High susceptibility of human cancer xenografts with higher levels of cytidine deaminase to a 2'-deoxycytidine antimetabolite, 2'-deoxy-2'-methylidenecytidine.

2'-Deoxy-2'-methylidenecytidine (DMDC) is a new 2'-deoxycytidine (dCyd) antimetabolite. The present study compared its antitumor activities with those of 2',2'-difluorodeoxy-cytidine (gemcitabine) in 15 human cancer xenograft models. DMDC was highly resistant to cytidine (Cyd) deaminase, which deaminates the dCyd analogues to inactive molecules, whereas gemcitabine was susceptible to the enzyme. Given p.o., high antitumor activity with therapeutic index of more than 10 was found with DMDC in 7 of 15 xenograft lines. In contrast, gemcitabine given i.v. or p.o. was highly effective in 4 of 15 human cancer xenograft lines. The antitumor spectrum of these compounds was quite different, although their molecular targets are reported to be similar. DMDC was highly effective in tumors with higher levels of Cyd deaminase activity, whereas it showed only slight activity in those with lower levels of Cyd deaminase. In contrast, gemcitabine appeared to be less effective in tumors with high levels of Cyd deaminase. We also investigated the correlation with the susceptibility to the two dCyd antimetabolites and dCyd kinase activity in tumors, but none was observed. Cyd deaminase activity was found to be high in tumor tissues from various types of human cancers thus far tested, such as colorectal cancer and non-small cell lung cancer. Such cancer types or individual patients who have tumors with high activity of the enzyme may be targets for DMDC therapy.

X-ray structure of a novel matrix metalloproteinase inhibitor complexed to stromelysin.

A new class of matrix metalloproteinase (MMP) inhibitors has been identified by screening a collection of compounds against stromelysin. The inhibitors, 2,4,6-pyrimidine triones, have proven to be potent inhibitors of gelatinases A and B. An X-ray crystal structure of one representative compound bound to the catalytic domain of stromelysin shows that the compounds bind at the active site and ligand the active-site zinc. The pyrimidine triones mimic substrates in forming hydrogen bonds to key residues in the active site, and provide opportunities for placing appropriately chosen groups into the S1' specificity pocket of MMPS: A number of compounds have been synthesized and assayed against stromelysin, and the variations in potency are explained in terms of the binding mode revealed in the X-ray crystal structure.

Novel 5,5-disubstitutedpyrimidine-2,4,6-triones as selective MMP inhibitors.

The 5,5-disubstitutedpyrimidine-2,4,6-triones represent a new class of MMP inhibitors showing selectivity for the gelatinases A and B, collagenase-3, and human neutrophil collagenase. The SAR presented here is in good agreement with an X-ray structure of compound 5 bound to the catalytic domain of stromelysin-1. While of the barbiturate structural class, compound 5 did not show any toxic or sedative effects.

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands.

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.

Inhibitors of farnesyltransferase and Ras processing peptidase.

Four analogs of the carboxy terminus of unprocessed p21Ras protein were evaluated as inhibitors of the p21Ras processing farnesyltransferase and peptidase. While three showed no crossover of inhibitory activity between the enzymes, the fourth (a naphthyl-substituted peptide) inhibited both farnesyltransferase and peptidase, with IC50s of 16 microM and 3 microM, respectively. Such inhibition of more than one step of Ras processing may complicate assessment of the mode of action for some inhibitors of Ras processing peptidase.

Covalent modification of the interleukin-5 receptor by isothiazolones leads to inhibition of the binding of interleukin-5.

Using a fusion protein of the human interleukin-5-receptor alpha chain (hIL5R alpha) and the human IgG C gamma 3 chain (hIL5R alpha-h gamma 3), we have developed a solid-phase assay for high-flux screening of a collection of synthetic compounds. We report on the identification of isothiazolone derivatives as potent inhibitors of binding of interleukin-5 (IL5) to the hIL5R alpha, as measured in a solid-phase assay (soluble hIL5R alpha or hIL5R alpha-h gamma 3) or on COS-1 cells expressing the hIL5R alpha on the cell membrane. The binding of hIL4 and human granulocyte macrophage colony-stimulating factor (hGM-CSF) to their respective receptors is not inhibited by the isothiazolones in similar assay systems. Scatchard analysis revealed that these compounds caused a decrease in affinity of the IL5R alpha for IL5. The inhibition of binding IL5 to its receptor by the isothiazolone derivatives is abrogated by free-sulfhydryl-containing compounds such as dithiothreitol, indicating that the isothiazolones react with the sulfhydryl group of free cysteine residues in the hIL5R alpha. Mutation of Cys66 led to a receptor which still binds hIL5, but which was insensitive to the inhibition by isothiazolones. Mutation of Cys249 and Cys296 to serine resulted in complete loss of IL-5-binding activity. The use of radio-labeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity.