Structural and biochemical insights into the inhibition of Mycobacterium tuberculosis cyclic dinucleotide phosphodiesterase by a sulfur-modified cyclic dinucleotide analog.

Cyclic dinucleotide (CDN) signaling plays a pivotal role in bacterial physiology and host-pathogen interactions. Mycobacterium tuberculosis (Mtb) releases a cyclic dinucleotide phosphodiesterase (CdnP) in the macrophages, which hydrolyzes Mtb-derived 3'3'-c-di-AMP and 3'3'-c-di-GMP, and a host-derived 2'3'-cGAMP STING agonist, to evade the host's innate immune response mediated by the STING protein. Therefore, by inhibiting CdnP released into host cells, the STING pathway can be potentiated, leading to improved bacterial clearance, which represents a potential novel approach for anti-tuberculosis (anti-TB) therapy. Here we report ES-2'3'-cAAMP, an analog of the host-derived STING agonist 2'3'-cGAMP, in which the phosphodiester bond is modified and the base is replaced, binds to CdnP with a micromolar binding affinity and competes with CdnP's substrates - 3'3'-c-di-AMP and 3'3'-c-di-GMP - binding to CdnP. Significantly, CdnP's phosphodiesterase catalytic activity is inhibited by ES-2'3'-cAAMP. Furthermore, the CdnP-ES-2'3'-cAAMP complex structure reported here is the first structure of the CdnP complexed with CDN, revealing the unique pose of ES-2'3'-cAAMP in the catalytic pocket of CdnP that is inaccessible to catalytic residues and Mn2+ ions for its hydrolysis, and in parallel blocks the binding of the natural substrates of CdnP that explains the structural basis of CdnP's catalytic activity inhibition by the inhibitor. Additionally, 2'3'-cGAMP in the STING receptor and ES-2'3'-cAAMP in the CdnP adopted an identical horseshoe conformation, suggesting that ES-2'3'-cAAMP, or an analogue thereof, can bind to and stimulate STING, thus acting as a synthetic STING agonist. These combined structural and biochemical findings provide new mechanistic insights into the inhibition of Mtb CdnP and offer a novel approach to host-directed anti-TB therapies that aim to enhance the host's own immune responses rather than directly killing the pathogen, which may help to mitigate the problem of antibiotic resistance.