Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Phase 2 Study of the HSP-90 Inhibitor AUY922 in Previously Treated and Molecularly Defined Patients with Advanced Non-Small Cell Lung Cancer.

INTRODUCTION: In this phase 2 study, we evaluated the activity of AUY922 in pretreated patients with stage IV NSCLC. METHODS: Patients with advanced NSCLC were divided into molecularly defined strata based on mutations in the EGFR gene, the ALK receptor tyrosine kinase gene (ALK), the KRAS gene, or the wild type of all three. All patients must have received more than two prior lines of therapy, except for those in a fifth stratum for a less pretreated EGFR cohort (EGFR<2). In the EGFR-mutant and ALK-rearranged strata, prior platinum therapy was not required. Patients with EGFR mutation must have received an EGFR tyrosine kinase inhibitor unless they had de novo resistance (e.g., T790M or exon 20 insertions). Eligible patients received weekly intravenous AUY922, 70 mg/m2. The primary objective was to estimate efficacy (complete or partial response, or in the absence of complete or partial response, stable disease) at 18 weeks, by the Response Criteria in Solid Tumors. RESULTS: A total of 153 patients from 21 global centers were enrolled from October 2010 to November 2014. The investigator-assessed overall response rate and stable disease rate at 18 weeks were 31.8% and 9.1% in the ALK-rearranged stratum, 17.1% and 8.6% in EGFR-mutant stratum, 9.7% and 22.6% in the EGFR<2 stratum, 0% and 7.1% in KRAS-mutant stratum, and 8.8% and 8.8% in wild-type stratum. Biomarker data showed activity of AUY922 in EGFR-mutant patients with exon 19 deletion, T790M mutation, and exon 20 insertion. The most common (≥40%) all-causality adverse events were diarrhea, nausea, and decreased appetite. Visual-related disorders were reported in 79.7% of patients (most were grade 1/2). Thirty-five patients (22.9%) reported night blindness. CONCLUSION: AUY922 is active in patients with NSCLC, particularly among patients with ALK rearrangements and EGFR mutations.

mGlu5R promotes glutamate AMPA receptor phosphorylation via activation of PKA/DARPP-32 signaling in striatopallidal medium spiny neurons.

Group I metabotropic glutamate receptors (mGluRs), which comprise mGlu1Rs and mGlu5Rs, are enriched in striatal medium spiny neurons (MSNs), where they modulate glutamatergic transmission. Here, we have examined the effect of group I mGluRs on the regulation of the state of phosphorylation of the GluA1 subunit of the AMPA glutamate receptor. We found that incubation of mouse striatal slices with the group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) promotes GluA1 phosphorylation at the cAMP-dependent protein kinase (PKA) site, Ser845. This effect is prevented by 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGlu5R antagonist. The increase in GluA1 phosphorylation produced by DHPG is also prevented by blockade of adenosine A2A receptors (A2ARs), which are known to promote cAMP signaling specifically in striatopallidal MSNs, as well as by enzymatic degradation of endogenous adenosine, achieved with adenosine deaminase. The ability of DHPG to increase PKA-dependent phosphorylation of GluA1 depends on concomitant activation of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32). Thus, inactivation of the PKA phosphorylation site of DARPP-32 abolishes the effect of DHPG. Moreover, cell-specific knock out of DARPP-32 in striatopallidal, but not in striatonigral, MSNs prevents the increase in Ser845 phosphorylation induced by DHPG. These results indicate that activation of mGlu5Rs promotes PKA/DARPP-32-dependent phosphorylation of downstream target proteins in striatopallidal MSNs and that this effect is exerted via potentiation of tonic A2AR transmission. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Geoditin A induces oxidative stress and apoptosis on human colon HT29 cells.

Geoditin A, an isomalabaricane triterpene isolated from the marine sponge Geodia japonica, has been demonstrated to dissipate mitochondrial membrane potential, activate caspase 3, decrease cytoplasmic proliferating cell nuclear antigen (PCNA), and induce apoptosis of leukemia cells, but the underlying mechanism remains unclear [1]. In this study, we found fragmentation of Golgi structure, suppression of transferrin receptor expression, production of oxidants, and DNA fragmentation in human colon cancer HT29 cells after treatment with geoditin A for 24 h. This apoptosis was not abrogated by chelation of intracellular iron with salicylaldehyde isonicotinoyl hydrazone (SIH), but suppressed by N-acetylcysteine (NAC), a thiol antioxidant and GSH precursor, indicating that the cytotoxic effect of geoditin A is likely mediated by a NAC-inhibitable oxidative stress. Our results provide a better understanding of the apoptotic properties and chemotherapeutical potential of this marine triterpene.

Interleukin-1 beta down-regulates the expression of metabotropic glutamate receptor 5 in cultured human astrocytes.

Expression of metabotropic glutamate receptor 5 (mGluR5) protein is known to be plastic and to depend critically on the astrocytes' microenvironment. In the present study we investigated whether interleukins, which are involved in the immune response following brain injury, could contribute to the regulation of mGluR5 protein in human astrocytes in culture. Using Western blotting and immunocytochemistry, no detectable changes in the expression of the mGluR5 protein were observed with both interleukin 1beta and interleukin 6 in undifferentiated cultures (growing in serum free media). In contrast, in cultures that had been morphologically differentiated by exposure to epidermal growth factor (EGF), addition of interleukin 1beta (but not interleukin 6) reduced mGluR5 protein expression. In addition, stimulation of phosphoinositide hydrolysis by the selective group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was reduced after exposure to interleukin 1beta. The suppressive effect on mGluR5 was prevented by the interleukin 1 receptor antagonist. Thus, interleukin 1beta may represent an additional pathway through which mGluR5 expression and function can be modulated in astrocytes under different pathological conditions associated with an inflammatory response.

Potentiation of synaptic transmission in the rat dentate gyrus in vitro by (S)-3,5-dihydroxyphenylglycine ((S)-DHPG).

The direct activation of metabotropic glutamate receptors (mGluRs) by 1S,3R-aminocyclopentane dicarboxylate (1S,3R-ACPD), has previously been shown to induce a relatively fast (maximum at 10 min) and slow (90 min) onset long-term potentiation (LTP) of synaptic transmission in the hippocampus. Here we report the first evidence for a relatively fast onset LTP of synaptic transmission in the immature male rat (50-100 g) dentate gyrus in vitro by application of the mGluR type I agonist, (S)-3,5-dihydroxyphenylglycine ((S)-DHPG; 20 microM). Bath application of (S)-DHPG caused a transient depression of the field excitatory postsynaptic potential (EPSP) slope, followed after washout by a relatively rapidly developing potentiation of synaptic transmission to a maximum increase at 12-15 min (161.1 +/- 11.4% compared to controls at 15 min; n = 8). This effect was not observed following perfusion with the enantiomer (R,S)-DHPG at the same dose. The (S)-DHPG potentiation occluded tetanically induced LTP and vice versa. The potentiation was antagonised by the non-specific mGluR antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine ((R,S)-MCPG) at high doses (500-1000 microM) but was unaffected in the presence of the N-methyl-D-aspartate (NMDA) receptor blocker, D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 microM). Our results demonstrate a robust NMDA-independent LTP induced by (S)-DHPG that occludes tetanically induced LTP.

Trypsin inhibitors and nutritive value in cereals.

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.