pharmaceuticals Article Scaffold Hopping of α-Rubromycin Enables Direct Access to FDA-Approved Cromoglicic Acid as a SARS-CoV-2 MPro Inhibitor Hani A. Alhadrami 1,2,† , Ahmed M. Sayed 3,† , Heba Al-Khatabi 1,4, Nabil A. Alhakamy 5 and Mostafa E. Rateb 6,* 1 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, P.O. Box 80402, Jeddah 21589, Saudi Arabia; [email protected] (H.A.A.); [email protected] (H.A.-K.) 2 Molecular Diagnostic Lab, King Abdulaziz University Hospital, King Abdulaziz University, P.O. Box 80402, Jeddah 21589, Saudi Arabia 3 Department of Pharmacognosy, Faculty of Pharmacy, Nahda University, Beni-Suef 62513, Egypt; [email protected] 4 Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah 21589, Saudi Arabia 5 Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] 6 School of Computing, Engineering & Physical Sciences, University of the West of Scotland, Paisley PA1 2BE, UK * Correspondence: [email protected]; Tel.: +44-141-848-3072 † These authors equally contributed to this work. Citation: Alhadrami, H.A.; Sayed, A.M.; Al-Khatabi, H.; Alhakamy, N.A.; Rateb, M.E. Scaffold Hopping Abstract: The COVID-19 pandemic is still active around the globe despite the newly introduced of α-Rubromycin Enables Direct vaccines. Hence, finding effective medications or repurposing available ones could offer great help Access to FDA-Approved during this serious situation. During our anti-COVID-19 investigation of microbial natural products Cromoglicic Acid as a SARS-CoV-2 (MNPs), we came across α-rubromycin, an antibiotic derived from Streptomyces collinus ATCC19743, Pro M Inhibitor. Pharmaceuticals 2021, which was able to suppress the catalytic activity (IC50 = 5.4 µM and Ki = 3.22 µM) of one of the viral 14, 541. https://doi.org/10.3390/ key enzymes (i.e., MPro). However, it showed high cytotoxicity toward normal human fibroblasts ph14060541 (CC50 = 16.7 µM). To reduce the cytotoxicity of this microbial metabolite, we utilized a number of in silico tools (ensemble docking, molecular dynamics simulation, binding free energy calculation) to Academic Editors: Jean propose a novel scaffold having the main pharmacophoric features to inhibit MPro with better drug- Jacques Vanden Eynde and like properties and reduced/minimal toxicity. Nevertheless, reaching this novel scaffold synthetically Annie Mayence is a time-consuming process, particularly at this critical time. Instead, this scaffold was used as a Received: 3 May 2021 template to explore similar molecules among the FDA-approved medications that share its main Accepted: 3 June 2021 pharmacophoric features with the aid of pharmacophore-based virtual screening software. As a Pro Published: 5 June 2021 result, cromoglicic acid (aka cromolyn) was found to be the best hit, which, upon in vitro M testing, was 4.5 times more potent (IC50 = 1.1 µM and Ki = 0.68 µM) than α-rubromycin, with minimal Publisher’s Note: MDPI stays neutral cytotoxicity toward normal human fibroblasts (CC50 > 100 µM). This report highlights the potential with regard to jurisdictional claims in of MNPs in providing unprecedented scaffolds with a wide range of therapeutic efficacy. It also published maps and institutional affil- revealed the importance of cheminformatics tools in speeding up the drug discovery process, which iations. is extremely important in such a critical situation. Keywords: COVID-19; Streptomyces collinus; α-rubromycin; in silico; MPro; cromolyn; cheminformatics Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and The severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) pandemic is still a conditions of the Creative Commons serious global concern, and hope is hanging on vaccines to provide enough protection [1,2]. Attribution (CC BY) license (https:// However, searching for suitable antiviral medications is also highly required to assist creativecommons.org/licenses/by/ vaccines in containing this rapidly evolving infectious disease. 4.0/). Pharmaceuticals 2021, 14, 541. https://doi.org/10.3390/ph14060541 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 15 Pharmaceuticals 2021, 14, 541 2 of 14 The SARS CoV-2 main protease (MPro) is a key hydrolytic enzyme that can activate the viral polyprotein replication complex (1ab) by recognizing and cleaving its specific amino-acidThe SARS sequences. CoV-2 main Moreover, protease (Mit isPro amon) is a keyg the hydrolytic conserved enzyme proteins that can in activate the coronavirus familythe viral [3]. polyprotein Hence, it has replication attracted complex many (1ab) research by recognizing groups and and cleavingpharmaceutical its specific companies developingamino-acid specific sequences. and Moreover, effective it an is amongti-SARS the CoV-2 conserved therapeutics proteins in [4,5]. the coronavirus familyStructurally, [3]. Hence, M it hasPro occurs attracted in many a dimeric research arrangement groups and pharmaceutical (Figure 1), and companies its hydrolytic ac- tivitydeveloping depends specific on this and structural effective anti-SARS assembly CoV-2 [6,7]. therapeutics Each monomer [4,5]. comprises three domains Structurally, MPro occurs in a dimeric arrangement (Figure1), and its hydrolytic (I, II, and III), where the catalytic active site occurs in a junction between domains I and activity depends on this structural assembly [6,7]. Each monomer comprises three domains II.(I, On II, the and other III), where hand, the domain catalytic III active mediates site occurs the en inzyme a junction dimerization between domains to reach I and its II.final active formOn the[7,8] other (Figure hand, 1). domain The enzyme III mediates active the enzymesite has dimerization a conserved to catalytic reach its final dyad active consisting of bothform HIS-41 [7,8] (Figure and CYS-145,1). The enzyme via which active sitethe hasenzy a conservedme hydrolyzes catalytic protein dyad consisting peptide ofbonds. Any mutationsboth HIS-41 or andmodifications CYS-145, via that which occur the enzyme in these hydrolyzes two catalytic protein residues peptide bonds.eventually Any lead to a completemutations loss or modificationsof enzyme hydrolytic that occur in activity these two [9]. catalytic Recently, residues a number eventually of covalent lead to a inhibitors complete loss of enzyme hydrolytic activity [9]. Recently, a number of covalent inhibitors (i.e.,(i.e., able able to to formform a a covalent covalent bond bond with with CYS-145) CYS have-145) been have reported been [reported10], while noncovalent[10], while noncova- lentcompetitive competitive inhibitors inhibitors are underexplored are underexplored [11]. [11]. FigureFigure 1. (A 1.) Monomeric(A) Monomeric structure structure of of SARS SARS CoV-2CoV-2 M MProPro(PDB: (PDB: 6LU7) 6LU7) showing showing its three its three main domainsmain domains (I, II, and (I, III; II, blue, and III; blue, green,green, and andorange, orange, respectively). respectively). (B (B))M MPro activeactive sitesite showing showing the the catalytic catalytic dyad dyad (HIS41–CYS145). (HIS41–CYS145). (C) The dimeric(C) The active dimeric active form formof SARS of SARS CoV-2 CoV-2 MPro M.Pro . Natural products have offered many anti-SARS CoV-2 agents so far. Among them, ivermectinNatural and products artemisinin have have offered shown many promising anti-SARS clinical efficacy CoV-2 [ 12agents–16]. Consequently, so far. Among them, ivermectinwe aimed and to extend artemisinin our investigation have shown of microbialpromising natural clinical products efficacy to [12–16]. find potential Consequently, weanti-SARS aimed to CoV-2 extend drug our candidates investigation by targeting of microb its MialPro natural. The fermentation products to products find potential of anti- SARSStreptomyces CoV-2 collinusdrug candidatesATCC19743 by afforded targeting the quinone its MPro antibiotic. The fermentationα-rubromycin products as a major of Strepto- Pro mycesmetabolite collinus [17 ATCC19743]. Testing of this afforded metabolite the against quinone the antibiotic SARS CoV-2 α-rubromycin M revealed thatas a itmajor me- has great potential as an inhibitor. However, it also showed significant toxicity toward tabolite [17]. Testing of this metabolite against the SARS CoV-2 MPro revealed that it has normal human cell lines. Accordingly, we decided to modify its core structure (i.e., scaffold greathopping) potential with theas an aid inhibitor. of molecular However, docking, molecularit also showed dynamics significant simulation, toxicity and binding toward normal humanfree energy cell lines. calculation Accordingly, to get a morewe decided appropriate to modify scaffold its in core terms structure of cellular (i.e., toxicity. scaffold hop- ping)Furthermore, with the weaid appliedof molecular a pharmacophore-based docking, molecular virtual dynamics screening simulation, of FDA-approved and binding free energydrugs calculation using this modified to get a scaffold more appropriate as a template scaffold to find structurally in terms of similar cellular candidates toxicity. Further- more, we applied a pharmacophore-based virtual screening of FDA-approved drugs us- ing this modified scaffold as a template to find structurally similar candidates that can inhibit the MPro catalytic activity in vitro, thus allowing