Heparan Sulfate Proteoglycan-Mediated Entry Pathway for Charged Tri-Platinum Compounds: Differential Cellular Accumulation Mechanisms for Platinum

Abstract

We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs), based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R9 (a nonarginine peptide, R9) coupled to the TAMRA fluorescent label 5-(and 6-)carboxytetramethylrhodamine) in Chinese Hamster Ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization, but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of “non-covalent” TriplatinNC, and to a lesser degree, the covalently-binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for rational design of platinum antitumor drugs with distinct biological profiles in comparison to the clinically-used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors.

A new approach to glycan targeting: enzyme inhibition by oligosaccharide metalloshielding

“…10 Given the measured affinity of TAMRA-R 9 to heparin is K d = 109 nM 9 , similar to typical receptor-ligand interactions, PPCs must have similar high affinity. 10 The interactions between the amine groups of the triplatinum compounds and the phosphate groups of the DNA backbone are very similar to those of the guanidine groups on arginine (Figure 1). Conceptualizing polynuclear platinum complexes as “polyarginine mimics’ has been very useful in drawing analogies between the DNA recognition modes of the arginine fork and the phosphate clamp, a third mode of ligand-DNA binding discrete from the classical intercalator and minor groove binders.…”

“…These results are consistent with the greater efficacy of TriplatinNC compared to AH44 to compete with TAMRA-R 9 for HSPG binding. 10 …”

“…The model further differentiates the biological profile of polynuclear and mononuclear platinums. 10,21,22 Glycans are aberrantly expressed in many tumors, regulating many important events in tumor progression including proliferation, invasion, angiogenesis and metastasis. 2,23 The model thus suggests the possibility of a systematic approach to tumor selectivity of platinum complexes through glycan targeting of those tumors with known glycan overexpression.…”

Dinuclear Platinum Complexes Containing Planar Aromatic Ligands to Enhance Stacking Interactions with Proteins

“…A 1:1 adduct of 12 :N-AcWLASW-OH is observed by ESI Mass Spectrometry. Compound 12 in standard MTT assays, [3], showed an IC 50 of approximately 1.5 μM in the highly cisplatin-sensitive A2780 ovarian cancer cell line while the corresponding value in OsACL, an osteosarcoma containing wtp53 and which overexpresses mdm2 protein, [24], is approximately 20 μM. The interpretation of these results with respect to the proposed target may be explored more fully with protein inhibition and expression assays [25].…”

“…Specifically from our own laboratory, non-covalent H-bonding interactions on polynuclear platinum compounds (PPCs) have described the phosphate clamp, a third mode of (ligand)-DNA interaction discrete from the classical minor groove binding and intercalation [1,2]. An unedited mode of cellular accumulation of PPCs identifying HeparanSulfateProteoGlycans (HSPGs) as receptors through initial non-covalent binding to oligosaccharides has significant implications for new target discovery for coordination compounds [3,4]. Stacking π-π interactions play a fundamental role in biology and especially DNA/RNA-protein selective recognition.…”