Sequence selectivity and its determination

Sequence selectivity can be defined as the ability o f a species to bind to a particular sequence of base pairs within a given piece o f DNA above any other sequences present. Traditional methods for the determination of sequence selectivity in the binding o f both drugs (small molecules) and proteins are reviewed in this section.

This process can be viewed on a purely statistical basis. As already described in section 1.3.2 on grounds o f pure intercalation there are 10 possible sites, all differing in their electrostatic potential and hydrogen bonding capabilities, to which a molecule could bind. When this is extended to cover the flanking sequences for the intercalation site this number rises rapidly.

If we take a compound which covers the intercalation site, and has contacts in the minor groove covering one more base pair we have 40 possibilities. For the X- ray diffraction and nmr data covered in the previous section we are considering compounds which cover between four and five base pairs which gives 160 or 640 possibilities respectively. For proteins such as the bZIP family described earlier we can consider a binding sites somewhere in the region o f ten base pair stretches of DNA. This leads to a possible million plus binding sites.

In addition, the ability o f a compound to bind to DNA is in çssence governed by geometric constraints which lead to a maximisation o f the interactions in the DNA-drug complex. However the overall three dimensional struçture of DNA is sequence dependent and therefore as well as the base pairs covered, the flanking sequences must also be accounted for.

With the number o f possible binding sites being so high, it becomes impractical to construct a library o f all the possible sequences an(^ then to test the binding characteristics on them all individually. Alternative methods for determining sequence selectivity need to be used which survey large pools of sequences simultaneously, to locate potential binding sites quickly and efficieqtly. Over the last few decades several possible techniques have been described and a few o f them have been applied to the anthracycline family o f antibiotics. Most o f these methods are summarised in a recent review article.^'

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3.1 Nmr and X-ray crvstallographv

As described previously, nmr and X-ray diffraction data give a valuable insight into any interactions which may exist in a preferred binding site. Nmr, since it is a dynamic technique, is able to investigate which, if any, sequences are adopted in solution by the drug. Nmr is also very useful in that it can simultaneously give kinetic information about the binding process, but it requires large amounts o f pure DNA (95-100 mg) and concentrations o f the duplex in the order o f 1 mM. Similarly X-ray crystallography can generate information which is vital for our understanding of the binding.

Both techniques are limited in as far that they do not give a definite sequence selectivity as all possible sequences cannot be surveyed. Both teçhniques rely on small pieces o f DNA, with only a few o f the ten intercalation sites çovered, let alone any flanking sequences. For the determination o f sequence selectivity, both can only give hints as to any preference, and then only for the smallest o f binding species such as anthracycline antibiotics.

Crystallography is further limited in that it depends on a crystal being grown. It is often found that a governing factor in crystal growth is symmetry and in organic chemistry it is often possible to separate enantiomers by crystallisation. It is therefore possible that it is not the preferred binding mode which is being studied but that which has the most favourable packing forces in the solid state. The results are very valid, however, in determining which interactions are important for binding for that particular sequence.

Both methods rely on small oligonucleotides, and it is oftep found that there is end fraying in such systems. As a result, potential binding sites should be placed at the centre o f the oligonucleotide being studied so that a realistic picture o f the binding is observed.

It must be noted that for those systems which have been studied by both nmr and X-ray crystallography, such as nogalamycin 13, there is very little difference between the structures obtained from both methods. However, it is the case that the structures based on refinement o f nOe distances use a template in the first instance to give an approximation. Since the crystal structure is often used as this template, it is perhaps not surprising that there is little difference found between the two methods.

Chapter 1 59

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3.2 Chemical attachm ent

The aim of this method is to covalently attach the DNA binding molecule under study to the DNA helix, and then to analyse the resulting addi^ct. This requires either chemical modification o f the binding drug (in a way which vyill not affect the binding itself), or the use of a second chemical reaction to link the DNA and the drug together.

Again, only small pieces o f DNA can be surveyed, siqce the resultant covalently attached adduct must be studied by chemical techniques such as nmr and mass spectrometry.

For a reaction to occur between the drug and the DNA, reaçtive components must be in close proximity. In essence this technique will be able to pick out hydrogen bond type interactions between nucleophilic species separated by distances o f the order of a few angstroms. However, there is no possibility o f determining other types o f electrostatic or van der Waals interactions between the binding molecule and the DNA as they occur between species that are non-rqactive.

This method has been used to study the DNA-daunomycin 9 interaction. When formaldehyde was added to a daunomycin 9-DNA crystallisation medium, a crosslink was formed with a methylene bridge joining the drug to the DNA which was 6 bp in length (dCGCGCG and dCGTDCG wherç D represents 2- aminoadenine).*^’*^ X-ray crystallography revealed that the link was^ between the N2 of guanine and the N3’ on the sugar o f daunomycin 9 forming an ^minal (fig 21). It is probable that this occurs via initial attack from the drug followed by elimination to the imine. The imine in proximity to the guanine base then reacts with the N2 nitrogen. This is consistent with the work on daunomycin analogues which found that the C3’ position modifications were the most biologically active"*^ and that the aglycone itself has no biological activity.

Chapter 1 60 OH NH- OH- O H OH CH. OCH

fig 21 Attachment product

The X-ray diffraction crystal structure o f the covalent complex also revealed that the formaldehyde cross-link had not altered the mode o f binding. It was found that only a 5’-GCG sequence has the structural prerequisites for bintjling, and that the structure was consistent with that obtained for the non-covalently linked daunomycin-DNA complex. The methylene bridge was found to fit yery well into the crystal structure with no defects observed.

This work has more recently been reexamined.*^ It was foupd that there was an absolute requirement for N2 o f guanine and N3’ o f daunosamine for the cross linking to occur For other derivatives without the N3’ amine group, such as hydroxyrubicin which has an OH group in the C3’ position, np reactions were observed. The requirement for the proximity o f reactive species was proved by substituting daunosamine with a sugar bearing a C4’ amine group. This can easily form the initial imine intermediate but no cross link was observed. These results indicate that the reaction is regiospecific.

This cross linking work was further validated by a potential in vivo

crosslinking reaction. It was found that, for adriamycin 8, transcriptional blockages could be observed due to alkylation o f the DNA.*^ By studying 5’-GCGCGCGC and adriamycin and daunomycin, in the presence of, not only formalcjehyde, but DTT and hydrogen peroxide, using HPLC, uv-visible spectroscopy and n^ass spectrometry it was discovered that all three gave the same adduct as in fig 2\. For the system which contains no formaldehyde, a formaldehyde equivalent mpst be produced within the system. With adriamycin this can be as a result o f peroxide oxidation.

Chapter I 61

leading to a Baeyer-Villiger reaction on the hydroxyketo side chain which generates formaldehyde (fig 22). This cannot be the case for daunomycin 9.

OH