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Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe-Pro) within the gag-pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K(i) values ranging from 5 to 18 microM when experiments were performed at high enzyme concentrations (above 5 x 10(-8) M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K(i) of 5 microM, whereas the non-boronated parental compound was inactive at concentrations up to 400 microM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.

We previously reported that the racemic mixture and both enantiomers of gossypol inhibit the replication of human immunodeficiency virus-type 1 (HIV-1) (Lin et al., Antimicrob Agents Chemother 33: 2149-2151, 1989). The present study evaluates the activities of a variety of analogs of gossypol as well as a few non-gossypol analogs. Compounds 2, 3, 10, and 13 were slightly more inhibitory than (-)-gossypol to the replication of HIV-1 in cell culture. Compounds 4 and 8 were cytotoxic to human peripheral blood monocyte (PBM) cells, and compounds 2 and 3 were cytotoxic to Vero cells but not PBM cells. The effects of the two enantiomers of gossypol on the cell volume and migration of H9 cells through the cell cycle were evaluated during 72 hr of incubation. The (-)-enantiomer of gossypol was more toxic to H9 cells than the (+)-enantiomer of gossypol as evidenced by cell destruction. Prior to cell destruction, there appeared to be no significant effect on cell cycle distribution with either enantiomer.

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.

Herpes simplex virus type-1 (HSV-1) was grown in the presence of 5-iodo-2'-deoxyuridine (IdUrd), and the virion-DNA was isolated by isopycnic centrifugation in CsCl. Irradiation of IdUrd-containing HSV DNA with either 302 nm or 254 nm ultraviolet (UV) light introduced strand breakage into the DNA in a dose-dependent manner when analyzed by alkaline sucrose density gradient sedimentation. Irradiation of unsubstituted HSV DNA under similar conditions produced little strand breakage. These observations are in agreement with the proposed mechanism for photochemical generation of strand breakage in 5-halo-2'-deoxyuridine-containing DNA. Irradiation of IdUrd-substituted virions followed by analysis of the isolated DNA indicated less strand breakage than irradiation of isolated IdUrd-substituted DNA under equivalent conditions. The dosage of irradiation required to introduce DNA strand breakage in IdUrd-substituted virions was equivalent to that employed to affect greater than 99% loss of infectious virus activity in both control and IdUrd-containing virions. It is suggested that the relative UV insensitivity of IdUrd-substituted HSV may be due to the microstructure environment of the substituted HSV DNA which may favor recombination of the photochemically formed halogen-uracil radical pairs.

The effect of 3'-deoxythymidin-2'-ene (d4T) on the metabolism of exogenously supplied radiolabeled nucleosides was investigated. Following a 24-hr exposure to 250 microM d4T, we observed no significant effect on the incorporation of [3H]thymidine or [3H]deoxycytidine into DNA. In contrast, the amounts of [3H]uridine, [3H]deoxyuridine, and [3H]cytidine were significantly lower than those incorporated by control cultures. d4T had no significant effect on the incorporation of [3H]uridine or [3H]cytidine into RNA, or the incorporation of 3H-labeled amino acids into protein. In d4T-treated cells the relative proportions of [3H]dTMP, [3H]dTDP, and [3H]dTTP formed did not change but their absolute concentrations were increased. d4T significantly reduced the level of [3H]dUMP, and a parallel decrease in [3H]dTMP derived from [3H]dUMP was also evident. d4T increased the amounts of labeled deoxycytidine metabolites formed, with increased dCMP levels the most prominent. In a cell-free extract, [3H]d4T was phosphorylated at a rate of 1.6 pmol/30 min. Increasing concentrations of both thymidine and deoxyuridine inhibited the phosphorylation of [3H]d4T with IC50 values of 5.7 and 35 microM respectively. d4T was found to be a weak substrate for purified H9 cytosolic thymidine kinase (Km = 138 microM) and a weak competitive inhibitor of thymidine and deoxyuridine phosphorylation by this enzyme (Ki = 1.37 and 0.33 mM respectively).

First generation progeny herpes simplex type 1 (HSV-1) virions grown in the presence of 5-iodo-2'-deoxyuridine (IdUrd) were irradiated with either 254 or 302 nm ultraviolet (u.v.) light. The kinetics of virus inactivation revealed decreased sensitivity of IdUrd-substituted virions to irradiation with 302 nm light under all conditions examined, and with 254 nm u.v. light when substituted and control virions were irradiated at equal particle concentrations. Comparison of virus survival after irradiation measured in Vero or Xeroderma Pigmentosum (complementation group A) cells indicated that cellular repair of ultraviolet-induced lesions was not a significant factor in the observed decrease in u.v. sensitivity. IdUrd substitution altered neither the formation of ultraviolet-induced thymidine photoproducts nor the ability of irradiated virions to express delayed early viral enzymes (thymidine kinase, DNA polymerase). It is suggested that nucleocapsid proteins or the highly ordered structure of IdUrd-substituted virions play a key role in u.v. desensitization, either by the formation of non-lethal photoproducts or by the prevention of the formation of DNA-uracilyl free radicals.

A new assay for carbamoylating activity using reversed phase liquid chromatography (HPLC) is described which offers several advantages over existing assays. A comparison was made of the extent of carbamoylation of the amino group of 5'-amino-5'-deoxythymidine (5'-AdThd) by chloroethyl isocyanate and a series of six nitrosoureas upon incubation (37 degrees) at pH 7.4 in phosphate-buffered saline. Determinations can be made in less than 30 min, whereas more than 24 hr are required in the paper chromatography method of Wheeler et al. [Cancer Res. 34, 194 (1974)]. There is a very high coefficient of correlation (r = 0.986, N = 7, P less than 0.001) between these two methods of analysis which allows compounds assayed by the new procedure to be compared to the data compiled through the earlier assay. The application of the new assay to the analysis of the rate of carbamoylating activity for these agents is presented, and a mechanistic basis for the marked differences among these structurally similar compounds is discussed.