Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing HormoneReceptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG, showed a significant advantage in tumor growth inhibition when compared to gemcitabine. 3 IntroductionDespite advancements in methods for early cancer detection and improved insights into the molecular mechanisms and treatment options, advanced prostate cancer (CaP) remains a major health problem for the aging man. 1,2 Hormonal therapy is usually the first line of defense for CaP treatment by using drugs that lead to chemical castration, suppression of testosterone and dihydrotestosterone (DHT) biosynthesis. 3,4 The hormonal ablation approach has been achieved successfully using agonist (through desensitization) or antagonist analogue drugs, of the native Gonadotropin Releasing Hormone (GnRH). These drugs exert their effects primarily on the pituitary gland through the GnRH-R by lowering gonadotropins and downstream gonadal sex steroids. Nevertheless, in many cases after treatment, following initial tumor regression, CaP progresses to an androgen-independent state with poor prognosis, which presents a major challenge for the physician and the patient. 3,[5][6][7][8][9][10] Research on the GnRH-R has shown that its expression is not confined solely to the pituitary but that is also present in several other tissues such as prostate, breast 11-13 and the GnRH-R level of expression along with cell context is critical for cell responses to either agonist or antagonist drugs of the receptor. 14 It is also well established that GnRH-R gene expression is upregulated in patients with androgenindependent CaP, making the GnRH-R an attractive target for the design of novel and specific therapeutics. 15 A modern approach to improve conventional chemotherapy is by direct targeting of chemotherapeutic agents to cancer cells in order to enhance the tumoricidal effect and reduce peripheral toxicity of a specific drug. Linking chemo...

Prav, in contrast to same-dose Sim or POC, reduces infarction in Chol rabbits independently of lipid lowering, potentially through eNOS activation and nitro-oxidative stress attenuation.

a b s t r a c tA series of symmetrically bis-substituted imidazole analogs bearing at the N-1 and N-3 two biphenyl moieties ortho substituted either with tetrazole or carboxylate functional groups was designed based on docking studies and utilizing for the first time an extra hydrophobic binding cleft of AT1 receptor. The synthesized analogs were evaluated for their in vitro antagonistic activities (pA 2 values) and binding affinities (elogIC 50 values) to the Angiotensin II AT1 receptor. Among them, the potassium (elogIC 50 ¼ 9.04) and the sodium (elogIC 50 ¼ 8.54) salts of 4-butyl-N,N 0 -bis{[2 0 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl} imidazolium bromide (12a and 12b, respectively) as well as its free acid 11 (elogIC 50 ¼ 9.46) and the 4-butyl-2-hydroxymethyl-N,N 0 -bis{[2 0 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (14) (elogIC 50 ¼ 8.37, pA 2 ¼ 8.58) showed high binding affinity to the AT1 receptor and high antagonistic activity (potency). The potency was similar or even superior to that of Losartan (elogIC 50 ¼ 8.25, pA 2 ¼ 8.25). On the contrary, 2-butyl-N,N 0 -bis{[2 0 -[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (27) (elogIC 50 ¼ 5.77) and 2-butyl-4-chloro-5-hydroxymethyl-N,N 0 -bis{[2 0 -[2H-tetrazol-5-yl)]biphenyl-4-yl] methyl}imidazolium bromide (30) (elogIC 50 ¼ 6.38) displayed very low binding affinity indicating that the orientation of the n-butyl group is of primary importance. Docking studies of the representative highly active 12b clearly showed that this molecule has an extra hydrophobic binding feature compared to prototype drug Losartan and it fits to the extra hydrophobic cavity. These results may contribute to the discovery and development of a new class of biologically active molecules through bis-alkylation of the imidazole ring by a convenient and cost effective synthetic strategy.

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Kir3 (or GIRK) channels have been known for nearly three decades to be activated by direct interactions with the βγ subunits of heterotrimeric G (Gαβγ) proteins in a membrane-delimited manner. Gα also interacts with GIRK channels and since PTX-sensitive Gα subunits show higher affinity of interaction they confer signaling specificity to G Protein-Coupled Receptors (GPCRs) that normally couple to these G protein subunits. In heterologous systems, overexpression of non PTX-sensitive Gα subunits scavenges the available Gβγ and biases GIRK activation through GPCRs that couple to these Gα subunits. Moreover, all Kir channels rely on their direct interactions with the phospholipid PIP2 to maintain their activity. Thus, signals that activate phospholipase C (e.g. through Gq signaling) to hydrolyze PIP2 result in inhibition of Kir channel activity. In this review, we illustrate with experiments performed in Xenopus oocytes that Kir channels can be used efficiently as reporters of GPCR function through Gi, Gs or Gq signaling. The membrane-delimited nature of this expression system makes it highly efficient for constructing dose-response curves yielding highly reproducible apparent affinities of different ligands for each GPCR tested.

Background:The molecular mechanisms underlying activation of CRF 1 receptor (CRF 1 R) were elusive. Results: We determined specific residues in the transmembrane domains (TMs) of CRF 1 R that are critical for receptor activation. Conclusion: A possible "transmission switch" involving TM interactions is important for CRF 1 R activation. Significance: This knowledge may aid in the development of nonpeptide CRF 1 R antagonists for use in stress-related disorders.

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