5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography–tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography–mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

A new class of anion transporter named 'perenosins' consisting of a pyrrole linked through an imine to either an indole, benzimidazole or indazole is reported. The indole containing members of the perenosin family function as effective transmembrane Cl(-)/NO3(-) antiporters and HCl cotransporters in a manner similar to the prodigiosenes. The compounds reduce the viability of MDA-MB-231 and MCF-7.

OBJECTIVEThe pan–histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG.METHODSThe authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 μM to the ventral pons of 2 juvenile Large White–Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED.RESULTSThere were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 μM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively.CONCLUSIONSCED of water-soluble panobinostat, up to a concentration of 30 μM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.