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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...

In-cell NMR spectroscopy has emerged as a powerful technique for monitoring biomolecular interactions at an atomic level inside intact cells. However, current methodologies are inadequate at charting intracellular interactions of nonlabeled proteins and require their prior isotopic labeling. Herein, we describe for the first time the monitoring of the quercetin-alanine bioconjugate interaction with the nonlabeled antiapoptotic protein Bcl-2 inside living human cancer cells. STD and Tr-NOESY in-cell NMR methodologies were successfully applied in the investigation of the binding, which was further validated in vitro. In-cell NMR proved a very promising strategy for the real-time probing of the interaction profile of potential drugs with their therapeutic targets in native cellular environments and could, thus, open a new avenue in drug discovery.

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [D-Lys 6 ]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site ($4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.

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Discovery of uncharted guanidinium peptide coupling reagent side reactions during peptide-drug conjugates synthesis.