The proteasome is considered an excellent drug target for many infectious diseases as well as cancer. Challenges with robust and safe supply of proteasomes from infectious agents, lack of structural information and complex pharmacology due to multiple active sites have hampered progress in the infectious disease space. We recombinantly expressed the proteasome of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, and demonstrate pharmacological equivalence to the native T. cruzi proteasome. Active-site mutant recombinant proteasomes reveal substrate promiscuity for wild-type proteasomes, with important implications for assessing pharmacological responses of active-site selective inhibitors. Using these mutant proteasomes, we show that some selective parasite proteasome inhibitors only partially inhibit the chymotrypsin-like activity, including a newly developed 5-(phenoxymethyl)furan-2-carboxamide-based proteasome inhibitor. In spite of partial inhibition, these compounds remain potent inhibitors of intracellular T. cruzi growth. Drug-resistant mutants provide further insights in drug mode-of-inhibition. We also present the high-resolution CryoEM structures of both native and recombinantly-expressed T. cruzi proteasomes which reveal pharmacologically-relevant differences in the ligand-binding site compared to the related Leishmania proteasome. Furthermore, we show that the trypanosomatid β4/β5 selectivity pocket is not present in the proteasome structures of other protozoan parasites. This work highlights the need, and provides approaches, to precisely assess proteasome substrate selectivity and pharmacology. It enables structure-guided drug discovery for this promising Chagas disease drug target, provides a new chemical starting point for drug discovery, and paves the road for development of robust proteasome drug discovery programmes for other eukaryotic infectious diseases.

中文翻译：

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对克氏锥虫蛋白酶体的药理学和结构理解为开发位点特异性抑制剂提供了关键见解。


蛋白酶体被认为是许多传染病和癌症的极好药物靶标。来自传染源的蛋白酶体可靠和安全供应的挑战、缺乏结构信息以及由于多个活性位点而导致的复杂药理学阻碍了传染病领域的进展。我们重组表达原生动物寄生虫克氏锥虫的蛋白酶体，这是恰加斯病的病原体，并证明了与天然克氏锥虫蛋白酶体的药理学等效性。活性位点突变重组蛋白酶体揭示了野生型蛋白酶体的底物混杂性，对评估活性位点选择性抑制剂的药理反应具有重要意义。使用这些突变蛋白酶体，我们表明一些选择性寄生虫蛋白酶体抑制剂仅部分抑制胰凝乳蛋白酶样活性，包括新开发的基于 5-（苯氧甲基）呋喃-2-羧酰胺的蛋白酶体抑制剂。尽管存在部分抑制，但这些化合物仍然是细胞内克氏锥虫生长的有效抑制剂。耐药突变体为药物抑制模式提供了进一步的见解。我们还展示了天然和重组表达的 T. cruzi 蛋白酶体的高分辨率 CryoEM 结构，这些结构揭示了与相关的利什曼原虫蛋白酶体相比，配体结合位点的药理学相关差异。此外，我们表明锥虫 β4/β5 选择性口袋不存在于其他原生动物寄生虫的蛋白酶体结构中。这项工作强调了精确评估蛋白酶体底物选择性和药理学的必要性，并提供了方法。 它能够为这一有前途的恰加斯病药物靶点进行结构引导的药物发现，为药物发现提供新的化学起点，并为开发针对其他真核传染病的稳健蛋白酶体药物发现计划铺平了道路。