Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

Identification of small molecule ceramide kinase inhibitors using a homogeneous chemiluminescence high throughput assay.

Lipid kinases have emerged as potentially important therapeutic targets in oncology and inflammation. Ceramide kinase (CERK) is a lipid kinase that catalyzes the formation of ceramide-1-phosphate from ceramide, a sphingolipid that is a key mediator of cellular apoptosis. Ceramide-1-phosphate has been shown to enhance the production of pro-inflammatory eicosonoids, to promote cell proliferation, and potentially to reduce intracellular ceramide levels by inhibition of acidic sphingomyelinases. Here we describe a homogeneous chemiluminescence assay that directly measures the ceramide-dependent ATP depletion by recombinant full-length human CERK. As compared to reported CERK assays that have limitations on compound throughput, the chemiluminescence assay has been miniaturized to a 1,536-well microtiter plate format and utilized to screen an ultra-large compound library (>4 million compounds). Multiple chemical scaffolds have been identified as CERK kinase inhibitors and characterized mechanistically, which to our knowledge represent the first known small molecule CERK inhibitors with nanomolar activities. These compounds can serve as tools to further elucidate the CERK pathway and its role in ceramide metabolism and human diseases.

Adenosine is the primary precursor of all purine nucleotides in Trichomonas vaginalis.

Trichomonas vaginalis, a parasitic protozoan and the causative agent of trichomoniasis, lacks de novo purine nucleotide synthesis and possesses a unique purine salvage pathway, consisting of a bacterial type purine nucleoside phosphorylase and a purine nucleoside kinase. It is generally believed that adenine and guanine are converted to their corresponding nucleosides and then further phosphorylated to form AMP and GMP, respectively, as the main as well as the essential pathway of replenishing the purine nucleotide pool in the organism. Formycin A, an analogue of adenosine, inhibits both enzymes as well as the in vitro growth of T. vaginalis with an estimated IC(50) of 0.27 microM. This growth inhibition was reversed by adding adenine to the culture medium but not by adding guanine or hypoxanthine. Furthermore, T. vaginalis can grow in semi-defined medium supplemented with only adenine but not with guanine or hypoxanthine. Radiolabeling experiments followed by HPLC analysis of the purine nucleotide pool in T. vaginalis demonstrated incorporation of [8-14C]adenine into both adenine and guanine nucleotides, whereas [8-14C]guanine was incorporated only into guanine nucleotides. Substantial adenosine deaminase activity and significant IMP dehydrogenase and GMP synthetase activities were identified in T. vaginalis lysate, suggesting a pathway capable of converting adenine to GMP via adenosine. This purine salvage scheme depicts adenosine the primary precursor of the entire purine nucleotide pool in T. vaginalis and the purine nucleoside kinase one of the most pivotal enzymes in purine salvage and a potential target for anti-trichomoniasis chemotherapy.

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme.

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.

The pivotal role of guanine phosphoribosyltransferase in purine salvage by Giardia lamblia.

Giardia lamblia is an anaerobic binucleate flagellated protozoan known to lack de novo synthesis of purine nucleotides. Our previous metabolic studies indicated two major parallel pathways mediated by adenine phosphoribosyltransferase (APRT) and guanine phosphoribosyltransferase (GPRT) that constitute the primary route of purine salvage in this organism. To verify further that these enzymes play a pivotal role in replenishing the purine ribonucleotide pool and are required for replicative growth of Giardia, a knock-out of GPRT gene expression in this organism was attempted. A hammerhead ribozyme flanked by two arms of GPRT antisense RNA (GPRZ) was designed, synthesized and found capable of cleaving a GPRT mRNA fragment in vitro at the designated site. GPRZ cDNA was then cloned into a viral vector pC631pac, derived from the genome of giardiavirus (GLV), and its transcript was introduced into GLV-infected Giardia trophozoites by electroporation. Stable transformants selected under increasing concentrations of puromycin displayed parallel increases in ribozyme levels and associated decreases in GPRT mRNA levels, GPRT enzyme activity and replicative cell growth to less than 10-20% of wild-type levels. Metabolite analyses showed specific depletion of the guanine ribonucleotide pools in parallel with slower cell growth. We conclude that GPRT plays an essential role in supplying guanine nucleotides required for growth and multiplication of Giardia, emphasizing its potential as a bona fide target for antigiardiasis chemotherapy.