Structure-activity screening of platinum intercalators and molecular mechanisms for the cytotoxicity of 56MESS - [Pt(5,6-Dimethyl-Phen)(1S,2S-DACH)]2+

Abstract

Efficacy of existing clinical anticancer drugs including cisplatin and its analogues is greatly limited by drug resistance and side effects. Anticancer compounds with novel structures and different mechanisms of action have been sought to expand anticancer activity and to minimise these limitations. First part of this project evaluated the structure-activity relationships for a class of novel platinum metallointercalators using cytotoxicity screening against cancer cell lines, and subsequently identified lead compounds. Second part elucidated the biological mechanisms underlying the cytotoxic action of a lead compound with a genome-wide gene expression profiling technology (or transcriptomics) using a eukaryotic model organism yeast Saccharomyces cerevisiae. These analyses identified the disruption of iron and copper metabolism as the major molecular mechanism of cytotoxic action of the lead compound. The novel platinum(II) metallointercalators studied are of the type of [Pt(IL)(AL)]2+, where IL is the intercalating ligand, such as 1,10-phenanthroline (phen), and AL is the ancillary ligand, either chiral 1,2-diaminocyclohexane (S,S-DACH, R,R-DACH), or achiral 1,2-diaminoethylene (EN). Nineteen (19) platinum metallointercalators, eleven (11) cucurbit[n]ural encapsulated forms of these complexes and three (3) non-platinum metal helicates were screened against cisplatin-sensitive (L1210) and resistant (L1210cisR) murine leukaemia cell lines using a standard methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. These metal complexes are water-soluble. They exhibited a wide range of cytotoxicities. The complex - [Pt(5,6-dimethyl-1,10-phen)(1S,2S-DACH)]2+ (56MESS) was found to be 163-fold more active than cisplatin against the L1210cisR cell line whereas complexes such as [Pt(4-methyl-phen)(EN)]2+ (4MEEN) are inactive. Correlation analysis of the cytotoxicity data with structures revealed three major structure-activity relationships. Chirality of the DACH ligand, the position and electronegativity of functional groups substituted in the intercalating ligand and type of ancillary ligands (i.e DACH or EN) profoundly influenced the cytotoxicity of these complexes. Encapsulation of metal complexes by different sized curcurbit[n]urals led to varied cytotoxicities. Analysis of these data resulted in the identification of three lead complexes including 56MESS, which has excellent activity in L1210 as well as in L1210cisR cell lines. To understand the biological mechanisms underlying the cytotoxicity of the lead complex 56MESS, genes and pathways in response to 56MESS treatment, in parallel to cisplatin treatment, were investigated using transcriptomics in yeast Saccharomyces cerevisiae. Ninety-three (93) genes were revealed to be important for the cytotoxicity of 56MESS as these genes altered their expressions in response to 56MESS challenge. Bioinformatics analysis of these genes showed that defective iron and copper metabolisms are implicated for the cytotoxic action of this compound. The suppression of biosynthesis of sulphur-containing amino acids and arginine and function of mitochondria could also play a role in the cytotoxicity of 56MESS. In comparison, one hundred and sixty-five (165) genes altered their expressions in response to cisplatin treatment. The genes involved in sulphur uptake and its assimilation and response to DNA damage were up-regulated while the genes involved in de novo purine biosynthesis and one carbon metabolism were suppressed by cisplatin. The differences in the major pathways in response to 56MESS and cisplatin suggest that these two platinum agents have different molecular mechanisms for their cytotoxicity. Cellular assays and gene deletion mutant screenings validated these transcriptomic findings. The results from inductively coupled plasma optical emission spectrometry (ICP-OES) revealed that 56MESS treatment reduced concentrations of the intracellular iron and copper, and increased that of manganese. Analysis of viability phenotypes of deletion mutants of the key genes including FTR1 in the iron and copper metabolisms further supported their involvement in the cytotoxicity of 56MESS. The data therefore suggested that iron and copper transporters were the molecular targets of 56MESS. Further biochemical and mutant analyses showed that glutathione, oxidative stress and mitochondria were important in 56MESS' cytotoxicity. 56MESS treatment arrested cells in G1 phase of the cell cycle. The cytotoxicity data, the structure-activity relationships and the elucidated molecular mechanisms of the most active platinum metallointercalator 56MESS suggested that the platinum metallointercalators of the type [Pt(IL)(AL)]2+ have potential as anticancer agents. The findings reported in this thesis can be used for further development of these complexes.

Date of Award	2011
Original language	English