Simulations of Shikimate Dehydrogenase from Mycobacterium tuberculosis in Complex with 3-Dehydroshikimate and NADPH Suggest Strategies for MtbSDH Inhibition

Shikimate dehydrogenase (SDH) from Mycobacterium tuberculosis ( MtbSDH), encoded by the aroE gene, is essential for viability of M. tuberculosis but absent from humans. Therefore, it is a potentially promising target for antituberculosis agent development. Molecular-level understanding of the interactions of MtbSDH with its 3-dehydroshikimate (DHS) substrate and NADPH cofactor will help in the design of novel and effective MtbSDH inhibitors. However, this is limited by the lack of relevant crystal structures for MtbSDH complexes. Here, molecular dynamics (MD) simulations were performed to generate these MtbSDH complexes and investigate interactions of MtbSDH with substrate and cofactor and the role of MtbSDH dynamics within these. The results indicate that, while structural rearrangements are not necessary for DHS binding, reorientation of individual side chains in the NADPH binding pocket is involved in ternary complex formation. The mechanistic roles for Lys69, Asp105, and Ala213 were investigated by generating Lys69Ala, Asp105Asn, and Ala213Leu mutants in silico and investigating their complexes with DHS and NADPH. Our results show that Lys69 plays a dual role, in positioning NADPH and in catalysis. Asp105 plays a crucial role in positioning both the ε-amino group of Lys69 and nicotinamide ring of NADPH for MtbSDH catalysis but makes no direct contribution to DHS binding. Ala213 is the selection key for NADPH binding with the nicotinamide ring in the proS, rather than proR, conformation in the MtbSDH complex. Our results identify three strategies for MtbSDH inhibition: prevention of MtbSDH binary and ternary complex formation by blocking DHS and NADPH binding (first and second strategies, respectively) and the prevention of MtbSDH complex formation with either DHS or NADPH by blocking both DHS and NADPH binding (third strategy). Further, based on this third strategy, we propose guidelines for the rational design of "hybrid" MtbSDH inhibitors able to bind in both the substrate (DHS) and cofactor (NADPH) pockets, providing a new avenue of exploration in the search for anti-TB therapeutics.