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Abstract

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multi-domain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO₂) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e. Cys and His), modulating protein functions and subcellular distribution in a reversible-manner. In accordance with the present invention, administration of OA-NO₂ inhibits kinase activity by covalently adducting PknG outside its catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO₂ mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO₂ induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. Altogether our results highlight the relevance of Rbx domain as an interesting new target for PknG inhibition. In addition, the reactivity of electrophilic fatty acids towards Rbx-Cys points to its potential as modulators of critical cell signaling activities and as model molecules for specific PknG inhibitors development.

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