MDL 74,695, a novel dipeptide-like compound containing the ‘difluorostatone type’ transition state mimic and a potent inhibitor of the human immunodeficiency virus (HIV) proteinase, was investigated for anti-HIV activity in vitro. The compound showed selective inhibition of both HIV-1 and HIV-2 in MT-4 cells. A potent antiviral effect against a range of clinical isolates of HIV-1 cultured in human peripheral blood mononuclear cells and primary monocytes was also demonstrated. The antiviral activity of MDL 74,695 against viruses resistant to a range of reverse transcriptase inhibitors was equivalent to the wild-type. In rats MDL 74,695 (30 mg kg−1) was 4.9% orally bioavailable and maintained levels above the in vitro 50% inhibitory concentration (IC50) for approximately 3 h. Viruses with reduced sensitivity to MDL 74,695 and saquinavir were selected in cell culture by continuous passage in increasing drug concentrations, and first appeared after 20 and 17 passages, respectively. Amino acid changes were identified at positions 48 (glycine to valine), 50 (isoleucine to valine) and 82 (valine to either isoleucine or alanine) in various combinations for MDL 74,695-resistant viruses. For saquinavir-resistant viruses changes were identified at positions 48 (glycine to valine) and 90 (leucine to methionine). Studies using MDL 74,695, saquinavir and a third proteinase inhibitor indinavir, indicated that virus selected in the presence of MDL 74,695, with amino acid exchanges at positions 48 and 82 showed cross-resistance to saquinavir. However, viruses selected in the presence of MDL 74,695 with amino acid exchanges at positions 50 and 82 showed no significant change in sensitivity to saquinavir. Likewise, viruses selected in the presence of saquinavir with amino acid exchanges at positions 48 and 90 remained sensitive to MDL 74,695. All viruses selected after growth in the presence of either MDL 74,695 or saquinavir showed little or no resistance to indinavir.
Anon (1996) New drugs for HIV infection. Medical Letters Drugs Therapy38:35–37.
2.
AshornPMcQuadeTJThaisrivongsSTomasselliAGTarpleyWG & MossB (1990) An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proceedings of the National Academy of Sciences, USA87:7472–7476.
BrennanTMTaylorDLBridgesCGLeydaJP & TymsAS (1995) The inhibition of human immunodeficiency virus type 1 in vitro by a non-nucleoside reverse transcriptase inhibitor MKC-442, alone and in combination with other anti-HIV compounds. Antiviral Research26:173–187.
5.
CollierACCoombsRWSchoenfeldDABassettRLTimponeJBaruchAJonesMFaceyKWhitacreCMcAuliffeVJFriedmanHMMeriganTCReichmanRCHooperC & CoreyL (1996) Treatment of human immunodeficiency virus infection with saquinavir, zidovudine and zalcitabine. New England Journal of Medicine334:1011–1017.
6.
CondraJHSchleifWABlahyOMGabryelskiLJGrahamDJQuinteroJCRhodesARobbinsHLRothEShivaprakashMTitusDYangTTepplerHSquiresKEDeutschPJ & EminiE (1995) In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature374:569–571.
7.
CraigJCDuncanIBHockleyDGriefCRobertsNA & MillsJS (1991) Antiviral properties of Ro-31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase. Antiviral Research16:295–305.
8.
CraigJCWhittakerLDuncanIB & RobertsN (1993) In vitro resistance to an inhibitor of HIV proteinase (Ro-31-8959) relative to inhibitors of reverse transcriptase (AZT and TIBO). Antiviral Chemistry and Chemotherapy4:335–339.
9.
DannerSACarrALeonardJMLehmanLMGudiolFGonzalesJRaventosARubioRBouzaEPintadoVAguadoAGde LomasJ GarciaDelgadoRBorleffsJCCHsuAValdesJMBoucherCAB & Cooper DA for the European-Australian Collaborative Ritonavir Study Group (1995) A short-term study of the safety, pharmocokinetics and efficacy of ritonavir, an inhibitor of HIV-1 protease. New England Journal of Medicine333:1528–1533.
10.
DarkePLNuttRFBradySFGaeskyVMCiceroneTLeuC-TLummaPKFriedingerRMVeberDF & SigalIS (1988) HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochemical and Biophysical Research Communications156:297–303.
11.
DebouckC (1992) The HIV-1 protease as a therapeutic target for AIDS. AIDS Research and Human Retroviruses8:153–164.
12.
DesJarlaisRLSeibelGLKuntzIDFurthPSAlavrezJCde MontellanoPR OrtizDeCampDLBabeLM & CraikCS (1990) Structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease. Proceedings of the National Academy of Sciences, USA87:6644–6648.
13.
DianzaniFAntonelliGTurrizianiORivaEDongG & ScBellarosaD (1993) In vitro selection of human immunodeficiency virus type 1 resistant to Ro-31-8959 proteinase inhibitor. Antiviral Chemistry and Chemotherapy4:329–333.
14.
DorseyBDLevinRBMcDanielSLVaccaJPGuareJPDarkePLZugayJAEminiEASchleifWA & QuinteroJC (1994) L-735,524: the design of a potent and orally bioavailable HIV protease inhibitor. Journal of Medicinal Chemistry37:3443–3451.
15.
EberleJBechowskyBRoseDHauserUVon der HelmKGurtlerL & NitschkoH (1995) Resistance of HIV type 1 to proteinase inhibitor Ro-31-8959. AIDS Research and Human Retroviruses11:671–677.
16.
El-FarrashMAKurodaMJKitazakiTMasudaTKatoKHatanakaM & HaradaS (1994) Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-protease inhibitor. Journal of Virology68:233–239.
17.
EricksonJNeidhartDJVanDrieJKempfDJWangX-CNorbeckDWPlattnerJJRittenhouseJWTuronMWideburgNKohlbrennerWESimmerRHelfrichRPaulDA & KniggeM (1990) Design, activity and 28Å crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease. Science249:527–533.
18.
HoDDToyoshimaTMoHKempfDJNorbeckDChemC-MWideburgNEBurtSKEricksonJW & SinghMK (1994) Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2 symmetric protease inhibitorJournal of Virology68:2016–2020.
19.
HuiKYManettaJVGygiTBowdonBJKeithKAShannonWM & LaiM-HT (1991) A rational approach in the search for potent inhibitors against HIV proteinase. FASEB Journal5:2606–2610.
20.
JacobsenHYasargilKWinslowDLCraigJCKrohnADuncanIB & MousJ (1995) Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro-31-8959. Virology206:527–534.
21.
JacobsenHHanggiMOttMDuncanIBOwenSAndreoniMVellaS & MousJ (1996) In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics and frequencies. Journal of Infectious Diseases173:1379–1387.
22.
KaplanAHMichaelSFWehbieRSKniggeMFPaulDAEverittLKempfDJNorbeckDWEricksonJW & SwanstromR (1994) Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of viral protease. Proceedings of the National Academy of Sciences, USA91:5597–5601.
23.
KempfDJNorbeckDWCodacoviLMWangXCKohlbrennerWEWideburgDAPaulDAKniggeMFVasavanondaSCraig-KennardASaldivarARosenbrockWMClementJJPlattnerJJ & EricksonJ (1990) Structure-based, C2 symmetric inhibitors of HIV protease. Journal of Medicinal Chemistry33:2687–2689.
24.
KempfDJMarshKCDenissenJFMcDonaldEVasavanondaSFlentgeCAGreenBEFinoLParkCHKongX-PWideburgNESaldivarARuizLKatiWMShamHLRobinsTStewartKDHsuAPlattnerJJLeonardJM & NorbeckDW (1995) ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. Proceedings of the National Academy of Sciences, USA92:2484–2488.
25.
KingRWGarberSWinslowDLReidCBachelerLTAntonE & OttoMJ (1995) Multiple mutations in the human immunodeficiency virus protease gene are responsible for decreased susceptibility to protease inhibitors. Antiviral Chemistry and Chemotherapy6:80–88.
26.
KitchenVSSkinnerCAriyoshiKLaneEADuncanIBBurckhardtJBurgerHUBragmanKPinchingAJ & WeberJN (1995) Safety and activity of saquinavir in HIV infection. Lancet345:952–955.
27.
KohlNEEminiEA & SchleifWA (1988) Active human immunodeficiency virus protease is required for viral infectivityProceedings of the National Academy of Sciences, USA85:4686–4690.
28.
KootMVosAHVKeetRPMde GoedeREYDercksenMWTerpstraFGCoutinhoRAMiedemaF & TersmetteM (1992) HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay. AIDS6:49–54.
29.
LarderBAKempSD & HarriganPR (1995) Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy. Science269:696–699.
30.
MarkowitzMSaagMPowderlyWGHurleyAHsuAValdesJMHenryDSattlerFLa MarcaALeonardJM & HoDD (1995a) A preliminary study of ritonavir, an inhibitor of HIV-1 protease, to treat HIV-1 infection. New England Journal of Medicine333:1534–1540.
31.
MarkowitzMMoHKempfDJNorbeckDWBhatTNEricksonJW & HoDD (1995b) Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor. Journal of Virology69:701–706.
32.
MascheraBFurfineE & BlairED (1995) Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors. Journal of Virology69:5431–5436.
33.
McQuadeTJTomasselliAGLiuLKaracostasVMossBSawyerTKHeinriksonRL & TarpleyWG (1990) A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science247:454–456.
34.
MeekTDLambertTDDreyerGBCarrTJTomaszekTAMooreMLStricklerJEDebouckCHylandLJMatthewsTJMetcalfBW & PettewaySR (1990) Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. Nature343:90–92.
35.
MellorsJWLarderBA & SchinaziRF (1995) Mutations in HIV-1 reverse transcriptase and protease associated with drug resistance. International Antiviral News3:8–13.
36.
MollaAKorneyevaMGaoQVasavanondaSSchipperPJMoHMMarkowitzMChernyavskiyTNiuPLyonsNHsuAGrannemanGRHoDDBoucherCALeonardJMNorbeckDW & KempfDJ (1996) Ordered accumulation of mutations in HIV protease confers resistance to ritonavir. Nature Medicine2:760–766.
37.
MyersGKorberBWain-HobsonSSmithRF & PavlakisGN (1993) Human Retroviruses and AIDS 1993: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos: Los Alamos National Laboratory.
38.
OttoMJReidCDGarberSLamPY-SScarnatiHBachelerLTRaynerMM & WinslowDL (1993a) In vitro anti-human immunodeficiency virus (HIV) activity of XM323, a novel HIV protease inhibitor. Antimicrobial Agents and Chemotherapy37:2606–2611.
39.
OttoMJGarberSWinslowDLReidCDAldrichPJadharPKPottersonCEHodgeCN & ChengY-SE (1993b) In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C2 symmetrical inhibitors of the HIV type-1 protease. Proceedings of the National Academy of Sciences, USA90:7543–7547.
40.
PartaledisJAYamaguchiKTisdaleMBlairEEFalcioneCMascheraBMyersREPazhanisamySFuterOCullinanABStuverCMByrnRA & LivingstonDJ (1995) In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease. Journal of Virology69:5228–5235.
41.
PauwelsRBalzafiniJScholsDBabaMDesmyterJRosenbergIHolyA & De ClercqE (1988) Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. Journal of Virological Methods20:309–321.
42.
PengCHoBKChangTW & ChangNT (1989) Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivityJournal of Virology63:2550–2556.
43.
RobertsNAMartinJAKinchingtonDBroadhurstAVCraigJCDuncanIBGalpinSAHandaBKKayJKKrohnALambertRWMorettJHMillsJSParkerKEBRedshawSRitchieAJTaylorDLThomasGJ & MachinPJ (1990) Rational design of peptide based HIV proteinase inhibitors. Science248:358–361.
44.
SchapiroJMWintersMAStewartFEfronBNorrisJKozalMJ & MeriganTC (1996) The effect of high-dose saquinavir on viral load and CD4+ T-cell counts in HIV-infected patients. Annals of Internal Medicine124:1039–1049.
45.
SchirlinDVan DorsselaerVTarnusCTaylorDLTymsDLBaltzerSWeberFRemyJMBrennanTMFarrR & JanowickD (1994) Beneficial replacement of the P1 phenylalanine side chain in HIV-1 protease inhibitors of the difluorostatone type. Bioorganic and Medicinal Chemistry Letters4:241–246.
46.
SchneiderJ & KentSBN (1988) Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV protease. Cell54:363–368.
47.
SchrammHJNakashimaHSchrammWWakayamaH & YamamotoN (1991) HIV-1 reproduction is inhibited by peptides derived from the N- and C-termini of HIV-1 protease. Biochemical and Biophysical Research Communications179:847–851.
48.
ShamHLBetebennerDAWideburgNESaldivarACKohlbrennerWEVasavanondaSKempfDJNorbeckDWZhaoCClementJJEricksonJE & PlattnerJJ (1991) Potent HIV-1 protease inhibitors with antiviral activities in vitro. Biochemical and Biophysical Research Communications175:914–919.
49.
TisdaleMMyersREMascheraBPartyNROliverNM & BlairED (1995) Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors. Antimicrobial Agents and Chemotherapy39:1704–1710.
50.
VaccaJPGuaraJPdeSohmsSJSanderWMGiulianiEAYoungSDDarkePLZugayJSigalISSchleifWAQuinteroJCEminiEAAndersonPS & HuffJR (1991) L-687,908 a potent hydroxyethylene-containing HIV protease inhibitor. Journal of Medicinal Chemistry34:1225–1228.
51.
VaccaJPDorseyBDSchleifWALevinRBMcDanielSLDarkePLZugayJQuinteroJCBlahyOMRothESardanaVVSchlabachAJGrahamPICondraJHGotlibLHollowayMKLinJChenI-WVastagKOstovicDAndersonPSEminiEA & HuffJR (1994) L-735,524: an orally bioavailable human immunodeficiency virus type 1 protease inhibitor. Proceedings of the National Academy of Sciences, USA91:4096–4100.
52.
Van DorsselaerVSchirlinDTarnusCTaylorDLTymsDLWeberFBaltzerSRemyJMBrennanTMJanowickD & FarrR (1994) Increased antiviral activity of HIV protease inhibitors of the difluorostatone type bearing (R)-valinol derivatives as novel C-termini. Bioorganic and Medicinal Chemistry Letters4:1213–1218.
53.
VellaS (1994) Update on a proteinase inhibitor. AIDS8: Supplement 3, 25–29.
54.
VellaS (1995) Rationale and experience with reverse transcriptase inhibitors and protease inhibitors. Journal of Acquired Immune Deficiency Syndromes10: Supplement 1, 58–61.
55.
WaynforthHB & FlecknellPA (1992) Experimental and Surgical Technique in the Rat, section 225. New York: Academic Press, pp. 230–234.
56.
WilliamsPEOMuirheadMJMadiganMJMitchellAM & ShawT (1992) Disposition and bioavailability of the HIV-proteinase inhibitor, Ro-31-8959, after single doses in healthy volunteers. British Journal of Clinical Pharmacology34:155.
57.
WlodawerA & EricksonJW (1993) Structure-based inhibitors of HIV-1 protease. Annual Review of Biochemistry62:543–585.
