Diisonitrile Chalkophore Structure and Function in Mycobacterial Copper Acquisition

Tuberculosis, the disease caused by Mycobacterium tuberculosis infection, is a leading cause of death worldwide. While first-line treatments are effective for many patients, multidrug-resistant tuberculosis poses a continued threat to global public health, and new approaches are needed to provide effective chemotherapy against drug-resistant disease. Diisonitrile lipopeptide chalkophores are a novel class of natural products that play critical roles in copper homeostasis and virulence in M. tuberculosis by supplying copper to the heme:Cu oxidase of the respiratory chain. However, the molecular mechanisms by which the bacteria use diisonitriles to mediate copper homeostasis are poorly understood. Herein, we investigate structure-activity relationships of synthetic diisonitriles to identify structural features required for biological function in mycobacterial copper acquisition. We have developed a modular synthetic approach that allows efficient construction of diisonitrile natural products and diverse analogues. We confirmed that a short-chain analogue of the natural products binds tightly to Cu(I) and Cu(II), but not Zn(II), and estimated its Cu(I) binding affinity. We then used these analogues to probe the impacts of diisonitrile acyl chain length, amino acid backbone, and C-terminal motif on the rescue of M. tuberculosis and Mycobacterium marinum growth in copper-restricted conditions. The observed structural preferences align with current knowledge of the corresponding natural product structures in these bacteria, but both species also exhibited surprising flexibility in accepting diverse diisonitrile analogues for this function. This work provides new insights into the structural features of diisonitrile chalkophores required for function in mycobacterial copper homeostasis, and the distinct structural preferences between species, while suggesting some structural plasticity in their cellular copper acquisition systems.