Motec. AspectsMed.Vol. 12, pp. 329-340, 1991
0098-2997/91 $0.00+ .50 ©1991 PergamonPressplc.
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PROTEINASE INHIBITORS Christina Baboonian and Angus G. Dalgleish C/inical Research Centre, Wafford Road, Harrow, Middlesex HA 1 3UJ, U. K.
Introduction
The increasing incidence of HIV infection has brought with it an urgent need to identify antiviral agents that are effective and safe. Molecular biological approaches have yielded rapid results in identifying viral factors essential for replication as well as characterising viral enzymes such as proteinase and reverse transcriptase. The information on the structure and function of viral proteins is now being used for the design and synthesis of highly selective inhibitors of HIV. Advances in AIDs chemotherapy have until recently concentrated on the development of zidovudine (AZT) and similar compounds targeting the viral reverse transcriptase for the treatment of patients with advanced disease. With the toxicities associated with AZT, research has been directed towards other targets for antiviral therapy, with major emphasis on proteinase inhibitors. Very little information is currently available on clinical studies with proteinase inhibitors. However, agents that target HIV proteinase are under development and data on in vitro studies of specifically designed inhibitors is gradually accumulating. Mode of Action
During the replication cycle of HIV, gag and pol genes of the virus are translated as polyproteins. These undergo post-transcriptional cleavage to yield structural proteins of the virus; p17, p24, p9 and p7 and enzymes essential for viral replication; proteinase, reverse transcriptase, endonuclease and integrase (Figure 1) (Mervis et al., 1988; Veronese et al., 1988). The processing is initiated by viral proteinase that itself is encoded within the pol open reading frame and is therefore part of the precursor polyprotein (Mous etal., 1988; DeBouck etal., 1987; Graves etal., 1988; Le Grice etal., 1988). This enzyme was first suggested as a potential target for AIDS therapy by Kramer et al. (1986) when it was shown that mutations in the proteinase region of the pol gene prevent processing of the gag polyprotein precursor. In the absence ofproteinase, non-infectious viral particles devoid of electron dense cores are produced (Kohl etal., 1988; Peng etal., 1989). Rational Design of Proteinase
Inhibitors
On the basis of its primary amino acid sequence and its crystal structure, HIV- 1 proteinase has been classified as aspartic proteinase (Lapatto et aL, 1989; Wlodawar et al., 1989). Aspartic proteinases share a highly conserved active site sequence Asp-Thr/Ser-Gly and include such enzymes as renin, 329
330
C. Baboonianand A. G. Dalgleish
~IWF~sF-jpol
]1
I
en,
]
J earl),trunnnripti.on regulatory proteins late transcription
full length mRNA translation
I gag pruuurner pUS J
MA
CA
~
NC p6
[ gug-lml I~nmqmr plee
l
1
t intogm..}endonueluse P32 p65/~51
proteiuase, revuPsetnnsorilAlU, pll RNase H
p7
Fig. 1. Diagrammatic representation of HIV-1 gone expresskm and protein processing. Structural proteins are encoded by an unspliced full length mRN/~ Gag and gas-Pol precursorproteins are cleaved by vitally e~codedpmteinase releasingviral matrixprotein (MA),the eapsidandnucleocapsidproteins (CA,NC), two subtmitsof~v easetranscriptase and the intcgrase/endonuclease. pepsin, gastrin, cathepsin D and cathepsin E (Jupp et al., 1990). Retroviral proteinases are smaller than mammalian aspartic proteinases and consist of a single domain structure. Since a single domain containing Asp-Thr-Gly would not be functional, an active binding site for interaction with peptide bonds of the substrate is formed by homodimerisation (Meek et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). It is thought that during viral assembly and budding, concentration of gag-pol precursor polyproteins at the membrane of the host cell allows dimerisation of the polyprotein, converting the inactive monomer of viral proteinase to a catalytically active dimer. It is likely that intermolecular cleavage involving two polyprotein dimers occurs, leading to the release of proteinase. Viral polyproteins then undergo limited proteolysis to yield more enzymatically active proteinase as well as reverse transcriptase and endonuclease.
Proteinase Inhibitors
331
The design of proteinase inhibitors initially proved difficult because purification of active proteinase from mature HIV-1 particles was unsuccessful (Lillehoj et aL, 1988). This problem has now been overcome by the expression of recombinant proteinase in Escherichia coli. large enough quantities of the enzyme for use as reagent in structural determinations and inhibition studies are now available (Hiral et al., 1990). Characterisation of HIV-1 proteinase has shown that there are close structural similarities between this enzyme and the mammalian aspartic proteinases, suggesting similar mechanism of action. Inhibitors of the mammalian proteinase renin, involved in the control of blood pressare, have already been described as anti hypertensive compounds. Methods similar to that used for renin can be used for the design and screening of potential HIV-1 aspartic proteinase inhibitors. Availability of purified viral proteinase has led to the determination of the structure of the natural substrate for proteinase (Figure 2) and the amino acid sequences of polyproteins recognised by this viral enzyme (Figure 3). It has therefore become possible to make synthetic oligopeptides that span each of the eight cleavage sites shown in Figure 3 for use as substrate in inhibition assays.
P1
H
H
0
O: PI" proteinase
Fig. 2. Schematic representation of the natural substrate for HIV-1 proteinase.
1. 2. 3. 4. 5. 6. 7. 8.
Val-Ser-Gln-Asn-Tyr Lys-Ala-Arg-Val-Leu Thr-Ala-Thr-Ile-Met Arg-Pro-Gly-Asn-Phe Val-Ser-Phe-Asn-Phe Cys-Thr-Leu-Asn-Phe Ile-Ala-Lys-Ile-Leu Gly-Ala-Glu-Thr-Phe
* * * * * * * *
Pro-Ile-Val-Gln-Asn Ala-Ghi-Ala-Met-Ser Met-Gln-Arg-Gly-Asn Leu-Gln-Ser-Arg-Pro Pro-Gln-Ile-Thr-Leu Pro-Ile-Ser-Pro-Ile Phe-Leu-Asp-Gly-Ile Tyr-Val-Asp-Lys-Arg
Fig. 3. Aminoacid sequencesrecognisedby HIV proteinase. Cleavage sites have the scissile bond representedby an asterisk(*) (HeUenet al., 1989; Graveset al., 1990).
Non Peptide Inhibitors Initial efforts towards finding an inhibitor of viral proteinase were limited to screening compounds which could be fortuitously active against HIV- 1. Pal et al., (1988) found that the antifungal antibiotic, cerulenin, which was characterised originally as an inhibitor of fatty acid biosynthesis, inhibits HIVpolyprotein processing. Cerulenin and related analogues are slow acting non specific inhibitors ofHIVproteinase (Blumenstein et al., 1989), which react with active site aspartic acid residues of the enzyme. Cerulenin is toxic at
concentrations required to be inhibitory for HIV.
332
C. Baboonian and A. G. Dalgleish
A different approach to finding anti-HIV proteinase inhibitors, taking into account the threedimensional structure of viral proteinase, was reported by Des Jarlais et al., (1990). A computer assisted search of compounds registered in a crystallographic database predicted bromoperidol to be active against HIV. Haloperidol, a closely related compound, is in use as an anti-psychotic agent. In vitro experiments showed that this compound selectively inhibits HIV-1 and HIV-2 proteinase with a Ki of 100/~tm. The concentrations used for treatment of psychological disorders is one thousand times lower than that required for inactivation of HIV. As high concentrations of this compound are very toxic, haloperidol cannot be used for treatment of AIDS. It is possible, however, to use this compound as a lead for development of less toxic antiviral agents.
Analogues of Statine Although compounds such as cerulenin and haloperidol have been found that are active against HIV, a more systematic approach to generating proteinase inhibitors is also in progress. Research carried out in the past few years in the study and design of inhibitors of renin has been a comerstone in the discovery of agents active against HIV. An example of such a compound is pepstatin, a naturally occurring inhibitor of all aspartic pmteinases. Pepstatin contains the dipeptide analogue statine (Figure 4) and in its most commonly available form ofisovaleryl pepstatin it is a very potent inhibitor of most aspartic proteinases binding with dissociation constants often in the range of 0.1-1 nM. It is, however, much less inhibitory for mammalian and retrovirai proteinases, probably due to the structural differences thatexist between such proteinases and pepstatin. In contrast acetyl-pepstatin (=Ac-Val-Val-Sta-Ala-Sta) is an effective inhibitor of HIV-1 (K i 20nM) and ofHIV-2 (K i 5nM) proteinase (Richards et al., 1989a;b). A large number of moderately potent proteinase inhibitors have been generated by insertion of statine (or its aromatic analogue PheSta, 4-amino-3-OH-5 phenyl pentanoic acid) in place of the scissile dipeptide unit in synthetic compounds(Mooreetal., 1989;Dreyeretal., 1989). More recently Grinde etal.(1990)testedapeptide Ac-Gln-Asn-Sta-Val-NHz with a polar group on the N-terminal side of statine. High concentrations of this compound (400~tm) were required in order to inhibit virus replication by 40%. Pepstatin and its analogues are poor candidates for treatment of HIV infection. The ability of these compounds to penetrate cells is limited and high intracellular concentrations are difficult to achieve. Acetyl pepstatin is also a good inhibitor of most aspartic proteinases and it is therefore not selective against the viral enzyme.
H
OH
0
Fig. 4. Diagrammatic representatico of statine, used in inhibitccs of HIV-1 proteinase.
Proteinase Inhibitors
333
Hydroxyethylene Isosteres Inhibitors of proteinase incorporating hydroxyethylene analogues (Figure 5) are considered more potent inhibitors than homologous compounds based on statine (Agarwal et al., 1986; Szelke et al., 1985; Holladay etal., 1987). The mode of action of both analogues is similar. X-ray structural analysis has shown that statine and hydroxyethylene isosteres structurally resemble transition state for peptide hydrolysis. It is thought that the inhibitor binds to the substrate binding region with statine or hydroxyethylene isostere coveting the scissile dipeptide binding site (James et al., 1982; Foundling et al., 1987). Several investigators have produced inhibitors with hydroxyethyleneisosteres. Richards et al. (1989b) described a compound H261 which has the dipeptide analogue Phe (-CHOH-CH 2)in a compound tB ocHis-Pro-Phe-His-Leu-Phe(CHOH-CH2)-Val-Ile-His, (tBoc=tert-butyloxycarbonyl).This is an analogue of angiotensinogen containing the residues flanking the peptide bond (-Leu*-Val-) cleaved by renin. H261 is a potent inhibitor of HIV-1 proteinase (Ki=5nM) in vitro. It is also able to inhibit HIV-2 proteinase, although much less effectively (Kt=35nM). This compound is not considered for use as an
H
_
0
A H
OH
B H
OH
C
Fig. 5. (A, B, C) - legend overlearj~.
334
C. Baboonian and A. G. Dalgleish
D O
OH
O
E H
OH
F
Fig. 5. Structures of hydroxyethylene isosteres incorporated into HIV-1 proteinase inhibitors. antiviral agent because of its inability to inhibit HIV-1 replication in C8166 cells. It is likely that the compound is unable to penetrate cells effectively and is therefore not available at high enough concentrations in the intracellular environment. Lack of effectiveness in infected cells is not a universal problem of hydroxyethylene isosteres. Many compounds are now available that can penetrate ceils and stop virus production. Examples of such compounds are those made by McQuade et al. (1990) and Meek et al. (1990). Merck, Sharp and Dohme Inc. have produced compounds (tBoc-Phe(CHOH-CH2)-Phe-Leu-Phe-NH2) (Durra and Kay, 1990) that have sub-nanomolar IC~ovalues against HIV- 1 proteinase and are effective against virus-infected cells at mieromolar concentrations. Data on the activity of these compounds in vivo is not available. The hydroxyethylene insert CH(OH)CH(OH) was used by Ashorn et al. (1990) in a compound U75875. U-75875 inhibited HIV-1 gag-pol processing in infected cells in an irreversible manner, resulting in immature virus particles. This compound also inhibited HIV-2 and SIV proteinase.
Proteinase Inhibitors
335
Preliminary studies with rodents were suggested to have shown that the compound is non toxic at higher than therapeutic concentrations for several hours. More recent studies of HIV-proteinase inhibitors have been directed towards the development of effective agents that would not be able to interact with any of the human aspartic proteinases. HIV proteinase is unusual in being able to cleave the Phe-Pro and Tyr-Pro sequences found in gag and gagpol polyproteins. Since mammalian endopeptides are unable to cleave amide bonds ofproline residues, Dreyer et al. (1990) generated compounds by introducing a transcyclopentyl ring in a hydroxyethylene isostere analogue of Phe-Pro replacing the prolyl pyroidine ring (Figure 5C and D). The transcyclopentyl ring proved not to be an optimal mimic for a prolyl residue and the compounds produced were weaker inhibitors than unsubstituted analogues. Overton et al. (1990) and Roberts et al. (1990) were more successful and showed that a hydroxyethylamine transition state mimic incorporating an analogue of the amide bond of Phe-Pro produced inhibitors that were potent at 1 ktm concentrations and demonstrated a very high level of selectivity. These inhibitors were effective against chronically infected cells. Data on the bioavailability of these compounds is not available, but it has been argued that the high selectivity associated with these compounds may result not only in reduced toxicity but low affinity for aspartic proteinases present in the gut and therefore enhance oral absorption.
Reduced Amide Groups The reduced amide isostere was used by Szelke and co-workers to generate inhibitors ofrenin (Figure 6; Szelke et al., 1982; Szeike et al., 1985). This group of inhibitors are thought to bind and inhibit aspartyl proteinase in a similar fashion to that of hydroxyethylene isosteres and statine analogues. The potency of compounds such as Ac-Ser-Gln-Asn-Phe-(CH2-NH)-Pro-Val-Val-NH2or Ac-Thr-Ile-NIe(CH2-NH)-Nle-Gln-Arg-NH2, that include the reduced amide group is poor (Moore et al., 1989); Miller et al., 1989).
H
OH
Fig. 6. Structureof reduced amide isostere.
Phosphinate Groups Phosphinic acids are modest inhibitors of HIV-1 proteinase (Figure 7; Dreyer et al., 1989). It has however, been reported that the substitution of the scissile amide bond with phosphinic acid isosteres of di' tetra and hexapeptides containing a side chain at the P1-P'I positions resulted in powerful inhibitors of HIV proteinase (Ki---0.4 riM; Grobelny et al., 1990). Although these compounds were considered to be selective for HIV, inhibition of cathepsin D at low pH values was substantial. The ability of these compounds to penetrate cells has not been tested.
336
C. Baboonian and A. G. Dalgleish
I
I
H
OH
I
H
I
OH
Fig. 7. Schematicrepresentationof phosphinateisosteres.
~-FIuoroketones These are inhibitors of several classes of enzymes. Pepsin inhibitors based on difluorostatone (Figure 8) have been described that are 20 times more potent than corresponding inhibitors containing statine (Gleb et al., 1985). Although competitive inhibitors of HIV proteinase have been studied by Dreyer et al. (1989) these are unlikely to be of use as chemotherapeutic agents because of the likelihood of interactions with a wide range of host enzymes.
Other Inhibitors Copeland et al. (1990) have also made use of the ability of viral enzyme to cleave amide bonds of proline residues. They have designed a nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2containing the retroviral Tyr-Pro cleavage site. This peptide is not readily hydrolysed by non viral proteinases including pepsin. Replacing the Pro residue with 2-piperidine carboxylic acid converts this substrate
Proteinase Inhibitors
H
0
337
0
Fig. 8. Structm~ of dJfluoro6tatone.
into an inhibitor of HIV-1 and HIV-2 proteinases with a high degree of selectivity. The ability of this compound to inhibit virus replication has not been assessed yet.
Conclusions Progress in the field of HIV research has been remarkable. A few years ago knowledge of viral proteinase was limited to the potential coding region in cDNA clones. Now not only can the enzyme be expressed in bacterial cells, it can also be chemically synthesised. Structure of the viral proteinase has been studied using X-ray analysis of the crystallised material and sequences of amino acids recognised by HIV proteinase as well as the length and location of these sequences have been determined. Since the significance of proteinase in virus maturation was established, several pharmaceutical companies have initiated molecular modelling studies in order to design proteinase inhibitors that can bind to the active site of the enzyme. It is now apparent that HIV proteinase inhibition can be achieved with compounds with a wide range of chemical smactures. This is because a range of residues contributing to the scissile peptide bond can be accommodated in the active site of the viral proteinase. Initial in vitro studies were encouraging and a large number of compounds were made that proved active against viral proteinase. These inhibitors were later found to be ineffective in cell culture either because of poor solubility or lack of cellular uptake. Antiviral agents designed later overcame these problems. Lack of selectivity of the inhibitors, however, remained until it was found that the HIV- 1 proteinase is able to cleave the amino terminal side of proline. The inclusion of proline mimics into the dipeptide analogues in response to the unusual cleavage pattern of HIV-1 proteinase introduced better selectivity to the inhibitors. Many problems still remain in the choice of therapeutically useful compounds for oral administration. Proteinase inhibitors which are active in cell culture may not necessarily be active in vivo. For antiviral agents to be effective an adequate in vivo pharmacokinetic profile must be achieved. Compounds have to be readily absorbed and able to penetrate infected cells. Sufficient concentrations of the antiviral agents must be maintained for an adequate length of time. At present there is very little information concerning the reversibility of the effect ofproteinase inhibitors. Immature virus particles may regain their proteinase activity and become infectious if the levels of antiviral compounds diminish. This will introduce a further constraint on maintaining a continuous inhibitory concentration of the antiviral agent. A final issue that has not been addressed is the activity of the compounds against clinical isolates. In most experiments carried out to date, strains ofHIV- 1 that are easy to propagate in the laboratory have been used. Activity of these inhibitors on fresh isolates of virus must be studied before clinical trials
338
C. Baboonian a n d A. G. Dalgleish
begin. Other strains of virus apart from HIV- 1 must also be considered. The spread of HIV-2 in Africa poses the possibility that in the not too distant future HIV-2 m a y have a major impact on the global AIDS epidemic. Therapeutic approaches to the disease must consider the likelihood that soon there may be both strains of virus in circulation in the population to be targeted for treatment.
References Agarwal, N.S. and Rich, D.H. (1986) J. Med. Chem. 29: 2519-2524. Ashom, P., McQuade, T.J., Thaisrivongs, S., Tomasselli, A.G., Tarpley, W.G. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87: 7472-7476. Blumenstein, J.J., Copeland, T.D., Oroszlan, S. and Michejda, C.J. (1989) Biochem. Biophys. Res. Comm. 163: 980-987. Copeland, T.D., Wondrak, E.M., Tozser, J., Roberts, M.M. and Oroszlan, S. (1990) Biochem. Biophys. Res. Comm. 169: 310-314. DeBouck, C., Gormiak, J.G., Striekler, J.E., Meek, T.D., Metcalf, B.W. and Rosenbexg, M. (1987) Proc. Natl. Acad. Sci. USA 84: 8903-8906. Des Jarlais, R.L., Seibel, G.L., Kuntz, I.D., Furth, P.S., Alvarez, J.C., Ottiz de Moatellano, P.R., DeCamp, D.L., Babe, L.M. and Craik, C.S. (1990) Proc. Natl. Acad. $ci. USA 87: 6644-6648. Dreyer, G.B., Metealf, B.W., Tomaszek, T.A. Jr., Carl T.I-L,Chandler HI, A.C., Hyland, L., Fakhoury, S.A., Magaard, V.W., Moore, M.L., Striekler, J.E., Debouek, C. and Meek, T.D. (1989) Proc. Natl. Acad. Sci. USA 86: 9752-9756. Duun, B.M. and Kay, J. (1990) Antiviral Chemistry and Chemotherapy 1: 3-8. Foundling, S.I., Cooper, J., Watson, F.E., Cleasby, A., Pearl, L.H., Sibanda, B.L., Hemmings, A., Wood, S.P., Blundell, T.L., Vallex, M.J., Norey, C.G., Kay, J., Boger, J., Dtmn, B.M., Leekie, B.J., Jones, D.M., Alrash, B., Hallett, A. and Szelke, M. (1987) Nature 327: 349-352. Gleb, M.H., Svaren, J.P. and Abeles, R.H. (1985) Biochemistry 24: 1813-1817. Graves, M.C., Lira, J.J., Heimer, E.P. and Kramer, R.A. (1988). Proc. Natl. Acad. Sci. USA 85: 2449-2553. Graves, M.C., Meidel, M.C., Pan, Y.C.E., Manneberg, M., Lahm, H.W. andGnminger-Leitch,F.(1990) Biochem. Biophys. Res. Comm. 168: 30-36. Grinde, B., Hungnes, O. and Tjotta, E. (1990) Arch. Virol. 114: 167-173. Grobelny, D., Wondrak, E.M., Galardy, R.E. and Oroszlan, S. (1990) Biochem. Biophys. Res. Comm. 169: 1111-1116. Hellen, C.U., Kransslich, H.G. and Wimmer, E. (1989) Biochemistry 28: 9881-9890. Henderson, L.E., Benveniste, R.E., Sowder, R., Copeland, T.D., Sehults, A.M. and Oroszlan, S. (1988) J. Virol. 62: 25872595. Hiral Ph, H., Parker, F., Boizian, J., Jung, G., Outervitch, D., Dugue, A., Peltiers, C., Giuliacci, C., Boulay, R., Lelievre, Y., Cambou, B., Mayat=, J.F. and Cartwright, T. (1990) Antiviral Chemistry and Chemotherapy 1: 9-15. Holladay, M.W., Salituro, F.G. and Rich, D.H. (1987) J. Med. Chem. 30: 374-383. James, M.N.G., Sielecki, A., Salituro, F., Rich, D.H. and Hofmann, T. (1982) Proc. Natl. Acad. Sci. USA. 79: 6137-6141. Jupp, R.A., Dunn, B.M., Jacobs, J.W., Vlasuk, G., Arcuri, K.E., Veber, D.F., Perlow, D.S., Payne, L.S., Roger, J., deLaszlo, S., Chakravarty, P.K., TenBroeke, J., Hangover, D. G., Ondeyka, D., Greealee, WJ. and Kay, J. (1990) Biochem. J. 265: 871-878.
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Kohl, N.E., Emini, E.A., Schleff, W.A., Davis, L.J., Heimbach, J.C., Dixon, R.A.F., Scolnick, KM. and Sigal, I.S. (1988) Proc. Natl. Acad. Sci. USA 85: 4686-4690. Kramer, R.A., Schaber, M.D., SIr, llr~ A.M., Ganguly, K., Wong-Staal, F. and Reddy, E.P. (1986) Science 231:1580-1584. Lapatto, R., BlundelL T.L., Hemmings, A., Overington, J., Wflderspin, A., Wood, S., Merson, J.1L, Whittle, P.J., Danley, D.F., Geogbegan, K.F., HawryllL SJ., Lee, S.E., Sfheld, K.G. and Hobart, P.M. (1989) Nature 342: 299-302. Le Ca'ice, S.F.J., Zehnle, R. and Mous, J. (1988) J. ViroL 62: 2525-2529. Lillehoj, E.P., Salazar, F.H.R., Mervis, RJ., Ramn, M.G., Chan, H.W., Abroad, N. and Venkatesan, S. (1988) J. Viroi 62: 3053-3058. McQuade, T.J., Tomasselli, A.G., Liu, L., Karacostas, V., Moss, B., Sawyer, T.K., Heinrikson, R.L. and Tarpley, W.G. (1990) Science 247: 454-456. Meek, T.D., Dayton, B.D., Metcalf, B.W., Dreyer, G.B., SUickler, J.E., Gornlak; J.G., Rosenberg, M., Moore, M.L., Magaard, V.W. and Debouck, C. (1989) Proc. Natl. Acad. Sci. USA 86: 1841-1845. Meek, T.D., Lambert, D.M., Dreyer, G.B., Cart, TJ., Tomaszek, T.A., Moore, M.L., Strickler, J.E., Debouck, C., Hyland, L.J., Matthews, T.J., Metcalf, B.W. and Petteway, S.R. (1990) Nature 343: 90-92. Mervis, R.J., Abroad, N., Lillehoj, E.P., Raum, M.G., Salazar, F.FLR.,Chart, H.W. and Venkatesan, S. (1988) J. Virol. 62: 3993-4002. Miller, M., Sathyanarayana, B.K., Wlodawer, A., Toth, M.V., Marshall, G.R., Clawson, L., Sell L., Schneider, J. andKent, S.B.H. (1989) Science 246:1149-1152. Moore, M.L., Bryan, M.W., Fakhoury, S.A., Magaard, V.W., Huffman, W.F., Dayton, B.D., Meek, T.D., Hyland, L., Dreyer, G.B., Metcalf, B.W., Strickler, J.E., Gomiak, J.G. and Debouck, C. (1989) Biochem. Biophys. Res. Comm. 159: 420-425. Mous, J., Heimer, E.P. and LcCaice, S.F.J. (1988) J. Virol. 62: 1433-1438. Navia, M.A., Fitzgerald, P.M.D., MeKeevcr, B.M., Leu, C.T., Heimbach, J.C., Herbcr, W.K., Sigal, I.S., Darl~, P.L. and Springer, J.P. (1989) Nature 337: 615-620. Overton, H.A., McMillan, D.J., Gridley, S.J., Brenner, J., Rcdshaw, S. and Mills, J.S. (1990) Virology 179: 508-511. Pal, R., Gallo, R.C. and Bamagadharan, M.G. (1988) Proc. Natl. Acad. Sci. USA 85: 9283-9286. Peng, C., Ho, B.K., Chang, T.W. and Chang, N.T. (1989) J. Virol. 63: 2550-2556. Richards, A.D., Roberts, R., Dunn, B.M., Graves, M.C. and Kay, J. (1989a) FEBS Letts. 247:113-117. Richards, A.D., Broadhurst, A., Ritehie, A.J., Durra, B.M. and Kay, J. (1989b) FEBS Letts 253: 214-216. Roberts, N.A., Martin, J.A., Kinchington, D., Broadhurst, A.V., Craig, J.C., Duncan, LB., Galpin, S.A., Handa, B.K., Kay, J., Krohn, A., Lambert, R.W., Merrett, J.H., Mills, J.S., Parkes, K.E.B., Redshaw, S., Ritchie, A.J., Tayler, D.L., Thomas, G.J. and Machin, P.J. (1990) Science 248: 358-361. Szelke, M., Leckie, B., Hallett, A., Jones, D.M., Sueiras, J., Atrash, B. and Lever, A.F. (1982) Nature 299: 555-557. Szelke M. (1985) In: Aspartic Proteinases and Their lnhibitors, ed. Kostlm V. (de Ca'uyter,Berlin), pp. 412--441. Veronese, F DiM, Copeland, T.D., Oroszlan, S., Gallo, R.C. and Samgadharan, M.G. (1988) J. Virol. 62: 795-801. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanalayana, B.K., Baldwin, E., Weber, LT., Selk, L., Clawson, L., Sdmeider, M. and Kent, S.B.H. (1989) Science 245: 616-621.
0098-2997/91 $0.00+ .50 ©1991 PergamonPressplc.
Printedin GreatBritain.All rightsreserved.
PROTEINASE INHIBITORS Christina Baboonian and Angus G. Dalgleish C/inical Research Centre, Wafford Road, Harrow, Middlesex HA 1 3UJ, U. K.
Introduction
The increasing incidence of HIV infection has brought with it an urgent need to identify antiviral agents that are effective and safe. Molecular biological approaches have yielded rapid results in identifying viral factors essential for replication as well as characterising viral enzymes such as proteinase and reverse transcriptase. The information on the structure and function of viral proteins is now being used for the design and synthesis of highly selective inhibitors of HIV. Advances in AIDs chemotherapy have until recently concentrated on the development of zidovudine (AZT) and similar compounds targeting the viral reverse transcriptase for the treatment of patients with advanced disease. With the toxicities associated with AZT, research has been directed towards other targets for antiviral therapy, with major emphasis on proteinase inhibitors. Very little information is currently available on clinical studies with proteinase inhibitors. However, agents that target HIV proteinase are under development and data on in vitro studies of specifically designed inhibitors is gradually accumulating. Mode of Action
During the replication cycle of HIV, gag and pol genes of the virus are translated as polyproteins. These undergo post-transcriptional cleavage to yield structural proteins of the virus; p17, p24, p9 and p7 and enzymes essential for viral replication; proteinase, reverse transcriptase, endonuclease and integrase (Figure 1) (Mervis et al., 1988; Veronese et al., 1988). The processing is initiated by viral proteinase that itself is encoded within the pol open reading frame and is therefore part of the precursor polyprotein (Mous etal., 1988; DeBouck etal., 1987; Graves etal., 1988; Le Grice etal., 1988). This enzyme was first suggested as a potential target for AIDS therapy by Kramer et al. (1986) when it was shown that mutations in the proteinase region of the pol gene prevent processing of the gag polyprotein precursor. In the absence ofproteinase, non-infectious viral particles devoid of electron dense cores are produced (Kohl etal., 1988; Peng etal., 1989). Rational Design of Proteinase
Inhibitors
On the basis of its primary amino acid sequence and its crystal structure, HIV- 1 proteinase has been classified as aspartic proteinase (Lapatto et aL, 1989; Wlodawar et al., 1989). Aspartic proteinases share a highly conserved active site sequence Asp-Thr/Ser-Gly and include such enzymes as renin, 329
330
C. Baboonianand A. G. Dalgleish
~IWF~sF-jpol
]1
I
en,
]
J earl),trunnnripti.on regulatory proteins late transcription
full length mRNA translation
I gag pruuurner pUS J
MA
CA
~
NC p6
[ gug-lml I~nmqmr plee
l
1
t intogm..}endonueluse P32 p65/~51
proteiuase, revuPsetnnsorilAlU, pll RNase H
p7
Fig. 1. Diagrammatic representation of HIV-1 gone expresskm and protein processing. Structural proteins are encoded by an unspliced full length mRN/~ Gag and gas-Pol precursorproteins are cleaved by vitally e~codedpmteinase releasingviral matrixprotein (MA),the eapsidandnucleocapsidproteins (CA,NC), two subtmitsof~v easetranscriptase and the intcgrase/endonuclease. pepsin, gastrin, cathepsin D and cathepsin E (Jupp et al., 1990). Retroviral proteinases are smaller than mammalian aspartic proteinases and consist of a single domain structure. Since a single domain containing Asp-Thr-Gly would not be functional, an active binding site for interaction with peptide bonds of the substrate is formed by homodimerisation (Meek et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). It is thought that during viral assembly and budding, concentration of gag-pol precursor polyproteins at the membrane of the host cell allows dimerisation of the polyprotein, converting the inactive monomer of viral proteinase to a catalytically active dimer. It is likely that intermolecular cleavage involving two polyprotein dimers occurs, leading to the release of proteinase. Viral polyproteins then undergo limited proteolysis to yield more enzymatically active proteinase as well as reverse transcriptase and endonuclease.
Proteinase Inhibitors
331
The design of proteinase inhibitors initially proved difficult because purification of active proteinase from mature HIV-1 particles was unsuccessful (Lillehoj et aL, 1988). This problem has now been overcome by the expression of recombinant proteinase in Escherichia coli. large enough quantities of the enzyme for use as reagent in structural determinations and inhibition studies are now available (Hiral et al., 1990). Characterisation of HIV-1 proteinase has shown that there are close structural similarities between this enzyme and the mammalian aspartic proteinases, suggesting similar mechanism of action. Inhibitors of the mammalian proteinase renin, involved in the control of blood pressare, have already been described as anti hypertensive compounds. Methods similar to that used for renin can be used for the design and screening of potential HIV-1 aspartic proteinase inhibitors. Availability of purified viral proteinase has led to the determination of the structure of the natural substrate for proteinase (Figure 2) and the amino acid sequences of polyproteins recognised by this viral enzyme (Figure 3). It has therefore become possible to make synthetic oligopeptides that span each of the eight cleavage sites shown in Figure 3 for use as substrate in inhibition assays.
P1
H
H
0
O: PI" proteinase
Fig. 2. Schematic representation of the natural substrate for HIV-1 proteinase.
1. 2. 3. 4. 5. 6. 7. 8.
Val-Ser-Gln-Asn-Tyr Lys-Ala-Arg-Val-Leu Thr-Ala-Thr-Ile-Met Arg-Pro-Gly-Asn-Phe Val-Ser-Phe-Asn-Phe Cys-Thr-Leu-Asn-Phe Ile-Ala-Lys-Ile-Leu Gly-Ala-Glu-Thr-Phe
* * * * * * * *
Pro-Ile-Val-Gln-Asn Ala-Ghi-Ala-Met-Ser Met-Gln-Arg-Gly-Asn Leu-Gln-Ser-Arg-Pro Pro-Gln-Ile-Thr-Leu Pro-Ile-Ser-Pro-Ile Phe-Leu-Asp-Gly-Ile Tyr-Val-Asp-Lys-Arg
Fig. 3. Aminoacid sequencesrecognisedby HIV proteinase. Cleavage sites have the scissile bond representedby an asterisk(*) (HeUenet al., 1989; Graveset al., 1990).
Non Peptide Inhibitors Initial efforts towards finding an inhibitor of viral proteinase were limited to screening compounds which could be fortuitously active against HIV- 1. Pal et al., (1988) found that the antifungal antibiotic, cerulenin, which was characterised originally as an inhibitor of fatty acid biosynthesis, inhibits HIVpolyprotein processing. Cerulenin and related analogues are slow acting non specific inhibitors ofHIVproteinase (Blumenstein et al., 1989), which react with active site aspartic acid residues of the enzyme. Cerulenin is toxic at
concentrations required to be inhibitory for HIV.
332
C. Baboonian and A. G. Dalgleish
A different approach to finding anti-HIV proteinase inhibitors, taking into account the threedimensional structure of viral proteinase, was reported by Des Jarlais et al., (1990). A computer assisted search of compounds registered in a crystallographic database predicted bromoperidol to be active against HIV. Haloperidol, a closely related compound, is in use as an anti-psychotic agent. In vitro experiments showed that this compound selectively inhibits HIV-1 and HIV-2 proteinase with a Ki of 100/~tm. The concentrations used for treatment of psychological disorders is one thousand times lower than that required for inactivation of HIV. As high concentrations of this compound are very toxic, haloperidol cannot be used for treatment of AIDS. It is possible, however, to use this compound as a lead for development of less toxic antiviral agents.
Analogues of Statine Although compounds such as cerulenin and haloperidol have been found that are active against HIV, a more systematic approach to generating proteinase inhibitors is also in progress. Research carried out in the past few years in the study and design of inhibitors of renin has been a comerstone in the discovery of agents active against HIV. An example of such a compound is pepstatin, a naturally occurring inhibitor of all aspartic pmteinases. Pepstatin contains the dipeptide analogue statine (Figure 4) and in its most commonly available form ofisovaleryl pepstatin it is a very potent inhibitor of most aspartic proteinases binding with dissociation constants often in the range of 0.1-1 nM. It is, however, much less inhibitory for mammalian and retrovirai proteinases, probably due to the structural differences thatexist between such proteinases and pepstatin. In contrast acetyl-pepstatin (=Ac-Val-Val-Sta-Ala-Sta) is an effective inhibitor of HIV-1 (K i 20nM) and ofHIV-2 (K i 5nM) proteinase (Richards et al., 1989a;b). A large number of moderately potent proteinase inhibitors have been generated by insertion of statine (or its aromatic analogue PheSta, 4-amino-3-OH-5 phenyl pentanoic acid) in place of the scissile dipeptide unit in synthetic compounds(Mooreetal., 1989;Dreyeretal., 1989). More recently Grinde etal.(1990)testedapeptide Ac-Gln-Asn-Sta-Val-NHz with a polar group on the N-terminal side of statine. High concentrations of this compound (400~tm) were required in order to inhibit virus replication by 40%. Pepstatin and its analogues are poor candidates for treatment of HIV infection. The ability of these compounds to penetrate cells is limited and high intracellular concentrations are difficult to achieve. Acetyl pepstatin is also a good inhibitor of most aspartic proteinases and it is therefore not selective against the viral enzyme.
H
OH
0
Fig. 4. Diagrammatic representatico of statine, used in inhibitccs of HIV-1 proteinase.
Proteinase Inhibitors
333
Hydroxyethylene Isosteres Inhibitors of proteinase incorporating hydroxyethylene analogues (Figure 5) are considered more potent inhibitors than homologous compounds based on statine (Agarwal et al., 1986; Szelke et al., 1985; Holladay etal., 1987). The mode of action of both analogues is similar. X-ray structural analysis has shown that statine and hydroxyethylene isosteres structurally resemble transition state for peptide hydrolysis. It is thought that the inhibitor binds to the substrate binding region with statine or hydroxyethylene isostere coveting the scissile dipeptide binding site (James et al., 1982; Foundling et al., 1987). Several investigators have produced inhibitors with hydroxyethyleneisosteres. Richards et al. (1989b) described a compound H261 which has the dipeptide analogue Phe (-CHOH-CH 2)in a compound tB ocHis-Pro-Phe-His-Leu-Phe(CHOH-CH2)-Val-Ile-His, (tBoc=tert-butyloxycarbonyl).This is an analogue of angiotensinogen containing the residues flanking the peptide bond (-Leu*-Val-) cleaved by renin. H261 is a potent inhibitor of HIV-1 proteinase (Ki=5nM) in vitro. It is also able to inhibit HIV-2 proteinase, although much less effectively (Kt=35nM). This compound is not considered for use as an
H
_
0
A H
OH
B H
OH
C
Fig. 5. (A, B, C) - legend overlearj~.
334
C. Baboonian and A. G. Dalgleish
D O
OH
O
E H
OH
F
Fig. 5. Structures of hydroxyethylene isosteres incorporated into HIV-1 proteinase inhibitors. antiviral agent because of its inability to inhibit HIV-1 replication in C8166 cells. It is likely that the compound is unable to penetrate cells effectively and is therefore not available at high enough concentrations in the intracellular environment. Lack of effectiveness in infected cells is not a universal problem of hydroxyethylene isosteres. Many compounds are now available that can penetrate ceils and stop virus production. Examples of such compounds are those made by McQuade et al. (1990) and Meek et al. (1990). Merck, Sharp and Dohme Inc. have produced compounds (tBoc-Phe(CHOH-CH2)-Phe-Leu-Phe-NH2) (Durra and Kay, 1990) that have sub-nanomolar IC~ovalues against HIV- 1 proteinase and are effective against virus-infected cells at mieromolar concentrations. Data on the activity of these compounds in vivo is not available. The hydroxyethylene insert CH(OH)CH(OH) was used by Ashorn et al. (1990) in a compound U75875. U-75875 inhibited HIV-1 gag-pol processing in infected cells in an irreversible manner, resulting in immature virus particles. This compound also inhibited HIV-2 and SIV proteinase.
Proteinase Inhibitors
335
Preliminary studies with rodents were suggested to have shown that the compound is non toxic at higher than therapeutic concentrations for several hours. More recent studies of HIV-proteinase inhibitors have been directed towards the development of effective agents that would not be able to interact with any of the human aspartic proteinases. HIV proteinase is unusual in being able to cleave the Phe-Pro and Tyr-Pro sequences found in gag and gagpol polyproteins. Since mammalian endopeptides are unable to cleave amide bonds ofproline residues, Dreyer et al. (1990) generated compounds by introducing a transcyclopentyl ring in a hydroxyethylene isostere analogue of Phe-Pro replacing the prolyl pyroidine ring (Figure 5C and D). The transcyclopentyl ring proved not to be an optimal mimic for a prolyl residue and the compounds produced were weaker inhibitors than unsubstituted analogues. Overton et al. (1990) and Roberts et al. (1990) were more successful and showed that a hydroxyethylamine transition state mimic incorporating an analogue of the amide bond of Phe-Pro produced inhibitors that were potent at 1 ktm concentrations and demonstrated a very high level of selectivity. These inhibitors were effective against chronically infected cells. Data on the bioavailability of these compounds is not available, but it has been argued that the high selectivity associated with these compounds may result not only in reduced toxicity but low affinity for aspartic proteinases present in the gut and therefore enhance oral absorption.
Reduced Amide Groups The reduced amide isostere was used by Szelke and co-workers to generate inhibitors ofrenin (Figure 6; Szelke et al., 1982; Szeike et al., 1985). This group of inhibitors are thought to bind and inhibit aspartyl proteinase in a similar fashion to that of hydroxyethylene isosteres and statine analogues. The potency of compounds such as Ac-Ser-Gln-Asn-Phe-(CH2-NH)-Pro-Val-Val-NH2or Ac-Thr-Ile-NIe(CH2-NH)-Nle-Gln-Arg-NH2, that include the reduced amide group is poor (Moore et al., 1989); Miller et al., 1989).
H
OH
Fig. 6. Structureof reduced amide isostere.
Phosphinate Groups Phosphinic acids are modest inhibitors of HIV-1 proteinase (Figure 7; Dreyer et al., 1989). It has however, been reported that the substitution of the scissile amide bond with phosphinic acid isosteres of di' tetra and hexapeptides containing a side chain at the P1-P'I positions resulted in powerful inhibitors of HIV proteinase (Ki---0.4 riM; Grobelny et al., 1990). Although these compounds were considered to be selective for HIV, inhibition of cathepsin D at low pH values was substantial. The ability of these compounds to penetrate cells has not been tested.
336
C. Baboonian and A. G. Dalgleish
I
I
H
OH
I
H
I
OH
Fig. 7. Schematicrepresentationof phosphinateisosteres.
~-FIuoroketones These are inhibitors of several classes of enzymes. Pepsin inhibitors based on difluorostatone (Figure 8) have been described that are 20 times more potent than corresponding inhibitors containing statine (Gleb et al., 1985). Although competitive inhibitors of HIV proteinase have been studied by Dreyer et al. (1989) these are unlikely to be of use as chemotherapeutic agents because of the likelihood of interactions with a wide range of host enzymes.
Other Inhibitors Copeland et al. (1990) have also made use of the ability of viral enzyme to cleave amide bonds of proline residues. They have designed a nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2containing the retroviral Tyr-Pro cleavage site. This peptide is not readily hydrolysed by non viral proteinases including pepsin. Replacing the Pro residue with 2-piperidine carboxylic acid converts this substrate
Proteinase Inhibitors
H
0
337
0
Fig. 8. Structm~ of dJfluoro6tatone.
into an inhibitor of HIV-1 and HIV-2 proteinases with a high degree of selectivity. The ability of this compound to inhibit virus replication has not been assessed yet.
Conclusions Progress in the field of HIV research has been remarkable. A few years ago knowledge of viral proteinase was limited to the potential coding region in cDNA clones. Now not only can the enzyme be expressed in bacterial cells, it can also be chemically synthesised. Structure of the viral proteinase has been studied using X-ray analysis of the crystallised material and sequences of amino acids recognised by HIV proteinase as well as the length and location of these sequences have been determined. Since the significance of proteinase in virus maturation was established, several pharmaceutical companies have initiated molecular modelling studies in order to design proteinase inhibitors that can bind to the active site of the enzyme. It is now apparent that HIV proteinase inhibition can be achieved with compounds with a wide range of chemical smactures. This is because a range of residues contributing to the scissile peptide bond can be accommodated in the active site of the viral proteinase. Initial in vitro studies were encouraging and a large number of compounds were made that proved active against viral proteinase. These inhibitors were later found to be ineffective in cell culture either because of poor solubility or lack of cellular uptake. Antiviral agents designed later overcame these problems. Lack of selectivity of the inhibitors, however, remained until it was found that the HIV- 1 proteinase is able to cleave the amino terminal side of proline. The inclusion of proline mimics into the dipeptide analogues in response to the unusual cleavage pattern of HIV-1 proteinase introduced better selectivity to the inhibitors. Many problems still remain in the choice of therapeutically useful compounds for oral administration. Proteinase inhibitors which are active in cell culture may not necessarily be active in vivo. For antiviral agents to be effective an adequate in vivo pharmacokinetic profile must be achieved. Compounds have to be readily absorbed and able to penetrate infected cells. Sufficient concentrations of the antiviral agents must be maintained for an adequate length of time. At present there is very little information concerning the reversibility of the effect ofproteinase inhibitors. Immature virus particles may regain their proteinase activity and become infectious if the levels of antiviral compounds diminish. This will introduce a further constraint on maintaining a continuous inhibitory concentration of the antiviral agent. A final issue that has not been addressed is the activity of the compounds against clinical isolates. In most experiments carried out to date, strains ofHIV- 1 that are easy to propagate in the laboratory have been used. Activity of these inhibitors on fresh isolates of virus must be studied before clinical trials
338
C. Baboonian a n d A. G. Dalgleish
begin. Other strains of virus apart from HIV- 1 must also be considered. The spread of HIV-2 in Africa poses the possibility that in the not too distant future HIV-2 m a y have a major impact on the global AIDS epidemic. Therapeutic approaches to the disease must consider the likelihood that soon there may be both strains of virus in circulation in the population to be targeted for treatment.
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