CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.

Transport of dicationic drugs pentamidine and furamidine by human organic cation transporters.

The antiparasitic activity of aromatic diamidine drugs, pentamidine and furamidine, depends on their entry into the pathogenic protozoa via membrane transporters. However, no such diamidine transporter has been identified in mammalian cells. The goal of this study is to investigate whether these dicationic drugs are substrates for human organic cation transporters (hOCTs, solute carrier family 22A1-3) and whether hOCTs play a role in their tissue distribution, elimination, and toxicity. Inhibitory and substrate activities of pentamidine and furamidine were studied in stably transfected Chinese hamster ovary (CHO) cells. The results of [(3)H]1-methyl-4-phenylpyridinium uptake study showed that pentamidine is a potent inhibitor for all three OCT isoforms (IC50 < 20 microM), whereas furamidine is a potent inhibitor for hOCT1 and hOCT3 (IC50 < 21 microM) but a less potent inhibitor for hOCT2 (IC50 = 189.2 microM). Both diamidines are good substrates for hOCT1 (Km = 36.4 and 6.1 microM, respectively), but neither is a substrate for hOCT2 or hOCT3. The cytotoxicity of pentamidine and furamidine was 4.4- and 9.3-fold greater, respectively, in CHO-hOCT1 cells compared with the mock cells. Ranitidine, an hOCT1 inhibitor, reversed this hOCT1-mediated potentiation of cytotoxicity. This is the first finding that dicationic drugs, such as pentamidine and furamidine, are substrates for hOCT1. In humans, aromatic diamidines are primarily eliminated in the bile but are distributed and cause toxicity in both liver and kidney. These transporters may play important roles in the disposition of aromatic diamidines in humans, as well as resultant drug-drug interactions and toxicity involving diamidine drugs.

Proteome-wide identification of family member-specific natural substrate repertoire of caspases.

Caspases are proteolytic enzymes that are essential for apoptosis. Understanding the many discrete and interacting signaling pathways mediated by caspases requires the identification of the natural substrate repertoire for each caspase of interest. Using an amplification-based protein selection technique called mRNA display, we developed a high-throughput screen platform for caspase family member specific substrates on a proteome-wide scale. A large number of both known and previously uncharacterized caspase-3 substrates were identified from the human proteome. The proteolytic features of these selected substrates, including their cleavage sites and specificities, were characterized. Substrates that were cleaved only by caspase-8 or granzyme B but not by caspase-3, were readily selected. The method can be widely applied for efficient and systematic identification of the family member specific natural substrate repertoire of any caspase in an organism of interest, in addition to that of numerous other proteases with high specificity.

Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases.

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.

Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1.

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine residue of heparan sulfate (HS) to form 3-O-sulfated HS. The 3-O-sulfated glucosamine residue contributes to two important biological functions of HS: binding to antithrombin and thereby carrying anticoagulant activity, and binding to herpes simplex viral envelope glycoprotein D to serve as an entry receptor for herpes simplex virus 1. A total of five HS 3-O-sulfotransferase isoforms were reported previously. Here we report the isolation and characterization of a novel HS 3-O-sulfotransferase isoform, designated as HS 3-O-sulfotransferase isoform 5 (3-OST-5). 3-OST-5 cDNA was isolated from a human placenta cDNA library and expressed in COS-7 cells. The disaccharide analysis of 3-OST-5-modified HS revealed that 3-OST-5 generated at least three 3-O-sulfated disaccharides as follows: IdoUA2S-AnMan3S, GlcUA-AnMan3S6S, and IdoUA2S-AnMan3S6S. Transfection of the plasmid expressing 3-OST-5 rendered wild type Chinese hamster ovary cells susceptible to the infection by herpes simplex virus 1, suggesting that 3-OST-5-modified HS serves as an entry receptor for herpes simplex virus 1. In addition, 3-OST-5-modified HS bound to herpes simplex viral envelope protein glycoprotein D. Furthermore, we found that 3-OST-5-modified HS also bound to antithrombin, suggesting that 3-OST-5 also produces anticoagulant HS. In summary, our results indicate that a new member of 3-OST family generates both anticoagulant HS and an entry receptor for herpes simplex virus 1. These results provide a new insight regarding the mechanism for the biosynthesis of biologically active HS.