The 1,2-intrastrand cross-links
It is thought that the 1,2-intrastrand cross-links are important to anticancer activity because they are the major adducts formed, and because clinically inactive compounds, such as the trans-dichlorodiammineplatinum(II) (transplatin), fail to form these cross-links.
The mechanism of the reaction of cisplatin with DNA is shown in Figure 21.
Kinetics is very important in this sequence of steps. The aquation of cisplatin is the slow, rate-limiting step, and the reaction of the cationic platinum complex with the DNA strand is fast.
Hydrogen-bonding from NH3 and OH2 ligands to the phosphate backbone of DNA is possibly important in orientating the platinum complex.
The structural studies of cisplatin binding to oligonucleotides (see the previous section) show that different adducts distort the DNA in different ways, as discussed in the next video.
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Which technique was used to determine the nature of the binding in DNA, and what practical issues did it present?
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X‑ray crystallography – you may recall this is a technique for determining the internal structure of solids. A specific arrangement of atoms will produce a unique diffraction pattern, which acts as a ‘fingerprint’ for a particular compound.
The necessity to produce single crystals of the cisplatin–DNA adduct proved a challenge, solved by the practical ingenuity of one of Professor Lippard’s students.
The main observed effects of 1,2-intrastrand cross-links are:
- a bend towards the major groove of about 35–40°, shown in Structure 12
Structure 12 - unwinding of the duplex by about 20°
- widening of the shallow minor groove
- distortion of the Watson–Crick base pairing.
This all leads to destabilisation of the duplex, which in turn blocks replication and inhibits transcription. Replication stops at sites corresponding to one nucleotide preceding the first Pt–G residue and at positions opposite the two Pt–G residues.
You will now look at some experimental evidence for this.
A method for separating macromolecules and their fragments is gel electrophoresis. This is based on the principle that molecules having different sizes or charges will move through a gel under the influence of an electric field to different extents – small molecules move more easily than large ones. In fact, you will see the technique being used in the laboratory in the next section.
Figure 22 shows gel electrophoresis data obtained during a kinetic study of the effect of a cis-GG adduct on DNA polymerisation by HIV-1 reverse transcriptase.
In Figure 22, panel A shows the fragments generated by enzymatic replication of a DNA duplex containing a site-specific cross-link at G(24)/G(25). Polymerisation is blocked by platination of the substrate. Panel B depicts results for an unmodified DNA probe.
OpenLearn - Metals in medicine
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