TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USA.
TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, USA
mSphere. 2020 Oct 14;5(5):e00985-20. doi: 10.1128/mSphere.00985-20.
The protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.
MmpL3 蛋白在细胞壁合成中起着至关重要的作用，因为它影响着海藻糖单胞苷脂穿过内膜的运输。大量结构多样的药效团已被鉴定为 MmpL3 的抑制剂，主要基于鉴定出 MmpL3 突变的耐药分离株。对于某些化合物，可能存在不同的主要或次要靶标。在这里，我们研究了对螺旋胺类化合物的耐药性。耐药突变体的分离和测序表明，所有突变都发生在 MmpL3 中。我们假设如果这个药效团存在其他靶点，那么连续几代产生耐药分离株可能会揭示其他基因座的突变。由于化合物仍然对耐药分离株有效，尽管效力降低，我们在较高浓度下在这个背景下分离出耐药突变体。在用螺旋胺进行第二轮分离后，我们在 MmpL3 中发现了其他突变。为了增加找到替代靶点的机会，我们使用不同的分子骨架（金刚烷脲 AU1235）进行了第三轮分离。令人惊讶的是，我们在 MmpL3 中获得了进一步的突变。MmpL3 的多重突变增加了对不同药效团的耐药水平和范围，但没有产生适应性成本。这些结果支持 MmpL3 是主要耐药机制和这些药效团的主要靶标的假设。
结核分枝杆菌是全球主要的人类病原体，迫切需要新的药物和新的药物靶点。细胞壁生物合成是目前抗结核药物和新开发药物的主要靶点。几种新的分子类别似乎具有相同的靶标，MmpL3，它参与分枝杆菌细胞壁的出口和合成。然而，对于这些类别，MmpL3 是主要或唯一靶标仍存在争议。我们想确认一个系列的耐药机制。我们确定了导致对螺旋胺系列耐药的 MmpL3 突变。相同蛋白（MmpL3）的多个突变赋予了对这些化合物和另外两个系列的高水平耐药性。这些突变没有降低培养物中的生长速度。这些结果支持 MmpL3 是主要耐药机制和这些药效团的主要靶标的假设。
