Zinc Ejection as a New Rationale for the Use of Cystamine and Related Disulfide-Containing Antiviral Agents in the Treatment of AIDS Nazli B. McDonnell, † Roberto N. De Guzman, † William G. Rice,* ,‡ Jim A. Turpin, ‡ and Michael F. Summers* ,† Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228, and Laboratory of Antiviral Drug Mechanisms, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21072 Received March 10, 1997 X The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1 H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein. In contrast, the reduced forms of disulfiram and cystamine, diethyl dithiocarbamate and cysteamine, respectively, were found to be ineffective at zinc ejection, although cysteamine formed a transient complex with the zinc fingers. Studies with HIV-1-infected human T-cells and monocyte/macrophage cultures revealed that cystamine and cysteamine possess significant antiviral properties at nontoxic concentrations, which warrant their consideration as thera- peutically useful anti-HIV agents. Introduction All retroviruses encode a Gag polyprotein that is produced in the host cell during the late stages of the infectious cycle and is critical for viral assembly. 1 Approximately 2000 copies of Gag assemble at the cell membrane and bud to form an immature viral particle. Concomitant with budding, the Gag polyproteins are cleaved by the viral protease into several smaller proteins, including the matrix (MA, p17), capsid (CA, p24), and nucleocapsid (NC, p7) proteins. 2,3 During this maturation process, the matrix proteins form an icosa- hedral-like shell that remains associated with the viral membrane and helps anchor the extraviral glycopro- teins, the capsid proteins condense to form the cone- shaped particle in the center of the virus that contains the viral RNA and essential enzymes, and the nucleo- capsid proteins form a ribonucleoprotein complex with the RNA. 4-6 As part of the Gag precursor protein, the NC domain functions in the recognition and packaging of the viral genome 7-11 and may be important for viral particle formation 12 and packaging of reverse transcription primer tRNA Lys 3 . 13 In the early stages of the virus replication cycle, the NC protein appears to be impor- tant for stabilizing the RNA, 14 recognition and anneal- ing of tRNA Lys 3 primer during reverse transcription, 15 and stabilizing proviral DNA. 16 NC is also capable of facilitating dimerization of nonhomologous viral RNA genomes. 17,18 Except for the spumaviruses, all retroviral NC pro- teins contain one or two copies of a conserved CCHC zinc finger motif (C-X 2 -C-X 4 -H-X 4 -C; C ) cysteine, H ) histidine, X ) variable amino acid residue). 19,20 Mu- tagenesis studies have demonstrated that the arrays play essential roles during several stages of the viral replication cycle (see, for example, refs 21-24), and spectroscopic studies of synthetic CCHC peptides 25-33 and native NC proteins 34-43 have shown that the arrays bind zinc with high affinity, leading to the formation of a compact, stable miniglobular fold. Furthermore, zinc- edge-extended X-ray absorption fine structure (EXAFS) studies have provided clear evidence that the arrays are populated with zinc in mature virions. 34,44 The arrays are thus attractive targets for potential drug interven- tion. Although these arrays are highly resistant to the removal of zinc by chelating agents such as EDTA, due to low dissociation constants (K d ca. 10 -12 -10 -14 M), 31,32,37 they are susceptible to modification by antiviral agents that function by chemically modifying the metal- coordinating cysteine residues. Thus, we first demon- strated that the two zinc finger domains of the HIV-1 nucleocapsid protein are susceptible to modification by nitroso-containing antiviral agents, 45,46 and more re- cently we identified several new classes of zinc-ejecting agents with potential chemotherapeutic utility. 47-49 One of these compounds, a derivative of disulfide benzamide (DIBA), 48 is currently in clinical trials in the United States, 50 and another is an azoic-based compound 51 that is in clinical trials in Europe for advanced AIDS. 52 In an attempt to expand the arsenal of retroviral zinc finger inhibitors, we searched for compounds that have been shown to inhibit HIV-1 replication by unknown mechanisms and that have functional groups that might electrophilically attack the metal-chelating sulfur atoms of the CCHC motif. This led to the identification of three disulfide-based compounds that fit these criteria, including disulfiram (also known as Antabuse or tetra- ethylthiuram disulfide, an FDA-approved drug that is widely used for the treatment of alcoholism), thiamine disulfide, and cystamine (see Figure 1). The present studies suggest that these antiviral agents function * To whom correspondence should be addressed. † Howard Hughes Medical Institute, University of Maryland Bal- timore County. ‡ SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center. X Abstract published in Advance ACS Abstracts, May 1, 1997. 1969 J. Med. Chem. 1997, 40, 1969-1976 S0022-2623(97)00147-7 CCC: $14.00 © 1997 American Chemical Society