Novel Crizotinib–GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems

• Main Authors: József Murányi, Attila Varga, Pál Gyulavári, Kinga Pénzes, Csilla E. Németh, Miklós Csala, Lilla Pethő, Antal Csámpai, Gábor Halmos, István Peták, István Vályi-Nagy
• Format: Article
• Language: English
• Published: MDPI AG 2019-11-01
• Series: International Journal of Molecular Sciences
• Subjects: targeted drug delivery 1; gnrh 2; gnrhr 3; lysosome 3; permeability 4; crizotinib 5; conjugate 6; nsclc 7; c-met 8; endocytosis 9; galectin 10
• Online Access: https://www.mdpi.com/1422-0067/20/22/5590

Several promising anti-cancer drug&#8722;GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [<span style="font-variant: small-caps;">d</span>-Lys⁶]&#8722;GnRH-I targeting peptide. Our most prominent crizotinib&#8722;GnRH conjugates, the amide-bond-containing [<span style="font-variant: small-caps;">d</span>-Lys⁶(crizotinib*)]&#8722;GnRH-I and the ester-bond-containing [<span style="font-variant: small-caps;">d</span>-Lys⁶(MJ55*)]&#8722;GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the <i>MET</i>-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib&#8722;GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets&#8212;the ATP-binding site of RTKs&#8212; and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib&#8722;GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug&#8722;GnRH conjugates.