key: cord-1013118-51db7c9c
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: 2021-03-11
journal: Heliyon
DOI: 10.1016/j.heliyon.2021.e06426
sha: aa41bf5f56d309886331af87690f91d49fa04c54
doc_id: 1013118
cord_uid: 51db7c9c

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease 2019 (COVID-19), has resulted in an ongoing pandemic. Presently, there are no clinically approved drugs for COVID-19. Hence, there is an urgent need to accelerate the development of effective antivirals. Herein, we discovered Clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a Food and Drug Administration (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 a 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 the clinical development of potential therapeutics for COVID-19.

between the human ACE2 receptor and the RBD in S protein of SARS-CoV-2 could 123 serve as a promising approach for the development of effective entry inhibitors for 124 potential prevention and/or treatment of In this study, we evaluated the effect of CLQ and two of its analogues (7-bromo-126 5-chloro-8-hydroxyquinoline (CLBQ14); and 5, 7-dichloro-8-hydroxyquinoline (CLCQ)) 127 on SARS-CoV-2 infection-induced cytopathic effect (CPE) in vitro. In addition, we 128 assessed the cytotoxicity of these compounds. Furthermore, we determined the impact 129 of all three compounds on recombinant human ACE2 (rhACE2) interaction with the RBD 130 on Spike protein of SARS-CoV-2; and independently assessed their effects on the virus infection, a 25µL aliquot of cells was added to columns 1-2 of each plate for the 183 cell only (no virus) controls. After incubating plates at 37°C/5%CO 2 and 90% humidity 184 for 72 hours, 30µL of Cell Titer-Glo (Promega) was added to each well. Luminescence 185 was read using a Perkin Elmer Envision or BMG CLARIOstar plate reader following 186 incubation at room temperature for 10 minutes to measure cell viability. Raw data from 187 each test well was normalized to the average (Avg) signal of non-infected cells (Avg 188

Cells; 100% inhibition) and virus infected cells only (Avg Virus; 0% inhibition) to 189 calculate % inhibition of CPE using the following formula: % inhibition = 100*(Test Cmpd Luminescent Cell Viability Assay (Severson et al., 2007) . Host cells in media were 199 added in 25µl aliquots (4000 cells/well) to each well of assay ready plates prepared with 200 test compounds as above. Cells only (100% viability) and cells treated with hyamine at 201 100µM final concentration (0% viability) serve as the high and low signal controls, 202 respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant 203 concentration for all wells (0.3%) as dictated by the dilution factor of stock test 204 compound concentrations. After incubating plates at 37°C/5%CO2 and 90% humidity 205 hr at room temperature (22°C) with shaking at 180rp m. Then, the solution was 252 discarded, and the wash step was repeated as described above. Next, the HRP-253 conjugated anti-goat IgG was added to each well, and the reaction plate was further 254 incubated for 1 hr at room temperature (22°C) with shaking at 180rpm. Again, the 255 solution was discarded, and the wash step was repeated as described above. Then, 256 100µL of 3,3',5,5'-tetramethylbenzidine (TMB) one-step substrate was added to each 257 well, and reaction mixtures were incubated in the dark at room temperature (22°C) with 258 shaking at 180rpm for an additional 30 mins and then stopped by the addition of 50µL 259 stop solution. The absorbance was read at 405 nm using a Beckman Coulter DTX880 260 multimode plate reader. The background hydrolysis was subtracted, and the data were 261 fitted to a special bell-shaped dose-response curve equation using GraphPad prism 262 software 8.4.3. 263

In our efforts to identify inhibitors of SARS-CoV-2 infection for the potential treatment of 268 COVID-19, we evaluated the in vitro antiviral activity of CLQ, and two of its derivatives, 269 CLBQ14 and CLCQ, using a standard luminescent-based high-throughput screening 270 inhibition at about 102.96% inhibition at 30µM (Table 1 ). In addition, we compared the 277 antiviral effects of CLBQ14 and its analogues with five other known inhibitors of SARS- of Aloxistatin (16.72µM); but moderately higher than Chloroquine (1.10µM), 284

Hydroxychloroquine (5.04µM), Remdesivir (4.42µM), and Calpain Inhibitor IV (0.41µM) 285 (Table 2) . These results suggest a potentially new mechanism of action for CLQ and its 286 congeners. Notably, this is the first report to our knowledge, revealing that CLQ and its 287 analogues effectively inhibit the novel SARS-CoV-2 infection-induced CPE. 288 289

We determined the preliminary cytotoxicity of CLQ and its analogues (CLBQ14 and 291 CLCQ), using a Cell Titer-Glo Luminescent Cell Viability Assay (Severson et al., 2007) . 292

We assessed the cytotoxic effects of the various compounds in Vero E6 cells and 293 observed that, the 50% cytotoxic concentration (CC 50 ) of CLQ and its derivatives were 294 all greater than 30 µM. However, in comparison to the other reference compounds 295 tested, CLQ and its analogues displayed lower percent minimum viability at higher 296 concentrations. On the other hand, we observed similar percent maximum viability for CLQ pharmacophore and the other reference compounds at lower concentrations 298 (Table 3) . This suggests that, the cytotoxic effects may not be a concern at lower 299 concentrations of CLQ and its analogues. Additional concentrations need to be tested in 300 future studies to determine the actual CC 50 value (Table 3) . 301

We determined the effect of CLQ, CLBQ14 and CLCQ on the exopeptidase activity of 303 rhACE2 using an adapted fluorometric assay (BPS Bioscience). We found that all three 304 compounds inhibited rhACE2 activity with similar IC 50 values in the low micromolar 305 concentration, with CLQ being the most potent amongst all three analogues tested at 306 IC 50 of 5.36µM (Table 4 ). To our knowledge, these results revealed for the first time that 307 rhACE2 is a biochemical target of CLQ and its analogues. Because the known metal 308 CLBQ14, we observed an increased shift in IC 50 value by over 28 fold compared to 315 CLBQ14 alone ( Figure 3 ). Interestingly, this data reveals that increasing concentrations 316 of ZnCl 2 titrates the inhibitory effect of CLBQ14 on rhACE2 from concentrations ranging 317 from above 5 -10µM, consistent with previous reports of the required optimal 318 concentration range of Zinc for the exopeptidase activity of ACE2 (Vickers et al., 2002) . 319

Taken together, these preliminary results reveal a new pharmacologic mode of action 320 and novel target for CLQ and its analogues. Table 4 . We found that all 333 three compounds had similar IC 50 values in the low micromolar concentration ranging 334 from 0.85 µM to 2.76 µM for IC 50_ 1; however, CLQ displayed a higher IC 50_2 at 18.15 335 µM ( Table 4 ). The unconventional dose response curve observed in this interaction 336 assay could be an indicator of additional binding site(s) and/or target(s) for the CLQ 337 pharmacophore, such as other sites on ACE2 or the Spike (RBD) protein. Again, these 338 findings are the first report to reveal that CLQ and its analogues inhibit and interfere with 339 the binding between human ACE2 receptor and SARS-CoV-2 Spike RBD protein in 340 vitro. These results suggest that the CLQ and its derivatives might be promising leads The availability of simple, rapid, cellular high throughput screening and well-361 characterized biochemical assays enabled us to quickly discover novel inhibitors of 362 SARS-CoV-2 infection in vitro. We successfully identified and characterized CLQ, a 363 known metal chelator, and Zinc ionophore as a novel inhibitor of SARS-CoV-2 infection-364 induced CPE. Using two structural analogues of CLQ (CLBQ14 and CLCQ) in hand, we 365 were able to further explore the impact of the active CLQ pharmacophore on the novel 366 coronavirus infection, the exopeptidase activity of rhACE2, and the interaction of rhACE2 367 with SARS-CoV-2 Spike (RBD) protein, all critical steps/processes in the pathogenesis of 368 COVID19. All three analogues displayed similar potent inhibition in the low micromolar 369 range against SARS-CoV-2 infection-induced CPE, rhACE2 activity, and its interaction 370 with Spike Protein. In this study, we also compared the dose-response curves of antiviral 371 effects of CLQ and its analogues with five other known inhibitors of SARS-CoV-2 in vitro: 372

Chloroquine, Hydroxychloroquine, Remdesivir, Aloxistatin, and Calpain Inhibitor IV and 373

found that CLQ's potency was better and comparable to Aloxistatin; but had lower 374 efficacy than the other reference inhibitors (Table 2) . It is important to note that the Vero 375 E6 cells used for the SARS-CoV-2 infection-induced CPE assay were first sorted by flow 376 cytometry by SRI for selection of cells that had higher levels of ACE2 expression to 377 increase the efficiency of infection. Therefore, the observed IC 50 values may be higher 378 than the actual IC 50 values in cells that do not have high levels of ACE2 expression. 379

Moreover, we observed that the IC 50 values of the compounds in the biochemical assays 380 were much lower than the IC 50 in the cellular antiviral assay. We also assessed the 381 cytotoxic effects of the compounds in Vero E6 cells and observed that CLQ and its 382 analogues displayed lower percent minimum viability at higher concentrations compared 383

to the other reference compounds tested. However, we observed similar percent 384 maximum viability for CLQ pharmacophore and the other reference compounds at lower 385 concentrations (Table 3 ). This suggests that the cytotoxic effects may not be a concern at 386 lower concentrations of CLQ and its analogues. In addition, the observed IC 50 values for 387 inhibition of rhACE2 exopeptidase activity and rhACE2-RBD interaction were in the low 388 micromolar range, suggesting that we may need lower concentrations for in vivo activity. 389

Furthermore, we have other preliminary cytotoxicity results from prior in vivo studies on 390 CLQ and its analogues that reveal no significant toxicity at much lower concentrations 391 below the nanomolar range (unpublished data). Therefore, additional in vivo cytotoxicity 392 studies for Vero E6 cells should be conducted at a wider range of concentrations. 393

Throughout our study, we consistently observed a correlation between the high 394 potency of CLQ compared to its other two analogues in the antiviral screen, inhibition of 395 rhACE2 metalloprotease activity, and its ability to disrupt the binding of rhACE2 with findings not only revealed a novel target (rhACE2) and mechanism of action for the CLQ 457 pharmacophore; but also provides insight into potential reversibility of inhibition and one 458 or more probable mode(s) of inhibition: 1) The concentration of CLBQ14 is titrated with 459 excess ZnCl 2 , thus pre-occupied and unavailable to inhibit rhACE2 exopeptidase activity; 460 and/or 2) potential competition for the similar binding sites on rhACE2. Additional 461 mechanistic kinetic studies will be required to ascertain this notion. properties, CLQ may temporarily or reversibly affect ACE2 function and prevent its 476 interaction with SARS-CoV-2 RBD protein; without permanently inhibiting its essential 477 exopeptidase function. Because rhACE2 is a novel host target for CLQ and its analogues, the potential effect of CLQ inhibition on heart and lung function needs to be 479 further explored in vivo and pre-clinical studies. 480

The crystal structure of full-length human ACE2 revealed that the RBD on SARS-481

CoV-2 S1 binds directly to the metallopeptidase domain (MPD) of the ACE2 receptor 482 affect ACE2 by reversibly chelating its Zinc ion, which is essential for ACE2 activity, as 496 well as interfere with ACE2-RBD interaction. Although Zinc is essential for stabilizing 497 protein structures and altering the substrate affinity of different metalloproteins (Ding et 498 al., 2005; Cox and McLendon, 2000) , the effects of Zinc chelation on the molecular catalytic site required for exopeptidase activity of ACE2 had no effect on Spike RBD 502 binding to ACE2 (Towler et al., 2004) . Howbeit, the unconventional dose-response bell 503

shaped curve that we observed in our studies suggests that there may be additional 504 binding sites and/or modes of action for CLQ and its congeners, resulting in the potent 505 inhibition of interaction at lower micromolar concentrations; compared to higher 506 concentrations. Although CLQ was found to be the most potent amongst all 3 analogues, 507 except for IC 50_2 , preliminary structure activity relationship studies (SAR) revealed that the 508 other two derivatives are comparable to CLQ, as they both show potent inhibition of 509 rhACE2-RBD interaction, as well as inhibition of antiviral and anti-rhACE2 activity. 510

Therefore, providing alternative analogues that might not have the same adverse effects 511 experienced with CLQ in the past, potentially alleviating some of the concerns with CLQ. 512

Additional biochemical and structural studies are required to explore other possible 513 mechanisms of action such as competition or interaction of CLQ with RBD for binding to 514 the MPD of ACE2, thereby preventing Zinc chelation and ionophore activity of CLQ. 515 Future X-ray structures could help to better understand the mode of inhibition of this 516 pharmacophore and the rational design of more potent drugs. 517

The strengths of our study includes the use of a rapid multi-prong approach via 518 three sensitive independent assays to identify and characterize an existing clinical drug 519 as a novel inhibitor of SARS-CoV-2 infection in vitro. In addition, the availability of 520 structural analogues of CLQ, made possible a preliminary SAR, which revealed 521 similarity between the IC 50 values of CLQ and its structural analogues. However, our 522 study has some limitations, such as the use of Vero E6 cells that were selected for high 523 expression of ACE2 in the antiviral assay, a HTS designed to rapidly screen for 524 inhibitors of infection-induced CPE. An additional limitation is that the amount of Zinc in 525 the purified rhACE2 supplied from BPS and RayBiotech assays was unknown. Future 526 metal dependent studies with apoenzymes will be required to determine the amount of 527

Zinc. Considering that CLQ is a known Zinc chelator and ionophore, an understanding 528 of the physiologic amount of Zinc required for inhibition will be critical for optimal 529 efficacy. Therefore, for these two limitations, the measured IC 50 values for the 530 compounds may not be representative of the actual in vitro IC 50 , which may be lower. 531

However, the remarkable consistency in the observed strong correlation between CLQ, 532 and its congener's antiviral activity, in vitro rhACE2 ,inhibition and disruption of ACE2-533 RBD protein interaction, reduce these concerns. analogues inhibit rhACE2 with antiviral activity but also suggests that CLQ 569 pharmacophore, potently disrupts the interaction of rhACE2 and Spike (RBD) protein.

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