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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-R(9) (a nonarginine peptide, R(9)) 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 noncovalent 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 the rational design of platinum antitumor drugs with distinct biological profiles in comparison to those of the clinically used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors.

SUBMITTER: Silva H

PROVIDER: S-EPMC3367083 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds: differential cellular accumulation mechanisms for platinum.

Silva Heveline H Frézard Frédéric F Peterson Erica J EJ Kabolizadeh Peyman P Ryan John J JJ Farrell Nicholas P NP

Molecular pharmaceutics 20120502 6

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 c ...[more]

PMID: 22494465

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Project description:The dependence of viruses on the host cell to complete their replicative cycle renders cellular functions potential targets for novel antivirals. We screened a panel of broadly acting cellular ion channel inhibitors for activity against human cytomegalovirus (HCMV) and identified the voltage-gated chloride ion channel inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) as a potent inhibitor of HCMV replication. Time-of-addition studies demonstrated that DIDS inhibited entry via direct interaction with the virion that impeded binding to the plasma membrane. Synthesis and analysis of pharmacological variants of DIDS suggested that intrinsic cysteine, and not lysine, reactivity was important for activity against HCMV. Although sequencing of DIDS-resistant HCMV revealed enrichment of a mutation within UL100 (encoding glycoprotein M) and a specific truncation of glycoprotein RL13, these did not explain the DIDS resistance phenotype. Specifically, only the introduction of the RL13 mutant partially phenocopied the DIDS resistance phenotype. Serendipitously, the entry of DIDS-resistant HCMV also became independent of heparan sulfate proteoglycans (HSPGs), suggesting that evasion of DIDS lowered dependence on an initial interaction with HSPGs. Intriguingly, the DIDS-resistant virus demonstrated increased sensitivity to antibody neutralization, which mapped, in part, to the presence of the gM mutation. Taken together the data characterize the antiviral activity of a novel HCMV inhibitor that drives HCMV infection to occur independently of HSPGs and the generation of increased sensitivity to humoral immunity. The data also demonstrate that compounds with cysteine reactivity have the potential to act as antiviral compounds against HCMV via direct engagement of virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen of nonimmunocompetent individuals that remains in need of new therapeutic options. Here, we identify a potent antiviral compound (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS]), its mechanism of action, and the chemical properties required for its activity. In doing so, the data argue that cysteine-reactive compounds could have the capacity to be developed for anti-HCMV activity. Importantly, the data show that entry of DIDS-resistant virus became independent of heparan sulfate proteoglycans (HSPGs) but, concomitantly, became more sensitive to neutralizing antibody responses. This serendipitous observation suggests that retention of an interaction with HSPGs during the entry process in vivo may be evolutionarily advantageous through better evasion of humoral responses directed against HCMV virions.

Dataset Information

Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds: differential cellular accumulation mechanisms for platinum.

Publications

Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds: differential cellular accumulation mechanisms for platinum.

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