AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 5, 1992 Mary Ann Fiebert, Inc., Publishers
Inhibition of the Reverse Transcriptase Activity and Replication of Human Immunodeficiency Virus Type 1 by AS 101 In Vitro AMI
VONSOVER,1 SHOSHANA LOYA,2 BENJAMIN SREDNI,' MICHAEL ALBECK,3 TAMAR GOTL1EB-STEMATSKY,1 ORLY ARAF,2 and AMNON HIZI2
ABSTRACT In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,0'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS 101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 u.M for 14 days. To elucidate a possible mechanism for the inhibition of AS 101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated ICS0 value of about 4 u.M. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 u.M. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.
INTRODUCTION
H etiologic agent (AIDS),1'2
type I (HIV-1), the of acquired immune deficiency syndrome is a human lymphotropic retrovirus, cytopathic for helper/inducer T cells.3'4 HIV-1, similar to other retroviruses. requires virion-associated reverse transcriptase (RT) for the replication of the virus and the production of infectious viral UMAN IMMUNODEFICIENCY VIRUS
particles. Viral replication
includes an early period in which the HIV virions attach to the cells and form proviral DNA, and a late phase, during which viral proteins are expressed and infectious virus particles are released from the infected cell. The crucial steps in the early phase are virus adsorption to the cell, followed by virus-cell fusion, penetration of the viral nucleocapsid, uncoating of the viral RNA genome and transcription of the viral
RNA into proviral DNA. Important steps in the late phase of HIV replication include transcription of proviral DNA into viral mRNA and its translation into viral precursor proteins. The precursor proteins are packaged to become infectious viral particles when released from the cell. Replication of the virus requires virion-associated reverse transcriptase activity. Thus, a therapy that interferes with RT activity might decrease the production of infectious virus and limit or prevent disease. Several chemical agents already have been tested for their inhibitory effect on HIV-1 replication in cell cultures in an effort to identify agents of potential therapeutic value for AIDS.5-1" Among these, the nucleoside analogues 3'-azido-2'deoxythymidine (AZT) and its active congeners 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddl) inhibit early HIV replication by interacting with RT, "~13 affecting both HIV infectivity and cytopathic effect in vitro.14 Two
'Central Virology Laboratory. The Chaim Sheba Medical Center. Tel-Hashomer, Israel.
2Department
of Cell
Tel-Aviv, Israel.
Biology
and
Histology,
and
Department
of Human
Microbiology.
Sackler School of Medicine, Tel-Aviv
'Department of Life Sciences and Department of Chemistry, The CAIR Institute. Bar-Ilan University, Ramat-Gan, Israel. 613
University.
VONSOVER ET AL.
614 new
lead
compounds
have been identified that
apparently
are
RT, l-(2-hydroxyethoxymethyl)-6-phenylthiothymine (HEPT) and a benzodiazepine derivative, the so-called TIBO derivative.1516 Both are highly specific against HIV-1
targeted and not
at
inhibitory to
HIV-2.
However, AZT, the only approved nucleoside analog currently in clinical use, is administered persistently and is associated with severe hematopoietic toxicity and appearance of
drug-resistant virus strains.I7IX
Our interest in ammonium trichloro(dioxyoethylene-0,0'-) tellurate (AS 101) (see structural formula in Fig. I) stems from the fact that it has immunomodulating properties and minimal ASIOI isanovel low-molecular weight organotel toxicity. lurium compound capable of inducing human lymphocytes to proliferate and to produce ¡nterleukin-2 (IL-2) in vitro; it also enhances the production of IL-2 and colony-stimulating factor (CSF) by mouse spleen cells. Splenocytes of Balb/c mice injected with AS 101 increased their production ol'IL-2 and CSF in vitro in the presence of mitogen.21 After incubation with AS 101. mononuclear cells from normal donors became responsive lo recombinant IL-2 and bound monoclonal antibody to IL-2 receptors. Splenocytes of mice treated in vivo with AS 101 expressed high levels of IL-2 receptor. AS 101 administered systemically to mice-mediated antitumor effects which could be attributed to its immunomodulatory properties. AS101 also enhanced the
ratio of OKT4 to OKT8+ cells in cultured mononuclear cells from AIDS patients.'1' In addition, results of Phase I clinical trials with ASI01 in cancer patients show an enhancement in the production of IL-2, 7-interferon (7-IFN) and tumor necrosis factor (TNF).22'21 Current Phase 1 clinical trials with AIDS patients show that no side effects were seen at the dosages of 2,3 and 5 p.g/m2 injected 3 times weekly (1 m2 50 kg). An enhancement of CD4 cells, and CD4/CD8 cell ratio and a simultaneous clinical and biological improvement occurred in most of the patients.24'25 These observations indicate that AS101 may be useful in the treatment of clinical conditions ~
involving immunosuppression. In an attempt to study whether this compound has defined antiviral activity, we investigated the inhibitory effect of AS 101 on the growth of HIV-1 in vitro. In an attempt to identify biochemically the nature of the inhibitory activity, we have studied the extent of inhibition of RT, utilizing purified recombinant HIV-1 RT, a key enzyme in the initial replication steps of HIV-1,2'' Retroviral reverse transcriptases are considered to be multifunctional enzymes. Thus, the same enzyme molecules act both RNA-dependent DNA polymerases (RDDP) and DNAdependent DNA polymerases (DDDP). In addition, they exhibit an activity of ribonuclease H (RNase H) that is capable of specific hydrolysis of RNA in RNA: DNA hcteroduplexes. In the current study, we report that AS10I suppressed HIV-1 replication in vitro at concentrations that do not affect cell as
>
b
ü ra
_
Virus control PhTeCl3 AS101 15nM
effectively
reduces HIV-l replication in PBMC in vitro under continuous inhibition conditions. Inhibition of virus production was confirmed by reduction of virion-associated RT activity and reduction of HIV antigen in culture fluids of treated cultures. As RT activity was not affected by the addition of ASIOl to control cell-free supernatants of HIV-1 -infected cultures prior to the concentration of virus particles, it can be concluded that the change in particulatc RT activity of treated infected cultures is indeed a reliable marker for production and release of viral particles. This is also evident from the dose-related inhibition of RT activity, and the amount of HIV-l antigen in supernatants of treated PBMC infected with HIV-l. In our system involving virus isolated from chronically infected cells and PBMC as the target cells, the HIV-induced cytopathicity could not be assessed: Such assays that have been useful in evaluating other compounds involve selected target cells either with high sensitivity to the lytic effects of HIV-l infection, or demonstrating remarkable cytopathic changes with
i: ¡z
D eg
AS101 15|iM PhTeCb
o
0
4
7
7.5
11
DAYS AFTER ADDITION OF COMPOUNDS
FIG. 6. Inhibition of extracellular HIV antigens production by ASIOl and phenyltrichlorotellurium, 9 days posttreatment. Each value represents the arithmetic mean of duplicate cultures.
15
30
120
CONCENTRATION OF INHIBITION
240
(nM)
Dose-response curves of HIV-1 RT-associated RDDP by AS101. and phenyltrichlorotellurium. The 100% enzymatic activity corresponds to 120 units. Assay conditions FIG. 7. inhibition
and unit definition
are as
described in Materials and Methods.
AS 101 INHIBITS HIV-l REPLICATION AND RT ACTIVITY IN VITRO
AS101 15u.M PhTeCl3
0
7.5
30
15
120
240
CONCENTRATION OF INHIBITIOR (nM)
Dose-response curves of HIV-l RT-associated DDDP by ASIOl, and phenyltrichlorotellurium. The 100% enzymatic activity corresponds to 10 units. Assay conditions FIG. 8. inhibition
and unit definition
are as
described in Materials and Methods.
FIG. 9. Survival and growth of CEM-SS cells (CD4+ cells) in the presence of various concentrations of ASIOl. 5 x 105 cells/ml were cultivated in growth medium and were exposed to multiple drug concentrations. At 4, 7, and 11 days, cell viability was determined by trypan blue exclusion method. Each value represents the arithmetic mean of duplicate cultures. Table 2. Kinetic Parameters for
the
by
621
the formation of giant syncytia that are quantifiable. Our virus stock, which was obtained from the culture supernatant of HUT-78 chronically infected with HIV induced only moderate cytopathic changes in the infected cells. AS101 appears to have partial activity against HIV with a narrow therapeutic window. Indeed, since proliferation of PBMC after stimulation with PHA involves a variety of cell types, while HIV replicates exclusively in CD4+ cells. To address this question we have tested the toxicity of AS101 in T-cell line (CEM-SS) and the results ruled out the possibility of the preferential toxicity of AS101 forCD4+ cells. (Fig. 9) Treatment of PBMC with AS101 prior to their HIV-l infection and propagation of infected cells in growth medium without the drug had no effect on virus production (data not shown). Similar treatment of viral particles with AS10I (10 p.g/ml) and their concentration by ultracentrifugation prior to the infection of PBMC. did not result in a change of enzymatic activity and infectivity (data not shown). These data suggest that AS101 does not affect virion binding to the cells. However, although we cannot exclude the possibility that at least some of the virions are directly inactivated by the inhibitor, we consider it more likely that inhibition of RT activity is the major, and possibly the only mechanism by which AS101 inhibits replication of HIV-l. This concept is further supported by the fact that ASIOl efficiently inhibited the different catalytic functions associated with HIV-l RT, namely, the RDDP, DDDP. and RNase H activities. The findings that the mode of inhibition of HIV-l RT by AS10I is noncompetitive with respect to dGTP, and of mixedtype inhibition with respect to primer template, suggest that the inhibitor binds to the enzyme at sites different from the binding sites of either one of the substrates. Thus, AS101 may effectively inhibit ongoing viral DNA synthesis. Such an inhibitory effect on the virus-associated RT could lower the infectivity of HIV-l, thus, ultimately reducing virus production. The fact that AS101 and phenyltrichlorotellurium showed a remarkable difference of activity against HIV-l RT clearly demonstrate that the antiviral activity of AS101 may be a result of interaction of the enzyme with the tellurium moiety itself, although the specific mode of action is still unknown. The anti HIV-l activity of ASIOl reflected by inhibition of virus replication and by inhibition of the different catalytic functions associated with the viral RT. in the absence of
Inhibition
of
HIV-l Reverse Transcriptase
AS101 Substrate
poly(rC)oligo(dG)
dGTP
Without inhibitor With I p.g/ml inhibitor With 2.5 p.g/ml inhibitor
K,„ (V-glml)
Km (p-Ai)
Reaction
75 48 37
4.5 5.5 5.5
78 49 35
0.9 0.9 2.4
"The Vmax and Km values were calculated from the double reciprocal curves of velocity rates substrate concentrations (at the different inhibitor concentrations). The maximal velocity rates are expressed in pmoles of dGMP incorporated into the TCA-insoluble material in 10 minutes at 37°C under the assay conditions described in materials and methods. versus
622
VONSOVER ET AL.
drug-related toxicity to lymphocytes, strongly argues in favor of its evaluation as a therapeutic agent for patients with AIDSrelatcd complex or AIDS.
lymphadenopathy-associated virus (HTLV-I1I/LAV) by 2'.3'dideoxynucleosides. Proc Nati Acad Sei (USA) 1986:83:19111915. 15. Babe M, Tanaka H.
REFERENCES 1.
PopovicM.Saryngadharan MG. Read E, and Gallo RC: Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science
16.
1984;224:497-500.
2. Barre-Sinoussi F, Chermann JC, Rey F. Nugeyre MT. Chamaret S. Gruesl J. Daugucl C. Axler-Blein C. Brun-Vezinet F. Rouzioux C. Rozenbaum W. and Montagnier L: Isolation of T lymphotropic retrovirus from a patient at risk for acquired immune deficiency
syndrome (AIDS). Science 1983;220:868-871. 3. Broder S and Gallo RC: A pathogenic retrovirus (HTLV-III) linked to AIDS. NEnglJMed 1984:311:1292-1297. 4. Weiss RA: In: RNA Tumor Viruses. Weiss RA, Teich NM. Varmus HE, and Coffin J (eds): Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. 1985: pp. 404-485. 5. Mitsuya H. Popovic M. Yarchoan R. Matsushita S. Gallo RC. and Broder S: Suramin protection of T cells in vitro against infecting and cytopathic effect of HTLV-III. Science 1984;226:172-174. 6. Rosenbaum W, Dormont D, Spire B, Vilmer E. Gentilini M. Griscelli C, Montagnier L, Barre-Sinoussi F, and Chermann JC: Antimoniotungistenate (MPA23) treatment of three patients with AIDS and one with prodrome. Lancet 1985:1:450-451. 7. McCormick JB. Getchell JP, Mitchell SW. and Hicks DR: Ribavirin suppresses replication of lymphadenopathy-associated virus in cultures of human adult T-lymphocytes. Lancet 1985:2:13671369. 8. Ho DD. Hartshorn KL. Rota RT. Andrews CA. Kaplan JC. Schooley RT, and Hirsch MS: Recombinant human inlerferon alpha-A suppresses HTLV-III replication in vitro. Lancet
17.
18.
19.
20.
21.
1985;1:602-604. 9. Sandstrom EG. Kaplan JC. Byington RE, and Hirsch MS: Inhibition of human T-cell lymphotropic virus type III in vitro by phosphonoformate. Lancet 1985;2:1480-1482. 10. Yusl I, Vardinon N. Skornick Y, Zakuth V. Hasner A. and Shinitzky M: Reduction of circulating HIV antigens in seropositive patients after treatment with AL-721. Isr J Med Sciences
1990;26:20-26. Mitsuya H, Jarret R. Malsukura M. Dimarzo-Veronese F. DeVico AL. Sarngadharan MG. Johns DG, Reitz MS. and Broder S: Long term inhibition of human T-lymphotropic virus type III/ lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2'.3'-dideoxynucleosides in vitro. Proc Nati Acad Sei (USA) 1987;84:2033-2037. 12. Fischl MA. Richman DD. Grieco MH. Gottlieb MS, Volberding
22.
23.
11.
PA. Laskin OL. Leedom JM. Groopman JE. Mildvan D. Schooley RT. Jackson GG. Durack DT. and King D: The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-relaled complex. A double blind, placebo controlled trial. N EnglJMed 1987:317:185-191. 13. Yarchoan R, Perno CF, Thomas RV, Klecket RW. Allain JP, Wills RJ. McAtee N. Fischl MA, Dubinsky R. McNeely MC. Mitsuya H. PludaJM, LawleyTJ. LeutherM, Safai B. Collins JM. Myers CE. and Broder S: Phase I studies of 2',3'-dideoxycytidine in a severe human immunodeficiency virus infection as a single agent and alternating with zidovudine (AZT). Lancet 1988;1:76-80. 14. Mitsuya H. and Broder S: Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotropic virus type III/
24.
25.
26.
27.
De-Clercq E, Pawels R, Balzarini J. Schols D. Nakashima H. Perno CF. Walker RT, and Miyasaka T: Highly specific inhibition of human immunodeficiency virus type 1 by a novel 6-substituted acyclouridine derivative. Biochem Biophys Res Commun 1989:165:1375-1381. Pawels R, Andries K, Desmyster J. Schols D, Kukla MJ, Breslin HJ, Raeymaeckerst A, Vandgelder J. Weocsternborghs R. Heykants J. Schellekens K, Janssen MAC. DeKlerck E. and Janseen PAJ: Potent and selective inhibition of HIV-l replication in vitro by a novel series ol'TIBO derivatives. Nature 1990;343:470-474. Richman DD. Fischl MA. Grieco MH. Gottlieb MS. Volberding PA. Laskin OL. Leedom JM, Groopman JE. Mildvan D. Hirsch MS, Jackson GG, Durack DT, Nusinoff-Lehrman S, and AZT Collaborative Working Group: The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex: a double-blind, placebo-controlled trial. N Engl J Med 1987:317:192-197. Rooke R. Tremblay M, Soudeyns H. DeStephano L, Yao XJ. Fanning M. Montaner JS, O'Shaughnessy M, Gelman K, and Tsoukas C: Isolation of drug-resistant variants of HIV-l from patients on long-term zidovudine therapy. AIDS 1989:3:411-415. Sredni B, Caspi RR. Klein A. Kalechman Y. Danziger Y. Ben Ya'akov M, Tamari T. Shalit F, and Albeck M: A new immunomodulating compound (AS-101 ) with potential therapeutic application. Nature 1987:330:173-176. Nyska A, Waner T, Pirak M. Albeck M. and Sredni B: Toxicity study in rats of a tellurium based ¡mmunomodulatingdrug, AS-101: A potential drug for AIDS and cancer patients. Arch Toxicol 1989:63:386-393. Sredni B. Caspi RR. Lustig S. Klein A, Kalechman Y, Danziger Y, Ben Ya'akov M. Tamari T. Shalit F. and Albeck M: The biological activity and immunotherapeutic properties of AS 101, a synthethic organotellurium compound. Nati Immun Cell Growth Reg 19X8:7:163-168. Sredni B, Catane R. Shani A. Gezin A, Levi E, Schlesinger M, Kalechman Y, Michlin H. Shalit F. Rosenzajn LA. Farbstein H. and Albeck M: Phase 1 study of ASIOl (an organotellurium compound) in patients with advanced malignancies. In: Recent Advances in Chemotherapy. E. Rubinstein and D. Adam (eds.), E. Lewin-Epstein, 1990, p. 851. Shani A. TichlerT. Catane R. Gurwith M, Rozenszajn LA, Gezin A, Levin E. Schlesinger M. Kalechman Y. Michlin H, Shalit F. Farbstein H. Farbstein M, Albeck M, and Sredni B: The immunologie effect of ASIOl in the treatment of cancer patients. Nat Immun Cell Growth Reg 1990;9:182-190. Laporte JP. Frottier J. Lebas J. Dormont D, Meyohas MC, Eliaszewicz M. Gonzals, Dubeaux D. Prugnaud JL, Sredni B. and Albeck M: A new immunomodulating compound (AS10I) in the treatment of AIDS. IV International Conference on AIDS, Stockholm. Sweden 1988; p. 399. Ruiz-PalaciosGM, Ponce de Leon A. Alarcon-SegoviaD. AlcocerVarela J. Cruz A. Nares F. Ponce de Leon A, Rojas G. and Sredni B: Tolerance and clinical response to AS10I. a new immunomodulator. in AIDS patients. IV International Conference on AIDS. Stockholm, Sweden 1988, p. 229. Hizi A, McGill C. and Hughes SH: Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in E. colt and analysis of mutants. Proc Nati Acad Sei (USA) 1988:85:1218-1222. Albeck M. Tamari T. and Sredni B: Synthesis and properties of ammonium trichloro (dioxyethylene-0.0') tellurate (AS101). A new immunomodulating compound. Synthesis 1989:8:635-636.
AS 101 INHIBITS HIV-l REPLICATION AND RT ACTIVITY IN VITRO 28. Yaniv A, Gotlieb-Stcmatsky T. Vonsover A. and Perk K: Evidence for type C retrovirus production by Burkitt's lymphoma-derived cell lines. Int. J. Cancer 1980;25:205-211. 29. WeislowOS. KiserR. FineDL. BaderJ. Shoemaker RH. and Boyd MR: Soluble-lormazan assay for HIV-l cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity. J Nati Cancer Inst 1989:81:577-586. 30. Schuderio DA. Shoemaker RH, and Alley MC: Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell line. Cancer Res
1988;48:4827-4833. 31. Clark PK. Ferris AL. Miller DA. Hizi A, Kim KW. Boyer S, Mellini ML. Clark Jr. AD. Arnold GF. Arnold E, Muschik GM, and Hughes SH: HIV-l reverse transcriptase purified from recombinant strain of E. coli. AIDS Res Human Retroviruse 1990:6:753764.
623
Broder S: Strategies for antiviral therapy of AIDS. Nature 1987:325:773-778. 33. Vrang L and Oberg B: PPi analogues as inhibitors of human T-lymphotropic virus type III reverse transcriptase. Antimicrob Agents Chemother 1986;29:867-872. 34. DeClerq E: Suramin in the treatment of AIDS: Mechanism of action. Antiviral Res 1987:7:1-10. 32.
Mitsuya H and
Address
reprint requests
to:
Ami Vonsover, Ph.D.
Central Virology Laboratory The Chaim Sheba Medical Center Tel-Hashomer, 52621, Israel
Inhibition of the Reverse Transcriptase Activity and Replication of Human Immunodeficiency Virus Type 1 by AS 101 In Vitro AMI
VONSOVER,1 SHOSHANA LOYA,2 BENJAMIN SREDNI,' MICHAEL ALBECK,3 TAMAR GOTL1EB-STEMATSKY,1 ORLY ARAF,2 and AMNON HIZI2
ABSTRACT In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,0'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS 101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 u.M for 14 days. To elucidate a possible mechanism for the inhibition of AS 101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated ICS0 value of about 4 u.M. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 u.M. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.
INTRODUCTION
H etiologic agent (AIDS),1'2
type I (HIV-1), the of acquired immune deficiency syndrome is a human lymphotropic retrovirus, cytopathic for helper/inducer T cells.3'4 HIV-1, similar to other retroviruses. requires virion-associated reverse transcriptase (RT) for the replication of the virus and the production of infectious viral UMAN IMMUNODEFICIENCY VIRUS
particles. Viral replication
includes an early period in which the HIV virions attach to the cells and form proviral DNA, and a late phase, during which viral proteins are expressed and infectious virus particles are released from the infected cell. The crucial steps in the early phase are virus adsorption to the cell, followed by virus-cell fusion, penetration of the viral nucleocapsid, uncoating of the viral RNA genome and transcription of the viral
RNA into proviral DNA. Important steps in the late phase of HIV replication include transcription of proviral DNA into viral mRNA and its translation into viral precursor proteins. The precursor proteins are packaged to become infectious viral particles when released from the cell. Replication of the virus requires virion-associated reverse transcriptase activity. Thus, a therapy that interferes with RT activity might decrease the production of infectious virus and limit or prevent disease. Several chemical agents already have been tested for their inhibitory effect on HIV-1 replication in cell cultures in an effort to identify agents of potential therapeutic value for AIDS.5-1" Among these, the nucleoside analogues 3'-azido-2'deoxythymidine (AZT) and its active congeners 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddl) inhibit early HIV replication by interacting with RT, "~13 affecting both HIV infectivity and cytopathic effect in vitro.14 Two
'Central Virology Laboratory. The Chaim Sheba Medical Center. Tel-Hashomer, Israel.
2Department
of Cell
Tel-Aviv, Israel.
Biology
and
Histology,
and
Department
of Human
Microbiology.
Sackler School of Medicine, Tel-Aviv
'Department of Life Sciences and Department of Chemistry, The CAIR Institute. Bar-Ilan University, Ramat-Gan, Israel. 613
University.
VONSOVER ET AL.
614 new
lead
compounds
have been identified that
apparently
are
RT, l-(2-hydroxyethoxymethyl)-6-phenylthiothymine (HEPT) and a benzodiazepine derivative, the so-called TIBO derivative.1516 Both are highly specific against HIV-1
targeted and not
at
inhibitory to
HIV-2.
However, AZT, the only approved nucleoside analog currently in clinical use, is administered persistently and is associated with severe hematopoietic toxicity and appearance of
drug-resistant virus strains.I7IX
Our interest in ammonium trichloro(dioxyoethylene-0,0'-) tellurate (AS 101) (see structural formula in Fig. I) stems from the fact that it has immunomodulating properties and minimal ASIOI isanovel low-molecular weight organotel toxicity. lurium compound capable of inducing human lymphocytes to proliferate and to produce ¡nterleukin-2 (IL-2) in vitro; it also enhances the production of IL-2 and colony-stimulating factor (CSF) by mouse spleen cells. Splenocytes of Balb/c mice injected with AS 101 increased their production ol'IL-2 and CSF in vitro in the presence of mitogen.21 After incubation with AS 101. mononuclear cells from normal donors became responsive lo recombinant IL-2 and bound monoclonal antibody to IL-2 receptors. Splenocytes of mice treated in vivo with AS 101 expressed high levels of IL-2 receptor. AS 101 administered systemically to mice-mediated antitumor effects which could be attributed to its immunomodulatory properties. AS101 also enhanced the
ratio of OKT4 to OKT8+ cells in cultured mononuclear cells from AIDS patients.'1' In addition, results of Phase I clinical trials with ASI01 in cancer patients show an enhancement in the production of IL-2, 7-interferon (7-IFN) and tumor necrosis factor (TNF).22'21 Current Phase 1 clinical trials with AIDS patients show that no side effects were seen at the dosages of 2,3 and 5 p.g/m2 injected 3 times weekly (1 m2 50 kg). An enhancement of CD4 cells, and CD4/CD8 cell ratio and a simultaneous clinical and biological improvement occurred in most of the patients.24'25 These observations indicate that AS101 may be useful in the treatment of clinical conditions ~
involving immunosuppression. In an attempt to study whether this compound has defined antiviral activity, we investigated the inhibitory effect of AS 101 on the growth of HIV-1 in vitro. In an attempt to identify biochemically the nature of the inhibitory activity, we have studied the extent of inhibition of RT, utilizing purified recombinant HIV-1 RT, a key enzyme in the initial replication steps of HIV-1,2'' Retroviral reverse transcriptases are considered to be multifunctional enzymes. Thus, the same enzyme molecules act both RNA-dependent DNA polymerases (RDDP) and DNAdependent DNA polymerases (DDDP). In addition, they exhibit an activity of ribonuclease H (RNase H) that is capable of specific hydrolysis of RNA in RNA: DNA hcteroduplexes. In the current study, we report that AS10I suppressed HIV-1 replication in vitro at concentrations that do not affect cell as
>
b
ü ra
_
Virus control PhTeCl3 AS101 15nM
effectively
reduces HIV-l replication in PBMC in vitro under continuous inhibition conditions. Inhibition of virus production was confirmed by reduction of virion-associated RT activity and reduction of HIV antigen in culture fluids of treated cultures. As RT activity was not affected by the addition of ASIOl to control cell-free supernatants of HIV-1 -infected cultures prior to the concentration of virus particles, it can be concluded that the change in particulatc RT activity of treated infected cultures is indeed a reliable marker for production and release of viral particles. This is also evident from the dose-related inhibition of RT activity, and the amount of HIV-l antigen in supernatants of treated PBMC infected with HIV-l. In our system involving virus isolated from chronically infected cells and PBMC as the target cells, the HIV-induced cytopathicity could not be assessed: Such assays that have been useful in evaluating other compounds involve selected target cells either with high sensitivity to the lytic effects of HIV-l infection, or demonstrating remarkable cytopathic changes with
i: ¡z
D eg
AS101 15|iM PhTeCb
o
0
4
7
7.5
11
DAYS AFTER ADDITION OF COMPOUNDS
FIG. 6. Inhibition of extracellular HIV antigens production by ASIOl and phenyltrichlorotellurium, 9 days posttreatment. Each value represents the arithmetic mean of duplicate cultures.
15
30
120
CONCENTRATION OF INHIBITION
240
(nM)
Dose-response curves of HIV-1 RT-associated RDDP by AS101. and phenyltrichlorotellurium. The 100% enzymatic activity corresponds to 120 units. Assay conditions FIG. 7. inhibition
and unit definition
are as
described in Materials and Methods.
AS 101 INHIBITS HIV-l REPLICATION AND RT ACTIVITY IN VITRO
AS101 15u.M PhTeCl3
0
7.5
30
15
120
240
CONCENTRATION OF INHIBITIOR (nM)
Dose-response curves of HIV-l RT-associated DDDP by ASIOl, and phenyltrichlorotellurium. The 100% enzymatic activity corresponds to 10 units. Assay conditions FIG. 8. inhibition
and unit definition
are as
described in Materials and Methods.
FIG. 9. Survival and growth of CEM-SS cells (CD4+ cells) in the presence of various concentrations of ASIOl. 5 x 105 cells/ml were cultivated in growth medium and were exposed to multiple drug concentrations. At 4, 7, and 11 days, cell viability was determined by trypan blue exclusion method. Each value represents the arithmetic mean of duplicate cultures. Table 2. Kinetic Parameters for
the
by
621
the formation of giant syncytia that are quantifiable. Our virus stock, which was obtained from the culture supernatant of HUT-78 chronically infected with HIV induced only moderate cytopathic changes in the infected cells. AS101 appears to have partial activity against HIV with a narrow therapeutic window. Indeed, since proliferation of PBMC after stimulation with PHA involves a variety of cell types, while HIV replicates exclusively in CD4+ cells. To address this question we have tested the toxicity of AS101 in T-cell line (CEM-SS) and the results ruled out the possibility of the preferential toxicity of AS101 forCD4+ cells. (Fig. 9) Treatment of PBMC with AS101 prior to their HIV-l infection and propagation of infected cells in growth medium without the drug had no effect on virus production (data not shown). Similar treatment of viral particles with AS10I (10 p.g/ml) and their concentration by ultracentrifugation prior to the infection of PBMC. did not result in a change of enzymatic activity and infectivity (data not shown). These data suggest that AS101 does not affect virion binding to the cells. However, although we cannot exclude the possibility that at least some of the virions are directly inactivated by the inhibitor, we consider it more likely that inhibition of RT activity is the major, and possibly the only mechanism by which AS101 inhibits replication of HIV-l. This concept is further supported by the fact that ASIOl efficiently inhibited the different catalytic functions associated with HIV-l RT, namely, the RDDP, DDDP. and RNase H activities. The findings that the mode of inhibition of HIV-l RT by AS10I is noncompetitive with respect to dGTP, and of mixedtype inhibition with respect to primer template, suggest that the inhibitor binds to the enzyme at sites different from the binding sites of either one of the substrates. Thus, AS101 may effectively inhibit ongoing viral DNA synthesis. Such an inhibitory effect on the virus-associated RT could lower the infectivity of HIV-l, thus, ultimately reducing virus production. The fact that AS101 and phenyltrichlorotellurium showed a remarkable difference of activity against HIV-l RT clearly demonstrate that the antiviral activity of AS101 may be a result of interaction of the enzyme with the tellurium moiety itself, although the specific mode of action is still unknown. The anti HIV-l activity of ASIOl reflected by inhibition of virus replication and by inhibition of the different catalytic functions associated with the viral RT. in the absence of
Inhibition
of
HIV-l Reverse Transcriptase
AS101 Substrate
poly(rC)oligo(dG)
dGTP
Without inhibitor With I p.g/ml inhibitor With 2.5 p.g/ml inhibitor
K,„ (V-glml)
Km (p-Ai)
Reaction
75 48 37
4.5 5.5 5.5
78 49 35
0.9 0.9 2.4
"The Vmax and Km values were calculated from the double reciprocal curves of velocity rates substrate concentrations (at the different inhibitor concentrations). The maximal velocity rates are expressed in pmoles of dGMP incorporated into the TCA-insoluble material in 10 minutes at 37°C under the assay conditions described in materials and methods. versus
622
VONSOVER ET AL.
drug-related toxicity to lymphocytes, strongly argues in favor of its evaluation as a therapeutic agent for patients with AIDSrelatcd complex or AIDS.
lymphadenopathy-associated virus (HTLV-I1I/LAV) by 2'.3'dideoxynucleosides. Proc Nati Acad Sei (USA) 1986:83:19111915. 15. Babe M, Tanaka H.
REFERENCES 1.
PopovicM.Saryngadharan MG. Read E, and Gallo RC: Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science
16.
1984;224:497-500.
2. Barre-Sinoussi F, Chermann JC, Rey F. Nugeyre MT. Chamaret S. Gruesl J. Daugucl C. Axler-Blein C. Brun-Vezinet F. Rouzioux C. Rozenbaum W. and Montagnier L: Isolation of T lymphotropic retrovirus from a patient at risk for acquired immune deficiency
syndrome (AIDS). Science 1983;220:868-871. 3. Broder S and Gallo RC: A pathogenic retrovirus (HTLV-III) linked to AIDS. NEnglJMed 1984:311:1292-1297. 4. Weiss RA: In: RNA Tumor Viruses. Weiss RA, Teich NM. Varmus HE, and Coffin J (eds): Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. 1985: pp. 404-485. 5. Mitsuya H. Popovic M. Yarchoan R. Matsushita S. Gallo RC. and Broder S: Suramin protection of T cells in vitro against infecting and cytopathic effect of HTLV-III. Science 1984;226:172-174. 6. Rosenbaum W, Dormont D, Spire B, Vilmer E. Gentilini M. Griscelli C, Montagnier L, Barre-Sinoussi F, and Chermann JC: Antimoniotungistenate (MPA23) treatment of three patients with AIDS and one with prodrome. Lancet 1985:1:450-451. 7. McCormick JB. Getchell JP, Mitchell SW. and Hicks DR: Ribavirin suppresses replication of lymphadenopathy-associated virus in cultures of human adult T-lymphocytes. Lancet 1985:2:13671369. 8. Ho DD. Hartshorn KL. Rota RT. Andrews CA. Kaplan JC. Schooley RT, and Hirsch MS: Recombinant human inlerferon alpha-A suppresses HTLV-III replication in vitro. Lancet
17.
18.
19.
20.
21.
1985;1:602-604. 9. Sandstrom EG. Kaplan JC. Byington RE, and Hirsch MS: Inhibition of human T-cell lymphotropic virus type III in vitro by phosphonoformate. Lancet 1985;2:1480-1482. 10. Yusl I, Vardinon N. Skornick Y, Zakuth V. Hasner A. and Shinitzky M: Reduction of circulating HIV antigens in seropositive patients after treatment with AL-721. Isr J Med Sciences
1990;26:20-26. Mitsuya H, Jarret R. Malsukura M. Dimarzo-Veronese F. DeVico AL. Sarngadharan MG. Johns DG, Reitz MS. and Broder S: Long term inhibition of human T-lymphotropic virus type III/ lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2'.3'-dideoxynucleosides in vitro. Proc Nati Acad Sei (USA) 1987;84:2033-2037. 12. Fischl MA. Richman DD. Grieco MH. Gottlieb MS, Volberding
22.
23.
11.
PA. Laskin OL. Leedom JM. Groopman JE. Mildvan D. Schooley RT. Jackson GG. Durack DT. and King D: The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-relaled complex. A double blind, placebo controlled trial. N EnglJMed 1987:317:185-191. 13. Yarchoan R, Perno CF, Thomas RV, Klecket RW. Allain JP, Wills RJ. McAtee N. Fischl MA, Dubinsky R. McNeely MC. Mitsuya H. PludaJM, LawleyTJ. LeutherM, Safai B. Collins JM. Myers CE. and Broder S: Phase I studies of 2',3'-dideoxycytidine in a severe human immunodeficiency virus infection as a single agent and alternating with zidovudine (AZT). Lancet 1988;1:76-80. 14. Mitsuya H. and Broder S: Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotropic virus type III/
24.
25.
26.
27.
De-Clercq E, Pawels R, Balzarini J. Schols D. Nakashima H. Perno CF. Walker RT, and Miyasaka T: Highly specific inhibition of human immunodeficiency virus type 1 by a novel 6-substituted acyclouridine derivative. Biochem Biophys Res Commun 1989:165:1375-1381. Pawels R, Andries K, Desmyster J. Schols D, Kukla MJ, Breslin HJ, Raeymaeckerst A, Vandgelder J. Weocsternborghs R. Heykants J. Schellekens K, Janssen MAC. DeKlerck E. and Janseen PAJ: Potent and selective inhibition of HIV-l replication in vitro by a novel series ol'TIBO derivatives. Nature 1990;343:470-474. Richman DD. Fischl MA. Grieco MH. Gottlieb MS. Volberding PA. Laskin OL. Leedom JM, Groopman JE. Mildvan D. Hirsch MS, Jackson GG, Durack DT, Nusinoff-Lehrman S, and AZT Collaborative Working Group: The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex: a double-blind, placebo-controlled trial. N Engl J Med 1987:317:192-197. Rooke R. Tremblay M, Soudeyns H. DeStephano L, Yao XJ. Fanning M. Montaner JS, O'Shaughnessy M, Gelman K, and Tsoukas C: Isolation of drug-resistant variants of HIV-l from patients on long-term zidovudine therapy. AIDS 1989:3:411-415. Sredni B, Caspi RR. Klein A. Kalechman Y. Danziger Y. Ben Ya'akov M, Tamari T. Shalit F, and Albeck M: A new immunomodulating compound (AS-101 ) with potential therapeutic application. Nature 1987:330:173-176. Nyska A, Waner T, Pirak M. Albeck M. and Sredni B: Toxicity study in rats of a tellurium based ¡mmunomodulatingdrug, AS-101: A potential drug for AIDS and cancer patients. Arch Toxicol 1989:63:386-393. Sredni B. Caspi RR. Lustig S. Klein A, Kalechman Y, Danziger Y, Ben Ya'akov M. Tamari T. Shalit F. and Albeck M: The biological activity and immunotherapeutic properties of AS 101, a synthethic organotellurium compound. Nati Immun Cell Growth Reg 19X8:7:163-168. Sredni B, Catane R. Shani A. Gezin A, Levi E, Schlesinger M, Kalechman Y, Michlin H. Shalit F. Rosenzajn LA. Farbstein H. and Albeck M: Phase 1 study of ASIOl (an organotellurium compound) in patients with advanced malignancies. In: Recent Advances in Chemotherapy. E. Rubinstein and D. Adam (eds.), E. Lewin-Epstein, 1990, p. 851. Shani A. TichlerT. Catane R. Gurwith M, Rozenszajn LA, Gezin A, Levin E. Schlesinger M. Kalechman Y. Michlin H, Shalit F. Farbstein H. Farbstein M, Albeck M, and Sredni B: The immunologie effect of ASIOl in the treatment of cancer patients. Nat Immun Cell Growth Reg 1990;9:182-190. Laporte JP. Frottier J. Lebas J. Dormont D, Meyohas MC, Eliaszewicz M. Gonzals, Dubeaux D. Prugnaud JL, Sredni B. and Albeck M: A new immunomodulating compound (AS10I) in the treatment of AIDS. IV International Conference on AIDS, Stockholm. Sweden 1988; p. 399. Ruiz-PalaciosGM, Ponce de Leon A. Alarcon-SegoviaD. AlcocerVarela J. Cruz A. Nares F. Ponce de Leon A, Rojas G. and Sredni B: Tolerance and clinical response to AS10I. a new immunomodulator. in AIDS patients. IV International Conference on AIDS. Stockholm, Sweden 1988, p. 229. Hizi A, McGill C. and Hughes SH: Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in E. colt and analysis of mutants. Proc Nati Acad Sei (USA) 1988:85:1218-1222. Albeck M. Tamari T. and Sredni B: Synthesis and properties of ammonium trichloro (dioxyethylene-0.0') tellurate (AS101). A new immunomodulating compound. Synthesis 1989:8:635-636.
AS 101 INHIBITS HIV-l REPLICATION AND RT ACTIVITY IN VITRO 28. Yaniv A, Gotlieb-Stcmatsky T. Vonsover A. and Perk K: Evidence for type C retrovirus production by Burkitt's lymphoma-derived cell lines. Int. J. Cancer 1980;25:205-211. 29. WeislowOS. KiserR. FineDL. BaderJ. Shoemaker RH. and Boyd MR: Soluble-lormazan assay for HIV-l cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity. J Nati Cancer Inst 1989:81:577-586. 30. Schuderio DA. Shoemaker RH, and Alley MC: Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell line. Cancer Res
1988;48:4827-4833. 31. Clark PK. Ferris AL. Miller DA. Hizi A, Kim KW. Boyer S, Mellini ML. Clark Jr. AD. Arnold GF. Arnold E, Muschik GM, and Hughes SH: HIV-l reverse transcriptase purified from recombinant strain of E. coli. AIDS Res Human Retroviruse 1990:6:753764.
623
Broder S: Strategies for antiviral therapy of AIDS. Nature 1987:325:773-778. 33. Vrang L and Oberg B: PPi analogues as inhibitors of human T-lymphotropic virus type III reverse transcriptase. Antimicrob Agents Chemother 1986;29:867-872. 34. DeClerq E: Suramin in the treatment of AIDS: Mechanism of action. Antiviral Res 1987:7:1-10. 32.
Mitsuya H and
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Central Virology Laboratory The Chaim Sheba Medical Center Tel-Hashomer, 52621, Israel
