The members of consortium are committed to Open Access Publishing. As part of its dissemination plan, E4C consortium is making a repository available here for publications originating from the project’s activities following green open access principles. This area will also host any other public documents and reports that the project will generate.


A Comprehensive Mapping of the Druggable Cavities within the SARS-CoV-2 Therapeutically Relevant Proteins by Combining Pocket and Docking Searches as Implemented in Pockets 2.0

Gervasoni, S.; Vistoli, G.; Talarico, C.; Manelfi, C.; Beccari, A.R.; Studer, G.; Tauriello, G.; Waterhouse, A.M.; Schwede, T.; Pedretti, A. A Comprehensive Mapping of the Druggable Cavities within the SARS-CoV-2 Therapeutically Relevant Proteins by Combining Pocket and Docking Searches as Implemented in Pockets 2.0. Int. J. Mol. Sci. 2020, 21, 5152.


(1) Background: Virtual screening studies on the therapeutically relevant proteins of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) require a detailed characterization of their druggable binding sites, and, more generally, a convenient pocket mapping represents a key step for structure-based in silico studies; (2) Methods: Along with a careful literature search on SARS-CoV-2 protein targets, the study presents a novel strategy for pocket mapping based on the combination of pocket (as performed by the well-known FPocket tool) and docking searches (as performed by PLANTS or AutoDock/Vina engines); such an approach is implemented by the Pockets 2.0 plug-in for the VEGA ZZ suite of programs; (3) Results: The literature analysis allowed the identification of 16 promising binding cavities within the SARS-CoV-2 proteins and the here proposed approach was able to recognize them showing performances clearly better than those reached by the sole pocket detection; and (4) Conclusions: Even though the presented strategy should require more extended validations, this proved successful in precisely characterizing a set of SARS-CoV-2 druggable binding pockets including both orthosteric and allosteric sites, which are clearly amenable for virtual screening campaigns and drug repurposing studies. All results generated by the study and the Pockets 2.0 plug-in are available for download.

Computational Studies of SARS-CoV-2 3CLpro: Insights from MD Simulations

Grottesi, A.; Bešker, N.; Emerson, A.; Manelfi, C.; Beccari, A.R.; Frigerio, F.; Lindahl, E.; Cerchia, C.; Talarico, C. Computational Studies of SARS-CoV-2 3CLpro: Insights from MD Simulations. Int. J. Mol. Sci. 2020, 21, 5346.


Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.

SARS-CoV-2 Entry Inhibitors: Small Molecules and Peptides Targeting Virus or Host Cells

Cannalire, R.; Stefanelli, I.; Cerchia, C.; Beccari, A.R.; Pelliccia, S.; Summa, V. SARS-CoV-2 Entry Inhibitors: Small Molecules and Peptides Targeting Virus or Host Cells. Int. J. Mol. Sci. 2020, 21, 5707.


The pandemic evolution of SARS-CoV-2 infection is forcing the scientific community to unprecedented efforts to explore all possible approaches against COVID-19. In this context, targeting virus entry is a promising antiviral strategy for controlling viral infections. The main strategies pursued to inhibit the viral entry are considering both the virus and the host factors involved in the process. Primarily, direct-acting antivirals rely on inhibition of the interaction between ACE2 and the receptor binding domain (RBD) of the Spike (S) protein or targeting the more conserved heptad repeats (HRs), involved in the membrane fusion process. The inhibition of host TMPRSS2 and cathepsins B/L may represent a complementary strategy to be investigated. In this review, we discuss the development entry inhibitors targeting the S protein, as well as the most promising host targeting strategies involving TMPRSS2 and CatB/L, which have been exploited so far against CoVs and other related viruses.

Trend of respiratory pathogens during the COVID-19 epidemic

Giuseppe Sberna, Alessandra Amendola, Maria Beatrice Valli, Fabrizio Carletti, Maria Rosaria Capobianchi, Licia Bordi, Eleonora Lalle, Trend of respiratory pathogens during the COVID-19 epidemic, Journal of Clinical Virology, Volume 129, 2020, 104470, ISSN 1386-6532


In Italy, the first SARS-CoV-2 infections were diagnosed in Rome, Lazio region, at the end of January 2020, but sustained transmission occurred later, since the end of February. From 1 February to 12 April 2020, 17,164 nasopharyngeal swabs were tested by real time PCR for the presence of SARS-CoV-2 at the Laboratory of Virology of National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI) in Rome. In the same period, coincident with the winter peak of influenza and other respiratory illnesses, 847 samples were analyzed by multiplex PCR assay for the presence of common respiratory pathogens. In our study the time trend of SARS-CoV-2 and that of other respiratory pathogens in the same observation period were analysed. Overall, results obtained suggest that the spread of the pandemic SARS-CoV-2 virus did not substantially affect the time trend of other respiratory infections in our region, highlighting no significant difference in rates of SARS-CoV-2 infection in patients with or without other respiratory pathogens. Therefore, in the present scenario of COVID-19 pandemic, differential diagnosis resulting positive for common respiratory pathogen(s) should not exclude testing of SARS-CoV-2.

Analysis of Covid-19 and non-Covid-19 viruses, including influenza viruses, to determine the influence of intensive preventive measures in Japan. J. Clin. Virol. 2020; 129: 104543. doi:10.1016/j.jcv.2020.104543

Giuseppe Sberna, Maria Rosaria Capobianchi, Licia Bordi, Eleonora Lalle, Letter of concern re: Analysis of Covid-19 and non-Covid-19 viruses, including influenza viruses, to determine the influence of intensive preventive measures in Japan. J. Clin. Virol. 2020; 129: 104543. doi:10.1016/j.jcv.2020.104543, Journal of Clinical Virology, Volume 132, 2020, 104635, ISSN 1386-6532


We read with great interest the paper by Hirotsu et al. [1], showing that, among 191 patients exhibiting cold-like symptoms during the period 10 March – 7 May 2020 in Japan, 8/191 (4 %) were infected with SARS-CoV-2, while 32/191 (17 %) with other respiratory viruses; among the 7 viral species detected, the most represented were Human Rhinovirus/Enterovirus (n = 11), while influenza A and B viruses were not detected at all. The Authors infer that the use of surgical mask to prevent SARS-CoV-2 infection had the favourable impact of substantially stopping influenza circulation. These findings are in agreement with previous studies, where influenza infections were virtually absent in patients from USA.
A different scenario was observed in Lazio region, in a study to establish the temporal trend non-SARS-CoV-2 respiratory pathogens from 1 February to 12 April 2020 based on 847 nasopharingeal swabs from patients of National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI) in Rome (Table 1, 3). Results obtained from our study showed that the most frequently detected viruses in this patients were not rhino/enteroviruses (accounting for up to 5.5 % of infections), but indeed influenza A and B viruses, with an overall frequency of 12.3 % and a temporal distribution that is described in the table, peaking at weeks 8–9 (25 %) and declining thereafter.

SARS-CoV-2 Isolation From Ocular Secretions of a Patient With COVID-19 in Italy With Prolonged Viral RNA Detection

Francesca Colavita, Daniele Lapa, Fabrizio Carletti, Eleonora Lalle, Licia Bordi, Patrizia Marsella, Emanuele Nicastri, Nazario Bevilacqua, Maria Letizia Giancola, Angela Corpolongo, Giuseppe Ippolito, Maria Rosaria Capobianchi, and Concetta Castilletti, SARS-CoV-2 Isolation From Ocular Secretions of a Patient With COVID-19 in Italy With Prolonged Viral RNA Detection, Annals of Internal Medicine 2020 173:3, 242-243


Background: Coronavirus disease 2019 (COVID-19), the disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that originated in China in December 2019, was recently recognized as pandemic threat by the World Health Organization, with the potential of rapidly overloading health care systems and causing substantial mortality worldwide. Human-to-human transmission occurs mainly through respiratory droplets, but other routes are under investigation, because SARS-CoV-2 has been detected in several body fluids (1). So far, few data are available on ocular samples from patients with COVID-19, although conjunctivitis has been occasionally reported among COVID-19 symptoms, similar to infections caused by other human coronaviruses. During the SARS epidemic, eye exposure to infectious fluids was associated with an increased risk for SARS-CoV transmission to health care workers. Although SARS-CoV RNA was occasionally found in ocular specimens during the early phase of illness, its infectivity is unknown.
With regard to COVID-19, unprotected ocular exposure was thought to be responsible for infections that occurred in the Wuhan Fever Clinic in January 2020; in addition, SARS-CoV-2 RNA was detected in conjunctival secretions collected from the only patient with conjunctivitis out of 30 patients with COVID-19 from a hospital in China. However, further studies are needed to evaluate the infectious potential of the SARS-CoV-2 RNA detected in the ocular specimens and to determine whether transmission may occur through ocular secretions.

Performance of a novel diagnostic assay for rapid SARS-CoV-2 antigen detection in nasopharynx samples

Liotti FM, Menchinelli G, Lalle E, Palucci I, Marchetti S, Colavita F, La Sorda M, Sberna G, Bordi L, Sanguinetti M, Cattani P, Capobianchi MR, Posteraro B, Performance of a novel diagnostic assay for rapid SARS-CoV-2 antigen detection in nasopharynx samples, Clinical Microbiology and Infection,


Our study shows that the STANDARD F COVID-19 Ag FIA assay had a good specificity for SARS-CoV-2 detection in nasopharynx swab samples but had a good sensitivity only for samples with Ct values lower than 25 (corresponding to higher viral loads). Thus, we believe that the STANDARD F COVID-19 Ag FIA assay might be reliably used in early phases of acute SARS-CoV-2 infection, e.g. within the first days after infection when Ct values are likely to still be below 25. This would be consistent with the viral load kinetics within the first days of SARS-CoV-2 infection, showing that nasopharynx swab samples obtained on day 7 may be persistently positive (Ct values, 23–24) for SARS-CoV-2 [5]. However, considering the current epidemiological scenario, a non-negligible proportion of symptomatic or, most commonly, asymptomatic patients, whose nasopharynx swab samples display Ct values of ≥25–<35 or ≥35, might be negative with STANDARD F COVID-19 Ag FIA (or similar) assays. This scenario would also encompass “new” patients who begin their SARS-CoV-2 infection course with a low viral load (resulting in Ct values of ≥35). In the light of these observations, it is presently difficult to envisage the correct, fruitful and safe use of these assays unless they are integrated in laboratory diagnostic algorithms based on both molecular and serological testing for SARS-CoV-2 infection.

SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February–March 2020

Bartolini B, Rueca M, Gruber C, Messina F, Carletti F, Giombini E, et al. SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February–March 2020. Emerg Infect Dis. 2020;26(8):1842-1845.


We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised serious concerns because of its rapid dissemination worldwide. Italy is one of the countries with the highest number of coronavirus disease (COVID-19) cases. Nevertheless, the information about the molecular epidemiology of SARS-CoV-2 strains circulating in Italy is still limited. The analysis of sequence data shown in GISAID indicates that the initial introduction of SARS-CoV-2 in Italy through 2 infected tourists in January was effectively contained, and no further circulation of similar clade V strains has been so far detected. An intense wave of infections occurred afterwards, initially affecting Lombardy and Veneto and later on all the other regions of Italy. The strains detected in Italy since February 20 belonged only to clade G. This clade, apparently originating in Shanghai, has been widely circulating in the European Union (EU) countries before reaching Italy.
Preliminary data suggested that multiple introductions of clade G strains have occurred in Italy, giving rise to contemporary circulation of different strains also detected in other EU countries; this pattern suggests that, after partially undetected introduction of the virus in EU from China, the circulation of travelers within EU ignited virus spread in Europe. We report the phylogenetic and mutational analysis of SARS-CoV-2 strains detected in the Lazio region of Italy, providing additional information on the dynamics of virus dissemination in this country.

Performance evaluation of Abbott ARCHITECT SARS-CoV-2 IgG immunoassay in comparison with indirect immunofluorescence and virus microneutralization test

Silvia Meschi, Francesca Colavita, Licia Bordi, Giulia Matusali, Daniele Lapa, Alessandra Amendola, Francesco Vairo, Giuseppe Ippolito, Maria Rosaria Capobianchi, Concetta Castilletti, Performance evaluation of Abbott ARCHITECT SARS-CoV-2 IgG immunoassay in comparison with indirect immunofluorescence and virus microneutralization test, Journal of Clinical Virology, Volume 129, 2020, 104539, ISSN 1386-6532


Serological tests for anti-SARS-CoV-2 antibodies are becoming of great interest to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic.
We evaluated the diagnostic performance of the Abbott ARCHITECT SARS-CoV-2 IgG test, a fully automated indirect immunoassay that detects antibodies directed to a recombinant SARS-CoV-2 Nucleocapsid antigen.
Study design
Abbott ARCHITECT SARS-CoV-2 IgG immunoassay was compared to an indirect immunofluorescence assay (IFA) on sera from patients with COVID-19 collected at different days after symptoms onset or infected by other human coronaviruses. Comparison with neutralization test was also performed.
After 7, 14 and >14 days after onset ARCHITECT was positive on 8.3 %; 61.9 % and 100 % of the tested samples compared to 58.3 %; 85.7 % and 100 % by IFA. The sensitivity was 72 % vs. IFA and 66.7 % vs. a real-time PCR, the specificity was 100 %. On 18 samples with neutralizing activity, 17 were positive by Abbott ARCHITECT SARS-CoV-2 IgG.
In our study, Abbott ARCHITECT SARS-CoV-2 IgG assay showed a satisfactory performance, with a very high specificity. IgG reactivity against SARSCoV-2 N antigen was detectable in all patients by two weeks after symptoms onset. In addition, concordance between this serological response and viral neutralization suggests that a strong humoral response may be predictive of a neutralization activity, regardless of the target antigens. This finding supports the use of this automated serological assay in diagnostic algorithm and public health intervention, especially for high loads of testing.

Unraveling the molecular basis of host cell receptor usage in SARS-CoV-2 and other human pathogenic β-CoVs

Camila Pontes, Victoria Ruiz-Serra, Rosalba Lepore, Alfonso Valencia bioRxiv 2020.08.21.260745; doi:


The recent emergence of the novel SARS-CoV-2 in China and its rapid spread in the human population has led to a public health crisis worldwide. Like in SARS-CoV, horseshoe bats currently represent the most likely candidate animal source for SARS-CoV-2. Yet, the specific mechanisms of cross-species transmission and adaptation to the human host remain unknown. Here we show that the unsupervised analysis of conservation patterns across the β-CoV spike protein family, using sequence information alone, can provide rich information on the molecular basis of the specificity of β-CoVs to different host cell receptors. More precisely, our results indicate that host cell receptor usage is encoded in the amino acid sequences of different CoV spike proteins in the form of a set of specificity determining positions (SDPs). Furthermore, by integrating structural data, in silico mutagenesis and coevolution analysis we could elucidate the role of SDPs in mediating ACE2 binding across the Sarbecovirus lineage, either by engaging the receptor through direct intermolecular interactions or by affecting the local environment of the receptor binding motif. Finally, by the analysis of coevolving mutations across a paired MSA we were able to identify key intermolecular contacts occurring at the spike-ACE2 interface. These results show that effective mining of the evolutionary records held in the sequence of the spike protein family can help tracing the molecular mechanisms behind the evolution and host-receptors adaptation of circulating and future novel β-CoVs.

Significance Unraveling the molecular basis for host cell receptor usage among β-CoVs is crucial to our understanding of cross-species transmission, adaptation and for molecular-guided epidemiological monitoring of potential outbreaks. In the present study, we survey the sequence conservation patterns of the β-CoV spike protein family to identify the evolutionary constraints shaping the functional specificity of the protein across the β-CoV lineage. We show that the unsupervised analysis of statistical patterns in a MSA of the spike protein family can help tracing the amino acid space encoding the specificity of β-CoVs to their cognate host cell receptors. We argue that the results obtained in this work can provide a framework for monitoring the evolution of SARS-CoV-2 specificity to the hACE2 receptor, as the virus continues spreading in the human population and differential virulence starts to arise.

Targeting SARS-CoV-2 Proteases and Polymerase for COVID-19 Treatment: State of the Art and Future Opportunities

Rolando Cannalire, Carmen Cerchia, Andrea R. Beccari, Francesco Saverio Di Leva, and Vincenzo Summa Journal of Medicinal Chemistry Article ASAP DOI: 10.1021/acs.jmedchem.0c01140


The newly emerged coronavirus, called SARS-CoV-2, is the causing pathogen of pandemic COVID-19. The identification of drugs to treat COVID-19 and other coronavirus diseases is an urgent global need, thus different strategies targeting either virus or host cell are still under investigation. Direct-acting agents, targeting protease and polymerase functionalities, represent a milestone in antiviral therapy. The 3C-like (or Main) protease (3CLpro) and the nsp12 RNA-dependent RNA-polymerase (RdRp) are the best characterized SARS-CoV-2 targets and show the highest degree of conservation across coronaviruses fostering the identification of broad-spectrum inhibitors. Coronaviruses also possess a papain-like protease, another essential enzyme, still poorly characterized and not equally conserved, limiting the identification of broad-spectrum agents. Herein, we provide an exhaustive comparative analysis of SARS-CoV-2 proteases and RdRp with respect to other coronavirus homologues. Moreover, we highlight the most promising inhibitors of these proteins reported so far, including the possible strategies for their further development.

Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a repurposing screen (“target confirmation of main protease”)

Maria Kuzikov*, Elisa Costanzi, Jeanette Reinshagen, Francesca Esposito, Laura Vangeel, Markus Wolf, Bernhard Ellinger, Carsten Claussen, Gerd Geisslinger, Angela Corona, Daniela Iaconis, Carmine Talarico, Candida Manelfi, Rolando Cannalire, Giulia Rossetti, Jonas Gossen, Simone Albani, Francesco Musiani, Katja Herzog, Yang Ye, Barbara Giabbai, Nicola Demitri, Dirk Jochmans, Steven De Jonghe, Jasper Rymenants, Vincenzo Summa, Enzo Tramontano, Andrea R. Beccari, Pieter Leyssen, Paola Storici, Johan Neyts, Philip Gribbon, and Andrea Zaliani. Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen. ACS Pharmacol. Transl. Sci. 2021;
Chembl link:


SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites.

Within our repurposing program we confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 μM. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line and the X-ray crystal structure of the complex of one of the inhibitors and SARS-Cov-2 3CL-Pro was solved, showing covalent binding to the catalytic Cys145.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease (“How do you find 37 new repurposed drugs that have direct 3D binding to SARSCoV2's main protease to knock out its ability to replicate”)

Sebastian Günther, Patrick Y. A. Reinke, Yaiza Fernández-García, Julia Lieske, Thomas J. Lane, Helen M. Ginn, Faisal H. M. Koua, Christiane Ehrt, Wiebke Ewert, Dominik Oberthuer, Oleksandr Yefanov, Susanne Meier, Kristina Lorenzen, Boris Krichel, Janine-Denise Kopicki, Luca Gelisio, Wolfgang Brehm, Ilona Dunkel, Brandon Seychell, Henry Gieseler, Brenna Norton-Baker, Beatriz Escudero-Pérez2 Martin Domaracky, Sofiane Saouane, Alexandra Tolstikova, Thomas A. White, Anna Hänle, Michael Groessler, Holger Fleckenstein, Fabian Trost, Marina Galchenkova, Yaroslav Gevorkov, Chufeng Li, Salah Awel, Ariana Peck, Miriam Barthelmess, Frank Schluenzen1 Paulraj Lourdu Xavier, Nadine Werner, Hina Andaleeb, Najeeb Ullah, Sven Falke, Vasundara Srinivasan, Bruno Alves França, Martin Schwinzer, Hévila Brognaro, Cromarte Rogers, Diogo Melo, Joanna J. Zaitseva-Doyle, Juraj Knoska, Gisel E. Peña-Murillo, Aida Rahmani Mashhour, Vincent Hennicke, Pontus Fischer, Johanna Hakanpää, Jan Meyer, Philip Gribbon, Bernhard Ellinger, Maria Kuzikov, Markus Wolf, Andrea R. Beccari, Gleb Bourenkov, David von Stetten, Guillaume Pompidor, Isabel Bento, Saravanan Panneerselvam, Ivars Karpics, Thomas R. Schneider, Maria Marta Garcia-Alai, Stephan Niebling, Christian Günther, Christina Schmidt, Robin Schubert, Huijong Han, Juliane Boger, Diana C. F. Monteiro, Linlin Zhang, Xinyuanyuan Sun, Jonathan Pletzer-Zelgert, Jan Wollenhaupt, Christian G. Feiler, Manfred S. Weiss, Eike-Christian Schulz, Pedram Mehrabi, Katarina Karničar, Aleksandra Usenik, Jure Loboda, Henning Tidow, Ashwin Chari, Rolf Hilgenfeld, Charlotte Uetrecht, Russell Cox, Andrea Zaliani, Tobias Beck, Matthias Rarey, Stephan Günther, Dusan Turk, Winfried Hinrichs, Henry N. Chapman, Arwen R. Pearson, Christian Betzel, Alke Meents
Science 02 Apr 2021;
DOI: 10.6019/CHEMBL4495565


Worldwide, there are intensive efforts to identify repurposed drugs as potential therapies against SARS-CoV-2 infection and the associated COVID-19 disease. To date, the anti-inflammatory drug dexamethasone and (to a lesser extent) the RNA-polymerase inhibitor remdesivir have been shown to be effective in reducing mortality and patient time to recovery, respectively, in patients.

Here, we report the results of a phenotypic screening campaign within an EU-funded project (H2020-EXSCALATE4COV) aimed at extending the repertoire of anti-COVID therapeutics through repurposing of available compounds and highlighting compounds with new mechanisms of action against viral infection. We screened 8702 molecules from different repurposing libraries, to reveal 110 compounds with an anti-cytopathic IC50 < 20 µM.

From this group, 18 with a safety index greater than 2 are also marketed drugs, making them suitable for further study as potential therapies against COVID-19. Our result supports the idea that a systematic approach to repurposing is a valid strategy to accelerate the necessary drug discovery process.

this is the link of the repository Exscalate4COV team is using to share the data coming from in silico simulations and that are related to produced scientific papers.

models used in the publication  free to download and produced by Swiss Institute of Bioinformatics team:


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