Projects

Background

                  Prodrugs play a crucial role in current treatment of numerous diseases.  Nucleoside analog prodrugs are only active in their triphosphate form by mimicking the physiological substrates of DNA polymerases, but since entry into cells is inefficient for highly charged molecules, these compounds are administered uncharged.  Therefore, activation by cellular kinases is required for their potency, a fact that in many cases limits the effectiveness of such drugs.  Such is the case for the anti-HIV drug AZT and the anti-neoplastic agent 6-thioguanine, which accumulate as the monophosphate, implicating nucleoside monophosphate kinases (thymidylate kinase in the case of AZT, guanylate kinase in case of thiopurines) as being the rate limiting enzyme in the activation pathway.  Increasing the activation of these prodrugs by the introduction of modified enzymes with enhanced activity towards the rate-limiting metabolite would enhance the efficacy of these compounds.  This is the long term goal of the work performed in this laboratory.

       3'-deoxy-3'-azidothymidine (AZT) was the first drug approved for the treatment of AIDS  and is still widely used as one of the two compounds that target reverse transcriptase (RT), the viral polymerase.  AZT monotherapy is characterized by a relatively low viral log decrease and relatively rapid failure due to emergence of resistant RT mutants.  This is in contrast to compounds that target the HIV protease that result in a 100 fold decrease in viral load (that decrease is only about 3 fold for AZT), and a longer time period needed for resistance to appear.  It is unlikely that this different effect on viral load (and the time span till resistance emerges) is due to the protease being a better target than reverse transcriptase, per se.  A possible explanation for these observations is the fact that the protease inhibitors are given in their active form while AZT is a prodrug that must be activated once it enters the cell.  Consistent with this interpretation is the fact that non-nucleoside-based RT inhibitors, which are administered in their active form, also result in a 100 fold decrease in viral load.  Since these family of compounds do not target RT's active site, it is relatively simple for the polymerase to mutate such that drug binding is diminished without affecting catalytic activity, severely limiting the usage of these compounds.  Nucleoside analogs, such as AZT, target the active site, and so pose the added restraint on the viral polymerase to select for mutants that discriminate against the drug while maintaining sufficient polymerase activity.

    The activation of AZT requires its phosphorylation to AZT-triphosphate (AZTTP), and this is catalyzed by human cellular kinases.  The first step in the activation pathway is the phosphorylation of AZT to AZT-monophosphate (AZTMP), which is catalyzed by thymidine kinase.  The bottleneck of this pathway occurs at the next phosphorylation step due to the very slow rate of AZTMP phosphorylation by thymidylate kinase (TMPK).  The addition of the third and final phosphate is catalyzed by still to be identified kinases (probably not by nucleoside diphosphate kinase (NDK)).

   The questions we want to address are: what makes AZTMP a bad substrate for TMPK? Based on this knowledge, can we design an improved TMPK mutant that has a much higher activity towards AZTMP? Will such a mutant shift the AZT nucleotide pools to the triphosphate (and active!) form? Last, could such a mutant play a role in a gene therapeutic approach to the treatment of HIV infection?

Thymidylate kinase

    Thymidylate kinase (E.C. 2.7.4.9; ATP:dTMP phosphotransferase) catalyzes the phosphorylation of thymidine monophosphate (dTMP) to thymidine diphosphate (dTDP) utilizing ATP as its preferred phosphoryl donor according to the scheme:

     Its location at the junction of the de-novo and salvage pathways for thymidine triphosphate (dTTP) synthesis makes thymidylate kinase (TMPK) an essential enzyme for cell proliferation, and thus an attractive target for the development of drugs against cancer.  In addition to its physiological role, TMPK is also involved in the activation of the AIDS drug 3'-azido-3'-deoxythymidine (AZT).  AZT is a prodrug that must be phosphorylated three times to its triphosphate form (AZTTP), since it is AZTTP that inhibits viral replication by DNA chain termination.  TmpK, which catalyzes the second phosphorylation step, from the monophosphate (AZTMP) to the diphosphate (AZTDP), has been shown to be the rate limiting enzyme in the AZT activation pathway .  This results in a toxic accumulation of AZTMP to millimolar concentration in cells exposed to AZT , and in a low concentration of the active compound AZTTP.  Slow activation rates of prodrugs have been implicated in allowing the replicating virus to select for resistant mutants.