their cleavage by the HIV-2 enzyme. Qualitatively speaking, the two proteases are identical in their cleavage of the Leu-Ala and Leu-Leu bonds in LysPE40 and at only the latter site in the chimeric CD4(178)PE40 (Fig. 1 (19, 36)). In both protein substrates, this Leu-Leu bond is hydrolyzed twice as fast by the HIV-2 protease than observed for the HIV-l enzyme. These kinetic data were obtained by scanning densitometry of SDS-polyacrylamide gel electrophoresis gels of reaction products at various times (data not shown). Although the rate of cleavage of the Leu-Ala bond by the HIV-l protease was shown by sequence analysis of the products to be about one-third that of the Leu-Leu bond (19), it was actually the preferred site of hydrolysis by the HIV-2 enzyme, being cleaved 1.5 times faster than Leu-Leu. Thus, the HIV-2 protease cleaves the Leu-Ala bond about 10 times faster than the HIV-l enzyme. We showed earlier that octa- and nonapeptides corresponding to these susceptable regions were cleaved by the HIV-l protease with kinetics mirroring those with the protein substrates (19). As shown in Table I (compounds 1 and 2), the general course of hydrolysis of the LysPE40 derivatives by both proteases is reflected in the kinetic analysis of peptide hydrolysis. The nonapeptide containing the Leu-Leu bond, and the octapeptide with the Leu- Ala bond are cleaved 2.5, and 17 times faster, respectively, by

(HANzJzh -Ala-Glu-Glu-(Ala-Phe-Lys-)Gly-

389

FIG. 1. Diagrammatic representation of two derivatives of the Pseudomonas exotoxin PE66 (19, 43, 44) in which domain I is missing (LysPE40, lower dashed line), or replaced by the 1’78 amino acids of the two NH2-terminal domains of CD4 (upper dashed line). That portion of the OmpA gene product remaining as part of the recombinant LysPE40 is indicated, together with Roman numerals referring to parts of, or whole domains defined crystallographically (44). Arrows above bonds indicate sites of cleavage of these protein substrates by proteases from HIV-l and HIV-2. The Tyr-Pro bond enclosed in a box is not cleaved by either enzyme. Residues in parentheses are present only in LysPE40 and not in the chimeric CD4(178)PE40 (36).

the HIV-2 protease as compared with the HIV-l enzyme. Interestingly, a peptide identical to compound 1 in Table I, but having His-Met in place of Asn-Leu, is cleaved only very slowly by the HIV-l protease. This peptide corresponds to the linkage region in the chimeric protein CD4(178)PE40 (Fig. l), and thus the failure to cleave the Met-Ala bond in the protein is, once again, reflected by results with the peptide substrate. Other parallels with the earlier study were docu- mented; neither enzyme was able to hydrolyze PE66, nor was cleavage seen at the Tyr-Pro bond (Fig. 1) originally expected to be the most likely site of hydrolysis ((19), Table I). Prein- cubation of the HIV proteases in the presence of 25 pM U- 855483, an active site titrant, completely abolished cleavage of any of the protein or synthetic peptide substrates.

Hydrolysis of Peptides Modeled after Viral Polyproteins- Comparison of HIV-l and HIV-2 proteases relative to their kinetics of hydrolysis of the HIV-l gag polyprotein-based model peptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, reveals that they have identical Km values, but the V,., for cleavage of the Tyr-Pro bond by the HIV-l enzyme is twice that of the HIV- 2 protease (Table I, compound 8). Thus, with respect to this substrate, and compound 9 in which the Tyr is replaced by Phe, the enzymes are quite similar. This also holds true for compounds 3 and 4 which are modeled from class 3 cleavage sites (31). However, replacement of the Tyr (or Phe) at P1 in the template of Val-Ser-Gln-Asn-Xaa-Yaa-Ile-Val by cyclo- hexylalanine (Cha) or Leu (compounds 10 and 11, respectively) or the P1’ Pro by Val (compound 12) yields peptide substrates much more efficiently cleaved by the HIV-l protease. In fact, the latter peptide is bound equally well by both enzymes (K, is lowered from 2.0 to 0.6 mM for both relative to the parent peptide, compound 8) but is hydrolyzed 64 times faster by HIV-l protease. Compounds 5, 6, and 13 (Table I) are not cleaved by either enzyme despite obvious similarities to other compounds listed that are good substrates. Compound 5 represents a sequence processed in the pol gene by avian myeloblastosis viral protease. It is difficult to say what subtle differences account for its lack of hydrolysis. Compound 6 is modeled after a putative site of cleavage (27,31) at the Phedb6- Prod47 bond in the HIV-2 gag region, but was not hydrolyzed by either enzyme. Since it is generally true that bonds cleaved in a protein are also hydrolyzed in model peptides (7, 19), this result was a surprise. Compound 14, containing an Ala-Ala scissile bond and modeled after an HIV-2 polyprotein processing site, represents one case of a peptide that is a substrate for the HIV-2 protease, but not for the HIV-l enzyme. How- ever, replacement of the Tyr-Pro sequence in compound 8 by Ala-Ala (compound 13) destroys the ability of the peptide to serve as substrate for either enzyme.

In summary, the ability of the HIV-2 protease to cleave Ala-Ala bonds in particular substrates underscores a basic difference from the HIV-l enzyme in that the former is able to cleave substrates with small substituents at P1 and PI’. Substrates having p-branched amino acids at P1’ (compound 12), however, exhibited significantly lower rates of hydrolysis by the HIV-2 protease as compared with the HIV-l enzyme. These findings would suggest that the HIV-2 protease binding pockets for Pi and P1’ side chains may be more occluded, and perhaps less efficient in the binding of bulky groups. In general, the HIV-2 enzyme appears to display a broader substrate specificity than the HIV-l protease. This conclusion finds further support from the course of hydrolysis of calmod- ulin by the two proteases described by Tomasselli et aL5

‘A. G. Tomasselli, W. J. Howe, J. 0. Hui, T. K. Sawyer, I. M. Reardon, D. DeCamp, C. S. Craik, and R. L. Heinrikson, manuscript submitted.

14678 Compared Specificities of HIV-l and HIV-2 Proteases

TABLE I

Comparison of substrate specificity of HIV-l and HIV-2proteases relative to hydrolysis of synthetic peptides based upon viral polyproteins from HIV and auian myeloblastosis virus (AMV) and from the non-viral protein substrate

Lys PE40

Compounds K”,

mM

HIV-1 protease HIV-2 protease

V mex Vm..lKm Km V msx Vm..lKn

firno x nin-’ rnM pm01 X min-’ X mg-’ x mg-’

1 H-Ala-Asn-Leu-Ala-Glu-Glu-Ala-Phe-OH’ 1.3 0.3 0.23 1.1 5.0 4.55

2 H-Ser-Gly-Asp-Ala-Leu-Leu-Clu-Arg-Asn-OH’ 1.6 1.2 0.71 1.1 3.0 2.73

3 H-Thr-Ala-Thr-Ile-Met-Met-Gin-Arg-Gly-OH’ 1.3 3.6 2.71 1.3 5.0 3.85

4 H-Thr-Ala-Thr-Ile-Nle-Nle-Gin-Arg-Gly-OHb 1.7 3.0 1.76 1.7 3.0 1.76

5 H-Thr-Phe-Glu-Ala-Tyr-Pro-Leu-Arg-Glu-Ala-OH’ 0 0

6 H-Lys-Pro-Arg-Asn-Phe-Pro-Val-Ala-OHd 0 0

7 H-Tyr-Val-Ser-Gln-Asn-Phe-Pro-Ile-Val-G1n-Asn-Arg-OHa 1.9 3.9 2.05 1.9 4.3 2.26

8 H-Val-Ser-Gin-Asn-Tyr-Pro-Ile-Val-OHb 2.0 4.9 2.45 2.0 2.5 1.25

9 H-Val-Ser.Gin-Asn-Phe-Pro-Ile-Val-OH’ 6.7 3.9 0.58 5.0 2.1 0.42

10 H-Val-Ser-Gin-Asn-Cha-Pro-Ile-Val-OH” 20 3.9 0.20 10 0.24 0.024

11 H-Val-Ser.Gin-Asn-Leu-Pro-Ile-Val-OHb 10 0.9 0.09 ND’ 0.01 ND

12 H-Val-Ser-Gin-Asn-Tyr-Val-Ile-Val-OHb 0.6 2.7 4.50 0.6 0.08 0.133

13 H-Val-Ser-Gln-Asn-Ala-Ala-Ile-Val-OHb 0 0

14 H-Ile-Pro-Phe-Ala-Ala-Ala-Gln-Gin-Arg-OHd 0 4.6 0.8 0.17

’ LysPE40. * HIV-l gag fragment (or analog). ’ AMV pol fragment. d HIV-2 gag fragment (or analog). ’ ND. not determined.

Entry

Pepstatin

U-85549E

U-84645E

U-85548E

U-71038

U-81749

TABLE II

Comparative inhibition of HIV-l and HIV-2proteases

Compound

Iva-Val-Val-Sta-Ala-Sta-OH

H-Val-Ser-Gln-Asn-Sta-Ile-Val-OH

H-Val-Ser-Gln-Asn-Phel[CH,N]Pro-Ile-Val-OH

H-Val-Ser-Gln-Asn-Leul[CH(OH)CH,]Val-Ile-Val-OH

Boc-Pro-Phe-We-His-Leu’@[CH(OH)CH,]Val-Ile-Amp

Tba-Chal[CH(OH)CH,]Val-Ile-Amp

HIV-1 HIV-2

K, (no) K, (nM)

362 720

3,690 9,000

3,520 26,100

-Cl 9

10 >l,OOO

80 1,000

Inhibitor Structure-Activity Relationships-Thus far, we have compared the HIV-l and HIV-2 proteases according to their cleavage site preferences in native proteins and modeled peptides. In Table II are shown K, values determined for a number of inhibitors relative to the two enzymes. The natural product, pepstatin, is only moderately inhibitory toward both HIV-l and HIV-2 proteases. Relative to pepstatin, we evaluated a series of HIV-gag/pal precursor-based inhibitors with the generic structure, Val-Ser-Gln-Asn-Xaa-Yaa-Ile-Val, having Pi-Pi’ substitutions by Sta, Phe\k[CHzNH]Pro, and Leu*[CH(OH)CHz]Val and found them to be of greater po- tency and selectivity. Noteworthy was U-85548E, a high affinity inhibitor of both enzymes (K, < 1 nM for HIV-l protease, and 9 nM for the HIV-2 enzyme). Previous studies by Richards et al. (21, 38) showed that the Leu\k[CH(OH)CHz] Vai-substituted renin inhibitor, H-261, is a strong inhibitor

of both HIV-l and HIV-2 proteases (K, = 15 and 90 nM,

respectively). To extend this study, we evaluated another high affinity renin inhibitor, U-71038, against both proteases (Table II). Overall, these findings show that U-85548E is the most potent inhibitor of both proteases yet described and that U-71038 is the most selective of those tested thus far.

The small inhibitor U-81749 (Tba-Cha\k[CH(OH)CH,] Val-Ile-Amp (25); M, = 572) is of special interest in this group. A Dixon plot for inhibition of HIV-l protease by this compound is shown in Fig. 2; values of 83 and 70 nM were

determined for HIV-l protease at pH 5.5 and 7.1, respectively. A replot of the slopes of the Dixon plots (inset, Fig. 2) gives a straight line through the origin, indicating that U-81749 is a competitive inhibitor of the protease. This inhibitory activity was demonstrated not only against the pure HIV-l protease but against viral maturation in a cell culture system consisting


of HIV-infected human peripheral blood lymphocytes (25). In this latter study, an I& value was determined to be between 0.1 and 1 pM concentrations of inhibitor (25). Processing of HIV-l gag and gag/p01 polyproteins to ~24 in cells infected with a recombinant vaccinia virus expressing the HIV-l pre-

FIG. 2. A Dixon plot for inhibition of HIV-l protease by U- 81749 shown on the figure. The substrate was Val-Ser-Gln-Asn- Tyr-Pro-Ile-Val at concentrations of 2.8 mM (B), 1.0 mM (Cl), and 0.5 mM (e). The inset shows a replot of the slopes from the Dixon plot uersus l/S; since this line passes through the origin, U-81749 is a competitive inhibitor of the protease.

1

HIV-l

HIV-2

SIV

HIV-1

HIV-2

SIV

FIG. 3. Comparison of the se- HIV-l

quences of proteases from HIV-l (33, 37, 46), HIV-Z (27), and SIV HIV-2

(45). SIV

HIV-1 Gln - Ile - Leu - Ile - cys - Gly - His -Ala - Ile - Gly - Val - Leu - Val - Pro _

HIV-2 Am - Val - Glu - Ile - Glu - Val - Leo -Am - Lyr - Lyr - Val - Arg - Ala - Th, - Ile - Met - Th, _ Gly -Asp - Th,

HIV-l - Vd - Ik Leu - Gln - Ile - Cyr - Th, Phe -

HIV.2 Pro - Ile - Am - Me - Phe - Gly - Arg - A*” - 11~ - Leo -Th,-Ala-Leu-Gly-Met-Scr-Leu-Am-Leu.OH

SIV

cursors (39) was also blocked by 10 FM concentrations of U- 81749. However, U-85548E, an inhibitor bound about 10 times more strongly to the enzyme (Table II) showed little activity in these cell culture assays. These findings underscore the importance of considerations relative to cell or particle delivery as well as K1 when designing protease-targeted drugs against HIV.

Structural Comparisons of HIV-l and HIV-2 Proteases- Comparison of the HIV-l and HIV-2 protease amino acid sequences (Fig. 3) reveals numerous similarities, including an S-residue stretch of exact identity in the 23-30 region sur- rounding the catalytic Asp-25. Overall, the identity between the two sequences is 50%, and when conservative substitutions are taken into account the similarity approaches 75%. Therefore, although the three-dimensional structure of HIV- 2 protease is not yet known, the structure of HIV-1 protease can serve as a useful starting point from which to examine the observed substrate preferences of the two enzymes.

In Fig. 4 is depicted a smoothed backbone representation of the complexed form of HIV-1 protease from which the MVTlOl inhibitor (Ac-Thr-Ile-Nle\k[CH2NH]Nle-Gln-Arg- NH,) has been removed. The orientation of the structure is such that the 2-fold symmetry of the molecule is readily apparent. The flap regions are at the top center of the diagram, and the view is down the length of the substrate binding cleft which is located just below the flaps. Color mapping of the

20

- lie -Th, Gin - Leu Me - Lyr - Gly Leu - Lyr

Pro - Gin - Phe - Ser - Leu - Trp - Lys - Arg -Pro - Val - Val - Th, -Ala - Ty, - Ilc - Glu - Gly - Gln - Pro - Val

Arg His

21 40

m Ala Th, - Val - Leu - Glu - Glu - Met- Se, - Pro - Gly

Glu - Val - Leu - Leu - Asp -Thr - Gly - Ala - Asp - Asp - Se, - Ile - Val - Ala - Gly - Ile - Glu - LCU - Gly - Am

Thr PI0

41 60

Arg - Trp - Lys - Met - lb? Lyr - val - Arg - Gin -Asp

Am - Tyr - Ser - Pro - Lys - Ile - Val - Gly - Gly - Ile - Gly - Gly - Phe - Ile -Am - Thr - Lys _ Glu - Tyr - Lys

His -Th,

61 80

Compared Specificities of HIV-I and HIV-2 Proteases

FIG 4. Backbone representation of HIV-I protease showing regions of homology with the HIV-2 enzyme. Areas of exact sequence match are shown in blue, eanssvative replacements are in .yellou, and nonconservative replacements are in red. Within the substrate binding cleft (upper middle) are three conservative replacements: He-47 (rll, Val-32 (B). and V&R2 (0. which are Val, Ile, and Ile, respectively in HIV-2 protease. .lust ourside the cleft is a fwrth replacement. Gin-7 to Lys Cd. foregrwnd~. Since the molecule is dimeric, there is a duplicate replacement for each of those indicated. located in symmetrically equivalent positions around the vertical C-2 axis. Protease coordinates are those of an inhibitor-complexed form of the enzyme. courtesy of Dr. A. Wlodawer

HIV-2 protease sequence has been applied to the structure to enable a comparison of the two enzymes. Regions of exact sequence match are shown in blue, conservative replacements are shown in yellow, and nonconservative replacements are in red. It should be stressed that this construction is not intended to be a model of HIV-2 protease; it is simply the HIV-l protease structure with sequence information for the HIV-2 enzyme mapped onto it. Nonetheless, it is a useful homology representation technique with which one can begin to infer some of the structural factors that may be influencing the relative specificities of the enzymes.

Of particular interest in this representation is the fact that most of the nonconservative replacements occur at the pe- riphery of the structure, far removed from the binding site, yet the residues which define the binding site surface are those which would be expected to have the greatest influence on substrate recognition. With regard to those residues actually in contact with the inhibitor in the x-ray structure (24),

the two enzymes differ in only three symmetrically located pairs. The first is Val-82 in the A and B chains of the HIV-l protease dimer, which is replaced by an Ile in the HIV-2 enzyme. In the crystal structure, this residue contributes surfaces which are in contact with inhibitor side chains at the P, and P,’ positions. The second replacement is Val-32 to Ile- 32 in HIV-2 protease; this residue is in contact with the Pi and PI’ side chains of the inhibitor. The third is Ile-47 to Val in the HIV-2 enzyme, a residue also in contact with the Px and Pr’ side chains. All three are conservative replacements which involve nothing more than addition or deletion of a single methyl group at each of the 3 pairs of residues. Assum- ing, then, that the binding site of HIV-2 enzyme is defined by the same residue positions as are seen in the HIV-l protease structure, it can be said that there are differences in the shape of the two binding sites, but they are likely to be subtle ones. Since this is a map and not a model, however, the possibility cannot be ignored that the more extreme sequence substitu-


FIG. 5. Stereo diagram showing interaction of a model peptide, Leu-Ala-Glu-Glu, with charged residues at one end of the HIV- 1 protease active site cleft. In this computer model, the two Glu residues of the peptide (upper right) are located approximately at the P2’- and P,‘-binding site positions and are able to form strong interactions with Arg-8 of HIV-l protease. During the minimization, Asp-29 has been displaced by the PB’ Glu side chain. Other protease residues which may interact with charged substrate residues at P:,’ and beyond are ASD-30 and Arp-87. In HIV-2 nrotease, Gln-7 (foreground) fs replaced by a Lys. Starting coordinates for the enzyme were obtained from the PDB entry 3HVP.

tions seen in other parts of the molecule could transmit their influence inward toward the binding site, affecting its shape in unknown ways.

While it is reasonable to assume that much of the substrate specificity demonstrated by HIV-l protease is due to interactions within the enzyme’s well-defined active site cavity, it is also likely that residues just outside this region play some role in substrate recognition. At each open end of its binding site cleft, HIV-l protease has 4 charged residues, Asp-30, Asp- 29, and Arg-8 at the cleft boundary, and Arg-87 just outside it. It has already been demonstrated (24) that an Arg in the Ps’ position of an inhibitor is able to interact strongly with Asp-29. In addition, the current experimental results dem- onstrate the ability of both proteases to hydrolyze substrates with Glu or Arg residues at the Pp’ and Ps’ positions. There- fore, we were interested in modeling the interactions of a negatively charged peptide fragment (Leu-Ala-Glu-Glu) with the charged protease region mentioned above. This peptide sequence represents a PE40 cleavage site that was observed for both proteases (Table I, compound 1).

For this part of the study, we used the uncomplexed HIV- 1 protease dimer structure since its charged residue positions had not been influenced by the presence of an inhibitor molecule. The qualitative tetrapeptide model generated by the procedure outlined earlier is shown in Fig. 5 which demon- strates that the two substrate Glus are well within reach of Arg-8. During the minimization, Asp-29 undergoes consider- able movement away from Arg-8 to accommodate the two additional salt bridges from the substrate Glus. Arg-8 does not have to project into the cleft at all to interact with these glutamyl residues. We now return to the mapping presented in Fig. 4 to examine this same region of the structure. In HIV- 2 protease, a Lys substitutes for Gln-7 which is in close proximity to Asp-29, Arg-8, and Arg-87. A closeup view of the Gln-7 position relative to the other charged residues is presented in Fig. 5. The addition of a positively charged residue serves to distinguish the surface of the HIV-2 protease from that of an already highly charged region in the HIV-l protease. If one assumes that this residue is located spatially in the same area in HIV-2 protease as it is in the HIV-l enzyme, it is possible for this substitution to influence substrate specificity, either by direct interaction of the Lys side chain with substrate or, more likely, by modification of the electric field developed by the other charged residues in this region. The nearly 20-fold increase in V,,,.,/K,,, shown toward Ala-Asn-

Leu-Ala-Glu-Glu-Ala-Phe (compound 1, Table I) may be due to enhanced binding of the Glu-Glu pair in the HIV-2 protease.

DISCUSSION

The present study has shown that the HIV-l and HIV-2 proteases may be distinguished according to their specificities toward particular substrates and inhibitors, despite the fact that their natural polyproteins are similar, and the enzymes display similar selectivities. A recent report based upon maturation of chimeric viral polyproteins came to essentially the same conclusions (40), although specific sites hydrolyzed were not identified and no kinetic analyses was undertaken. In defining sites of cleavage shared in common, it is important to consider kinetic parameters for hydrolysis as well. With LysPE40, both proteases cleave the same two bonds, but with dramatically different preferences (Table I, compounds 1 and 2). This difference cannot be attributed solely to the relative sizes of the P1’ residues (Ala versus Leu) since the compounds differ at other positions as well. However, the modeling comparison of the HIV-l and HIV-2 protease-binding sites does suggest a greater occlusion at the SJS, subsites in HIV-2 protease, which could favor smaller P,’ side chains in corresponding substrates. This would be in keeping with the processing function of this enzyme in cleaving Ala-Ala bonds in HIV-2 polyproteins (31). Such processing sites do not exist in the HIV-l polyproteins. Accordingly, we have demonstrated in the present paper that a peptide substrate based upon the HIV-2 gag polyprotein-processing site that contains an Ala- Ala scissile bond is cleaved by HIV-2 protease, but not by the HIV-l enzyme (Table I, compound 14). However, it is of interest that one cannot replace the Tyr-Pro bond in a class 1 substrate (31) by an Ala-Ala sequence characteristic of class 3 substrates; the resulting peptide (Table I, compound 13) is cleaved by neither enzyme.

Both proteases are able to hydrolyze particular peptide bonds having bulky, hydrophobic amino acids in P, and P1’ (Table I). Thus, the subtle differences at S,/S,’ suggested by the binding site models certainly do not rule out HIV-2 protease binding of bulky side chains at those positions, but they may have an effect on the efficiency of hydrolysis. For example, changing the Phe at P1 (compound 9) to a Cha (compound 10) causes a substantial reduction in hydrolysis by HIV-2 protease. A similar effect is seen when a P1’ Pro (compound 8) is changed to the P-branched Val (compound 12). Interpretation of this latter observation, however, is complicated by the possibility that the P1’ Pro may be induc- ing a backbone conformation in the P1’ to PB’ positions different from that which exists in compound 12. This possibility deserves further study, especially in light of the fact that substrate activity of the class 1 compound 8 (Tyr-Pro insert) is abolished in compound 13 (Ala-Ala insert) for both enzymes, even though the two peptides are identical in all other respects. Additionally, our modeling results suggest that class 3 substrates, which often contain charged residues at PZ, Pz’, Ps, or P3’, bind to both proteases in a manner that involves salt bridging with one or more of the charged protease residues at the ends of the cleft. Such interactions may not be possible when a Pro is at the P1’ position and could explain the general lack of charged residues downstream from the Pro in class 1 substrates.

As an approach to rationalizing the differences we have observed between the proteases in specificity and inhibitor binding, we have made extensive use of structural models of the HIV-l enzyme. We were particularly interested in the observation (24) that the MVTlOl peptide binds in an ex-


tended conformation. This fact is easily reconciled with results of studies in solution. Our observations from protease hydrolysis of PE40 derivatives are in accord with the view that cleavage takes place at flexible, extended structures in the protein substrates that behave, essentially, the same as small modeled peptides (19). Indeed, restriction of processing by the protease to interdomain or interprotein regions of the natural viral polyproteins provides further support for the idea that the enzyme prefers substrates with an extended conformation. A similar conclusion was reached in modeling studies of HIV-l and HIV-2 protease cleavage site in calmod- ulin, described by Tomasselli et ~1.~

of such compounds as AIDS therapeutics in the monkey model.

From the foregoing discussion, it would appear that if one were to design inhibitors of the HIV-2 enzyme with selectivity over the HIV-l protease, scissile dipeptide substitutions with small amino acids such as Ala might contribute in an important way to their differential activity. This is certainly an interesting topic to pursue from the point of view of deline- ating differences in binding of specific compounds to the two proteases and in elucidating facets of their enzymology. In a more practical sense, the increasing impact of HIV-2 on the global AIDS epidemic makes it clear that therapeutic approaches to the disease must take into account whatever similarities or differences may exist between targets in the two major viral forms. With our focus on the protease as an intervention point, it appears that we already have inhibitors that may prove effective against HIV-l in a clinical setting, and it could be that a single inhibitor will serve to block viral maturation in both HIV-l and HIV-2. However, in the event that such a universal drug lacks the activity required to block maturation in HIV-2, the present work provides the basis for development of inhibitors with improved binding character- istics relative to the HIV-2 protease. In any case, it is now clear that tightly bound inhibitors of the protease in vitro are not necessarily effective antiviral agents in cell culture. If inhibitors are unable to penetrate cells or budded viral parti- cles, they will not find their way to the target. Such considerations underscore the importance of the delivery character- istics of candidate compounds as well as KI in drug design.

Finally, it should be stressed that therapeutic approaches to AIDS treatment that are based upon recombinant CD4 strategies or “suicide substrates” such as AZT have already been validated in monkeys or humans (41, 42). Such is not the case for protease inhibitors as drugs, and it would be desirable to test the concept in an animal model such as SIV. Because of the close similarity between the genomic structures of SIV and HIV-2, it is logical that the latter disease should provide more definitive clues about compounds for testing in SIV. In considering the virally encoded proteases, it is interesting to note that although the HIV-l and HIV-2 enzymes are about 50% identical in sequence, the latter is about 90% identical to the SIV protease (Fig. 3). Moreover, the processing sites in SIV and HIV-2 gag polyproteins are remarkably similar (31). One would infer, based upon sequence homology, that the tertiary structures of proteases from SIV and HIV-2 will be essentially identical. Indeed, we find that there are no differences in sequence between the HIV-2 and SIV proteases that would translate to a structural difference anywhere within 9 A of the active site cleft, except for a very conservative Lys-to-Arg replacement at residue 7. One might well expect that this active site similarity between the SIV and HIV-2 proteases will extend, as well, to their substrate specificity. In any case, what we have learned about the substrate preferences of the HIV-2 protease suggests strategies for design of inhibitors that could be used to validate the concept

Acknowledgments-We thank Drs. A. Wlodawer of the Frederick Cancer Research Facility, and Drs. D. B. McKay of Stanford Univer- sity for providing the x-ray crystallographic coordinates for HIV-l protease and Pseudomonas exotoxin, respectively. We wish to ac- knowledge Drs. D. J. FitzGerald and I. Pastan of the National Cancer Institute for their generous gifts of exotoxin and recombinant derivatives, and for helpful discussions of the work.

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