Switching the three-component Biginelli-like reaction conditions for the regioselective synthesis of new 2-amino[1,2,4]triazolo[1,5-a]pyrimidines.

Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.

Discovery of 2-phenylquinazolines as potent Mycobacterium avium efflux pump inhibitors able to synergize with clarithromycin against clinical isolate.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.

Privileged Scaffolds for Potent and Specific Inhibitors of Mono-ADP-Ribosylating PARPs.

The identification of new targets to address unmet medical needs, better in a personalized way, is an urgent necessity. The introduction of PARP1 inhibitors into therapy, almost ten years ago, has represented a step forward this need being an innovate cancer treatment through a precision medicine approach. The PARP family consists of 17 members of which PARP1 that works by poly-ADP ribosylating the substrate is the sole enzyme so far exploited as therapeutic target. Most of the other members are mono-ADP-ribosylating (mono-ARTs) enzymes, and recent studies have deciphered their pathophysiological roles which appear to be very extensive with various potential therapeutic applications. In parallel, a handful of mono-ARTs inhibitors emerged that have been collected in a perspective on 2022. After that, additional very interesting compounds were identified highlighting the hot-topic nature of this research field and prompting an update. From the present review, where we have reported only mono-ARTs inhibitors endowed with the appropriate profile of pharmacological tools or drug candidate, four privileged scaffolds clearly stood out that constitute the basis for further drug discovery campaigns.

C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity.

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors.

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors.

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 µM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.

Efficient and regioselective one-step synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives.

Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-triazole-3-carboxylate was used. [1,2,4]Triazolo[1,5-a]pyrimidine being a privileged scaffold, the procedures herein reported may be useful for the preparation of biologically active compounds. In this study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA polymerase PA-PB1 subunit heterodimerization.

The 6-Aminoquinolone WC5 inhibits different functions of the immediate-early 2 (IE2) protein of human cytomegalovirus that are essential for viral replication.

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional factor essential for viral replication. IE2 modulates both viral and host gene expression, deregulates cell cycle progression, acts as an immunomodulator, and antagonizes cellular antiviral responses. Based on these facts, IE2 has been proposed as an important target for the development of innovative antiviral approaches. We previously identified the 6-aminoquinolone WC5 as a promising inhibitor of HCMV replication, and here, we report the dissection of its mechanism of action against the viral IE2 protein. Using glutathione S-transferase (GST) pulldown assays, mutagenesis, cell-based assays, and electrophoretic mobility shift assays, we demonstrated that WC5 does not interfere with IE2 dimerization, its interaction with TATA-binding protein (TBP), and the expression of a set of cellular genes that are stimulated by IE2. On the contrary, WC5 targets the regulatory activity exerted by IE2 on different responsive viral promoters. Indeed, WC5 blocked the IE2-dependent negative regulation of the major immediate-early promoter by preventing IE2 binding to the crs element. Moreover, WC5 reduced the IE2-dependent transactivation of a series of indicator constructs driven by different portions of the early UL54 gene promoter, and it also inhibited the transactivation of the murine CMV early E1 promoter by the IE3 protein, the murine cytomegalovirus (MCMV) IE2 homolog. In conclusion, our results indicate that the overall anti-HCMV activity of WC5 depends on its ability to specifically interfere with the IE2-dependent regulation of viral promoters. Importantly, our results suggest that this mechanism is conserved in murine CMV, thus paving the way for further preclinical evaluation in an animal model.

Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information gathered from this work confirms the primary role of water molecules and protein flexibility in docking-based studies and can be exploited to aid NS5B-directed HCV drug discovery efforts.

Exploiting the anti-HIV 6-desfluoroquinolones to design multiple ligands.

It is getting clearer that many drugs effective in different therapeutic areas act on multiple rather than single targets. The application of polypharmacology concepts might have numerous advantages especially for disease such as HIV/AIDS, where the rapid emergence of resistance requires a complex combination of more than one drug. In this paper, we have designed three hybrid molecules combining WM5, a quinolone derivative we previously identified as HIV Tat-mediated transcription (TMT) inhibitor, with the tricyclic core of nevirapine and BILR 355BS (BILR) non-nucleoside reverse transcriptase inhibitors (NNRTIs) to investigate whether it could be possible to obtain molecules acting on both transcription steps of the HIV replicative cycle. One among the three designed multiple ligands, reached this goal. Indeed, compound 1 inhibited both TMT and reverse transcriptase (RT) activity. Unexpectedly, while the anti-TMT activity exerted by compound 1 resulted into a selective inhibition of HIV-1 reactivation from latently infected OM10.1 cells, the anti-RT properties shown by all of the synthesized compounds did not translate into an anti-HIV activity in acutely infected cells. Thus, we have herein produced the proof of concept that the design of dual TMT-RT inhibitors is indeed possible, but optimization efforts are needed to obtain more potent derivatives.

Recent Progress toward the Discovery of Small Molecules as Novel Anti-Respiratory Syncytial Virus Agents.

Respiratory syncytial virus (RSV) stands as the foremost cause of infant hospitalization globally, ranking second only to malaria in terms of infant mortality. Although three vaccines have recently been approved for the prophylaxis of adults aged 60 and above, and pregnant women, there is currently no effective antiviral drug for treating RSV infections. The only preventive measure for infants at high risk of severe RSV disease is passive immunization through monoclonal antibodies. This Perspective offers an overview of the latest advancements in RSV drug discovery of small molecule antivirals, with particular focus on the promising findings from agents targeting the fusion and polymerase proteins. A comprehensive reflection on the current state of RSV research is also given, drawing inspiration from the lessons gleaned from HCV and HIV, while also considering the impact of the recent approval of the three vaccines.

Synthetically accessible de novo design using reaction vectors: Application to PARP1 inhibitors.

De novo design has been a hotly pursued topic for many years. Most recent developments have involved the use of deep learning methods for generative molecular design. Despite increasing levels of algorithmic sophistication, the design of molecules that are synthetically accessible remains a major challenge. Reaction-based de novo design takes a conceptually simpler approach and aims to address synthesisability directly by mimicking synthetic chemistry and driving structural transformations by known reactions that are applied in a stepwise manner. However, the use of a small number of hand-coded transformations restricts the chemical space that can be accessed and there are few examples in the literature where molecules and their synthetic routes have been designed and executed successfully. Here we describe the application of reaction-based de novo design to the design of synthetically accessible and biologically active compounds as proof-of-concept of our reaction vector-based software. Reaction vectors are derived automatically from known reactions and allow access to a wide region of synthetically accessible chemical space. The design was aimed at producing molecules that are active against PARP1 and which have improved brain penetration properties compared to existing PARP1 inhibitors. We synthesised a selection of the designed molecules according to the provided synthetic routes and tested them experimentally. The results demonstrate that reaction vectors can be applied to the design of novel molecules of biological relevance that are also synthetically accessible.

[1,2,4]Triazolo[3,4-b]benzothiazole Scaffold as Versatile Nicotinamide Mimic Allowing Nanomolar Inhibition of Different PARP Enzymes.

We report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogues and cocrystal structures with TNKS2, PARP2, PARP14, and PARP15. Based on the substitution pattern, we were able to identify 3-amino derivatives 21 (OUL243) and 27 (OUL232) as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14, and PARP15 at nM potencies, with 27 being the most potent PARP10 inhibitor described to date (IC50 of 7.8 nM) and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity toward PARP2. The scaffold does not possess inherent cell toxicity, and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of TBT scaffold for future drug development efforts toward selective inhibitors against specific enzymes.

Medicinal Chemistry Perspective on Targeting Mono-ADP-Ribosylating PARPs with Small Molecules.

Major advances have recently defined functions for human mono-ADP-ribosylating PARP enzymes (mono-ARTs), also opening up potential applications for targeting them to treat diseases. Structural biology combined with medicinal chemistry has allowed the design of potent small molecule inhibitors which typically bind to the catalytic domain. Most of these inhibitors are at the early stages, but some have already a suitable profile to be used as chemical tools. One compound targeting PARP7 has even progressed to clinical trials. In this review, we collect inhibitors of mono-ARTs with a typical "H-Y-Φ" motif (Φ = hydrophobic residue) and focus on compounds that have been reported as active against one or a restricted number of enzymes. We discuss them from a medicinal chemistry point of view and include an analysis of the available crystal structures, allowing us to craft a pharmacophore model that lays the foundation for obtaining new potent and more specific inhibitors.

Triazolopyrimidine Nuclei: Privileged Scaffolds for Developing Antiviral Agents with a Proper Pharmacokinetic Profile.

Viruses are a continuing threat to global health. The lack or limited therapeutic armamentarium against some viral infections and increasing drug resistance issues make the search for new antiviral agents urgent. In recent years, a growing literature highlighted the use of triazolopyrimidine (TZP) heterocycles in the development of antiviral agents, with numerous compounds that showed potent antiviral activities against different RNA and DNA viruses. TZP core represents a privileged scaffold for achieving biologically active molecules, thanks to: i) the synthetic feasibility that allows to variously functionalize TZPs in the different positions of the nucleus, ii) the ability of TZP core to establish multiple interactions with the molecular target, and iii) its favorable pharmacokinetic properties. In the present review, after mentioning selected examples of TZP-based compounds with varied biological activities, we will focus on those antivirals that appeared in the literature in the last 10 years. Approaches used for their identification, the hit-to-lead studies, and the emerged structure-activity relationship will be described. A mention of the synthetic methodologies to prepare TZP nuclei will also be given. In addition, their mechanism of action, the binding mode within the biological target, and pharmacokinetic properties will be analyzed, highlighting the strengths and weaknesses of compounds based on the TZP scaffold, which is increasingly used in medicinal chemistry.

Potent 2,3-dihydrophthalazine-1,4-dione derivatives as dual inhibitors for mono-ADP-ribosyltransferases PARP10 and PARP15.

While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130-160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.

New C-6 functionalized quinoline NorA inhibitors strongly synergize with ciprofloxacin against planktonic and biofilm growing resistant Staphylococcus aureus strains.

Antimicrobial resistance (AMR) represents a global health issue threatening our social lifestyle and the world economy. Efflux pumps are widely involved in AMR by playing a primary role in the development of specific mechanisms of resistance. In addition, they seem to be involved in the process of biofilm formation and maintenance, contributing to enhance the risk of creating superbugs difficult to treat. Accordingly, the identification of non-antibiotic molecules able to block efflux pumps, namely efflux pump inhibitors (EPIs), could be a promising strategy to counteract AMR and restore the antimicrobial activity of ineffective antibiotics. Herein, we enlarge the knowledge about the structure-activity relationship of 2-phenylquinoline Staphylococcus aureus NorA EPIs by reporting a new series of very potent C-6 functionalized derivatives. Best compounds significantly inhibited ethidium bromide efflux in a NorA-overexpressing S. aureus strain (SA-1199B) and strongly synergized at very low concentrations (0.20-0.78 µg/mL) with ciprofloxacin (CPX) against CPX-resistant S. aureus strains (SA-1199B and SA-K2378), as proved by checkerboard and time-kill experiments. In addition, some of these EPIs (9b and 10a) produced a post-antibiotic effect of 1.2 h and strongly enhanced antibiofilm activity of CPX against SA-1199B strain. Interestingly, at the concentrations used to reach synergy with CPX against resistant S. aureus strains, most of the EPI compounds did not show any human cell toxicity. Finally, by exploiting the recent released crystal structure of NorA, we observed that best EPI 9b highlighted a favourable docking pose, establishing some interesting interactions with key residues.

From Serendipity to Rational Identification of the 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one Core as a New Chemotype of AKT1 Inhibitors for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy whose prognosis is globally poor. In more than 60% of AML patients, the PI3K/AKTs/mTOR signaling pathway is aberrantly activated because of oncogenic driver alterations and further enhanced by chemotherapy as a mechanism of drug resistance. Against this backdrop, very recently we have started a multidisciplinary research project focused on AKT1 as a pharmacological target to identify novel anti-AML agents. Indeed, the serendipitous finding of the in-house compound T187 as an AKT1 inhibitor has paved the way to the rational identification of new active small molecules, among which T126 has emerged as the most interesting compound with IC50 = 1.99 ± 0.11 µM, ligand efficiency of 0.35, and a clear effect at low micromolar concentrations on growth inhibition and induction of apoptosis in AML cells. The collected results together with preliminary SAR data strongly indicate that the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one derivative T126 is worthy of future biological experiments and medicinal chemistry efforts aimed at developing a novel chemical class of AKT1 inhibitors as anti-AML agents.

Discovery of 2-Phenylquinolines with Broad-Spectrum Anti-coronavirus Activity.

A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 µM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low µM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 µM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 µM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.