Human polymerase alpha inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives.

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Targeting human DNA polymerase alpha for the inhibition of keratinocyte proliferation. Part 1. Homology model, active site architecture and ligand binding.

In order to understand the binding modes of human DNA polymerase alpha (pol alpha) inhibitors on a molecular level, a 3D homology model of the active site of the enzyme was proposed based on the application of molecular modelling methods and molecular dynamic simulations using available crystal coordinates of pol alpha relatives. Docking results for a series of known nucleotide analogue inhibitors were consistent with reported experimental binding data and offered the possibility to elucidate structure-activity relationships via investigations of active site-inhibitor interactions. Furthermore, the study could explain, at least partially, the inhibitory effect of aphidicolin on pol alpha. In molecular dynamics simulations, aphidicolin occupied the catalytic centre, but acted in a not truly competitive manner with respect to nucleotides. It destabilized the replicating "closed" form of the pol alpha and transferred the enzyme into the inactive "open" conformation. This result is consistent with recent experiments on the binding mode of aphidicolin.