Rational Design and Synthesis of Covalent Inhibitors for Mycobacterium tuberculosis Isocitrate Lyase

During latent tuberculosis infection, Mycobacterium tuberculosis (Mtb), the causative bacterial agent, survives within the intracellular spaces of macrophages by rerouting its metabolic flux towards the glyoxylate shunt. Isocitrate lyase (ICL), the first enzyme of this alternative pathway is intrinsic for the bacteria’s persistence in the body. As such, it presents as an attractive drug target for combatting the disease during its latent phase. However, despite this fact, there are currently no available drugs on the market specifically targeting Mtb ICL.

This report describes the synthesis of covalent inactivators of ICL.

These compounds are isocitrate or succinate analogues bearing electrophilic groups and were designed to interact with ICL’s nucleophilic cysteine residue.  

Chapter Two outlines the synthesis of nitroisocitrate (1) -a des-carboxy isocitrate analogue bearing a nitro functionality on the 4- carbon. Upon the enzyme’s retro-aldol cleavage of 1, the generated 3-Nitropropionate (3-NP) results in covalent inactivation through the formation of a stable thiohydroximate adduct. Given the neurotoxicity of 3-NP, we developed nitroisocitrate (1) to limit its reactivity towards ICL. Using an isocitrate-based precursor, we installed the nitro functionality through Hofmann rearrangement of a primary amide intermediate followed by oxidation of the resulting amine. A prodrug form of NIC (1) was also synthesised by derivatisation of the carboxyl groups into methyl esters 2. Further, an attempt towards the synthesis of nitrile isocitrate was also carried out.  

Chapter Three describes the synthesis of three succinate analogues containing an epoxide ring as the main electrophilic moiety. Based on the recent findings outlining cis-2,3 epoxysuccinic acid (cis-EpS) as a covalent inhibitor of Mtb ICL1, we synthesised cis-EpS analogues containing a mono-amide group in place of one of the carboxyl groups.  

Kinetic assays establish NIC (1) as an ICL1 inactivator. However, further data needs to be collected for all the synthesised substrates to investigate their biological relevance. Overall, the findings in this report contribute towards an improved design and synthesis of ICL inactivators. Accordingly, these compounds have the potential to attenuate Mtb in its persistent stage.