New structure reveals how an inhibitor blocks mycobacterial energy enzyme

Tuberculosis and other mycobacterial infections persist in part because the bacteria have essential energy pathways that are different from ours. One enzyme at the center of those pathways is the mycobacterial type II NADH dehydrogenase, known as NDH-2. NDH-2 is considered a promising drug target because it plays a central role in energy metabolism in Mycobacterium tuberculosis and other pathogens, and because it lacks a known mammalian homologue, reducing the chance of directly hitting human proteins. Until now, though, drug developers faced a major gap: the absence of structural information showing how inhibitors bind NDH-2. That gap made it hard to optimize lead compounds. To close it, a team led by corresponding author John L. Rubinstein turned to a fast-growing, non-pathogenic model species, Mycobacterium smegmatis, which is commonly used to study respiration relevant to M. tuberculosis. Using that model, the researchers determined the structure of mycobacterial NDH-2 both by itself and while it was bound to a 2-mercapto-quinazolinone inhibitor, providing the first detailed snapshot of the enzyme in its inhibited form.

The team applied electron cryomicroscopy (cryo-EM) to capture high-resolution images of NDH-2 from Mycobacterium smegmatis, collecting structures of the enzyme alone and in complex with a 2-mercapto-quinazolinone inhibitor. The structures revealed that active mycobacterial NDH-2 is dimeric: two monomers associate to form the functional unit. Importantly, the dimerization interface is stabilized by an extended C-terminal α helix, a feature the authors note is not found in NDH-2 from other bacterial genera. The way the two monomers arrange in the mycobacterial dimer differs from arrangements described for other prokaryotic NDH-2 dimers and instead resembles dimers formed by NDH-2 in eukaryotes. In the inhibitor-bound map, density for the 2-mercapto-quinazolinone sits in the menaquinone-binding site and shows that the inhibitor blocks menaquinone reduction through a direct interaction with the flavin adenine dinucleotide cofactor. Those precise contacts explain how this chemical class interferes with the enzyme's chemistry.

These structural insights carry clear implications for drug discovery against mycobacteria. By revealing the shape of the menaquinone-binding site, the dimer interface and the role of an extended C-terminal α helix specific to mycobacterial NDH-2, the work identifies concrete structural elements that medicinal chemists can target when designing or improving inhibitors. The direct interaction observed between the 2-mercapto-quinazolinone and the flavin adenine dinucleotide cofactor explains a mechanism for blocking the enzyme's activity and provides a template for optimizing potency and specificity. Because NDH-2 has a central role in mycobacterial energy metabolism and lacks a known mammalian homologue, inhibitors crafted using this new structural information have the potential to be selective for bacterial enzymes and to avoid off-target effects on human proteins. Overall, the cryo-EM structures reported by John L. Rubinstein and colleagues convert a long-standing blind spot into actionable detail for rational inhibitor design.