Enhanced In Vitro Antitumor Activity of GnRH-III-Daunorubicin Bioconjugates Influenced by Sequence Modification.

Sabine Schuster,Gábor Halmos,Zsuzsanna Szabó,Gábor Mező,László Buday,János Gardi,Bálint Szeder,Beáta Biri-Kovács

doi:10.3390/pharmaceutics10040223

Abstract

Receptors for gonadotropin releasing hormone (GnRH) are highly expressed in various human cancers including breast, ovarian, endometrial, prostate and colorectal cancer. Ligands like human GnRH-I or the sea lamprey analogue GnRH-III represent a promising approach for the development of efficient drug delivery systems for targeted tumor therapy. Here, we report on the synthesis and cytostatic effect of 14 oxime bond-linked daunorubicin GnRH-III conjugates containing a variety of unnatural amino acids within the peptide sequence. All compounds demonstrated a reduced cell viability in vitro on estrogen receptor α (ERα) positive and ERα negative cancer cells. The best candidate revealed an increased cancer cell growth inhibitory effect compared to our lead-compound GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)]. Flow cytometry and fluorescence microscopy studies showed that the cellular uptake of the novel conjugate is substantially improved leading to an accelerated delivery of the drug to its site of action. However, the release of the active drug-metabolite by lysosomal enzymes was not negatively affected by amino acid substitution, while the compound provided a high stability in human blood plasma. Receptor binding studies were carried out to ensure a high binding affinity of the new compound for the GnRH-receptor. It was demonstrated that GnRH-III-[2ΔHis,3d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] is a highly potent and promising anticancer drug delivery system for targeted tumor therapy.

Highlights

Targeted cancer therapy is a promising tool to overcome the drawbacks of classical chemotherapy like the lack of selectivity, toxicity to healthy tissue and the development of multidrug resistance forced by high dose treatments
The native ligand of this seven-transmembrane G-protein coupled receptor regulates and stimulates the synthesis and release of gonadotropins like luteinizing hormone (LH) and follicle stimulating hormone (FSH), whereby the application of gonadotropin-releasing hormone (GnRH) analogs causes inhibition of gonadotropin release, which can occur by two different mechanisms of action [12]
A constant exposure to GnRH agonists triggers a desensitization of the gonadotropic cells as well as the downregulation of the receptor level on the cell surface followed by a reduced LH and FSH release [15]

Summary

Introduction

Targeted cancer therapy is a promising tool to overcome the drawbacks of classical chemotherapy like the lack of selectivity, toxicity to healthy tissue and the development of multidrug resistance forced by high dose treatments. Prominent examples hereby are gonadotropin-releasing hormone (GnRH) agonists and antagonists like triptorelin and cetrorelix, which have been developed by Schally and coworkers [8,9] Both peptide-based pharmaceutics were approved by the Food and Drug Administration (FDA) at the beginning of this century [10,11]. Cetrorelix acetate was the first GnRH antagonist on the market and is used to prevent premature luteinizing hormone (LH) surges in women undergoing controlled ovarian stimulation, whereas triptorelin pamoate received approval for the palliative treatment of advanced prostate cancer [10,11,12] These potent GnRH analogs bind like the natural peptide hormone GnRH-I (

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