Jerome Mendeleyev

Potential chemotherapeutic activity of 4-iodo-3-nitrobenzamide. Metabolic reduction to the 3-nitroso derivative and induction of cell death in tumor cells in culture.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function.

6‐Nitroso‐ 1,2‐benzopyrone, an oxidation product of 6‐amino‐ 1,2‐benzopyrone, binds to the DNA‐recognizing domain of the ADP‐ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn2+‐isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc‐deficient enzyme still binds to a DNA template. consisting of a 17‐mer DNA primer annealed to M 13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I, Auto‐poly‐ADP‐ribosylated ADP‐ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer‐template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP‐ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

Inhibition of HIV-1 IIIb replication in AA-2 and NT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.

Inhibition of the replication of native and 3′-azido-2′,3′-dideoxy-thymidine (AZT)-resistant simian immunodeficiency virus (SIV) by 3-nitrosobenzamide

The 3‐nitrosobenzamide (NOBA) drug abolishes SIV replication sharply at 20 μM concentration when CEM × 174 cells are preincubated for 1 h with the drug prior to viral infection. Treatment of CEM × 174 cells with 20 μM NOBA resulted in the inhibition of the synthesis of the DNA sequence coding for the gag gene, as determined by the PCR technique. Cell viability was directly proportional to the antiviral action of NOBA. Replication of AZT‐resistant SIV 23740 in MMU 23740 cells in vitro was suppressed by NOBA in a concentration‐dependent manner without significant effects on cell viability. Reverse transcriptase activity of SIVmac239 was unaffected by NOBA up to 800 μM concentration. Preincubation of two SIV strains with NOBA completely abolished their infectivity in human PHA‐PBL cells. Replication of two strains of SIV in PHA‐PBL cells was also inhibited by NOBA.

Enzymatic mechanism of the tumoricidal action of 4-iodo-3-nitrobenzamide.

Activation of the prodrug 4-iodo-3-nitrobenzamide critically depends on the cellular reducing system specific to cancer cells. In non-malignant cells, reduction of this prodrug to the non-toxic amine occurs by the flavoprotein of complex?I of mitochondria receiving Mg2+-ATP-dependent reducing equivalents from NADH to NADPH via pyridine nucleotide transhydrogenation. This hydride transfer is deficient in malignant cells; therefore, the lethal synthesis of 4-iodo-3-nitrosobenzamide takes place selectively. Enzymatic evidence for this mechanism has been provided by cellular studies with lysolecithin-permeabilized cells and cell fractions, which have identified the defect in transhydrogenation in neoplastic cells to be located at the energy transfer site. Confirming previous results, the present study demonstrates the validity of the selective tumoricidal action of the prodrug in cell cultures.

Inactivation of the Polymerase but not the DNA Binding Function of ADPRT by Destabilization of one of its Zn2+ Coordination Centers by 6-Nitroso-1,2-Benzopyrone

In the course of studies on the metabolism of inhibitors of poly (ADP- ribose) transferase (ADPRT), we observed that the inhibitor 6-amino-1,2-benzopyrone (6-ABP) (1) when metabolized by rat liver microsomal preparations is oxidized to the corresponding 6-nitroso derivative (6-NOBP) as the main metabolite (2). Since ADPRT is located in the nuclear matrix (3,4) and most of the cytochrome P450 resides in the endoplasmic reticulum which is contiguous with the nuclear membrane (5), generation of 6-NOBP near ADPRT is likely. This suggested that the exact cellular significance of this oxidation path be investigated with respect to ADPRT.