With a Viral Uncoating Event
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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5286-5290, June 1992 Medical Sciences Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event ERIK DE CLERCQ*t, NAOHIKO YAMAMOTO*, RUDI PAUWELS*, MASANORI BABA*t, DOMINIQUE SCHOLS*, HIDEKI NAKASHIMA*§, JAN BALZARINI*, ZEGER DEBYSER*, BARRY A. MURRER¶, DAVID SCHWARTZ"I, DAVID THORNTON¶, GARY BRIDGERII, SIMON FRICKER¶, GEOFFREY HENSONII, MICHAEL ABRAMS II AND DONALD PICKER"1 *Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium; 1Johnson Matthey Technology Centre, Blount's Court, Sonning Common, Reading RG4 9NH, Great Britain; and IlJohnson Matthey Pharmaceutical Research, 1401 King Road, West Chester, PA 19380 Communicated by M. F. Perutz, February 24, 1992 (receivedfor review November 22, 1991) ABSTRACT A series of bicyclams have been shown to be RT step. To date, no compounds have been shown to act at potent and selective inhibitors of human immunodeficiency the uncoating of HIV or retroviruses in general. In a review virus (HIV). The compounds are inhibitory to the replication article (10), it was mentioned that the aromatic polycyclic of various HIV-1 and HIV-2 strains in various human T-cell diones hypericin and pseudohypericin may inhibit the pro- systems, including peripheral blood lymphocytes, at 0.14-1.4 cess of uncoating. However, in the original publications (19, ,uM, without being toxic to the host cells at 2.2 mM. The 20), these compounds were reported to directly inactivate bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine retrovirions as well as to inhibit their assembly. Here we (zidovudine; AZT)-resistant HIV-1 strains and acts additively report on a newly discovered class of potent and selective with AZT. Mechanism of action studies revealed that the HIV inhibitors, which seem to be targeted at a virus uncoat- bicyclams (i.e., JM2763) interact with an early event of tbe ing-associated process. retrovirus replicative cycle, which could be tentatively identi- fied as a viral uncoating event. MATERIALS AND METHODS The compounds that have been most extensively studied in Compounds. Cyclam (JM1498) was supplied by Aldrich. the chemotherapy of human immunodeficiency virus (HIV) The isolation of the free base ofJM1657 as an impurity in the infections are the 2',3'-dideoxynucleoside (ddN) analogues large scale preparation of cyclam has been described (21). 3'-azido-3'-deoxythymidine (zidovudine; AZT), dideoxycy- JM2762 and JM2763 were synthesized by published proce- tidine (ddC), and dideoxyinosine (ddl) (1). These compounds dures (22). JM2849 and JM2936 were prepared at Johnson interfere with the virus-associated reverse transcriptase Matthey. The cyclam JM1498 was tested as the free base. (RT). To this end, they need to be phosphorylated intracel- Other compounds were tested as the octahydrochlorides. lularly by host enzymes to their 5'-triphosphate (ddNTP), Viruses, Cells, Antiviral Activity Assays, and Cytotoxicity terminators in the RT Assays. Anti-HIV activity and cytotoxicity measurements in which then interact as DNA chain MT-4 cells (origin of the MT-4 cells is described in ref. 23) reaction (2). Other, nonnucleoside inhibitors ofthe HIV-1 RT were based on viability of cells that had been infected or not have been recently identified (3-5) that directly interact with infected with HIV and then exposed to various concentra- the enzyme at a specific target site, provisionally designated tions of the test compounds. After the MT-4 cells were as the TIBO {tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin- allowed to proliferate for 5 days, the number of viable cells 2(1H)-one and -thione; see ref. 6} site. was quantified by a tetrazolium-based colorimetric 3-(4,5- In addition to the RT, several other stages in the HIV dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide replicative cycle, starting from the virion binding to the cells (MTT) method in 96-well microtrays (24). Cytotoxicity mea- to the ultimate release (budding) of the virus particles from surements were also based on inhibition of the growth of the cells, have been envisaged as targets for therapeutic human embryonic lung (HEL) cells and microscopically intervention (7-10). One such target is the HIV protease, and detectable alteration of the morphology of various confluent several HIV-1 protease inhibitors have been designed that cell cultures-i.e., human embryonic skin-muscle fibroblasts appear to inhibit the enzyme, and consequently virus repli- and HeLa, Vero, and Madin-Darby canine kidney (MDCK) cation, with high specificity (11-16). The viral protease is cells. essential for proper assembly of mature, fully infectious HIV While the cytopathic effect of HIV-1 and HIV-2 in MT-4 particles. The uncoating process has also been proposed as a cells was measured by the MTT procedure (24), for simian possible target for anti-HIV agents (17). This suggestion was immunodeficiency virus (SIV) in MT4 cells it was based on based on the possible similarity in tertiary structure between trypan blue exclusion (25). Viral antigen expression in the HIV capsid protein(s) and the capsid protein of picorna- viruses, which have indeed proved sensitive to uncoating Abbreviations: HIV, human immunodeficiency virus; AZT, 3'- inhibitors (18). azido-3'-deoxythymidine; RT, reverse transcriptase; TIBO, tetrahy- Therapeutic strategies targeted at the HIV uncoating pro- droimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione; SIV, cess would be worth pursuing, as they should block the simian immunodeficiency virus; PBL, peripheral blood lymphocyte; release of the functional RNA inside the cells and thus MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)- interrupt the HIV replicative cycle before it proceeds to the 2,5-diphenyltetrazolium bromide. tTo whom reprint requests should be addressed. tPresent address: Department of Microbiology, Fukushima Medical The publication costs of this article were defrayed in part by page charge College, Fukushima 960-12, Japan. payment. This article must therefore be hereby marked "advertisement" §Present address: Department of Microbiology, Tokyo Medical and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Dental University, School of Medicine, Tokyo 113, Japan. 5286 Downloaded by guest on October 2, 2021 Medical Sciences: De Clercq et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5287 HUT-78 or CEM cells was revealed by an indirect immuno- Drug Combination. The combined effect of JM2763 and fluorescence procedure with polyclonal antibodies; the per- AZT was examined in MT-4 cells infected with HIV-1(IIIB) centage ofantigen-positive cells was determined by laserflow under the experimental conditions used for determination of cytofluorometry (25). For the viral plaque formation in MT-4 the IC50 values (see above). Checkerboard 1:5 drug dilutions cells and viral focus immunoassay in CD41 HeLa cells, see were prepared in three individual 96-well trays. After addi- refs. 26 and 27, respectively. For the p24 antigen assays with tion of virus and MT-4 cells, the trays were incubated for 5 peripheral blood lymphocytes (PBLs), see ref. 4; for the p24 days at 370C. Cell viability was determined by the MTT antigen assays with MT-4 cells, see ref. 28. In both cases, p24 procedure (24) and is expressed in OD units. To assess antigen was quantified by a sandwich ELISA. Murine sar- whether the drug combination resulted in a synergistic, coma virus infection was monitored by scoring cell transfor- additive, or antagonistic effect, a three-dimensional method mation (29). In all assays, virus multiplicity of infection (moi) of analysis [MacSynergy (35)] was used (R.P., unpublished was -0.01. data). Theoretical additive interactions were calculated from The origin ofthe different virus strains was as follows: "InB the dose-response curves of the individual drugs. The re- (LAI), ref. 30; RF, ref. 31; HE, isolated in our laboratory sulting dose-response surface was then compared with the from an AIDS patient; ROD, ref. 32; EHO, ref. 33; MAC-251, actual dose-response surface. For an additive interaction the ref. 34. The A012B, A012D, A018A, and A018C strains (28) actual dose-response surface would coincide with the theo- were obtained through the AIDS Research and Reference retical one, but any peaks above or below this surface would Reagent Program, AIDS Program, National Institute of Al- be indicative of synergy or antagonism, respectively. Signif- lergy and Infectious Diseases (NIAID) (Bethesda, MD) (con- icance was assessed by 95% confidence intervals around the tributors were D. D. Richman for the viruses and the Divi- experimentally obtained dose-response surface. sion of AIDS/NIAID for the RT). The original stocks of HIV-1(IIIB), HIV-2(ROD), HIV-2(EHO), and SIV(MAC- RESULTS AND DISCUSSION 251) were provided by R. C. Gallo, L. Montagnier, L. Montagnier, and C. Bruck, respectively. A variety of macrocyclic polyamines, with the cyclam Time of Addition Experiment. MT-4 cells were infected 1,4,8,11-tetraazacyclotetradecane (JM1498) used as the pro- with HIV-1(IIIB) at a moi of >1, and the test compounds were totype, were synthesized and analyzed for anti-HIV activity. added at different times (0, 1, 2, 3, .. 22, 23, or 24 hr) after The monocyclam had slight activity against HIV-1 and infection. Viral p24 antigen production was determined 29 hr HIV-2. Compounds in which two cyclam units were linked in postinfection by a sandwich ELISA. The compounds were various ways (bicyclams), in particular JM1657 and JM2763, added at a standardized concentration-that is, 100 times were found to be active against HIV-1 and HIV-2 at a their 50o inhibitory concentration (IC50) required to reduce concentration as low as 0.14 kLM (Table 1), with a selectivity by 50% the cytopathicity of HIV-1(IIIB) (moi, 0.01) in MT-4 index of >5000 (Table 2).