Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity

Abstract

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.

Biological profile of FCE 24517, a novel benzoyl mustard analogue of distamycin A

“…Alkylation of DNA by FCE 24517 is however weak, and in contrast to known nitrogen mustards, this compound does not induce alkylation at guanine N7 but at selected adenines (Broggini et al, 1991). The exact molecular mechanisms of this molecule are however still a matter of speculation also in consideration of the finding that this compound has additionally been found to display a very potent inhibitory activity for DNA ligase in conditions not affecting DNA polymerases (Montecucco et al, 1991). In view of its mode of action that on the basis of available evidence seems different from known cytototoxic agents, and its broad spectrum of activity in experimental conditions, FCE 24517 appears to be an interesting candidate for clinical evaluation and phase I studies are already ongoing.…”

DNA Ligase I, the Replicative DNA Ligase

“…These loops and helices arrange to create a relatively flat surface of approximately 2000 Å 2 that interacts almost exclusively with the phosphodiester backbone of the DNA substrate. The DBD interacts with the minor groove of the DNA backbone on both sides of the nick, explaining how DNA ligase I binds to DNA in a sequence-independent binding manner and why chemicals that bind to the minor groove of DNA, such as distamycin, inhibit DNA ligase activity (Montecucco, et al, 1991). Notably, the DBD stimulates the weak DNA joining activity of the DNA ligase I catalytic core containing the AdD and OBD when added in trans , indicating that contacts between the DBD and both AdD and the OBD observed in the crystal structure stabilize the folding of the catalytic core around the DNA nick (Pascal, et al, 2004).…”

Mammalian DNA Ligases

“…DNA ligase I is much more effective at blunt-end joining than mammalian DNA ligases II and III (41, 42a), but is less efficient in this regard than T4 DNA ligase. Similarly to microbial DNA ligases and Drosophila DNA ligase I (43), mammalian DNA ligase I can act at low efficiency as a topoisomerase, relaxing supercoiled DNA in an AMP-dependent reversal of the last step of the ligation reaction followed by re-ligation (44) .…”