chemosensors Review Current Trends in SPR Biosensing of SARS-CoV-2 Entry Inhibitors Elba Mauriz 1,2, * and Laura M. Lechuga 3 1 Department of Nursing and Physiotherapy, Campus de Vegazana, Universidad de León, s/n, 24071 León, Spain 2 Institute of Food Science and Technology (ICTAL), La Serna 58, 24007 León, Spain 3 Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST, and CIBER-BBN, Campus UAB, 08193 Barcelona, Spain; laura.lechuga@icn2.cat * Correspondence: elba.mauriz@unileon.es; Tel.: +34-987-293617 Abstract: The emerging risk of viral diseases has triggered the search for preventive and therapeutic agents. Since the beginning of the COVID-19 pandemic, greater efforts have been devoted to investigating virus entry mechanisms into host cells. The feasibility of plasmonic sensing technologies for screening interactions of small molecules in real time, while providing the pharmacokinetic drug profiling of potential antiviral compounds, offers an advantageous approach over other biophysical methods. This review summarizes recent advancements in the drug discovery process of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) inhibitors using Surface Plasmon Resonance (SPR) biosensors. A variety of SPR assay formats are discussed according to the binding kinetics and drug efficacies of both natural products and repurposed drugs. Special attention has been given to the targeting of antiviral agents that block the receptor binding domain of the spike protein (RBD-S)   and the main protease (3CLpro) of SARS-CoV-2. The functionality of plasmonic biosensors for high- Citation: Mauriz, E.; Lechuga, L.M. throughput screening of entry virus inhibitors was also reviewed taking into account experimental Current Trends in SPR Biosensing of parameters (binding affinities, selectivity, stability), potential limitations and future applications. SARS-CoV-2 Entry Inhibitors. Chemosensors 2021, 9, 330. https:// Keywords: plasmonic; SPR biosensor; viral diagnostics; virus entry inhibitors; drug screening; doi.org/10.3390/chemosensors9120330 antiviral agents; SARS-CoV-2; COVID-19 Academic Editors: Xudong Wang and Hongshang Peng 1. Introduction Received: 31 October 2021 Past pandemics of severe acute respiratory syndrome (SARS-CoV-1) in 2003, influenza Accepted: 22 November 2021 (H1N1, 1918 and 2009; H2N2, 1958; H3N2, 1968), Middle East respiratory syndrome (MERS) Published: 25 November 2021 in 2013, and Ebola virus disease in 2014, had previously menaced global health. However, the immense impact of the COVID-19 pandemic outburst in the health condition and so- Publisher’s Note: MDPI stays neutral cioeconomic prospects of the world population has reached unknown proportions [1]. The with regard to jurisdictional claims in published maps and institutional affil- unexpected risks of recurrent virus outbreaks have stressed the need for searching efficient iations. strategies to prevent viral infections [2]. Generally, antiviral therapeutic interventions rely on the development of candidate drugs following the ‘one bug, one drug’ approach [2,3]. This paradigm is insufficient to lead the fight against viruses due to: (i) the increasing number of emerging viruses; (ii) the presence of undiscovered mammalian virus in the wildlife reservoir; (iii) the lack of broad-spectrum antiviral agents; and (iv) the appearance Copyright: © 2021 by the authors. of drug resistances [4]. Licensee MDPI, Basel, Switzerland. Thus, the amelioration of viral infections requires the enlargement of drug candidates This article is an open access article distributed under the terms and with potent antiviral effect and broad-spectrum activities [3]. Novel drug discovery strate- conditions of the Creative Commons gies rely on the high throughput screening of different classes of antiviral agents [5]. For Attribution (CC BY) license (https:// example, the increase of antibody drug candidates has provided an efficient approach creativecommons.org/licenses/by/ to treat viral infections due to their specificity [6,7]. However, this selectivity is directed 4.0/). against specific antigens thus limiting their spectrum of activity. To overcome this problem, Chemosensors 2021, 9, 330. https://doi.org/10.3390/chemosensors9120330 https://www.mdpi.com/journal/chemosensors Chemosensors 2021, 9, 330 2 of 25 the design of antiviral agents has concentrated on the screening of low-molecular-weight fragments that can bind to membrane protein drug targets [5,8]. Fragment-based drug dis- covery has enabled the identification of a significant number of drugs approved for clinical use [9]. Owing to their low molecular mass, the binding with their target of interest may re- sult in low affinity interactions that require the employment of highly sensitive biophysical techniques for their estimation [8]. Among them, nuclear magnetic resonance (NMR) [10], X-ray crystallography [11], mass spectroscopy [12], isothermal titration calorimetry [13], protein thermal shift [14], affinity capillary electrophoresis [15], weak affinity chromatog- raphy [16], have been commonly used to assess protein–drug interactions. More recently, label-free biosensors based on Optical Waveguide Grating (OWG) [17] and reflectomet- ric interference spectroscopy (RIfS) [18] have emerged as valuable tools for evaluating biomolecular interactions [8]. From this perspective, surface plasmon resonance (SPR) biosensors seem to be ideally suited to address this challenge. The application of SPR biosensor technology in the pharmaceutical field has been extensively employed to investigate the binding properties of a broad range of drug candidates from nucleic acids to antibodies [19]. SPR biosensors provide label-free evaluations of molecular interactions between viral targets and their specific recognition element immobilized on the biosensor surface [8,20]. Specifically, SPR sensor devices detect the refraction index variations resulting from the interaction between the analyte and the immobilized bioreceptor occurring on the sensing layer after recognizing the target analyte (Figure 1) [21,22]. The main advantages of SPR biosensor over others biophysical techniques rely on both the short-time response and the capacity to monitor binding activities in real time. SPR analysis offers precise data on the kinetics and thermodynamics of the binding between virus target and the immobilized bioreceptor. SPR sensorgrams provide the association, equilibrium and dissociation phases observed after the injection of the analyte onto the immobilized bioreceptor surface [5,23]. By monitoring binding interactions, SPR biosensors inform on the association (Ka) and dissociation rates (Kd), equilibrium dissociation constants (KD) and stoichiometry of the interacting biomolecules depending on their binding affinity [6,8,24]. The kinetic information supplied by SPR biosensors is particularly useful for the rapid screening of drug candidates such as virus entry inhibitors. To date, SPR strategies have been successfully applied to the label-free sensitive detection of a wide variety of biomolecules including G-protein coupled receptors, antibodies, aptamers and enzymes [8,24,25]. The role of SPR biosensors in pharmaceutical analysis and viral diagnosis has been previously explored [5,8,19,20,26]. However, the specific utility of SPR biosensors for high-throughput screening of viral inhibitors in the current pandemic context has not been sufficiently addressed [5,6]. Therefore, the aim of this work is to compile recent develop- ments of SPR biosensor approaches in targeting SARS-CoV-2 entry into host cells. This review specifically concentrates on the identification and prioritization of viral inhibitors through the characterization of their binding and kinetic parameters. Current research in SPR antiviral drug development involving fragment-based drug discovery screening of COVID-19 candidate drugs is also reviewed. Chemosensors Chemosensors 2021,9,9,x330 2021, FOR PEER REVIEW 3 of 25 Figure1.1.(a)(a) Figure Surface Surface plasmon plasmon resonance resonance biosensing biosensing configuration. configuration. (b) Sensorgram (b) Sensorgram representat representation of the theinteraction interaction between the analyte between and immobilized the analyte biomolecules. and immobilized Reprinted with biomolecules. permission Reprinted withfrom permission Kumar Kumaretet al.al. [5][5] Copyright © 2021 Copyright © Wiley. 2021 Wiley. 2. Viral Entry and Drug Screening The role of SPR biosensors in pharmaceutical analysis and viral diagnosis has Viral entry is an indispensable condition to initiate the infection cycle by releasing the previously viral genome intoexplored [5,8,19,20,26]. the infected However, cell [2,5,27]. The the specific entry process in envelopedutility of involves viruses SPR biosensor high-throughput first screening the specific binding of the viralofsurface viral proteins inhibitors to theinreceptors the current pandemic of the cell context ha to be infected been sufficiently addressed [5,6]. Therefore, the aim of this work is to compile and the subsequent viral fusion with the host cell membrane [2,5,27]. Depending on the recen virus class, theof velopments activation of one type SPR biosensor or a set ofin approaches viral envelope targeting glycoproteins,entry SARS-CoV-2 triggers into host conformational This review specifically concentrates on the identification and prioritization A changes in the membrane proteins of both, the virus and the host [4]. of viral in series of intermediate stages comprising pre-fusion, pre-hairpin and post-fusion membrane itors through the characterization of their binding and kinetic parameters. Curren structural changes complete the viral fusion into the host cell (Figure 2). search in SPR Despite antiviral the process drug development complexity, the vast majority involving of virusesfragment-based present a similar drugcell disco screening entry of COVID-19 route regardless of theircandidate morphology, drugs is also genome reviewed. structure and life cycle. This feature is especially interesting for the design of universal drug strategies that contain viral infections. Tackling 2. Viralviral Entryentry andguarantees easier access to the virus target sites while preserving the Drug Screening integrity of the host cell [5]. Particularly, blocking the entry process into host cells seems to Viral entry be a promising way is to an indispensable protect against viral condition infection [2].toAmong initiate theentry viral infection cycle inhibitors, theby relea the viral genome into the infected cell [2,5,27]. The entry process in most common approaches for inhibiting virus fusion to host cells are based on peptides and enveloped vir involves small first the molecules. specific Although binding of inhibitors peptide-based the viral have surface beenproteins extensivelyto used the receptors to block of th viral fusion, recent strategies have set the focus on the screening of low-molecular-weight to be infected and the subsequent viral fusion with the host cell membrane [2,5,27] compounds pending on known as fragments the virus [8,28]. class, the activation of one type or a set of viral envelope glyco teins, triggers conformational changes in the membrane proteins of both, the virus the host [4]. A series of intermediate stages comprising pre-fusion, pre-hairpin and fusion membrane structural changes complete the viral fusion into the host cell (Figu Chemosensors 2021, Chemosensors 9, x9,FOR 2021, 330 PEER REVIEW 4 of 25 Figure 2.2.Scheme Figure Schemeof viral protein of viral binding protein to host receptor binding to hostand membrane receptor andfusion mediated membrane by viral fusion mediated proteins (hemagglutinin, HA). (a) Viral surface protein binding to the host cell receptor. (b) Viral proteins (hemagglutinin, HA). (a) Viral surface protein binding to the host cell receptor. protein initiates membrane fusion process with host cell membrane. Reprinted with permission from protein initiates membrane fusion process with host cell membrane. Reprinted with pe Kumar et al. [5] Copyright © 2021 Wiley. from Kumar et al. [5] Copyright © 2021 Wiley. Fragment screening has led to the discovery of a considerable number, more than Despite 30 clinical drugthe processincomplexity, candidates the[24]. the last 20 years vastThe majority of of utilization viruses present a similar fragment-derived compounds has produced significant benefits in drug discovery try route regardless of their morphology, genome structure and life cycle. since it provides large This fe areas of chemical space [29] and lesser probability of undesired interaction. Additionally, especially interesting for the design of universal drug strategies that contain vir the optimization of the molecular size may lead to improve the affinity and selectivity of the tions. Tackling fragments. However,viral entry binding guarantees between easier weight low molecular accesscompounds to the virus andtarget sites while p their ligands ing oftenthe integrity results of the host in low-affinity cell [5].Therefore, interactions. Particularly, blocking detection of low the entry requires fragments process into h the employment of highly sensitive biophysical methods (NMR, X-ray seems to be a promising way to protect against viral infection [2]. Among viral e crystallography, spectrometry, and so on). hibitors, the most common approaches for inhibiting virus fusion to host cells ar In this context, the SPR biosensor approach has contributed to the characterization and on peptides and pharmacokinetic small profiling of amolecules. Although peptide-based variety of fragment-based virus inhibitors ininhibitors have been the last decade. sively The roleused of SPRto block viral biosensors is notfusion, recent only limited strategies have to complementing otherset the focus screening on the scree techniques but also to informing on thecompounds low-molecular-weight kinetics and binding knownconstants of the low-affinity as fragments [8,28]. fragments. RecentFragment advances inscreening SPR biosensor technology have permitted the rapid has led to the discovery of a considerable screening,number, identifi- more cation, validation and confirmation of fragments directed against different types of targets clinical drug candidates in the last 20 years [24]. The utilization of fragment-deriv (proteases) [30], kinases [29], GPCRs [8] and protein–protein interactions (PPIs) [31]. The pounds following has produced sections significant considerations describe experimental benefits in drug as welldiscovery as strengthssince it provides larg and limitations of chemical in the detectionspace of virus[29] and entry lesser against inhibitors probability of undesired interaction. Additiona SARS-CoV-2. optimization of the molecular size may lead to improve the affinity and selectivit fragments. However, binding between low molecular weight compounds and t ands often results in low-affinity interactions. Therefore, detection of low fragm quires the employment of highly sensitive biophysical methods (NMR, X-Ray cry raphy, spectrometry, and so on). Chemosensors 2021, 9, x FOR PEER REVIEW 5 of 27 types of targets (proteases) [30], kinases [29], GPCRs [8] and protein–protein interactions Chemosensors 2021, 9, 330 (PPIs) [31]. The following sections describe experimental considerations as well 5 of 25 as strengths and limitations in the detection of virus entry inhibitors against SARS-CoV-2. 3. SPR 3. SPR Analysis Analysis of of SARS-CoV-2 SARS-CoV-2Entry EntryInhibitors Inhibitors Coronaviruses (order Coronaviruses (order Nidovirales, Nidovirales, family family Coronaviridae) Coronaviridae) are are positive-sense positive-sense single- single- stranded RNA (ssRNA) with large genomes (27 to 32 Kb stranded RNA (ssRNA) with large genomes (27 to 32 Kb in length) coiled inside a helicalin length) coiled inside a helical nucleocapsid. They belong to the subfamily Coronavirinae nucleocapsid. They belong to the subfamily Coronavirinae that includes four genera that includes four genera (al- pha (α)-, beta (β)-, gamma (γ)- and delta (δ)-CoVs) [32,33]. (alpha (α)-, beta (β)-, gamma (γ)- and delta (δ)-CoVs) [32,33]. Coronaviruses usually Coronaviruses usually contain canonical contain structural canonical proteinsproteins structural open reading frames (ORF) open reading frames1a(ORF)and 1b, 1a Eand(envelope protein), 1b, E (envelope M (membrane protein), protein),protein), M (membrane N (nucleocapsid protein) protein) N (nucleocapsid and S (spike and Sprotein) and some (spike protein) andaccesso- some ries proteins such as the membrane-anchored HE (hemagglutinin–esterase) accessories proteins such as the membrane-anchored HE (hemagglutinin–esterase) protein protein ex- pressed by betacoronaviruses [6]. Amongst this genus, expressed by betacoronaviruses [6]. Amongst this genus, SARS-CoV, MERS-CoV and SARS-CoV, MERS-CoV and SARS- CoV-2 are considered SARS-CoV-2 are consideredthe most thepathogenic most pathogenic humanhuman betacoronaviruses. betacoronaviruses. These new These corona- new virus types are primarily transmitted by aerosols and cause coronavirus types are primarily transmitted by aerosols and cause infections of the res- infections of the respiratory tract andtract piratory atypical and pneumonia. Their recurrent atypical pneumonia. break intobreak Their recurrent human intopopulations in 2002–2003 human populations in (severe acute 2002–2003 (severerespiratory syndrome, acute respiratory SARS-CoV) syndrome, and 2012and SARS-CoV) (Middle East respiratory 2012 (Middle syn- East respira- drome tory (MERS-CoV)) syndrome has ended (MERS-CoV)) haswith ended thewith ongoing global pandemic the ongoing global pandemic of coronavirus disease of coronavirus 2019 (COVID-19) caused by SARS-CoV-2 [32]. Coronaviruses disease 2019 (COVID-19) caused by SARS-CoV-2 [32]. Coronaviruses enter the infected enter the infected cellcell by attaching to cell surface receptors. Particularly, by attaching to cell surface receptors. Particularly, the fusion between the fusion between the virus and the virus and the cell cell membrane occurs membrane occurs through through the the binding binding of of the the virus virus SS protein protein and and thethe ACE2 ACE2 (angiotensin- (angiotensin- convertingenzyme converting enzyme2) 2) expressed expressedon on thethe membrane membrane of of host host cells cells from from the the respiratory respiratory tract, tract, myocardial tissue, myocardial tissue, kidney, kidney,intestine, intestine,male malereproductive reproductivecells, cells,eyes, eyes,vasculature vasculatureand andbrain brain amygdala amygdala [34–37](Figure 3). 3). The The SSprotein proteinisisnot notonly onlyresponsible responsible forfor giving giving thethe virus virus the the appearance of a crown [35] but also in facilitating both the appearance of a crown [35] but also in facilitating both the attachment and the entry into attachment and the entry into the host the host cells.cells. Specifically, Specifically, subunitsubunit S1 plays S1 plays an important an important role inrole in recognizing recognizing ACE2ACE2 recep- receptors tors sincesince it contains it contains the N-terminal the N-terminal domain domain (NTD), (NTD), the receptor the receptor binding binding domain domain (RBD) (RBD) and two andconserved two conserved subdomains subdomains (SD1 and (SD1SD2) and[6]. SD2)On[6]. theOn the other other hand, hand, subunit subunit S2 medi-S2 mediates the process ates the process of membrane of membrane fusion. fusion. The The characterization characterization of the of the binding binding affinities affinities be- between tween the theS1S1 main main domains domains (NTD (NTD andand RBD)and RBD) andthe theACE2 ACE2human humanmembrane membranereceptorreceptoris isofof primary importance to understand virus infectivity. For primary importance to understand virus infectivity. For example, binding affinities be- example, binding affinities between tween the theRBDRBDofofSARS-CoV-2 SARS-CoV-2and andhuman humanACE2 ACE2are are 22 22 times times higher higher thanthan those those ofof SARS- SARS- CoV, thus explaining the stronger infectious capacity of SARS-CoV2. CoV, thus explaining the stronger infectious capacity of SARS-CoV2. Therefore, inhibiting Therefore, inhibiting SARS-CoV-2 SARS-CoV-2entry entryintointohost hostcells cellsseemed seemedtotobe beessential essentialtotoblocking blockingviral viralinfection infection[2].[2]. Figure 3. The invasion model of SARS-CoV-2. SARS-CoV-2 first attaches to the cell surface with the aid of heparan sulfate, and then enters the host cell by interacting with ACE2. Reprinted with permission from Wang et al. [6] Copyright © 2021 Elsevier (under CC BY-NC-ND 4.0 license). Chemosensors 2021, 9, 330 6 of 25 3.1. SPR Strategies for Studying the Blocking of ACE2 Receptors Several SPR approaches have been involved in both pharmaceutical profiling of SARS- CoV-2 entry inhibitors and characterization of the kinetics during the entry process of SARS-CoV-2 into host cells. Most strategies focus on the ability of drug candidates to block ACE2-S-RBD interactions by examining their binding affinities with both ACE2 and RBD proteins. However, a limited number of studies have come forward to assume the detection of affinity constants while screening potential viral inhibitors. Table 1 summarizes the most singular approaches involving either the kinetic analysis or the screening of SARS-CoV-2 inhibitors capable of blocking the ACE2 receptor. The analytical performance characteristics of each method are specifically discussed below. 3.1.1. SPR Biosensor as Primary Screening Method Zhu et al. [38] investigated protein–protein interactions between human ACE2 and SARS-CoV-2 RBD proteins in search of drug inhibitors. The targeting of surface interactions allowed the discovery of demethylzeylasteral among a library of 960 compounds. First, the binding ability of the proteins was evaluated using ACE2-His as the captured ligand on the SPR sensor chip and S-RBD-mFc as the analyte. To improve the screening efficiency, several models established the contact and dissociation times according to the molecular weights of both the target protein and the screened compound. The screening and affinity analysis of low-molecular-weight compounds was performed following three steps, namely: clean screen, binding level screen and affinity screen. Clean screen permitted the exclusion of 13 compounds more likely to bind residually on the sensor chip surface due to their viscosity. Subsequently, binding level screen enabled the identification of compounds that could bind to the targets of interest and finally, a dose–response curve was assayed to determine their binding affinity. Since residence time was crucial for evaluating the drug efficacy, the association rate constant (kon ) and the duration of target occupancy measured by the dissociation rate constant (koff ) were examined through a kinetic fitting model. The results confirmed by Isothermal Titration Calorimetry (ITC), showed that demethylzeylasteral could bind to ACE2 and S-RBD with KD values of 1.736 and 1.039 M, respectively. The capacity for blocking ACE2-S-RBD interactions in a dose-dependent manner was also demonstrated by demethylzeylasteral via an SPR-based competitive assay (Figure 4). These results were confirmed by virtual docking, thereby showing that demethylzeylasteral could dock into the ACE2 binding area of S-RBD protein. Therefore, the feasibility of SPR technology as the main method to screen drug inhibitors without the support of traditional screening methods was demonstrated. Moreover, the utility of SPR analysis as a primary screening method has paved the way to acquire information on kinetics and affinity constants more easily by using a single-step format. Chemosensors 2021, 9, 330 7 of 25 Table 1. Key analytical features of SPR biosensor approaches for screening of SARS-CoV-2 inhibitors that block ACE2 receptor classified according to the characteristics of target analyte, analytical approach (namely sensing scheme or biological receptor) and binding affinities (KD equilibrium dissociation constant). Binding Affinities (KD: Equilibrium Target Analyte (MW, Common Uses) Analytical Approach SPR Instrument Reference Dissociation Constant) Protein–protein interaction testing Demethylzeylasteral (480.59 (g/mol); Screening and kinetic analysis (binding to Biacore T200 (Washington, DC, USA) KD = 1.736 mM; kon (1/Ms) = 1989; immunosuppressor, anti-inflammatory, ACE2 and S-RBD) or S200 instrument (GE Healthcare koff (×10− 3 1/s) = 3.345 (ACE2) [38] anti-tumoral) Competition assay (blocking Life Sciences). KD = 1.039 mM (S-RBD) RBD-ACE2 interactions) SPR screening combined approach Sodium lifitegrast (among Biacore KD = 1.92 nM (sodium lifitegrast) Kinetic analysis (binding to S-RBD) 21 screened compounds) S200 system (GE Healthcare (KD < 3 mM: rest of compounds) [39] Competition assay (blocking (637.5 g/mol; keratoconjunctivitis) Life Sciences) (kon and koff values N/A) RBD-ACE2 interactions) Biacore T200 Thymoquinone (164,201 g/mol; antioxidant, KD = 32.140 mM (kon and koff Binding affinity to ACE2 receptors System (GE Healthcare, [40] anti-inflammatory, chemotherapic) values N/A) Uppsala, Sweden) Ginsenoside Ra2, ginsenoside Rb1, KD = 55.6 mM (ginsenoside Ra2); ginsenoside Rb3, glycyrrhizic acid and KD = 29.7 mM (ginsenoside Rb3); BIAcore T200 instrument berberine chloride (1211.38, 1109.29, 1079.27, Binding activity of TCM-derived KD = 2.0 mM (ginsenoside Rb1); (BIAcore T200, GE [41] 822.94 and 371.81 g/mol respectively; components with SARS-CoV-2 S1 subunit KD = 66.8 mM (glycyrrhizic acid); Healthcare, Chicago, IL, USA) herbs within clinically KD = 23.9 mM (berberine chloride) effective TCM schemes) (kon and koff values N/A) Quinoline-2-carboxylic acids (3 compounds) Binding affinity constants (to S-RBD) BIAcore T200 instrument (GE KD = 0.60–5.37 mM (173.168 g/mol, Ephedra sinica extracts; lung Competition assay (blocking [42] Healthcare Life Sciences) (kon and koff values N/A) diseases treatment) RBD-ACE2 interactions) Radix Scutellariae extract: Oroxylin A Open SPR™ (Nicoya Lifesciences, KD = 9.72 × 10−6 M (284.26 g/mol; respiratory diseases, diabetes, Binding affinity constants (to ACE2) [43] Waterloo, Canada) (kon and koff values N/A) diarrhea treatment) Binding affinity constants (to S-RBD) Polyphenols: corilagin and TGG (636.46 and Binding affinity of TGG and corilagin KD = 1.8 nM (corilagin) 423.40 g/mol, respectively; antioxidant, with RBD mutations of three main Biacore T200 instrument KD = 1.3 nM (TGG) [44] anti-inflammatory, and SARS-CoV-2 variants (GE Healthcare) (kon and koff values N/A) antidiabetic treatments) Competition assay (blocking RBD-ACE2 interactions) Chemosensors 2021, 9, 330 8 of 25 Table 1. Cont. Binding Affinities (KD: Equilibrium Target Analyte (MW, Common Uses) Analytical Approach SPR Instrument Reference Dissociation Constant) Erythrodiol (442.7 g/mol; Momordica charantia component: treatment of Binding affinity constants (to SARS-CoV-2 Open SPR instrument KD = 1.15 µM [45] nephropathy, neuropathy, S1 subunit) (Nicoya Lifescience, ON, Canada) (kon and koff values N/A) gastroparesis, cataracts and atherosclerosis) Histamine H1 receptor antagonists: doxepin, KD = 9.54 mM (doxepin); chlorpheniramine, and doxylamine (279.376, Analysis of bimolecular interactions with Open SPRTM (Nicoya, KD = 0.30 mM (chlorpheniramine); 274.788 and 270.369 g/mol respectively; [46] ACE2 receptor Waterloo, Canada) KD = 47.3 mM (doxylamine) allergic rhinitis, allergic conjunctivitis and (kon and koff values N/A) allergic dermatitis) Azelastine (381,898 g/mol; Kinetic analysis (binding to Open SPRTM (Nicoya, KD = 0.258 µM [47] allergic conjunctivitis) ACE2 receptor) Waterloo, Canada) (kon and koff values N/A) KD = 9.13 µM (desloratadine); Desloratadine and loratadine (310.82 and Kinetic analysis (binding to Open SPRTM (Nicoya, KD = 0.1.02 µM (loratadine) [48] 382.88 g/mol; treatment of allergic disease) ACE2 receptor) Waterloo, Canada) (kon and koff values N/A) Chloroquine (CQ) and KD = (7.31 ± 0.62) × 10−7 M (CQ) hydroxychloroquine (HCQ) Kinetic analysis (binding to Open SPRTM (Nicoya, KD = (4.82 ± 0.87) × 10−7 M (HCQ) [49] (319.872 and 335.872 g/mol; ACE2 receptor) Waterloo, Canada) (kon and koff values N/A) antimalarial drugs) KD = (7.03 ± 3.28) × 10−6 M (Tri); Trifluoperazine (Tri); thioridazine (Thi); KD = (8.91 ± 5.25) × 10−5 M (Thi); chlorpromazine (Chl), aripiprazole (Ari), Kinetic analysis (binding to Open SPRTM (Nicoya, KD = (1.38 ± 0.38) × 10−5 M (Chl); tiapride (Tia) (407.497, 370.6, 318.86, 448.385 [50] ACE2 receptor) Waterloo, Canada) KD = (7.88 ± 0.49) × 10−6 M (Ari), and 328,427 g/mol, respectively; and (3.33 ± 3.13) × 10−5 M (Tia) antipsychotic drugs) (kon and koff values N/A) MW: Molecular weight; kon = association rate; koff = dissociation rate; KD = equilibrium dissociation constants; N/A = not available; TCM: traditional Chinese medicine. pendent manner was also demonstrated by demethylzeylasteral via an SPR-based com- petitive assay (Figure 4). These results were confirmed by virtual docking, thereby show- ing that demethylzeylasteral could dock into the ACE2 binding area of S-RBD protein. Therefore, the feasibility of SPR technology as the main method to screen drug inhibitors Chemosensors 2021, 9, 330 9 of 25 without the support of traditional screening methods was demonstrated. Moreover, the utility of SPR analysis as a primary screening method has paved the way to acquire infor- mation on kinetics and affinity constants more easily by using a single-step format. Figure 4. (a) Sensorgrams of compounds identified from the affinity screening bound to ACE2; (b) sensorgrams of compounds identified from the affinity screening bound to S-RBD. (c) Chemical structures of compounds discovered during SPR affinity screening. Reprinted with permission from Zhu et al. [38]. Copyright © 2021 MDPI. Following the same trend, a multidisciplinary approach took advantage of a dual strategy involving both molecular docking and SPR combined screens [39]. Day et al. reported a blended procedure comprising molecular modeling and SPR interrogation to identify potential blocking candidates of RBD SARS-CoV-2–ACE2 interactions, from two libraries of 57,641 and 3141 compounds, respectively. Primarily, the role of SPR biosensing in these experiments consisted of the initial screening of compounds that bound ACE2, followed by the analysis of binding affinities of either the compounds identified by molecular docking or SPR. Measured affinities were in agreement with those predicted by molecular docking. The combination of both screening techniques allowed the characterization of the binding affinities of 17 compounds for ACE2 and 6 for SARS- CoV-2 RBD that showed <3 mM affinity to their respective protein targets. An SPR-based competition assay was also implemented to examine the ability of 12 ACE2 and 6 RBD binders for blocking RBD–ACE2 interactions. By using immobilized ACE2, SPR analysis demonstrated the ability of 11 of the 12 compounds to inhibit the interaction between a RBD mimic-peptide and recombinant human ACE2. Among them, two compounds, Evans blue and Irinotecan, could block completely the RBD–mimic peptide–ACE2 interaction. Virus-like particles (VPL) expressing SARS-CoV-2 spike protein were also immobilized in the SPR sensor chips to study the blockade of human ACE2–VLP spike protein interactions. Using this immobilization format one compound, sodium lifitegrast, demonstrated the elimination of 99.8% of the RBD–ACE2 interaction. Finally, 11 compounds were selected to prove their antiviral potency against SARS-CoV-2 infection of Vero-E6 cells. These Chemosensors 2021, 9, 330 10 of 25 experiments demonstrated that three compounds (Evans blue, lifitegras and lumacaftor) could block SARS-CoV-2 activity and exhibited dose-dependent antiviral in vitro potency without showing cytotoxicity. In this work, SPR biosensing proved its practical functionality not only to repurpose and identify potential drugs as SARS-CoV-2 entry inhibitors but also to design chemical scaffolds for drug development. 3.1.2. Kinetic Analysis of Natural Products Obtained from Plants Despite these advances, the number of studies that report combined screens of molec- ular docking and SPR is still scarce. Most of the strategies make use of SPR studies to determine binding affinities between drug candidates and protein receptors. Several works have focused on the characterization of SARS-CoV2 potential inhibitors by examining the affinity with their target proteins. In this sense, the discovery of new inhibitors from the repurposing of commercial medicines has played a significant role. For instance, Xu et al. confirmed the binding of thymoquinone, a phytochemical compound obtained from the plant Nigella sativa to ACE2 receptor [40]. SPR analysis revealed the capacity of thymo- quinone as a potential drug candidate to block ACE2 receptor. By flowing thymoquinone over the sensor surface immobilized with ACE2 protein, thymoquinone–ACE2 interactions showed a strong dependence on the dosage with an equilibrium dissociation constant (KD) of 32.140 mM. These results suggest that thymoquinone may block the virus entry into the host cell by interfering the S1-ACE2 binding. This assumption was confirmed by half maximal inhibitory concentration (IC50) and half maximal cytotoxic concentration (CC50) values for SARS-CoV-2 pseudovirus. However, the specificity of the assays should be further studied because of the pan-assay interference properties of thymoquinone. The search for spike protein inhibitors among bioactive compounds of traditional Chinese medicine (TCM) has also been explored [41]. SPR analysis was applied to the determination of the binding activity of 66 TCM-derived components with SARS-CoV-2 S1 subunit. From the 66 components selected by molecular docking virtual screening, 19 were excluded due to their insolubility in the assay buffer (HBS-EP+). The binding activity of the remaining 44 compounds was verified using S1 subunits of SARS-CoV-2, MERS-CoV and SARS-CoV as immobilized receptors. The characterization of the binding activities demonstrated that five compounds: ginsenoside Ra2 (ZZY-8), ginsenoside Rb1 (ZZY-9), ginsenoside Rb3 (ZZY-13), glycyrrhizic acid (ZZY-44) and berberine chloride (ZZY-54) showed high affinity for the SARS-CoV-2 S1 subunit, being concentration-dependent, with KD values between 0.47 and 9.44 mM. Similar binding activity was found with S1 subunits of MERS-CoV while those obtained for SARS-CoV were reduced. The high affininities of these five active compounds with the binding interface of SARS-CoV-2 S1 could be exploited to block the interaction between the S protein and human ACE2 protein. The five compounds discovered by SPR were further investigated to determine their activity in the interaction between the RBD of SARS-CoV-2 and ACE2, by a NanoBit assay. The results showed that glycyrrhizic acid (ZZY-44) was the best candidate to disrupt the interaction between the RBD of SARS-CoV-2 and ACE2, exhibiting no toxicity at high concentrations. Although SPR analysis did not contribute to the primary screening of TCM compounds, the characterization of the binding activities with S1 subunits of SARS-CoV-2, led to the discovery of glycyrrhizic acid as a potential nontoxic broad-spectrum anti-coronavirus multitarget inhibitor. Mei et al. followed a similar approach for discovering active compounds in Ephedra sinica, a broadly used medicinal plant in traditional Chinese medicine [42]. Particularly, it was investigated with regards to its capacity to disrupt the interaction between ACE2 and the SARS-CoV-2 spike protein RBD. First, SPR biosensing was used to validate the inhibition activity of SARS-CoV-2 RBD/ACE2 interaction. By immobilizing RBD on the sensor chip, ACE2 could bind to RBD in a dose-dependent manner with an affinity of 20.19 nM. Similarly, the kinetics and binding activity of the compounds previously identified by HPLC-Q-TOF-MS/MS and NMR were also evaluated. Finally, SPR competition inhibition assays were performed to explore the inhibitory effect of the selected compounds (Figure 5). Chemosensors 2021, 9, 330 11 of 25 The interference of these compounds with the binding interface of SARS-CoV-2 RBD was demonstrated by flowing the RBD–compound mixture over immobilized ACE2. The results showed that three compounds (quinoline-2-carboxylic acids) could block the ACE2–RBD interaction, thus suggesting their potential to inhibit SARS-CoV-2 virus infection. The inhibitory efficacy on ACE2-RBD interaction of these quinoline-2-carboxylic acids was verified by molecular docking. This work exemplifies the contribution of SPR technology Chemosensors 2021, 9, x FOR PEER REVIEW 12 of in 27 supporting other techniques to discover potential therapeutic compounds in the treatment of COVID-19. Figure 5. Quinoline-2-carboxylic acids inhibited ACE2-RBD ACE2-RBD interaction interaction by by directly binding binding to SARS-CoV-2 RBD. (a) The time-of-addition experimental scheme. (b) The relative binding amount under different time-of-addition experimental scheme. (b) The relative binding amount under different conditions.conditions. Compound Compound 2 were 2 were in- injected in two-fold serial dilutions over SARS-CoV-2 RBD (c) or ACE2 (d) immobilized on the biosensor chip. Compound jected in two-fold serial dilutions over SARS-CoV-2 RBD (c) or ACE2 (d) immobilized on the biosensor chip. Compound 1 (e) 1 (e) or 3 (f) were injected in two-fold serial dilutions over SARS-CoV-2 RBD immobilized on the biosensor chip. (g) Vari- or 3 (f) were injected in two-fold serial dilutions over SARS-CoV-2 RBD immobilized on the biosensor chip. (g) Various ous amounts of compound 2 were incubated with 22.5 nM SARS-CoV-2 RBD◦ at 37 °C for 1 h, then each mixture was amounts of compound 2 were incubated with 22.5 nM SARS-CoV-2 RBD at 37 C for 1 h, then each mixture was injected injected over ACE2 immobilized on the biosensor chip. TCM-derived components. Reprinted with permission from Mei over et al. ACE2 immobilized [41]. Copyright on the © 2021 biosensor chip. TCM-derived components. Reprinted with permission from Mei et al. [41]. Elsevier. Copyright © 2021 Elsevier. Gao Gao et etal. al.reported reportedthethe combined combined useuse of molecular docking of molecular and and docking SPR analysis for eval- SPR analysis for uating evaluating the binding properties of oroxylin A (another TCM-derived compound)ACE2 the binding properties of oroxylin A (another TCM-derived compound) with with protein [43]. CMC ACE2 protein method [43]. CMC was was method usedused as aasscreening method a screening method totoisolate isolateoroxylin oroxylinAA from from Radix Scutellariae extract. After proving the anti-viropexis effect of the screened Radix Scutellariae extract. After proving the anti-viropexis effect of the screened compound compound in in HEK293T HEK293T cells cells expressing expressing ACE2 ACE2 receptors, receptors, SPR SPR assays assays confirmed confirmed that that oroxylin oroxylin A A could could bind bind to to ACE2 ACE2 (KD(KD ==9.72 −6 − 9.72××1010 M),M), 6 thereby paving thereby thethe paving waywayto further to furtherinvestigation on the investigation on antiviral properties the antiviral of this properties plant of this extract. plant extract. Polyphenols Polyphenols havehavealso alsobeen beenconsidered consideredasaspotential antiviral potential antiviraldrug candidates drug for for candidates in- hibiting SARS-CoV2 infection. The therapeutic potential of polyphenols inhibiting SARS-CoV2 infection. The therapeutic potential of polyphenols obtained obtained from plants have been investigated by molecular docking and molecular dynamics (MD) sim- ulations. Several natural compounds which can block the interaction with ACE2 have been recently identified. Among them, the interaction of corilagin (C27H22O18) and 1,3,6- tri-Ogalloy-b-D-glucose (TGG) (C27H24O18) with Spike protein/ACE2 interface have been examined by numerical methods to further evaluate their binding affinities with SPR Chemosensors 2021, 9, 330 12 of 25 plants have been investigated by molecular docking and molecular dynamics (MD) simula- tions. Several natural compounds which can block the interaction with ACE2 have been recently identified. Among them, the interaction of corilagin (C27H22O18) and 1,3,6-tri- Ogalloy-b-D-glucose (TGG) (C27H24O18) with Spike protein/ACE2 interface have been examined by numerical methods to further evaluate their binding affinities with SPR and ELISA [44]. SPR experiments indicated that both polyphenols could bind to the immobi- lized RBD, showing equilibrium dissociation constants in the nanomolar range, 1.8 nM for corilagin/RBD and 1.3 nM for TGG/RBD, respectively. On the other hand, a significant binding to ACE2 was not observed of either corilagin, or TGG in the 1 to 80 nM range (Figure 6). The interactions between ACE2 and RBD were also explored, displaying KD values of 41 nM (ACE2 to immobilized RBD) and 63 nM (RBD to immobilized ACE2), as cor- roborated by other studies. Finally, the ability of these compounds to prevent RBD/ACE2 interactions were evaluated flowing TGG/corilagin mixtures previously incubated with RBD over ACE2 immobilized sensor chips. SPR assays demonstrated that both TGG (12.5 nM) and corilagin (50 nM) could inhibit RBD/ACE2 binding. Furthermore, the bind- ing affinity of TGG and corilagin with RBD mutations of three main SARS-CoV-2 variants (E484K, N501Y and E484K/N501Y) was determined using MD simulations. Overall, the numerical approach suggested that the RBD residues remain accessible at the interface with ACE2 despite these mutations. Although SPR experiments only served to validate the numerical protocol, these results show that corilagin did not interfere with the recognition of ACE2 by RBD for the structures with the N501Y mutant while TGG was more effective in these mutants. Therefore, further investigation is needed to monitor the selectivity of these compounds with regards to other virus-binding sites and their in vivo behavior. The interest in natural remedies has led to the investigation of the anitviral properties of other compounds obtained from vegetables. For instance, erythrodiol, an extract compo- nent of Momordica charantia, commonly known as bitter melon, was identified by molecular docking to study their capacity to inhibit SARS-CoV-2 infections [45]. In this work, SPR biosensor was used to examine the interaction of erythrodiol and S1 and S2 domains of the SARS-CoV-2 spike protein. SPR results revealed that erythrodiol has a strong binding affinity (equilibrium dissociation constant, KD = 1.15 µM) with S2, the active site of the SARS-CoV-2 spike protein. Therefore, the combination of docking, ADME (absorption, distribution, metabolism and excretion) properties, and SPR contributed to confirm the inhibition activity of erythrodiol to block the binding of the spike protein. 3.1.3. SPR Role in Drug Repurposing Other interesting approaches have made use of SPR biosensing for drug repurposing of histamine H1 receptor antagonists [46,47]. Antihistamine drugs are commonly used to treat allergic diseases by downregulating allergic inflammation. However, several studies have recently described their effect as inhibitors of Ebola, Marburg [51], and influenza viruses [52] into host cells. By following this hypothesis, Ge et al. proved the antiviral properties of histamine H1 receptor antagonists through the evaluation of their binding affinities with ACE2 receptor. On that premise, a series of histamine H1 receptor an- tagonists with ACE2 binding activity were screened by cell membrane chromatography (CMC). SPR assays were performed to detect biomolecular interactions of the ACE2 pro- tein immobilized on the sensor chip and three compounds (doxepin, chlorpheniramine, doxylamine) identified by CMC. The calculation of the kinetic values showed equilibrium dissociation constants (KD) of 9.54 ± 0.30, 10.5 ± 2.1 and 47.3 ± 5.2 µM for fordoxepin, chlorpheniramine and doxylamine, respectively. The smallest value was obtained for doxepin, being in accordance with the activity showed for inhibiting pseudovirus entry. SPR measurements were further confirmed by analyzing the docking conformation of doxepin with ACE2, demonstrating that the binding to the S protein prevented the binding with ACE2 protein leading to the subsequent inhibition of the virus entry. A similar con- cept was applied by the same authors for monitoring the binding of azelastine, another antihistamine, to ACE2. Direct binding of azelastine to immobilized ACE2 showed Kd Chemosensors 2021, 9, 330 13 of 25 values of 0.258 ± 0.48 mM, exhibiting a stronger inhibitory effect at low concentrations. Chemosensors 2021, 9, x FOR PEER REVIEW Therefore, 14 of SPR analysis contributed to the discovery of dual-target clinical drugs that can27 inhibit virus entry while reducing the inflammatory response evoked by COVID-19. Time (sec) Time (sec) Figure 6. Figure 6. Characterization Characterization of of molecular molecular interactions interactions by by surface surface plasmon plasmon resonance. resonance. (A (A and and B) B) Binding Binding kinetics kinetics of of corilagin corilagin and TGG on immobilized (a) RBD and (b) ACE2. The recombinant proteins RBD and ACE2 are respectively and TGG on immobilized (a) RBD and (b) ACE2. The recombinant proteins RBD and ACE2 are respectively immobilized on a immobilized on a CM5 sensor chip and increasing concentrations of polyphenols are injected to evaluate binding kinetics. (c) Kinetics CM5 sensor chip and increasing concentrations of polyphenols are injected to evaluate binding kinetics. (c) Kinetics of ACE2 of ACE2 binding to immobilized RBD (left panel) and kinetics of RBD binding to immobilized ACE2 (right panel). (d) Pre- binding to immobilized RBD (left panel) and kinetics of RBD binding to immobilized ACE2 (right panel). (d) Pre-incubation incubation of RBD (50 nM) for 30 min with increasing concentrations of corilagin or TGG inhibit the binding of RBD to of RBD (50 nM) immobilized for 30 ACE2. min withwith Reprinted increasing concentrations permission of corilagin from Binette et al. [44] or TGG inhibit Copyright the Royal © 2021 binding of RBD Society. Chemical to immobilized ACE2. Reprinted with permission from Binette et al. [44] Copyright © 2021 Royal Chemical Society. The interest in natural remedies has led to the investigation of the anitviral properties of other compounds obtained from vegetables. For instance, erythrodiol, an extract com- ponent of Momordica charantia, commonly known as bitter melon, was identified by mo- lecular docking to study their capacity to inhibit SARS-CoV-2 infections [45]. In this work, SPR biosensor was used to examine the interaction of erythrodiol and S1 and S2 domains Chemosensors 2021, 9, 330 14 of 25 In this framework, the antiviral effect of histamine H1 antagonist approved drugs loratadine (LOR) and desloratadine (DES) has also been investigated. A combined approach involving cell membrane chromatography and SPR sensing was developed to determine the binding affinity of loratadine and desloratadine to ACE2 receptor [48]. On the one hand, CMC showed that the specific binding durations of LOR and DES to ACE2 receptor were 2.49 and 51.8 min, respectively, showing that desloratadine could bind to ACE2 receptor more strongly than loratadine. These findings were confirmed by SPR assays where desloratadine exhibited higher binding constants (KD = 9.13 ± 0.67 × 10−6 M) than loratadine (1.02 ± 0.38 × 10−7 M). Similarly, molecular docking determinations proved that desloratadine could bind to ACE2 through one hydrogen bond with LYS31 while loratadine did not form hydrogen bonds, thus validating the SPR results. Since the blocking effect was only measured on the entry of SARS-CoV-2 spike pseudotyped virus into ACE2h cells, the efficacy of loratadine and desloratadine may need further evaluation in native viruses and animal models. Chloroquine (CQ) and hydroxychloroquine (HCQ) are well-known compounds for their antimalarial properties. However, their use as anti-COVID drugs remains controver- sial due to the risk of cardiotoxicity reported in some clinical trials [53]. At the same time, other studies suggested that CQ and HCQ could disrupt ACE2 terminal glycosylation thus preventing the binding with viral S protein. The ability to block ACE2 binding and inhibit virus entry into host cells has been explored by using an SPR approach [49]. Wang et al. performed SPR assays to assess the binding activity of CH and HCQ with ACE protein immobilized on the sensor chips. SPR studies confirmed virtual molecular docking tests, showing binding constants KD of (7.31 ± 0.62) × 10−7 M and (4.82 ± 0.87) × 10−7 M, for CQ and HCQ, respectively. These results demonstrated that CQ and HCQ could bind to ACE2 protein. Despite these findings, the unwanted effects caused by CQ and HCQ together with the inefficiency to reduce deaths from COVID-19 suggest that the role of CQ and HCQ as inhibitory drugs in suppressing the entrance of the SARS-CoV-2 virus while blocking infection of human cells will need further confirmation. The search for new uses of already existing drugs has led to the investigation of the antiviral properties of antipsychotic drugs [50]. Previous studies have suggested that antipsychotic drugs can inhibit replication of SARS-CoV or MERSCoV viruses, Hepatitis B Virus (HBV), measles virus germination, and HIV infection [54]. On the other hand, preclinical studies have reported that psychotropic medical treatments can protect from severe forms of COVID-19. Based on these considerations, Lu et al. examined the affinity for ACE2 of five antipsychotic drugs previously screened by cell membrane chromatogra- phy. The binding of these compounds (trifluoperazine (Tri); thioridazine (Thi); Chlorpro- mazine (Chl), aripiprazole (Ari), tiapride (Tia)) with ACE2 protein, showed high affinity as demonstrated by their KD values: (7.03 ± 3.28) × 10−6 M, (8.91 ± 5.25) × 10−5 M, (1.38 ± 0.38) × 10−5 M, (7.88 ± 0.49) × 10−6 M and (3.33 ± 3.13) × 10−5 M, respectively (Figure 7). SPR studies were complemented with molecular docking to identify modified regions. This work suggested that antipsychotic drugs with phenothiazine scaffolds had potential antiviral ability. However, they were not completely effective for preventing cell invasion, since these antipsychotic drugs bind to different amino acids, thereby blocking small areas of SARSCoV-2 binding to ACE2. As a result, the use of combined cocktails of these drugs could contribute to preventing virus infections. were complemented with molecular docking to identify modified regions. This work sug- gested that antipsychotic drugs with phenothiazine scaffolds had potential antiviral abil- ity. However, they were not completely effective for preventing cell invasion, since these antipsychotic drugs bind to different amino acids, thereby blocking small areas of Chemosensors 2021, 9, 330 SARSCoV-2 binding to ACE2. As a result, the use of combined cocktails of these drugs 15 of 25 could contribute to preventing virus infections. (a) (b) Figure 7. Figure 7. Study Study of of the the binding binding activity activity of of five five antipsychotic antipsychoticdrugs: drugs:trifluoperazine trifluoperazine(Tri); (Tri);thioridazine thioridazine(Thi); (Thi);chlorpromazine chlorpromazine (Chl), aripiprazole (Ari), tiapride (Tia). (a) The SPR analysis of the five antipsychotic drugs. (b) The virtual (Chl), aripiprazole (Ari), tiapride (Tia). (a) The SPR analysis of the five antipsychotic drugs. (b) The virtual molecular molecular docking analysis of the five APDs. Reprinted with permission from Lu et al. [50]. Copyright © 2021 Elsevier. docking analysis of the five APDs. Reprinted with permission from Lu et al. [50]. Copyright © 2021 Elsevier. 3.1.4. SPR Biosensors as Diagnostic Tools to Detect Neutralizing Antibodies 3.1.4. SPR Biosensors as Diagnostic Tools to Detect Neutralizing Antibodies Alternatively, SPR biosensing has been tested as a diagnostic method to validate the Alternatively, SPR biosensing has been tested as a diagnostic method to validate the results obtained by enzyme-linked immunosorbent assay (ELISA) for detecting receptor- results obtained by enzyme-linked immunosorbent assay (ELISA) for detecting receptor- binding antibodies in sera. SPR serological assays were conducted to demonstrate anti- binding antibodies in sera. SPR serological assays were conducted to demonstrate antibody body binding to the SARS-CoV-2 spike protein while blocking the ACE2 receptor [55]. binding to the SARS-CoV-2 spike protein while blocking the ACE2 receptor [55]. Specif- Specifically, the role of SPR biosensing consisted of the assessment of serological re- ically, the role of SPR biosensing consisted of the assessment of serological responses of sponses of different antibodies’ epitopes when competing with ACE2 or blocking the different antibodies’ epitopes when competing with ACE2 or blocking the binding to SARS- binding to SARS-CoV-2. Therefore, by evaluating the neutralizing activity of antibodies, CoV-2. Therefore, by evaluating the neutralizing activity of antibodies, SPR assays proved SPR assays proved their utility to target viral surface proteins and inhibit SARS-CoV-2 their utility to target viral surface proteins and inhibit SARS-CoV-2 spike interaction with spike interaction with its receptor. The binding of scFv, Fab, and IgG (immunoglobulin G) its receptor. The binding of scFv, Fab, and IgG (immunoglobulin G) of CR3022 antibodies of CR3022 antibodies to the RBD of SARS-CoV-2 has also been investigated by SPR in a to the RBD of SARS-CoV-2 has also been investigated by SPR in a silkworm-baculovirus expression system [56]. SPR analysis demonstrated that the IgG antibodies produced by silkworms had almost the same affinity to S protein that those produced in mammalian expression system. This is another singular approach in which an SPR sensor can contribute to determine antibody responses when examining vaccine candidates against SARS-CoV-2 viral spike protein [57]. On the one hand, SPR measurements confirmed that rabbit sera contained anti-spike antibodies: 80% IgG, 15% IgA (immunoglobulin A) antibodies and minimal IgM (immunoglobulin M). At the same time, the generation of antibodies by the three antigens (S1 + S2 ectodomain, S1 domain and RBD) was also investigated. The kinetic analysis performed by SPR revealed that all three domains, except for the S2, induced the fabrication of neutralizing antibodies against SARS-CoV-2 (Figure 8). In the same way, Ye et al. made use of SPR biosensing technology to characterize the binding affinity between single-domain antibodies (i.e., nanobody), Nanosota-1 and SARS-CoV-2 RBD, using recombinant ACE2 as a comparison [58]. Nanobodies were identified by screening a camelid nanobody phage display library. The greater ability to block ACE2 receptor was demonstrated by the drug candidate that possessed an Fc tag (Nanosota-1C-Fc), show- ing a KD value 15.7 pM with RBD. This finding suggested that the binding affinity was ~3000 times tighter when compared to the RBD-binding affinity of ACE2. Additionally, the association rates indicated faster binding to the RBD than ACE2, as well as slower dissociation rates. As a result, it was proved that Nanosota-1C-Fc could inhibit SARS- CoV-2 pseudovirus ~160 times more efficiently than ACE2. The efficacy as preventing and therapeutic agent against live SARS-CoV-2 infection was confirmed by hamster and mouse models. Therefore, SPR biosensing demonstrated its functionality in monitoring the immune response generated by the different antigens of the spike proteins after vaccination against SARS or SARS-CoV-2 infection. emosensors 2021, 9, x 2021, Chemosensors FOR9,PEER 330 REVIEW 16 of 25 18 o Figure 8. SARS-CoV-2 spike binding and SARS-CoV-2 neutralization by serum antibodies generated after rabbit immuniza- Figure 8. tion SARS-CoV-2 spike binding with spike antigens. and SARS-CoV-2 (a) Schematic representationneutralization of the SARS-CoV-2 byspike serum antibodies protein generated and subdomains. after Spike S1 rabbit + S2 immun- ization with spike antigens. ectodomain (a)16 (amino acids Schematic to 1213), S1representation domain (amino acidsof the SARS-CoV-2 16 to 685), receptor spike bindingprotein domainand(RBD)subdomains. (amino acids 319 Spike S1 + S2 ectodomain to 541) and S2 domain (amino acids 686 to 1213). (b) Binding of purified proteins to human ACE2 (hACE2) proteins in SPR. acids 319 (amino acids 16 to 1213), S1 domain (amino acids 16 to 685), receptor binding domain (RBD) (amino to 541) and S2 domain Sensorgrams (amino represent acidsof686 binding to 1213). purified (b) Binding spike proteins of purified on low-density proteins His-captured to human chips to hACE2ACE2 protein(hACE2) (5 µg/mL).proteins in SPR. Sensorgrams represent (c) SPR binding binding of antibodies fromoftwo purified rabbits spike proteins twice each immunized on low-density with SARS-CoV-2His-captured antigens tochips to hACE2 spike protein and protein (5 μg/mL). (c) SPR binding of antibodies from two rabbits each immunized twice with SARS-CoV-2 antigens to spike domains from SARS-CoV-2 (S1 + S2, black; S1, blue; RBD, red; and S2, purple). Total antibody binding is represented in protein maximum resonance units (RU) in this figure for 10-fold serum dilution. (d) Antibody off-rate and domains from SARS-CoV-2 (S1 + S2, black; S1, blue; RBD, red; and S2, purple). Total antibody binding is represented constants were directly in maximum determined from the resonance serum units (RU)sample interaction in this figurewith SARS-CoV-2 for 10-fold spikedilution. serum ectodomain (d)(S1Antibody + S2), S1, S2, and RBD off-rate using SPRwere constants in directly the dissociation phase. (e) RBD-hACE2 competition assay. Percent inhibition of hACE2 binding determined from the serum sample interaction with SARS-CoV-2 spike ectodomain (S1 + S2), S1, S2, and RBD using SPR to RBD in the presence of 1:50 dilutions of post-second. Reprinted with permission from Ravichandran et al. [57]. Copyright © 2020 AMER ASSOC in the dissociation phase. (e) RBD-hACE2 competition assay. Percent inhibition of hACE2 binding to RBD in the presence ADVANCEMENT SCIENCE (Distributed under a Creative Commons Attribution License 4.0 (CC BY)). of 1:50 dilutions of post-second. Reprinted with permission from Ravichandran et al. [57]. Copyright © 2020 AMER ASSOC ADVANCEMENT SCIENCE (Distributed under a Creative Commons Attribution License 4.0 (CC BY)). 3.2. SPR Strategies for the Development of SARS-CoV-2 Main Protease Inhibitors Although most of the SPR approaches rely on the monitoring of binding affini between the RBD of the spike S protein and ACE2 receptors, SARS-CoV-2 possesses nonstructural proteins. Among them, the main protease (Mpro), also known as 3-chym Chemosensors 2021, 9, 330 17 of 25 3.2. SPR Strategies for the Development of SARS-CoV-2 Main Protease Inhibitors Although most of the SPR approaches rely on the monitoring of binding affinities between the RBD of the spike S protein and ACE2 receptors, SARS-CoV-2 possesses Chemosensors 2021, 9, x FOR PEER REVIEW 16 nonstructural proteins. Among them, the main protease (Mpro), also known as 3- chymotrypsin-like protease (3CLpro) or chymotrypsin-like protease, is an essential enzyme that plays an important role in mediating the virus replication and processes the polypro- teins that are translated from CoV RNA [59–61]. Mpro consists of three domains (I to III), with the active could be a site located between promising domains strategy to Iblock and II (3CLpro) (Figure 9) [59,60]. viral replication andOwing to prevent viru the importance of 3CLpro on the life cycle of the virus, the development of Mpro inhibitors addition, could the main be a promising protease strategy is highly to block conserved viral replication in coronavirus and prevent andInhas no virus infection. homologue addition, in protease the main humans with conserved is highly a similarin cleavage coronavirusspecificity, andrelated and has no closely then 3CLp homologue in humans with a similar cleavage specificity, and then an attractive target for the design of broad antiviral drugs (Table 2). 3CLpro has become an attractive target for the design of broad antiviral drugs (Table 2). Figure Figure Ensemble 9. 9. SARS-CoV-2 Ensemble Mpro 3D structures SARS-CoV-2 Mpro 3D shown in cartoonshown structures representation with different in cartoon representatio colors. Domain I (residues 10 to 99), Domain II (residues 100 to 182) and Domain III (residues 198 colors. Domain I (residues 10 to 99), Domain II (residues 100 to 182) and Domain III to 303) of the protease are labeled. The substrate-binding site (active site) of Mpro composed of 303) of the protease are labeled. The substrate-binding site (active site) of Mpro co four subsites (S1, S3, S3, and S4) marked by the gray surface. Reprinted with permission from subsites Yang (S1,Copyright et al. [62]. S3, S3, and S4)Elsevier. © 2021 marked by the gray surface. Reprinted with permission [62]. Copyright © 2021 Elsevier. Chemosensors 2021, 9, 330 18 of 25 Table 2. Key analytical features of SPR approaches for screening of SARS-CoV-2 inhibitors that bind to Mpro classified according to the characteristics of target analyte, analytical approach (namely, sensing scheme or biological receptor) and binding affinities (KD equilibrium dissociation constant). Binding Affinities Target Analyte (MW; Common Uses) Analytical Approach SPR Instrument Reference (KD = Equilibrium Dissociation Constant) Quercetin, Luteolin, kaempferol, KD = 1.24 µM; naringenin and epigallocatechin-3-gallate Open SPR instrument KD = 1.63 µM (luteolin); Kinetic analysis (binding (302.236, 286.24, 286.23, 272.257 and (Nicoya Life KD = 1.70 µM (kaempferol); KD = 2.87 µM (naringenin); [63] to 3CLpro) 458,372 g/mol, respectively; antioxidant, Science, Inc., Kitchener, Canada) KD = 6.17 µM (epigallocatechin-3-gallate). anti-inflammatory) (kon and koff values N/A) Punicalagin: PA; ellagic acid: EA; tannic KD = 6.8 × 10−6 ; kon (1/Ms) = 697.3; koff (1/s) = 0.0047 (PA) acid: TA, pentagalloyl glucose: PGG; KD = 2.7 × 10−6 M kon (1/Ms) = 4755.2; koff (1/s) = 0.0130 (EA); ginnalin A, and gallic acid: GA; urolithins: Kinetic analysis and Biacore T200 instrument KD = 1.13 × 10−6 M kon (1/Ms) = 6309.0; koff (1/s) = 0.0071 (TA); UB and pyrogallol: PYG (1.084.71, binding affinities (GE Healthcare; KD = 4.33 × 10−6 M kon (1/Ms) = 3991.0; koff (1/s) = 0.0173 (PGG); [64] 302.297, 1701.19, 940.67, 468.4, 170.12, (binding to 3CLpro) Marlborough, MA, USA) KD = 1.18 × 10−6 M kon (1/Ms) = 2657; koff (1/s) = 0.0031 (GA); 212.20 and 126.11 g/mol respectively; KD = 5.27 × 10−5 M kon (1/Ms) = 1874.0; koff (1/s) = 0.0988 (UB); antioxidant, anti-inflammatory) KD = 3.59 × 10−6 M kon (1/Ms) = 661.8; koff (1/s) = 0.0024 (PYG) Suramin and quinacrine (1.297.29 and Biacore T200 instrument KD = 59.7 µM (suramin) and KD = 227.9 µM (quinacrine) 399.957 g/mol, respectively; treatment of Binding affinity to 3CLpro (GE Healthcare, [65] (kon and koff values N/A) protozoal infection) Uppsala, Sweden) Teicoplanin (1709.4 g/mol; KD = 1.6 mM Binding affinity to 3CLpro Biacore 3000 (GE Healthcare.) [66] glycopeptide antibiotic) kon (1/Ms) = 7.8 × 103 ; koff (1/s) = 0.012 KD = 2.1 × 10−6 (cobicistat); Cobicistat and cangrelor and denufosol BIAcore-3000 (Biacore Inc., KD = 6.4 × 10−4 M (cangrelor); (776.023, 776.35 and 773.323 g/mol, Binding affinity to 3CLpro [67] Uppsala, Sweden) KD = 1.4 × 10−3 M (denufosol) respectively; pulmonary diseases) (kon and koff values N/A) Nonpeptide inhibitors (compounds Biacore KD = 465 µM (Z1244904919); Z1244904919 and Z1759961356, MW and Binding affinity to 3CLpro 8K device (Cytiva, Previously KD = 133 µM (Z1244904919) [62] common uses N/A) GE Healthcare Life Sciences) (kon and koff values N/A) MW: Molecular weight; kon = association rate; koff = dissociation rate; KD = equilibrium dissociation constants; N/A = not available; TCM: traditional Chinese medicine. Chemosensors 2021, 9, 330 19 of 25 Chemosensors 2021, 9, x FOR PEER REVIEW 21 of 27 3.2.1. SPR Analysis of Mpro Inhibitors from Plant Origin Amongst the different ways to develop new drugs, the selection of natural compounds from plants 3.2.1. SPR offersofaMpro Analysis promising Inhibitorsapproach from PlanttoOrigin obtaining potential drug candidates. In this context, the functionality Amongst the different waysof SPR to biosensors develop newisdrugs, mainly thelimited toof selection the monitoring natural com- of binding interaction pounds frombetween plants offers thea target promising compound approach toand the immobilized obtaining potential drugprotein. Following this candidates. In this context, detection scheme,the functionality of SPR biosensors Du et al. determined is mainly limited the inhibitory activitytoof theactive monitoring of ingredients derived binding from interaction TCMs against between the target3CLpro SARS-CoV-2 compound[63].and the immobilized Screening of activeprotein. Follow- through net- ingredients ing this detection scheme, Du et al. determined the inhibitory activity of active ingredients work pharmacology and molecular docking resulted in the selection of seven compounds derived from TCMs against SARS-CoV-2 3CLpro [63]. Screening of active ingredients that were responsible for three TCMs. Particularly, SPR studies revealed high binding affini- through network pharmacology and molecular docking resulted in the selection of seven ties compounds thatto of 3CLpro quercetin were responsible(1.24 three luteolin forµM), (1.63 µM),SPR TCMs. Particularly, kaempferol (1.70 high studies revealed µM), naringenin (2.87 bindingµM), and epigallocatechin-3-gallate affinities of 3CLpro to quercetin (1.24 μM), (6.17luteolin µM) (Figure (1.63 μM),10). The best(1.70 kaempferol inhibitory effect was μM),further examined naringenin (2.87 μM), byandfluorescence resonance energy epigallocatechin-3-gallate (6.17 μM) transfer (FRET)-based (Figure 10). The best cleavage inhibitory assay, effect was showing thatfurther examined by fluorescence(EGCG) epigallocatechin-3-gallate resonancehasenergy thetransfer lowest(FRET)- median inhibitory based cleavage assay, concentration (IC50)showing that epigallocatechin-3-gallate value (0.847 µM). Therefore, SPR(EGCG) assayshas the lowest me- contributed to elucidate the dian inhibitory concentration (IC50) value (0.847 μM). Therefore, SPR assays contributed effect of EGCG, an active ingredient abundant in tea plants, for inhibiting the enzymatic to elucidate the effect of EGCG, an active ingredient abundant in tea plants, for inhibiting activity of 3CLpro. the enzymatic activity of 3CLpro. Figure 10. SPR assay of specific binding affinities of several active compounds to immobilized 3CLpro of SARS-CoV-2. Different concentrations of the compounds (1–80 µM) were injected separately on the surface of the ligand chip, and the analyte was sampled at 20 µL/min. The binding time of the analyte to the ligand was 240 s; and the natural dissociation was carried out for 180 s. Data are representative of three independent experiments. Reprinted with permission from Du et al. [63] Copyright © 2021 Elsevier. Chemosensors 2021, 9, 330 20 of 25 A similar approach made use of phenolic compounds to test their anti-SARS-CoV-2 Mpro activity [64]. This study offers an interesting procedure comprising the combination of enzyme inhibition, SPR-, and docking-based assays to evaluate the inhibition capacity of dietary tannins (ellagitannins (punicalagin: PA and ellagic acid: EA) and gallotannins (tannic acid: TA, pentagalloyl glucose: PGG, ginnalin A and gallic acid: GA), and their gut microbial metabolites, urolithins UB and pyrogallol PYG. The optimization of the SPR conditions include the analysis of binding parameters, flow rate and regeneration buffers. Ellagitannins PA and EA showed high binding affinity to the SARS-CoV-2 Mpro protein with KD values of 6.8 × 10−6 and 2.7 × 10−6 M, respectively. Similarly, gallotannins TA, PGG and GA also exhibited a strong binding capacity with high KD values of 1.13, 4.33 and 1.18 × 10−6 M, respectively. Lastly, KD of UB and PYG were 5.27 × 10−5 and 3.59 × 10−6 M, respectively. Although SPR experiments could not predict the specific binding to the catalytic or allosteric domain to the protein, this work demonstrated that biochemical assays, biophysical-based binding assays, and computational approach can be combined successfully for evaluating the inhibition and binding affinities of drug candidates. 3.2.2. Discovery of Mpro Inhibitors via Drug Repurposing Another way to obtain antiviral drugs relies on the identification of the inhibitory effect of small molecules on 3CLpro protein by repurposing clinically approved drugs. From this perspective, several antiparasitic drugs have demonstrated their antiviral activity against SARS-CoV-2 in cell cultures. In this sense, the inhibitory effect of chloroquine, quinacrine and suramin for blocking SARS-CoV-2 3CLpro has been recently studied by Eberle et al. [65]. Since chloroquine did not affect the SARS-CoV-2 3CLpro activity, SPR biosensing was used to probe the interaction between both suramin and quinacrine with SARS-CoV-2 3CLpro immobilized on the sensor chip. Suramin exhibited stronger interactions with SARS-CoV-2 than quinacrine, exhibiting equilibrium dissociation constants of 59.7 ± 4.5 mM and 227.9 ± 7.9 mM for suramin and quinacrine, respectively. These results were confirmed by molecular docking and MD simulation, indicating that quinacrine blocks the active site entrance through hydrophobic interactions while suramin induces conformational changes in the protein due to allosteric interactions. Additionally, the cooperative effect of suramin and quinacrine to inhibit SARS-CoV-2 3CLpro was evaluated by fluorescence spectroscopy and SPR. SPR experiments indicated that the affinity of quinacrine increased significantly from 230 to 30 mM after the interaction of suramin with SARS-CoV-2 3CLpro, thereby suggesting that suramin binding facilitates quinacrine interaction through a structural change in one active site in the dimer. Following the same strategy, the effect of teicoplanin, a glycopeptide antibiotic used in the treatment of infections caused by gram-positive bacteria was evaluated as a potential inhibitor of SARS-CoV-2 3CLPro [66]. Protein–drug interactions were examined by SPR involving the immobilization of the 3CLPro protein on the sensor chip and the teicoplanin drug as analyte. The binding affinity was good showing an KD value of 1.6 × 10−6 M. SPR studies also suggest that 1:1 binding occurred at lower teicoplanin concentrations, whereas the saturation level was not reached when increasing teicoplanin concentration, thus indicating possible nonspecific bindings. Overall, the main asset of this work is the demonstration of the reduction of proteolytic activity of 3CLPro. Although these results were confirmed by molecular docking studies and Thermofluor®® Assay, SPR measurements were limited to the analysis of the bimolecular interaction between 3CLPro and Teicoplanin. Other studies have taken advantage of computational methods to screen approved drugs in search of potential inhibitors against Mpro. For example, Gupta et al. validated the effect of three FDA-approved compounds, cobicistat, cangrelor and denufosol [67]. The latter one was identified from the Drug Bank, while the other two had been previously tested. Biochemical studies were carried out to perform the binding analysis, whereas molecular interactions were evaluated by SPR sensing. The dissociation constants (KD) of cobicistat, cangrelor and denufosol were 2.1 × 10−6 , 6.4 × 10−4 , and 1.4 × 10−3 M, Chemosensors 2021, 9, 330 21 of 25 respectively, being in agreement with the values of the binding free energy values. The inhibitory effect was examined in vitro by enzyme activity and the results validated by biochemical studies. Since cobicistat is already an approved drug for treating HIV infection and a clinical trial is underway to prove its efficiency against COVID-19 disease, further investigation is needed to consider these compounds as therapeutic options against SARS- CoV-2 infection. Finally, Yang et al. evaluated 49 compounds obtained by multiple conformational- based virtual screening from a protein mimetics library with 8960 compounds [62]. SPR analysis examined the binding profile of the selected candidates showing that six com- pounds could bind to Mpro. Subsequently, the alteration of the enzyme biochemical function was studied by fluorescence resonance energy transfer assay and bioluminescence resonance energy transfer (BRET) to investigate the inhibition activities against the cell lines of SARS-CoV-2 Mpro. Although the values of dissociation constants were not shown, SPR contributed to the selection of the compounds with potential inhibitory activity. 4. Conclusions and Future Perspectives This work compiles recent advancements on the discovery of SARS-CoV2 inhibitors by SPR analysis. One of the major benefits of SPR biosensing is the possibility of easily investigating molecular interactions. Owing to this quality, SPR biosensor technology has become a first-line tool for analyzing the binding properties of potential drug candidates against COVID-19. Primarily, SPR biosensors have been used to measure the binding affinities between an immobilized protein and the selected compound with antiviral properties. Most of the SPR approaches follow this route to characterize the binding specificity between interactants. Recent progress in SPR technology has enabled the detection of weak interactions occurring between small molecules and immobilized proteins with equilibrium dissociation constants in the mM range. In general, SPR-based assays make use of the immobilization of either ACE2 receptor expressed on the membrane of host cells or the main protease Mpro. By uti- lizing this detection scheme, SPR biosensors are capable of generating a full kinetic profile of the interaction with SARS-CoV2 entry inhibitors. When monitoring drug-ACE2 binding constants, SPR experiments usually focused on the capacity of the antiviral compound to block the interaction with the RBD of the virus spike protein. Alternatively, Mpro-based immobilization formats addressed drug discovery from the perspective of the inhibitory compound ability to prevent virus replication and disrupt the virus life cycle. In both cases, target molecules subjected to SPR analysis commonly come from natural origin, mainly fruits and vegetables, or pharmaceutical drug synthesis. Among the latter, commercially approved drugs are normally selected for drug repurposing studies according to previously reported therapeutic uses. Thus, a broad range of compounds, including antiparasitic, antibiotics or antipsychotic drugs, have been repositioned as possible anti- COVID 19 drug candidates. Following this path, the principal role of SPR biosensors relies on the recognition of drug targets that can interact with ACE2 receptors of infected cells or inhibit the S protein and MPro main protease of coronaviruses. The identification of both natural compounds and authorized drugs requires the development of highly specialized biophysical methods. Although SPR configurations are perfectly suited for the screening of small molecules, the number of SPR applications in fragment-based drug discovery is still limited. SPR analysis can provide rapid and sensitive measurements with high selectivity and low protein consumption. Additionally, SPR biosensing can neutralize mass transport effects derived from protein immobilization thanks to the rapid dissociation and the low molecular weight of the analytes. Despite these remarkable advantages, several limitations may arise from the calculation of binding constants due to the low solubility of compounds, thereby requiring previous calibration in the solvent buffer. The multiplexing capacity is also lower in SPR devices in comparison with other biophysical methods such as NMR or X-ray crystallography. However, this limi- Chemosensors 2021, 9, 330 22 of 25 tation can be easily overcome by the low time of response of SPR biosensors. The structural information on binding sites can also be obtained by using competitive assay formats. Overall, SPR biosensors seems to offer a single methodology for attaining full kinetic profiles while providing the screening of fragment libraries and the validation of fragment hits. Nevertheless, to date, the complementarity with other biophysical methods is unques- tionable since NMR produces highly sensitive detection of binding sites and validation whereas X-ray crystallization contributes to select the optimal candidates for fragment enhancement. Consequently, the consolidation of SPR devices as primary screening meth- ods depends on the reduction of false positive/negative rates as well as the optimization of the protein immobilization conditions to obtain the maximum binding response. 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