From M. tuberculosis thymidylate kinase inhibitors towards TK-2 inhibitors


In search for better TB drugs we  have synthesized a number of promising inhibitors of M. tuberculosis thymidine monophosphate (thymidylate) kinase (TMPKmt), an attractive target for blocking mycobacterial DNA synthesis. The 3D structure of this enzyme proved a valuable tool for inhibitor design. Unfortunately, translating these inhibitors into leads with in vitro antimycobacterial activity was not obvious.

Recently, two series of thymidine analogues that were originally designed as TMPKmt inhibitors, were evaluated for their inhibitory activity against a panel of other nucleoside kinases (TK-1, TK-2, HSV-1 and VZV TK). Several substituted 3’-thiourea derivatives of β-dThd proved highly inhibitory to and exquisitely selective for human mitochondrial TK-2 (IC50: 0.15-3.1 µM) compared to the other enzymes. In fact, TK-2 was inhibited at concentrations at least 3 orders of magnitude lower than those required to inhibit cytosolic TK-1. These analogues showed competitive inhibition of TK-2 when dThd was used as the variable substrate, but uncompetitive inhibition of the enzyme in the presence of variable concentrations of ATP, which suggests specific binding of the inhibitor to an enzyme–ATP complex. These kinetic properties against TK-2 could be accounted for by molecular modeling results showing that two hydrogen bonds can be formed between the thiourea nitrogens and the oxygens of the γ-phosphate of the co-substrate ATP.


Proposed binding mode of a 3’-thiourea derivative of β-thymidine (C atoms in white)  in the active site of human TK-2 (pink ribbon).

Human mitochondrial TK-2 catalyses the phosphorylation of pyrimidine deoxynucleosides to the corresponding deoyxnucleoside 5’-monophosphate analogues.  In resting cells TK-2 is the most important thymidine phosphorylating enzyme. TK-2  is suggested to play a key role in the mitochondrial salvage pathway in which nucleotides are provided for mtDNA synthesis. Since long-term treatment with antiviral nucleoside analogues such as AZT and FIAU has been associated with severe mitochondrial toxicity, it is assumed that TK-2 is involved in their toxicity. In this respect, TK-2 inhibitors could be valuable tools to unravel the role of TK-2 in mitochondrial dNTP pools and homeostasis and may also help to clarify the contribution of TK-2 activity to mitochondrial toxicity of certain antivirals.

In addition, a striking close correlation was found between the inhibitory activities of the test compounds against TK-2 and Mycobacterium tuberculosis thymidylate kinase that is strongly indicative of close structural and/or functional similarities between both enzymes in relation to their mode of interaction with these nucleoside analogue inhibitors.

Currently, we are constructing new thymidine analogues to optimize TK-2 inhibition, to further explore the SAR for TK-2 inhibition and to validate the homology models of TK-2.

Researchers: Charlotte Courtens
TK-2: Jan Balzarini (Rega Institute, Leuven),
Federico Gago (Universidad de Alcalá, Madrid, Spain), Anna Karlsson (The Karolinska Institute, Huddinge, Sweden)
Hélène Munier-Lehmann (Institut Pasteur, Paris), Mathy Froeyen (Rega Institute, Leuven)