The Mycobacterium tuberculosis 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 in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

中文翻译：

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结核分枝杆菌 MmpL3 的多重突变增加对 MmpL3 抑制剂的抗性

该结核杆菌蛋白质 MmpL3 在细胞壁合成中发挥重要作用，因为它影响海藻糖单分枝杆菌跨内膜的转运。许多结构不同的药效团已被鉴定为 MmpL3 的抑制剂，这主要是基于对 MmpL3 突变的耐药菌株的鉴定。对于某些化合物，可能存在不同的主要或次要目标。在这里，我们研究了对螺旋胺类化合物的抵抗力。抗性突变体的分离和测序表明所有突变体都在 MmpL3 中发生突变。我们假设，如果该药效团的其他目标存在，那么连续轮次产生抗性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菌株有活性，尽管效力降低，我们在此背景下以较高浓度分离了抗性突变体。在用螺旋胺进行第二轮分离后，我们在 MmpL3 中发现了额外的突变。为了增加我们找到替代目标的机会，我们使用不同的分子支架（AU1235，一种金刚烷基尿素）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 中的多个突变增加了对不同药效团的抗性水平和谱，但不会产生健康成本 我们在 MmpL3 中获得了进一步的突变。MmpL3 中的多个突变增加了对不同药效团的抗性水平和谱，但不会产生健康成本 我们在 MmpL3 中获得了进一步的突变。MmpL3 中的多个突变增加了对不同药效团的抗性水平和谱，但不会产生健康成本体外。这些结果支持 MmpL3 是耐药性的主要机制和这些药效团的可能目标的假设。