Two novel classes of enzymes are required for the biosynthesis of aurofusarin in Fusarium graminearum.

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.

Novel cadaverine non-invasive biosensor technology for the prediction of shelf life of modified atmosphere packed pork cutlets.

Food waste in perishable products calls for the development of cost-efficient and real-time freshness and shelf life assessment tools. The current study evaluated a newly developed cadaverine biosensor for its ability to assess the sensory freshness stage and microbial quality of modified atmosphere packed (MAP) pork cutlets under a realistic supply chain scenario. The experiment compared the cadaverine levels measured by the biosensor to liquid chromatography - tandem mass spectrometry (LC-MS/MS) cadaverine concentrations, and associated these to the shelf life estimation and freshness states determined by sensory and microbial evaluations during an 18-day storage period (5 °C). Results underlined the potential of cadaverine as a freshness biomarker as well as the applicability of the biosensor as a shelf life prediction tool. This is supported by the correlations obtained between sensory odour freshness evaluation and total viable counts with biosensor cadaverine levels for which the r obtained were 0.97 (<0.001) and 0.95 (<0.001), respectively.

Identification of the first synthetic steroidogenic factor 1 inverse agonists: pharmacological modulation of steroidogenic enzymes.

Steroidogenic factor SF-1, a constitutively active nuclear hormone receptor, is essential to the development of adrenal and gonadal glands and acts as a shaping factor of sexual determination and differentiation. Its effects are exerted primarily through the control of the synthesis of steroid hormones. The functional cell-based assay Receptor Selection and Amplification Technology (R-SAT) was used to identify potent and selective SF-1 inverse agonists through the screening of a chemical library of drug-like small-molecule entities. Among them, 4-(heptyloxy)phenol (AC-45594), a prototype inverse agonist lead, was used to show that SF-1 constitutive activity can be pharmacologically modulated by a synthetic ligand. In a physiological system of endocrine function, the expression of several reported SF-1 target genes, including SF-1 itself, was inhibited by treatment with AC-45594 and analogs. Thus, pharmacological modulation of SF-1 is critical to its function as an endocrine master regulator and has potentially important consequences to diseases in which SF-1 activity is critical.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator.

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

Differential modulation of inflammatory pain by a selective estrogen receptor beta agonist.

To understand the contribution of the estrogen receptor beta, the potent and selective agonist ERb-131 was evaluated in animal models of inflammatory pain. In paradigms of acute and persistent inflammatory pain, ERb-131 did not alleviate the nociception induced by either carrageenan or formalin. However, in the chronic complete Freund's adjuvant model, ERb-131 resolved both inflammatory and hyperalgesic components. Thus, ERb-131 is sufficient to alleviate chronic but not acute inflammatory pain states.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist.

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

Discovery of a potent, orally available, and isoform-selective retinoic acid beta2 receptor agonist.

4'-Octyl-4-biphenylcarboxylic acid (1g, AC-55649) was identified as a highly isoform-selective agonist at the human RARbeta2 receptor in a functional intact cell-based screening assay. The subsequent hit to lead optimization transformed the lipophilic, poorly soluble hit into a more potent and orally available compound (2, AC-261066) with retained beta2 selectivity and greatly improved physiochemical properties. Being an isoform-selective RARbeta2 receptor agonist that discriminates between nuclear receptor isoforms having identical ligand binding domains, 2 will be useful as a pharmacological research tool but also a valuable starting point for drug development.

Identification of novel subtype selective RAR agonists.

Drugs targeting retinoid receptors have been developed to treat a variety of therapeutic indications, but their success has been limited in part due to lack of selectivity. A novel functional cell-based assay R-SATtrade mark was employed to screen a small molecule chemical library and identify a variety of novel RAR agonists with various subtype selectivities, including RARbeta/gamma and RARgamma selective agonists. A novel class of synthetic compounds that distinguishes between the different RARbeta isoforms is described. This pharmacophore displays anti-proliferative activity and induces differentiation in a neuronal cell line, consistent with a classical retinoid mechanism of action while providing unique subtype selectivity. These novel subtype selective RAR agonists could serve as powerful tools to probe into subtype and isoform-specific retinoid function.

Design, synthesis, and structure-activity analysis of isoform-selective retinoic acid receptor beta ligands.

We recently discovered the isoform selective RAR beta 2 ligand 4'-octyl-4-biphenylcarboxylic acid (3, AC-55649). Although 3 is highly potent at RAR beta 2 and displays excellent selectivity, solubility issues make it unsuitable for drug development. Herein we describe the exploration of the SAR in a biphenyl and a phenylthiazole series of analogues of 3. This ultimately led to the design of 28, a novel, orally available ligand with excellent isoform selectivity for the RAR beta 2.

Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators.

Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.