Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

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Nitric oxide (NO), an important mediator of both physiological and pathological processes [1], is derived from l-arginine by a family of enzymes termed NO synthases [2]. At least three isoforms of NO synthase (NOS) have been detected. Of these, endothelial (eNOS) and neuronal (nNOS) enzymes are constitutive and regulated by Ca 2+ /calmodulin. The inducible NOS (iNOS) originally detected in macrophages and in the endothelium is produced in response to cytokines and cellular debris of microbial origin. This inducible form can produce 10-to 50-fold more NO than the constitutive NOS. In addition, Diabetologia (1999) Abstract Aims/hypothesis. The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. Methods. Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting.Results. In the presence of extracellular l-arginine (100 mmol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of larginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of l-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. Conclusion/interpretation. Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes. [Diabetologia (1999) 42: 539± 544]

The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose-and calcium-mediated insulin secretion from ␤TC3 cells. Both 3-allyl-and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80 -95%) inhibited glucoseand KCl-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCl. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucoseand KCl-induced insulin secretion to comparable degrees. Treatment of ␤TC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.