Mechanism-based rational design of cisplatin analogues

Abstract

The success of cisplatin as an anticancer drug is attributed to the ability of the platinum compound to damage DNA and subsequently induce apoptosis. Details of the cellular processing of cisplatin-damaged DNA can provide invaluable insight into the rational design of cisplatin analogues or combination therapies. Chapter I provides a survey of recent developments in the understanding of the mechanism of cisplatin action and summarizes relevant platinum-based anticancer compounds. Chapter 2 describes a series of estrogen-tethered platinum(IV) complexes (BEPn, n=l -5) that were synthesized, evaluated for their ability to upregulate HMGB1 and screened for cytotoxicity against human breast cancer cell lines. All BEPn complexes induced the overexpression of HMGB I in ER(+) MCF-7 cells. BEP3 was nearly twice as cytotoxic in ER(+) MC'F-7 cells than in ER(-) HCC-1937 cells. This result suggests the possibility of using compounds in this class specifically to target ER(+) malignancies, such as breast and ovarian cancers. In addition, the series of BEPn compounds provide an example of a useful strategy in the development of platinum-containing anticancer agents, namely, using mechanistic insights to aid in the rational design of new complexes.

(cont.) The strategy of exploiting estrogen-induced HMGBI upregulation to sensitize ER(+) cells to platinum was further pursued in work described in chapters 3 and 4. Chapter 3 reports the synthesis and characterization of a series of platinum(IV)-estrogen conjugates derived from carboplatin. Although these BECPn complexes were moderately cytotoxic in ER(+) MCF-7 human breast cancer cells, no differential cytotoxicity was observed as compared to ER(-) HCC- 1937 cells. However, these compounds represent the first example of a biomolecule-tethered platinum(IV) complex that reduces to yield carboplatin in cells. The platinum estrogen conjugate described in chapter 4 was designed not only to induce upregulation of HMGB I but also to enter ER(+) cells selectively. Unlike the BEP and BECP compounds, BEEP was designed to maintain affinity for the estrogen receptor and by tethering platinum to estradiol through the 17c-position of the steroid ring. Compounds with affinity for the estrogen receptor, which is overexpressed in breast and ovarian cancers, are selectively taken up into ER(+) cells. Unexpectedly, BEEP had very low affinity for the estrogen receptor and was therefore equally cytotoxic in ER(+) and ER(-) human breast cancer cell lines.

(cont.) A common feature of many cancers is overexpression of the folate receptor, which is responsible for the uptake of folic acid. Therefore targeting the folic receptor is an attractive method for achieving selective uptake in cancer cells. Chapter 5 describes the synthesis and biological activity of a folic acid-tethered platinum(lV) compound, which demonstrates the validity of this premise. The nuclear protein HMGBI has recently been discovered to function as an extracellular signaling agent. Because of oxygen deprivation, the core of a solid tumor dies by necrosis and passively releases HMGB I into the extracellular environment. This characteristic of solid tumors leads to the hypothesis that extracellular HMGB I is taken up by surrounding viable tumor tissue and mediates cisplatin sensitivity. The final chapter investigates the capability of exogenously administered HMGB to modulate the cytotoxicity of cisplatin and trans-DDP in human cancer cells. The Appendix sections provide detailed experimental protocols for several useful laboratory methods. In Appendix A, a procedure for isolation of nuclei from cisplatin-treated cells is presented.

(cont.) The nuclei were subsequently used by our collaborators to examine the post- translational modifications of histones induced by cisplatin exposure. A protocol for isolation of protein extracts from formalin-fixed paraffin-embedded tissue is described in Appendix B. In addition, the extracts were probed by western blot analysis to examine the expression levels of HMGB1 in clinical testicular seminoma samples. Appendix C provides a solid-phase synthetic methodology for the preparation of peptide-conjugated platinum(IV) compounds.