An Optimized Route to the Syringolin Natural Products Enables Combinatorial Synthesis of Selective, Bioactive Inhibitors of the Plasmodium falciparum 20S Proteasome

The syringolin natural products are covalent inhibitors of the 20S proteasome that inspire therapeutic development. Here, we report a new route to the syringolins amenable to solution and solid-phase synthesis that overcomes a problematic macrocyclization. Exploiting our synthetic approach and substrate mimicry models for proteasome inhibition by the syringolins, we generated a collection of hypothetically selective inhibitors of the Plasmodium falciparum proteasome, which is an emerging target for antimalarial drugs. We identified compounds from the library having high second-order rate constants for Plasmodium proteasome inhibition and nanomolar antiparasitic activity. They exhibited selectivity for the Plasmodium proteasome over the human proteasome. We solved cryo-EM structures of an inhibitor bound to both 20S proteasomes, revealing key contacts favoring species-selective inhibition. Together, this work provides an improved route to syringolin analogs, sheds new light on substrate mimicry by the syringolins, and provides a structural basis for the pursuit of new antimalarial drugs.丁香菌素（syringolin）类天然产物是一类靶向20S蛋白酶体的共价抑制剂，其相关研究可为治疗药物开发提供重要借鉴。本研究报道了一条适配溶液相及固相合成的丁香菌素全新合成路线，攻克了长期制约该类化合物合成的大环化反应难题。依托本研究建立的合成方法，以及丁香菌素介导蛋白酶体抑制的底物模拟模型，我们构建了一系列针对恶性疟原虫（Plasmodium falciparum）蛋白酶体的假想选择性抑制剂库；而恶性疟原虫蛋白酶体正是当前抗疟药物研发的新兴靶点。本研究从该化合物库中筛选得到了对疟原虫蛋白酶体抑制具有高二级速率常数、且具备纳摩尔级抗寄生虫活性的活性化合物。相较于人源蛋白酶体，此类化合物对疟原虫蛋白酶体展现出优异的物种选择性。我们解析了一种抑制剂分别结合两种20S蛋白酶体的冷冻电镜（cryo-EM）结构，揭示了助力物种选择性抑制的关键相互作用界面。综上，本研究为丁香菌素类似物的合成提供了更优化的途径，阐明了丁香菌素介导底物模拟的全新机制，并为新型抗疟药物的研发提供了结构生物学基础。