Triazole Substitution of a Labile Amide Bond Stabilizes Pantothenamides and Improves Their Antiplasmodial Potency

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo. In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase

Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1′,2′]isoxazolo[4′,5′:2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na2SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17β-hydroxy-2α,17α-dimethyl-5α-androstan-3-one) was performed as a prediction of the in vivo metabolic profile.Australian Research Council LP12020044

Construction and validation of a mammalian expression vector for in utero electroporation study of miR-3099 in the mouse neocortex

Introduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p<0.001) in cells transfected with miR-3099 vector as compared to both negative and empty plasmid control groups. In addition, the expression of eGFP was noted in the brain section indicating that the vectors with or without miR-3099 transgene were successfully transfected into and expressed in the neocortex upon electroporation. Conclusion: The bicistronic expression vector of miR-3099 which was driven by the CAG promoter was successfully constructed, validated and sufficiently delivered to brain cells via the in utero electroporation approach. The regulatory roles of miR-3099 in embryonic brain development can be manipulated using similar approach

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)