key: cord-280198-bhjw6xc5 authors: Olaleye, Omonike A.; Kaur, Manvir; Onyenaka, Collins; Adebusuyi, Tolu title: Discovery of Clioquinol and Analogues as Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, ACE2 and ACE2 - Spike Protein Interaction In Vitro date: 2020-08-14 journal: bioRxiv DOI: 10.1101/2020.08.14.250480 sha: doc_id: 280198 cord_uid: bhjw6xc5 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease 2019 (COVID-19), has emerged as an ongoing global pandemic. Presently, there are no clinically approved vaccines nor drugs for COVID-19. Hence, there is an urgent need to accelerate the development of effective antivirals. Here in, we discovered Clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a FDA approved drug and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14); and 5, 7-Dichloro-8-hydroxyquinoline (CLCQ)) as potent inhibitors of SARS-CoV-2 infection induced cytopathic effect in vitro. In addition, all three compounds showed potent anti-exopeptidase activity against recombinant human angiotensin converting enzyme 2 (rhACE2) and inhibited the binding of rhACE2 with SARS-CoV-2 Spike (RBD) protein. CLQ displayed the highest potency in the low micromolar range, with its antiviral activity showing strong correlation with inhibition of rhACE2 and rhACE2-RBD interaction. Altogether, our findings provide a new mode of action and molecular target for CLQ and validates this pharmacophore as a promising lead series for clinical development of potential therapeutics for COVID-19. shedding of S1 and transition of the S2 subunit to expose a hydrophobic fusion peptide 42,55,56 . The initial priming at S1/S2 boundary promotes subsequent cleavage at the S2 site by host 105 proteases, which is critical for membrane fusion and viral infectivity 54,57,58 . Therefore, targeting 106 the interaction between human ACE2 receptor and the RBD in S protein of SARS-CoV-2 could 107 serve as a promising approach for the development of effective entry inhibitors for potential 108 prevention and/or treatment of COVID-19. In this study, we evaluated the effect of CLQ, and two of its analogues (7-bromo-5- Thereafter, the solution was discarded and the plate was washed consecutively four times with 222 300 µL 1X wash buffer, followed by the addition of the detection antibody (anti-ACE2 goat 223 antibody). The reaction was allowed to go on for 1 hr at room temperature (22°C) with shaking 224 at 180rpm. Then, the solution was discarded and the wash step was repeated as described 225 above. Next, the HRP-conjugated anti-goat IgG was added to each well, and the reaction plate 226 was further incubated for 1 hr at room temperature (22°C) with shaking at 180rpm. Again, the 227 solution was discarded and the wash step was repeated as described above. Then, 100µL of 228 3,3',5,5'-tetramethylbenzidine (TMB) one-step substrate was added to each well, and reaction Compared to its counterparts, CLBQ14 exhibited the highest maximum inhibition at about 246 102.96% inhibition at 30µM (Table 1 ). In addition, we compared the antiviral effects of CLBQ14 Table 2 ). These results 254 suggest a potential new mechanism of action for CLQ and its congeners. Notably, this is the first 255 report to our knowledge, revealing that CLQ and its analogues effectively inhibit the novel SARS- We determined the preliminary cytotoxicity of CLQ and its analogues (CLBQ14 and CLCQ), 260 using a Cell Titer-Glo Luminescent Cell Viability Assay 60 . We assessed the cytotoxic effects of 261 the various compounds in Vero E6 cells and observed that, the 50% cytotoxic concentration 262 (CC 50 ) of CLQ and its derivatives were all greater than 30 µM. However, in comparison to the 263 other reference compounds tested, CLQ and its analogues displayed lower percent minimum 264 viability at higher concentrations. On the other hand, we observed similar percent maximum 265 viability for CLQ pharmacophore and the other reference compounds at lower concentrations 266 (Table 3) . This suggests that, the cytotoxic effects may not be a concern at lower concentrations 267 of CLQ and its analogues. Additional concentrations need to be tested in future studies to 268 determine the actual CC 50 value (Table 3) . We determined the effect of CLQ, CLBQ14 and CLCQ on the exopeptidase activity of rhACE2 272 using an adapted fluorometric assay (https://bpsbioscience.com/pub/media/wysiwyg/79923.pdf). We found that all three compounds inhibited rhACE2 activity with similar IC 50 values in the low 274 micromolar concentration, with CLQ being the most potent amongst all three analogues tested, 275 at IC 50 of 5.36µM (Table 4) . To our knowledge, these results revealed for the first time that, 276 rhACE2 is a biochemical target of CLQ and its analogues. Because, the known metal cofactor 277 for ACE2 is Zinc 48, 61 , using the same fluorometric assay described above in the methods 333 activity and its interaction with Spike Protein. In this study, we also compared the dose-response 334 curves of antiviral effects of CLQ and its analogues with five other known inhibitors of SARS-CoV-found that CLQ's potency was better and comparable to Aloxistatin; but had lower efficacy than 337 the other reference inhibitors (Table 2) . It is important to note that the Vero E6 cells used for the 338 SARS-CoV-2 infection induced CPE assay were first sorted by flow cytometry by SRI for selection 339 of cells that had higher levels of ACE2 expression to increase the efficiency of infection. (Table 3) . This suggests that, the cytotoxic effects may not be a concern at lower 348 concentrations of CLQ and its analogues. In addition, the observed IC 50 values for inhibition of 349 rhACE2 exopeptidase activity and rhACE2-RBD interaction were in the low micromolar range, Recent studies have also shown that ACE2 plays a key role in protecting the lungs from 409 ARDS 67,68 , a severe complication of COVID-19 disease 4 . Therefore, one has to proceed 410 cautiously when targeting ACE2 66 ; without permanently inactivating its exopeptidase or other 411 cellular functions, to avoid potential adverse effects to heart and/or lung function. Our lead 412 compound CLQ is a weak metal chelator and zinc ionophore, that can shuttle free zinc across the function and prevent its interaction with SARS-CoV-2 RBD protein; without permanently inhibiting 415 its essential exopeptidase function. Because rhACE2 is a novel host target for CLQ and its 416 analogues, the potential effect of CLQ inhibition on heart and lung function needs to be further 417 explored in vivo and pre-clinical studies. The crystal structure of full length human ACE2 revealed that the RBD on SARS-CoV-2 419 S1 binds directly to the metallopeptidase domain (MPD) of ACE2 receptor 40,41 , that consists of 420 amino acid residues that coordinates zinc, providing further support for the utility of zinc chelators The strengths of our study includes, the use of a rapid multi-prong approach via three World Health Organization (WHO) Director-General's opening remarks at the media briefing on 520 COVID-19 Coronavirus disease 2019 (COVID-19) Situation Report A novel coronavirus from patients with pneumonia in China Clinical features of patients infected with 2019 novel coronavirus in Wuhan Potential Effects of Coronaviruses 527 on the Cardiovascular System: A Review Neurological Manifestations of Hospitalized Patients with COVID-19 in Wuhan, 530 China: A Retrospective Case Series Study Clinical characteristics of coronavirus disease 2019 in China Potential Antiviral Drugs Under Evaluation for the Treatment of COVID-19 COVID-19 Treatment Guidelines Current updates on the European and WHO registered clinical trials of 544 coronavirus disease 2019 (COVID-19) Clioquinol, an alternative antimicrobial agent against 550 common pathogenic microbe Effect of clioquinol, an 8-hydroxyquinoline derivative, on 552 rotavirus infection in mice RNA dependent DNA polymerase 554 (reverse transcriptase) from avian myeloblastosis virus: a zinc metalloenzyme Characterization of Clioquinol and Analogs as Novel Inhibitors of Methionine 557 Aminopeptidases from Mycobacterium tuberculosis Killing of non-replicating Mycobacterium tuberculosis by 8-33 Structure and function of the methionine aminopeptidases Control of protein life-span by N-terminal methionine 599 excision Zinc supplementation for the treatment or prevention of 601 disease: Current status and future perspectives Pyrithione and 8-hydroxyquinolines transport lead 603 across erythrocyte membranes Clioquinol and pyrithione activate TRPA1 605 by increasing intracellular Zn2+ Zinc-binding compounds induce cancer cell death via 608 distinct modes of action Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 Quantitative mRNA expression profiling of 621 ACE 2, a novel homologue of angiotensin converting enzyme Angiotensin converting 624 enzyme-2 (ACE2) and its possible roles in hypertension, diabetes and cardiac function. 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