In vivo Reduction

It is known that a variety o f mild reducing agents can cause the cleavage o f anthracyclines via a radical pathway (fig 14). In vivo, molecular oxygen can promote a similar pathway which produces radicals within the cell. This can pqtentially cause DNA scission as in the enediyne class of antibiotics."^^

OH OH

e" from

COCH3 m ild reducing

agent OH OH OH OR OH OR sem iquinone H + OH OH OH OH COCH: OH OH Or hydroquinone quinone methide OH RSH OH 7-deoxyaklavinone OH OH OH COCH OH SH OCH OH

[O] M olecular O xygen OH

OH SH

Chapter 1 43

The pathway goes via a semiquinone and a hydroquinone st^te at which time it is thought that the aglycone cleaves leaving a quinone methide. This quinone methide is susceptible to attack from variety o f nucleophiles whiclt exist in the cell including -SH, -NH2 and -OH groups. This quinone methide can, therefore, feasibly

be a reactive intermediate which attaches itself to the DNA and then the resultant adduct acts as an inhibitor o f cellular processes. Indeed, some worl^ers propose that metabolic activation is a prerequisite for cross linking o f anthracyclines to DNA by means o f a radical pathway although no structure was given for thç adduct, just the results o f dénaturation studies.

Initial studies using a daunomycin 9 and an oxidising agent as a treatment for leukaemia in vitro have proved to be successful, and this suggests that the mechanism as a proposed mode o f action is a valid one. With, a derivative of nogalamycin 13 called menogaril (nogalamycin aglycone withouf sugars at C l - replaced by OMe) it has been shown that, with in vivo reduction it is possible to get addition to N2 of guanine.^ ^ However, it must be noted, that for in vivo models to succeed, a method o f delivery which protects the agents from molecular oxygen must be devised. This will most probably take the form o f liposomes which are hydrophobic and may also aid delivery into the cell.

Whatever the mode o f action, there is a pronounced effect when these compounds are used in the fight against cancer. The drug aclacinopiycin 14, one of the less well studied anthracyclines, has been marketed for nearly twenty years as aclacin/aclarubicin. It has efficacy as a single agent, or combined with other drugs and can be used in a wide variety of illnesses including acute lymphocytic leukaemia and solid gastric, lung, breast and ovarian tumours. It is clear that there are very important effects in vivo associated with the anthracyclines, anc^ the majority o f evidence points to an effect as a result o f binding to dsDNA.

2.3 The binding of anthracyclines to DNA 2.3.1 Kinetics and thermodynamics

It has been shown that the nature of the anthraquinone and glycosidic components have a pronounced effect on the binding o f a particular anthracycline to DNA.^^’^^ This not only manifests itself in the binding constant but also in the

_____________________________________________________________________________ Chapter 1 44

binding kinetics as well. DuVemay noted an apparent two fold, increase in the binding constant per sugar residue added, with ATgpp being in the ran^e 1 - 6 x 10^ M*

1 52

This increase in binding constant with the number o f sugar residues has been mooted as grounds for increased sequence selectivity. However, sequence selectivity cannot be just a factor o f the final DNA-drug complex but rather q combination of kinetic and thermodynamic factors for both the drug in its bound conformation and the local DNA structure. For a drug to show sequence selectivity if must show fast association kinetics and slow dissociation kinetics as a result of favourable interactions in the binding site.

For intercalation to occur, in the case o f nogalamyciq 13, molecular modelling has shown that DNA breathing has to occur to allow the bqse pairs to open 10Â for the aglycone to thread through the DNA helix.^"^ This process is necessarily sequence dependent. However the binding o f the sugars in the mino^ groove o f DNA will be dependent on the local groove geometry, and may play a moi^e important role than the intercalation in the overall thermodynamics of the binding process.

CH 4 ' OH OCH OH" CH: CH5 OH OH CH: Nogalamycin 13

Kinetic studies have been carried out by several worker^ on the binding process by both stopped-flow and temperature jump-relaxation methods on the binding o f daunomycin 9 to DNA.^^’^^

______________________________________________________ Chapter 1 45

These separate pieces of work seem to have a consensus opinion in that the binding to DNA is a multistep process although the precise number o f steps remains unclear. Chaires postulates that that a three step process occurs ip the binding of daunomycin 9 to DNA (fig 15).^^

D + S

Cl

C

D = DNA

S = Substrate

C = Complex

fig 15 Mechanism for the kinetics o f binding (1)

Kinetics and thermodynamics point to the first step being thp formation of a loosely bound outside complex. This is seen to be endothermie and driven by the entropically favourable condensation of water from the phosphate backbone (AH = +2 . 1 kcal mol'% E ^ i = +1 2 . 8 kcal mof^) which is consistent with qther data for this

type of p r o c e s s . T h i s step as expected is found to be bimoleoular with a rate constant o f 2 . 8 x 1 0^ M '^s'\

The equilibrium constant for the second step (8.3) is equivalent to that found in the intercalation of proflavine l / ^ This most probably occurs^ via the thermal breathing o f the DNA having a large enough amplitude to accommodate the aglycone between the base pairs.

The third step is then postulated to be some form o f organisation within the complex with the rate seen to be similar to those found from studying the mode of binding of actinomycin.^^

This proposed mechanism fits in well with the kinetics foupd for propidium and ethidium 2 .^ Krishnamoorthy found kinetics in this serieg to satisfy the following equilibria where C \ relates to an outside condensed s^ate and C ] the intercalated complex (fig 16).

Chapter 1 46

D + S

Cl

C

fig 16 Mechanism for the kinetics o f binding (2)

For the proposed mechanism of Chaires/^ the formation o f C l and C2 should

be dependent on the ionic strength of the solvent whilst the organisation o f the complex should be relatively indifferent to ionic strength. However this was not found to be the case, suggesting a different method of binding showp below (fig 17).

D + S

C

fig 17 Mechanism for the kinetics o f binding (3)

In this proposed mechanism both C2 and Cg are intercalate^ forms from the

outside complex C i and some form o f interplay can exist between the two. This could correspond to binding at two different sites and imply sequence specificity if the thermodynamics o f binding in mode C2 outweigh those for C3 ^nd both of these

are not just simply a different conformation of a complex bound to the same binding site.

The exact mechanism for the general binding of anthracyclines to DNA is still somewhat unclear. Nevertheless, since an overall negative enthalpy o f binding for daunomycin 9 to DNA was observed there must be a total negative enthalpy for the formation o f states C2 and C3 .

It seems reasonable to assume that if one step is an intercalation step and the other an organisation step (fig 17), then the sugars can, and will, play a role in this. Thus, if there is a situation whereby the intercalation process has gimilar energetics and kinetics at two sites, it is feasible that the sugars play a role in determining which one is the most favourable from their subsequent organisation in the groove and expulsion of any water present on the groove floor.

C h a p t e r 1 4 7

2.3.2 Structural aspects o f binding