New A-ring pyridine fused androstanes in 17a-homo-17-oxa (d-homo lactone), 17α-picolyl or 17()-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds ,, and were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione d-modified androstane derivatives with propargylamine catalyzed by Cu(ii), and evaluated for potential anticancer activity using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal d-homo lactones led to identification of new inhibitors of aldo-keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine d-lactone steroid also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.