Targeting Urokinase and the Transferrin Receptor with Novel, Anti-Mitotic N-Alkylisatin Cytotoxin Conjugates Causes Selective Cancer Cell Death and Reduces Tumor Growth

Abstract

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

Recent Highlights in the Development of Isatin-Based Anticancer Agents

“…Both Tf-and PAI-2-N-AIE conjugates exhibited impressive tumour growth delay in a metastatic, orthotopic human breast tumour xenograft mouse model and were efficacious at 1/20th and 1/10th of the dose of the free drug respectively, with no observable signs of toxicity [171].…”

“…Vine et al was the first to publish this approach using isatin whereby two potent N-alkylisatin-based microtubule destabilisers were coupled to the delivery agents, plasminogen activator inhibitor type-2 (PAI-2) or transferrin (Tf) [170][171], to achieve site-specific drug delivery. Tf is an iron ferrying glycoprotein that has commonly been exploited to deliver potent chemotherapeutics to the intracellular space of cancer cells via the Tf receptor (TfR/CD71) [172], while PAI-2 is an irreversible, specific inhibitor of the proven metastatic marker urokinase plasminogen activator (uPA) [173].…”

“…In one report, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (80) was functionalised for attachment to Tf and PAI-2 via an esterase-labile succinate linker to form Tf-and PAI-2-N-alkylisatin conjugates (Tf-N-AIE and PAI-2-N-AIE, respectively) (83, Fig. (25)) [171]. Conjugation of the Nhydroxysuccinimide-derived active ester (82) to the targeting ligands Tf and PAI-2 was achieved by forming stable amide bonds between the ε-amino group of available surface lysine residues on the protein and the carboxylic acid group of the succinate linker on the functionalised N-alkylisatin (81).…”

(Z)-2-(2-Oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide

“…For various biological activities of Schiff bases, see: Bhandari et al (2008); Bhardwaj et al (2010); Pandeya et al (1999); Sridhar et al (2002); Suryavanshi & Pai (2006). For the cytotoxic and anticancer activities of isatin and its derivatives, see: Vine et al (2009). For graph-set analysis, see Bernstein et al (1995 Table 1 Hydrogen-bond geometry (Å , ).…”

(Z)-2-(5-Chloro-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbothioamide

“…For various biological activities of Schiff bases, see: Bhandari et al (2008); Bhardwaj et al (2010); Pandeya et al (1999); Sridhar et al (2002); Suryavanshi & Pai (2006). For cytotoxic and anticancer activities of isatin and its derivatives, see: Vine et al (2009). For graph-set analysis, see Bernstein et al (1995).…”