Introduction to Platinum Chemistry 1.1. Overview
1.2. The Anticancer Activity of Platinum Complexes
1.2.1. The Mechanism of Action of Cisplatin
1.2.1.1. Reactivity of Cisplatin with DNA
Cisplatin is administered into human body intravenously as a sterile saline solution at a dose
(phase one) of about 50-120 mg/m2 (m2 = body surface area) per course over 0.5 to 2 hours.[4, 23] The drug is often used in combination with other adjuvants to minimise the side
effects. Once the drug enters into the blood stream, it is subjected to other compounds in the blood such as sugars, proteins, salts and water. High chloride concentration (~100 mM) in the bloodstream suppresses the hydrolysis of the molecule thus most of it remains unchanged.[12, 23] The neutral compound enters the cell by passive diffusion or active uptake.
Inside the cell, due to the low chloride concentration (ca. 20 mM), the neutral cisplatin molecule undergoes hydrolysis producing a charged species (Scheme 1.2).[5, 9, 12] Since water is a better leaving group than chloride, the resulting charged species are more reactive towards biomolecules.[57] This hydrolysis of cisplatin lowers the pKa of the complex. When comparing the pKa values at 25 oC, the monoaqua complex has a pKa of 6.49 and the pKa1 and pKa2 of the diaquatic complex are 5.39 and 7.1 respectively.[4] Research has shown that the rate of reaction of cisplatin with DNA depends on the hydrolysis of the drug.[58] Any side reactions of cis-Pt(NH3)22+ other than to DNA leads potential deactivation or resistance of the drug and increased toxicity[53] (Scheme 1.2).
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Scheme 1. 2 Schematic diagram representing hydrolysis of cisplatin and attack of cisplatin to DNA.
The k values and t1/2 values were obtained from a 195Pt NMR platination kinetic study of platination of cisplatin to chicken erythrocyte DNA at 37 oC and pH 6.5.[12]
The hydrated species then bind to the DNA in multiple steps (Figure 1.4). The steps involved are:[59]
a. Aquation: The binding of cisplatin to DNA starts with the formation of the mono aquated species. Experimental evidence shows that this aquation step follows a first- order kinetics and this was assumed to be the rate determining step.[56, 60]. However, there is a degree of uncertainty about this since the aquation involves many species.[61]
b. Preassociation: This involves an irreversible binding of platinum to DNA and is therefore kinetically controlled.[62]
c. Monofunctional adduct formation: Irreversible monofunctional Pt-DNA adducts are formed in this step. The cross-links are formed mainly between 1,2 adjacent guanine-guanine (60-65%)[58] while the cross-link between adjacent adenine-guanine was found to be 20-25%[63] and this binding unwinds DNA by 13o. Minor bindings involve 1,3-intrastrand bindings which unwind DNA by 23o.
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d. Second aquation: Hydrolysis of the second chlorine. This diaqua species has double reactivity towards DNA in comparison to the monoaqua species.
e. Ring closure and formation of bifuntional adduct: Binding of the second DNA strand to the platinum complex. There is some degree of uncertainty about the formation of the bifuntional adduct being directly from the monochloro complex or whether it is via aquation.
f. DNA distortion and recognition of DNA distortion by a variety of proteins
Figure 1. 4 The sequence of cisplatin binding to DNA and the structurally different adducts formed.[59] The k values and t1/2 values were obtained from a kinetic study of hydrolysis of cisplatin by short lengths of single- or double-strand DNA containing adjacent guanosines using 15N NMR at pH 7.1 in water containing 10 mol dm-3 sodium phosphate.[12]
Although inside the cell, cisplatin can react with many cellular components such as proteins, RNA and sulphur donor groups (Figure 1.3), it was proven that the binding involves the coordination of cisplatin with the DNA bases viz. cytosine (C), adenine(A), thymine(T) and guanine(G), showing a preference for the N7-position of guanine.[4, 9, 12, 19, 53, 63-70] This is because the N7 position of the imidazole ring in guanine is more accessible for platinum binding as it is located in the major groove of DNA. Also the binding of N7 with cisplatin is enhanced due to the high nucleophilicity of the N7 centre. The basicity of guanine N7 allows to form hydrogen bonding with the ligands of platinum complex.[65]
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As can be seen from Figure 1.4, the binding of the complexes may form mono- or di- Pt-DNA adducts. Often the monofunctional adducts react to form either interstrand or intrastrand adducts which can then stop DNA multiplication resulting in antitumor activity.[71] However, the di-Pt-DNA adducts are assumed to be the major contributor to the cisplatin anticancer activity.[65] The possible modes of cisplatin binding to DNA are shown in Figure 1.5.
Figure 1. 5 Some possible cisplatin-DNA binding modes.[12]
In DNA, the main target of cisplatin is the telomeric regions of chromosomes.[23] The telomeric region lies at the ends of eukaryotic chromosomes and consists of 5´-TTAGGG-3´
tandem repeat units.[72] These telomeric units are rich in guanosines and their main function is to protect the ends of chromosomes from degradation and to transfer the genetic information during cell division.[23, 73] On average, during one cell division, the telomeres get shortened by 50-200 bp (where bp = base pairs).[74] Cell death occurs when they get critically shortened. In over 90% of the tumor cell lines, the telomeric length is maintained by telomerase.[75] Cisplatin degrades the telomeric regions in the chromosomes and suppresses cell division. With low doses of cisplatin, telomere degradation was reported to be 61%
effective.[23] Consequences of telomeres degradation are significant damages in the DNA and DNA replication by apoptosis, often known as “programmed cell death”.[23].
The binding of cisplatin to DNA alters the DNA conformation and brings distortion to the structure. This results in DNA unwinding, bending and flattening of the minor grooves in the DNA helix.[4, 67] Research has shown that cisplatin binding at adjacent guanine-guanine sequence causes the bending of DNA by 32-40o.[76, 77] These changes result in the inhibition
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of DNA transcription which is an important step for protein synthesis and cell division.
Synthesis of anticancer drugs which can inhibit the telomerase activity is therefore important.