Top Poisons 19311 Topi poisons

Relatively few topi poisons have been thus far described. A clinically useful drug known for a long time is camptothecin (CPT), discovered as an antitumor alkaloid from the Chinese tree Camptotheca acuminata approximately 30 years ago. CPT was later shown to stabilize the covalent topi-DNA cleavage complex by preventing DNA religation. In fact, CPT both inhibits DNA relaxation and induces cleavage complexes (protein-linked DNA single-strand breaks) [38]. The drug-stabilized cleavable complex formation is a reversible molecular event. In addition, the single-strand breaks resulting from topl-CPT interaction should in principle be easily repaired. Hence, it is difficult to explain the outstanding antiproliferative activity exhibited by topi poisons. In fact, to account for cell death stimulation, it is believed that collisions between ternary (drug-top-DNA) complexes and advancing replication forks generate double-strand breaks. These lesions are much more toxic than single-stranded breaks and. by recruiting other factors like p53, are able to trigger the cell death program [39].

Topi is the primary target of CPT as shown by CPT-resistance in yeast cells, in which the topi gene is deleted, and sensitivity is restored upon expression of wild-type topi. Also, eukaryotic cells selected for high levels of CPT resistance show mutations that render the enzyme insensitive to the drug. Due to its poor pharmacokinetic properties, analogs need to be developed, and some of them are now in clinical trials. Topotecan, irinotecan, 9-aminocamptothecin, and 7-N-methylpiperazinomethyl-10,ll-ethylenedioxy-20-S-camptothecin are important examples (Fig. 19.6).

Since CPT is the only drug for which a detailed molecular model for the ternary cleavage complex is available, we will consider this drug (and its family) in more detail.


rj= oh, oco-tfytfy hCPT

rj= oh, oco-tfytfy camptothecins

bls(9-methylphenazine-1-carboxamldes) Fig. 19.6. Chemical structure of selected topi poisons.

CPT is a planar molecule with a bent shape. In spite of its planarity, it does not appreciably intercalate into duplex DNA. Interaction with guanine (possibly from the DNA minor groove) can, however, be detected using UV activation, and a recent study suggests a direct interaction between CPT carboxylate form and DNA [40]. Another study in solution using flow linear dichroism, circular dichroism, and Raman spectroscopy suggests that the lactone (rather than the carboxylate) form of CPT derivative topotecan interacts with DNA within the DNA minor groove and confirms the preferential binding to GC-rich DNA [41].

Testing of a number of camptothecin derivatives with purified topi has suggested the existence of a stereospecific drug-binding site in the topi cleavage complex. Salient points of the CPT structure-activity are as follows:

1. Substitutions at positions 7 and 9 do not generally affect topi inhibition, suggesting the absence of tight interaction between the receptor site and part of the A and B rings of CPT around positions 7 and 9. For instance, 7-ethyl substitution in SN-38 contributes to the remarkable potency of this agent.

2. Hydroxyl substitutions at position 10 increase topi targeting.

3. Bulky substitutions at position 11 and even small substituents at position 12 inactivate CPT derivatives, suggesting a tight interaction between the topi cleavage complex and the concave region of the drug. By contrast, addition of a methylenedioxy (or ethylenedioxy) ring increases topi inhibition.

4. Natural CPT is in the 20-S configuration with the hydroxyl group pointing upward. The fact that 20-R-camptothecin is inactive suggests that the 20 hydroxyl residue must interact closely, possibly by hydrogen bonding with the topi cleavage complex. Other substitutions in the E-ring generally abolish CPT activity. The 21-lactam derivative is inactive in spite of similar geometry. This difference is probably related to the resistance of the lactam derivative to ring opening.

Based on drug crosslinking experiments and on the observations described above, CPT likely interacts both with the enzyme and the DNA in the ternary complex. Fan et al. [42] have recently used molecular modeling and computer simulation to dock camptothecin into a hypothetical cleavage complex. In this model, camptothecin is stacked inside the topl-mediated DNA cleavage site with its concave region facing the DNA major groove. A second model has been proposed by Redinbo et al [43] on the basis of the crystal structure of the topl-DNA cleavage complex. In this model CPT is stacked against the +1 purine, the +1 base is flipped out of the DNA duplex and the CPT molecule is structurally altered.

A third proposal, obtained by docking CPT into the topl-DNA cleavage complex obtained from X-ray data upon the crystal structure of the topl-DNA complex, is consistent with intercalation of the drug at the cleavage site, with the A-ring directed toward the major groove and the E-ring pointing into the minor groove. Here, the drug's orientation differs remarkably with the previous intercalation model, while it is similar in the guanine-flipping model [44].

More recently, solution of the X-ray structure of a ternary complex with top-

otecan has been announced [45]. This will perhaps give the final answer to the issue. Of course, the relevance of the solid-state structure of the cleavage complex to the real situation occurring in cell systems has to be assessed in the future.

Besides structural approaches to drug design, another powerful technique is a predictive QSAR analysis using the most advanced statistical methods and the availability of large database information (for example the NCI Drug Database). Principal methods include stepwise linear regression, principal component regression, partial least-squares regression, and fully cross-validated genetic function approximation. Hierarchical clustering, considering over 150 compounds, was successfully used for the CPT family [46].

Novel CPT congeners have been recently prepared. BN 80915, a difluoro-derivative where the six-membered alpha-hydroxylactone ring of CPT is replaced by a seven-membered beta-hydroxylactone ring to give the homocamptothecin (hCPT Fig. 19.6) family, is a lead compound that recently entered clinical trials [47, 48]. In spite of the modification to the crucial E-ring, it retains effective topl-poisoning activity, and is also very potent as evidenced by IC50 values consistently lower than those of the corresponding CPT analogs in sensitive cell lines as well as in related multidrug-resistant lines. In addition, BN80915 is a potent inducer of apoptosis in HL-60 cells. This is associated with cell cycle changes, intracellular pH, activation of caspase-3 and caspase-8, DNA fragmentation, and externalization of phosphatidylserine lipids but no significant changes in the mitochondrial membrane potential or in the expression of oncogenes like Bcl-2. A nice finding is that homologation of the lactone ring slows down hydrolysis, which, in contrast to CPT analogs, is irreversible, leading to improved plasma stability and reduced toxicity related to ring closure during excretion.

Interesting topl-poisoning potency was found in another hCPT derivative, 12-C1-hCPT, which bears a chlorine substituent at position 12 [49]. Again, homologation of the E-ring lactone produced a noticeable loss of cross-resistance with CPT in cancer cells presenting multidrug resistance phenotype.

Among 7-substituted derivatives, a novel series of lipophilic analogs of CPT was recently described. The authors' hypothesis is that lipophilicity could promote a rapid cellular accumulation and stabilization of drug-tar get interactions. In particular, derivative ST1481, suitable for oral administration, exhibited an impressive response in terms of efficacy, potency, and therapeutic index when compared with the reference topotecan [50]. Even in this case, no cross-resistance to ST1481 was found in ovarian carcinoma cells overexpressing P-glycoprotein.

Other important members of the topi poison family are the indolocarbazoles derived from rebeccamycin (Fig. 19.6), and minor groove binders such as the bis-benzimidazoles Hoechst 33258 and Hoechst 33342 [5]. The indolocarbazole family are dealt with in another chapter of this book. The Hoechst derivatives are more interesting as biochemical probes than as drug candidates.

Besides monomeric ring systems, a family of dimeric bis(9-methylphenazine-l-carboxamides), reported in Fig. 19.6, joined by several dicationic linkers of varying length and rigidity showed effective topl-poisoning activity [51]. Analogs joined by a (CH2)2NR(CH2)2NR(CH2)2 linker were extremely potent and showed selectivity toward a panel of human cancer cell lines. Moreover, two selected compounds showed in vivo activity in murine and human colon tumor xenograft models.

In general, interference with topi is characterized by reversible interactions with the drug. More complex appears to be the situation in which chemically reactive groups, such as alkylating functions, characterize the drug. In this case the cytotoxic response may arise from the sum of enzyme-mediated and non-mediated effects. A typical example is represented by the potent antitumor agent presently in clinical trials ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt [52]. It selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. Et743 DNA adducts have been claimed to suppress gene expression selectively and to induce topi cleavage complexes in vitro and topi-DNA complexes in cell culture. The DNA damage consisted of comparable amounts of DNA-protein cross-links and DNA single-strand breaks, not associated with detectable DNA double-strand breaks. Hence, the drug was suggested to act by poisoning topi. However, by contrast with CPT, the DNA lesions persisted for several hours after drug removal. In addition, the sensitivity of CPT-resistant cells, which fail to express detectable topi, was similar to the sensitivity of the wild-type cells, suggesting that topi was not a critical target for the drug. Indeed, it was recently demonstrated that what appeared a topi-mediated process depended instead upon a new cell-killing mechanism mediated by transcription-coupled DNA nucleotide excision repair [53]. This fact should make us careful in assessing modes of drug action.

In any event, alkylating agents can promote to pi-mediated DNA damage by affecting the stability of a phosphodiester linkage close to the site of damage. In fact methylation of position 6 of guanine by N-methyl-N'-nitro-N-nitrosoguanidine produced an 8- to 10-fold enhancement of topi cleavage by affecting both the rate of topi-mediated DNA religation and that of the enzyme cleavage step. Enzymatic removal of the methyl groups from guanine abolished this enhancement [54]. These results suggest a role for topi poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.

As mentioned before, a deeper insight into the mechanism of drug action can be gained by having a more precise knowledge of the underlying biochemical processes. Two interesting examples deserve attention.

The first consists of an investigation on the effects of topi-targeting drugs on Flp recombinase, an enzyme of the lambda- Int family able to introduce transient single-strand breaks in DNA by similar chemical processes [55]. Indeed, agents like CPT and NSC-314622 interfere specifically with the enzyme. Hence, Flp and other Int family recombinases may provide further model systems for dissecting the molecular mechanisms of topoisomerase I-directed anticancer therapeutic agents.

The second example deals with the amino acid residue(s) involved in topi interaction with the drug [56]. In this study, Capranico et al were able to select human topi mutants about 30-fold more CPT-sensitive than wild-type topi in S. cerevisiae cells by directed evolution of a C-terminal portion. A topi mutant had only two amino acid changes in domain II, one of which mapped at the domain II-III contact surface. The information arising from such drug-hypersensitive topoisomerases might prove useful in developing DNA cutters with high cell lethality.

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