Platinum on the road: the activation and transport of novel platinum anticancer drugs by the extracellulardomain of human copper transporter I (HCTR1)

Platinum on the road: the activation and transport of novel platinum anticancer drugs by the extracellulardomain of human copper transporter I (HCTR1)

王星昊(Xinghao Wang)

香港大學/Doctor of Philosophy/Chemistry/博士(2012年)

並列摘要

Platinum-based anticancer drugs such as cisplatin, carboplatin and nedaplatin have

been widely used in the chemotherapy of a variety of solid tumours for several

decades. However, the development of both inherent and acquired resistance has

greatly limited the efficacy of all of these drugs. Several mechanisms were

proposed to explain the cellular resistance to these platinum drugs, including

decreased drug accumulation. Previously, it was suggested that cisplatin enters

cells via passive diffusion, followed by intracellular hydrolysis and activation

prior to targeting DNA. However, recent in vivo and in vitro studies confirmed

that transporters and carriers involved in copper homeostasis play important roles

on the transport as well as cellular resistance to the platinum drugs. CTR1, a major

plasma-membrane transporter involved in intracellular copper(I) homeostasis, was

found to facilitate the uptake of several platinum drugs although the molecular

mechanism remains unclear. The extracellular N-terminal domain of human CTR1

(hCTR1) with two methionine(Met)-rich and two histidine(His)-rich motifs has

been proved to be essential for the uptake of both copper and platinum drugs by

the transporter.

In this thesis, the extracellular domain of hCTR1 (hCTR1_N, residues 1-55) was

overexpressed and the role of the Met- and His-rich motifs on cisplatin binding

was examined by either mutagenesis or chemical modification. Cisplatin was

found to directly and rapidly bind to the Met residues of hCTR1_N by the

formation of monofunctional cisplatin-thioether adducts. The kinetics of the

binding process was found to correlate with the number of Met residues,

indicating that all Met residues are exposed to solvents and capable for cisplatin

binding. Such a non-sequence-specific binding may increase the likelihood of

capturing the anticancer drug in extracellular fluid by the N-terminus of hCTR1.

The effect of hCTR_N on the binding and activation of second-generation

platinum anticancer drugs, e.g. carboplatin and nedaplatin, were subsequently

investigated. hCTR1_N was found to significantly facilitate the activation of these

platinum drugs by the formation of ring-opened monofunctional Pt-thioether

species through Met residues. Although the activities of platinum drugs against

hCTR1_N are significantly different, their monofunctional protein-bound species

demonstrated great similarity in both structure and kinetic aspects, suggesting the

uptake of these platinum drugs by hCTR1 might follow the same mechanism. The

formation of active ring-opened species of carboplatin and nedaplatin by

chloride/bicarbonate was observed, indicating these nucleophiles may play a

critical role in the pre-activation of the drugs prior to their reaching cellular targets.

Pt-thioether species were proposed as intermediates for the platination of other

biomolecules. The monofunctional cisplatin adduct of hCTR1_N was proved to

further transfer its active platinum species to either cysteine- or guaninecontaining

biomolecules which mimic the C-ternimus of hCTR1 and DNA.

Methionine residues of hCTR1 may therefore serve as key residues for the

activation and transport of platinum anticancer drugs in the form of

monofunctional Pt-thioether species through the pole of trimeric hCTR1 and

eventually to their final target – DNA.

並列關鍵字

Platinum compounds. ； Antineoplastic agents. ； Copper - Physiological transport.