Abstract Lung cancer patients are more vulnerable to COVID-19 infection. Treatment of patients with lung cancer during the current COVID-19 pandemic is challenging and development of drugs for COVID-19 and lung cancer is urgently needed. Cathepsin L plays key role in lung cancer progression, invasion and more so, endocytosis of SARS-Cov-2 virus into the lung epithelial cells. Importantly, patients with KrasG12V mutation show agreessive disease and high-resistance to chemotherapy due to over-expression of Cathepsin L. Thus, Cathepsin L is a common target for lung cancer and COVID-19 infection. Chymostatin is a known cathepsin L inhibitor, here we screened it for dual inhibition of COVID-19 Mpro and lung cancer patients with COVID-19. In vitro COVID-19 Mpro fluorometric assay was performed to evaluate its inhibitory efficacy by chymostatin. Chymostatin showed dose-dependent inhibition of COVID-19 Mpro activity with IC50 of 15.81 µM (P<0.0001). Isothermal titration calorimetry based binding studies determined that chymostatin strongly bound to COVID-19 Mpro (KD=22.45 µM). CD spectrum analysis demonstrated that chymostatin (10 µM)-induced changes in the secondary structure of COVID-19 Mpro. A unit cell crystal parameter COVID-19 Mpro co-crystalized with chymostatin (a = 49.926 Å, b = 108.71 Å, c = 56.49 Å, α = 90.0°, β = 102.7553°, γ = 90.0°; unit cell volume: 299.03 Ao) was determined using X-ray diffractometer. Molecular docking studies demonstrated that chymostatin interacts with active site amino acids (Cys145). Pro-apoptotic effect of chymostatin was evaluated on human H441 lung cancer cells and mouse lung normal epithelial cells using Annexin V/propidium iodide staining. Chymostatin did not show any cytotoxicity on normal mouse lung epithelial cells up to 100 µM, where as it inhibited proliferation of human H441 lung cancer cells (KrasG12V mutant) with IC50 of 1.2 µM. In conclusion, preliminary in vitro studies demonstrated that naturally occurring cathepsin L inhibitor chymostatin is a SARS-CoV-2 Mpro inhibitor and exhibited anticancer activity against lung cancer cells. The results of the study warranted detailed studies to develop as drugs for lung cancer patients during COVID-19 pandemic (supported by VPR-PHF, the University of Oklahoma Health Sciences Centre for financial support through COVID-19 seed grant program). Citation Format: Nagendra Sastry Yarla, Gopal Pathuri, Simon Terzyan, Yuting Zhang, Anil Singh, Marcus T. Scotti, Venkateshwar Madka, Chinthalapally V. Rao. Chymostatin, a cathepsin L inhibitor, inhibits lung cancer cell proliferation and COVID-19 Mpro in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB074.

中文翻译：

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摘要 LB074：组织蛋白酶 L 抑制剂凝乳抑制素在体外抑制肺癌细胞增殖和 COVID-19 Mpro

摘要 肺癌患者更容易感染 COVID-19。在当前 COVID-19 大流行期间治疗肺癌患者具有挑战性，迫切需要开发 COVID-19 和肺癌药物。组织蛋白酶 L 在肺癌进展、侵袭以及 SARS-Cov-2 病毒内吞进入肺上皮细胞中起关键作用。重要的是，具有 KrasG12V 突变的患者由于 Cathepsin L 的过度表达而表现出一致的疾病和对化疗的高耐药性。因此，Cathepsin L 是肺癌和 COVID-19 感染的常见靶标。凝乳抑制素是一种已知的组织蛋白酶 L 抑制剂，在这里我们筛选了它对 COVID-19 Mpro 和 COVID-19 肺癌患者的双重抑制作用。进行了体外 COVID-19 Mpro 荧光测定，以评估其对凝乳酶的抑制效果。凝乳酶抑制 COVID-19 Mpro 活性呈剂量依赖性，IC50 为 15.81 µM (P<0.0001)。基于等温滴定量热法的结合研究确定，凝乳酶与 COVID-19 Mpro (KD=22.45 µM) 紧密结合。CD 光谱分析表明，促凝乳酶 (10 µM) 诱导了 COVID-19 Mpro 二级结构的变化。晶胞晶体参数 COVID-19 Mpro 与凝乳酶共结晶（a = 49.926 Å，b = 108.71 Å，c = 56.49 Å，α = 90.0°，β = 102.7553°，γ = 90.0°；晶胞体积：299.03 Ao) 使用 X 射线衍射仪测定。分子对接研究表明，糜蛋白酶与活性位点氨基酸 (Cys145) 相互作用。使用膜联蛋白 V/碘化丙啶染色评估了凝乳抑制素对人 H441 肺癌细胞和小鼠肺正常上皮细胞的促凋亡作用。凝乳抑制素在高达 100 µM 的正常小鼠肺上皮细胞上没有表现出任何细胞毒性，因为它抑制人 H441 肺癌细胞（KrasG12V 突变体）的增殖，IC50 为 1.2 µM。总之，初步体外研究表明，天然组织蛋白酶 L 抑制剂 chymostatin 是一种 SARS-CoV-2 Mpro 抑制剂，对肺癌细胞具有抗癌活性。该研究的结果需要进行详细研究，以便在 COVID-19 大流行期间开发用于肺癌患者的药物（由俄克拉荷马大学健康科学中心 VPR-PHF 支持，通过 COVID-19 种子赠款计划提供财政支持）。引文格式：Nagendra Sastry Yarla、Gopal Pathuri、Simon Terzyan、Yuting Zhang、Anil Singh、Marcus T. Scotti、Venkateshwar Madka、Chinthalapally V. Rao。凝乳抑制素是一种组织蛋白酶 L 抑制剂，可在体外抑制肺癌细胞增殖和 COVID-19 Mpro [摘要]。在：美国癌症研究协会 2021 年年会论文集；2021 年 4 月 10 日至 15 日和 5 月 17 日至 21 日。费城 (PA)：AACR；癌症研究 2021；81(13_Suppl)：摘要 nr LB074。