Retroviral nucleocapsid and gag-precursor proteins from all known strains of retroviruses contain one or two copies of an invariant sequence, Cys-X2-Cys-X4-His-X4-Cys, that is populated with zinc in mature particles. Modification of cysteine or histidine residues results in defective packaging of genomic viral RNA and formation of non-infectious particles, making these structures potentially attractive targets for antiviral therapy. We recently reported that aromatic C-nitroso ligands of poly(ADP-ribose) polymerase preferentially destabilize one of the two (Cys-X2-Cys-X28-His-X2-Cys) zinc-fingers with concomitant loss of enzymatic activity, coincidental with selective cytocidal action of the C-nitroso substituted ligands on cancer cells. Based on the occurrence of (3Cys, 1His) zinc-binding sites in both retroviral nucleocapsid and gag proteins and in poly(ADP-ribose) polymerase, we reasoned that the C-nitroso compounds may also have antiretroviral effects. We show here that two such compounds, 3-nitrosobenzamide and 6-nitroso-1,2-benzopyrone, inhibit infection of human immunodeficiency virus HIV-1 in human lymphocytes and also eject zinc from isoalted HIV-1 nucleocapsid zinc fingers and from intact HIV-1 virions. Thus the design of zinc-ejecting agents that target retroviral zinc fingers represents a new approach to the chemotherapy of AIDS.

The enzymatic mechanism of poly(ADP-ribose) polymerase (PARP-1) has been analyzed in two in vitro systems: (a) in solution and (b) when the acceptor histones were attached to a solid surface. In system (a), it was established that the coenzymatic function of dsDNAs was sequence-independent. However, it is apparent from the calculated specificity constants that the AT homopolymer is by far the most effective coenzyme and randomly damaged DNA is the poorest. Rates of auto(poly-ADP-ribosylation) with dsDNAs as coenzymes were nearly linear for 20 min, in contrast to rates with dcDNA, which showed product [(ADPR)n] inhibition. An allosteric activation of auto(poly-ADP-ribosylation) by physiologic cellular components, Mg2+, Ca2+, and polyamines, was demonstrated, with spermine as the most powerful activator. On a molar basis, histones H(1) and H(3) were the most effective PARP-1 activators, and their action was abolished by acetylation of lysine end groups. It was shown in system (b) that oligo(ADP-ribosyl) transfer to histone H(1) is 1% of that of auto(poly-ADP-ribosylation) of PARP-1, and this trans(ADP-ribosylation) is selectively regulated by putrescine (activator). Physiologic cellular concentrations of ATP inhibit PARP-1 auto(poly-ADP-ribosylation) but less so the transfer of oligo(ADP-ribose) to histones, indicating that PARP-1 auto(ADP-ribosylation) activity is dormant in bioenergetically intact cells, allowing only trans(ADP-ribosylation) to take place. The inhibitory mechanism of ATP on PARP-1 consists of a noncompetitive interaction with the NAD site and competition with the coenzymic DNA binding site. A novel regulation of PARP-1 activity and its chromatin-related functions by cellular bioenergetics is proposed that occurs in functional cells not exposed to catastrophic DNA damage.

ABSTRACT6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-ringer site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesiumdependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.In pursuing the biological consequences of the inhibition of poly(ADP-ribose) polymerase [NAD+ :poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] within intact cells we have developed a group of C-nitroso-substituted molecules that are the oxidation products of previously described ligands containing amino groups (1). These C-nitroso ligands uniquely oxidize one of the zinc fingers of ADPRT, resulting in zinc ion ejection and concomitant inactivation of the poly(ADP-ribosyl)ation activity of ADPRT without abrogating its binding to DNA (1). In the present paper we demonstrate a cytostatic and apoptotic action of two of these ADPRT ligands, 6-nitroso-1,2-benzopyrone (NOBP) and 3-nitrosobenzamide (NOBA), ¶ toward malignant human cells. The apoptotic effect was traced to the derepression of a calcium/magnesiumdependent endonuclease that under resting conditions is known to be inhibited by poly(ADP-ribosyl)ation (2). (85 Ci/mmol; 1 Ci = 37 GBq) was added per well and its incorporation into DNA following an 18-hr incubation was determined radiochemically (6). Proliferation was also monitored by direct cell counting. Since both assays were in agreement, the more convenient radiochemical test was used routinely. The tetrazolium reduction assay for cell viability (7), which was originally developed as a test for mitochondrial succinate dehydrogenase (8), was compared with trypan blue uptake and plating efficiency, and on the basis of positive correlation among the three assays, the more quantitative dye reduction method was adopted. MATERIALS AND METHODSClonigenic Studies. Human 855-2 and HL-60 leukemic cells were plated at 105 per ml and human and rhesus bone marrow mononuclear cells and human peripheral stem cells at 4 x 105 per ml, and assays for colony-forming units were performed as reported (5). A semiquantitative assessment of malignant cells in bone marrow specimens was done by flow cytometry with labeled fluorescein-conjugated antibodies against ...

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