Novel antitubercular agents are urgently needed to combat the emergence of global drug resistance to human tuberculosis. Mycobacterial membrane protein Large 3 (MmpL3) is a promising drug target because its activity is essential and required for cell-wall biosynthesis. Several classes of MmpL3 inhibitors have been developed against Mycobacterium tuberculosis (Mtb) with potent anti-tuberculosis activity. These include the drug candidate SQ109, which has progressed to phase IIb/III clinical trials. Here, we have determined the crystal structures of MmpL3 in complex with NITD-349 and SPIRO. Both inhibitors bind deep in the central channel of transmembrane domain and cause conformational changes to the protein. The amide nitrogen and indole nitrogen of NITD-349 and the piperidine nitrogen of SPIRO interact and clamp Asp645. Structural analysis of the two structures reveals that these inhibitors target the proton relay pathway to block the activity of MmpL3. The findings presented here enrich our understanding of the binding modes of MmpL3 inhibitors and provide directions to enable further rational drug design targeting MmpL3.

迫切需要新型抗结核药来对抗全球对人类结核病的耐药性的出现。分枝杆菌膜蛋白大分子3（MmpL3）是一种有前途的药物靶标，因为它的活性对于细胞壁生物合成至关重要，也是必需的。已经开发了几种针对结核分枝杆菌的MmpL3抑制剂（Mtb）具有有效的抗结核活性。其中包括候选药物SQ109，该药物已进入IIb / III期临床试验。在这里，我们确定了与NITD-349和SPIRO复合的MmpL3的晶体结构。两种抑制剂都在跨膜结构域的中央通道深处结合，并引起蛋白质的构象变化。NITD-349的酰胺氮和吲哚氮与SPIRO的哌啶氮相互作用并钳制Asp645。两种结构的结构分析表明，这些抑制剂靶向质子传递途径，以阻断MmpL3的活性。此处提出的发现丰富了我们对MmpL3抑制剂结合模式的理解，并为进一步针对MmpL3进行合理药物设计提供了指导。