C‑2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV‑2 through Interaction with the Viral Spike (S) Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
causes COVID-19, by infecting cells via the interaction of its spike
protein (S) with the primary cell receptor angiotensin-converting
enzyme (ACE2). To search for inhibitors of this key step in viral
infection, we screened an in-house library of multivalent tryptophan
derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical
optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell
entry. Thermofluor and microscale thermophoresis studies revealed
their binding to S and to its isolated receptor binding domain (RBD),
interfering with the interaction with ACE2. High-resolution cryoelectron
microscopy structure of S, free or bound to 2, shed light
on cell entry inhibition mechanisms by these compounds. Overall, this
work identifies and characterizes a new class of SARS-CoV-2 entry
inhibitors with clear potential for preventing and/or fighting COVID-19.

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）可引发新型冠状病毒肺炎（COVID-19），该病毒通过其刺突蛋白（spike protein，S）与宿主细胞表面主要受体血管紧张素转换酶2（angiotensin-converting enzyme 2，ACE2）的相互作用侵染宿主细胞。为筛选靶向病毒感染这一关键步骤的抑制剂，我们对内部构建的多价色氨酸衍生物文库进行了筛选。利用VSV-S假病毒颗粒（VSV-S pseudoparticles），我们鉴定出化合物2是一种强效病毒进入抑制剂，且无细胞毒性。对化合物2进行化学优化后，得到化合物63与65，二者同样可强效抑制天然完整SARS-CoV-2的细胞侵染过程。热位移荧光法（Thermofluor）与微量热泳动（microscale thermophoresis）实验结果显示，这些化合物可结合S蛋白及其分离得到的受体结合域（receptor binding domain，RBD），从而干扰其与ACE2的相互作用。游离状态或与化合物2结合的S蛋白的高分辨率冷冻电镜（cryoelectron microscopy）结构，为阐明这类化合物的病毒进入抑制机制提供了关键依据。综上，本研究鉴定并表征了一类新型SARS-CoV-2病毒进入抑制剂，其在预防和/或治疗新型冠状病毒肺炎方面具有明确的应用潜力。