Abstract：
The discovery of SARS-CoV-2 RNA in the periodontal tissues of patients who tested positive for COVID-19, 24 days post the initial symptom onset, indicates the oral cavity could serve as a viral reservoir. This research aims to investigate the antiviral capabilities of Ovatodiolide, introducing a novel periodontal ligament organoid model for the study of SARS-CoV-2. We have successfully established a reliable and expandable organoid culture from the human periodontal ligament, showcasing characteristics typical of epithelial stem cells. This organoid model enables us to delve into the lesser-known aspects of dental epithelial stem cell biology and their interactions with viruses and oral tissues. We conducted a series of in vitro and ex vivo studies to examine the inhibitory impacts of Ova on SARS-CoV-2. Our findings indicate that Ovatodiolide molecules can bind effectively to the NRP1 active domain. Our study identifies potential interaction sites for Ovatodiolide (OVA) within the b1 domain of the NRP1 receptor. We generated point mutations at this site, resulting in three variants: Y25A, T44A, and a double mutation Y25A/T44A. While these mutations did not alter the binding activity of the spike protein, they did impact the concentration of OVA required for inhibition. The inhibitory concentrations for these variants are 15 μM for Y25A, 15.2 μM for T44A, and 25 μM for the double mutant Y25A/T44A. In addition, in vitro inhibition experiments demonstrate that the EC50 of Ova against the main protease (Mpro) of the SARS-CoV-2 virus is 7.316 μM. Our in vitro studies and the use of the periodontal ligament organoid model highlight Ovatodiolide\'s potential as a small molecule therapeutic agent that impedes the virus\'s ability to bind to the Neuropilin-1 receptor on host cells. The research uncovers various pathways and biochemical strategies through which Ovatodiolide may function as an effective antiviral small molecule drug.
摘要：
在最初症状发作后24天，在COVID-19检测呈阳性的患者的牙周组织中发现SARS-CoV-2RNA，表明口腔可以作为病毒储库。这项研究旨在调查Ovatdiolide的抗病毒能力，介绍了一种用于SARS-CoV-2研究的新型牙周膜类器官模型。我们已经成功地建立了一个可靠的和可扩展的类器官培养人牙周膜，展示上皮干细胞的典型特征。这种类器官模型使我们能够深入研究牙齿上皮干细胞生物学的鲜为人知的方面及其与病毒和口腔组织的相互作用。我们进行了一系列体外和离体研究，以检查Ova对SARS-CoV-2的抑制作用。我们的发现表明Ovatdiolide分子可以有效地结合NRP1活性域。我们的研究确定了NRP1受体b1域内Ovatdiolide（OVA）的潜在相互作用位点。我们在这个位点产生了点突变,产生三个变体：Y25A，T44A,和双突变Y25A/T44A。虽然这些突变不会改变刺突蛋白的结合活性，它们确实影响了抑制所需的OVA浓度.对于Y25A，这些变体的抑制浓度为15μM，T44A为15.2μM，和25μM的双突变体Y25A/T44A。此外,体外抑制实验表明，Ova对SARS-CoV-2病毒主要蛋白酶（Mpro）的EC50为7.316μM。我们的体外研究和牙周膜类器官模型的使用强调了Ovatdiolide作为小分子治疗剂的潜力，阻碍了病毒与宿主细胞上Neuropilin-1受体结合的能力。该研究揭示了Ovatdiolide可以作为有效的抗病毒小分子药物的各种途径和生化策略。
