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Antiviral chemotherapy often relies on nucleoside analogues, which, once phophorylated by intracellular kinases, target viral polymerases impeding DNA synthesis. In contrast, nucleoside analogues are much less explored as antibacterial drugs. Thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), which is essential to DNA replication, was selected as a promising target for the design of new inhibitors. This review describes stepwise modifications of the TMPKmt substrate, guided by the feedback of enzyme assays and crystallographic analysis to afford potent enzyme inhibitors some of which also exhibited antitubercular activity. More importantly, several of the reported thymidine analogues provided a deeper understanding of the structure and catalytic mechanism of this intriguing enzyme.

A series of N(1)-(4-substituted-benzyl)-pyrimidines were synthesized as potential inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). Key SAR parameters included the chain length substitution in para position of the benzyl ring, the functional group terminating the alkyl chain, and the substituent on the C-5 pyrimidine ring. Synthesized molecules were assayed against both recombinant enzyme and mycobacteria cultures. The most potent compounds have K(i) values in the micromolar range and an MIC(50) of 50microg/mL against Mycobacterium bovis. These results will guide the design of a new generation of lead compounds.

The affinity of a series of 2', 3'- and 5-modified thymidine analogues for Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) was evaluated. The affinities of several non-phosphorylated analogues are in the same order of magnitude as those of their phosphorylated congeners. In view of drug delivery problems associated with phosphorylated compounds, these 'free' nucleosides seem more promising leads in the search of TMPKmt inhibitors as novel anti-tuberculosis agents.

Thymidine monophosphate kinase (TMPK) of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for blocking the bacterial DNA synthesis. In an attempt to find high-affinity inhibitors of TMPKmt, a cavity in the enzyme at the 3'-position was explored via the introduction of various substituents at the 3'-position of the thymidine monophosphate (dTMP) scaffold. Various 3'-C-branched chain substituted nucleotides in the 2'-deoxyribo (3-6) and ribo series (7, 8) were synthesized from one key intermediate (23). 2'-Deoxy analogues proved to be potent inhibitors of TMPKmt: 3'-CH(2)NH(2) (4), 3'-CH(2)N(3) (3), and 3'-CH(2)F (5) nucleotides exhibit the highest affinities within this series, with K(i) values of 10.5, 12, and 15 microM, respectively. These results show that TMPKmt tolerates the introduction of sterically demanding substituents at the 3'-position. Ribo analogues experience a significant affinity decrease, which is probably due to steric hindrance of Tyr103 in close vicinity of the 2'-position. Although the 5'-O-phosphorylated compounds have somewhat higher affinities for the enzyme, the parent nucleosides generally exhibit affinities for TMPKmt in the same order of magnitude and display a superior selectivity profile versus human TMPK. This series of inhibitors holds promise for the development of a new class of antituberculosis agents.

A number of 2'- and 3'-modified thymidine 5'-O-monophosphate analogues were synthesized as potential leads for new anti-mycobacterial drugs. Evaluation of their affinity for Mycobacterium tuberculosis thymidine monophosphate kinase showed that a 2'-halogeno substituent and a 3'-azido function are the most favorable leads for further development of potent inhibitors of this enzyme.