Targeting aldehyde dehydrogenase: a potential approach for cell labeling.

INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV.

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

Structure-based design of potent retinoid X receptor alpha agonists.

A series of tetrahydrobenzofuranyl and tetrahydrobenzothienyl propenoic acids that showed potent agonist activity against RXRalpha were synthesized via a structure-based design approach. Among the compounds studied, 46a,b showed not only very good potency against RXRalpha (K(i) = 6 nM) but was also found to be greater than 167-fold selective vs RARalpha (K(i) > 1000 nM). This compound profiled out as a full agonist in a cell-based transient transfection assay (EC(50) = 3 nM). The two antipodes were separated via chiral chromatography, and 46b was found to be 40-fold more potent than 46a. Interestingly, cocrystallization of 46a,b with the RXRalpha protein generated a liganded structure whereby the (S)-antipode was found in the binding pocket. Given orally in db/db mice or ZDF rats, 46a,b showed a significant glucose-lowering effect and an increase in liver mass. Triglycerides decreased significantly in db/db mice but increased in the ZDF rats. A dose-dependent decrease of nonesterified free fatty acids was seen in ZDF rats but not in db/db mice. These differences indicate a species specific effect of RXR agonists on lipid metabolism.

CYP3A induction by N-hydroxyformamide tumor necrosis factor-alpha converting enzyme/matrix metalloproteinase inhibitors use of a pregname X receptor activation assay and primary hepatocyte culture for assessing induction potential in humans.

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.