Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2–P1 residues, allowing the development of broad-spectrum inhibitors. The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2–P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the β-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low μM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics

approccio integrativo in drug discovey contro il SARS-CoV2: studio biochimico e biofisico sulla proteasi Papain-like

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), l'agente eziologico della sindrome Covid-19, ha causato una delle pandemie più impattanti della storia umana. Le campagne di vaccinazione hanno offerto la soluzione all'emergenza globale, ma finora sono stati approvati solo pochi trattamenti della malattia. Tra i potenziali bersagli farmacologici del SARS-CoV2, le cistein-proteasi sono tra le più promettenti, grazie al loro ruolo essenziale nella replicazione virale e per le loro funzioni aggiuntive nello sviluppo dell'infezione: la più studiata Main protease (Mpro) e la Papain-like protease (PLpro). La seconda è un dominio della proteina non strutturale 3 (nsp3) e, oltre al clivaggio della poli-proteina virale pp1a in tre siti, mostra attività deISGilasica e deUbiquitinasica. La PLpro rappresenta infatti uno dei principali sistemi di protezione del coronavirus contro la risposta immunitaria, interferendo con le vie delle citochine regolate dal sistema di modificazione dell'Ubiquitina e dell'ISG15. Nonostante il numero di screening farmacologici effettuati su questo target, solo poche molecole sono state validate come inibitori. In questa tesi vengono presentati i nostri sforzi per identificare inibitori accertati di questo enzima e la caratterizzazione delle differenze tra il costrutto comunemente usato della PLpro e il costrutto a doppio dominio PLpro_NAB, contenente il Nucleic-Acid Binding domain dell’nsp3, entrambi prodotti nella protein facility di Elettra. Il progetto è stato sviluppato in collaborazione con validi partner che hanno messo a disposizione le loro competenze, portando all'identificazione di interessanti inibitori di riposizionamento. Dopo aver valutato criticamente i primi risultati, applicando un approccio integrativo di tecniche biochimiche, computazionali e biofisiche, abbiamo delucidato il meccanismo d'azione di composti falsi-positivi che all'inizio sembravano promettenti, evidenziando invece un vero inibitore, il CPI-169. Grazie all'affinità μM della molecola, è stato possibile studiare l’interazione con tecniche di ligand-based NMR, che hanno permesso di validare i risultati computazionali e biochimici. In questo progetto è stata investigata anche l'interazione con il substrato umano di elezione della PLpro, l'ISG15. La produzione di ISG15 e del suo precursore (proISG15) è stata necessaria per evidenziare interessanti differenze nel legame e nell'attività catalitica dei mutanti S466R e T467K della PLpro_NAB, due mutazioni scoperte nella variante Delta del SARS-CoV2. Le nostre osservazioni dimostrano che le mutazioni puntiformi localizzate sul dominio NAB alterano il dominio vicino, la PLpro, e come una singola mutazione puntiforme su un altro dominio dell'nsp3 possa influenzare anche il PLpro.Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the etiological agent of Covid-19 syndrome, caused one of the most impacting pandemics in human history. Vaccination campaigns offered the solution to the global emergency, but up to now only a few treatments of the disease have been approved. Among potential pharmacological targets of the SARS-CoV2, the Cysteine proteases are one of the most promising, due to their essential role in viral replication and additional functions in the infection development: the more explored Main protease (Mpro) and the Papain-like protease (PLpro). The second one is a domain of the sizeable non-structural protein 3 (nsp3) and besides the cleavage of the viral polyprotein pp1a in three sites, shows deISGylating and deUbiquitinating activity. PLpro represents indeed one of the main viral protecting systems against the immune response, interfering with the cytokines pathways regulated by the Ubiquitin and ISG15 modification system. Despite the number of drug screenings performed on this target, only a few molecules were validated as inhibitors. In this thesis are presented our efforts to identify ensured repurposing inhibitors of this enzyme and the characterization of differences between the commonly used construct of the PLpro and the double-domain construct PLpro_NAB, containing the Nucleic-Acid Binding domain of nsp3, both produced in the Elettra Protein Facility. The project was developed in collaboration with valuable partners who provided their skills, leading to the identification of interesting repurposing inhibitors. After critically evaluating the first results, applying an integrative approach of biochemical, computer-aided and biophysical techniques, we elucidated the mechanism of action of false-positive compounds which at first looked promising, highlighting instead a real inhibitor, the CPI-169. The μM affinity was suitable to be studied by ligand-based NMR techniques, which allowed the validation of the computational and biochemical readouts. The interaction with the preferred human substrate of the PLpro, the ISG15, was also investigated in this project. The production of ISG15 and its precursor (proISG15) was necessary to highlight interesting discrepancies in the binding and catalytic activity of the PLpro_NAB mutants S466R and T467K, two mutations discovered in the Delta-variant of SARS-CoV2. Our results show that the point mutations located on the NAB domain alter the neighbour domain, the PLpro, and how a single point mutation on another domain of the nsp3 can also affect the PLpro

Dossier Día de África 2019

El 25 de mayo de 1963 fue creada la Organización de la Unidad Africana (OUA), devenida en el año 2001 en la actual Unión Africana (UA). Por este motivo, cada 25 de mayo se celebra internacionalmente el “Día de África”. En conmemoración a este día especial, desde el Programa de Estudios América Latina - África (PEALA) confeccionamos este dossier que nos invita a reflexionar sobre la unidad y la identidad africana a partir de la discusión de las problemáticas actuales que atraviesan al continente. El dossier reúne una serie de comentarios sobre artículos seleccionados por estudiantes y jóvenes investigadores que conforman el grupo de estudios sobre África en el marco del Programa de Relaciones y Cooperación Sur-Sur (PRECSUR) radicado en el Instituto de Investigaciones de la Facultad de Ciencia Política y Relaciones Internacionales de la Universidad Nacional de Rosario.Fil: Morasso, Carla. Universidad Nacional de Rosario. Facultad de Ciencia Política y Relaciones Internacionales; Argentina.Fil: Marchetti, Agustina. Universidad Nacional de Rosario. Facultad de Ciencia Política y Relaciones Internacionales; Argentina

Dataset for Surface Enhanced Raman Spectroscopy for quantitative analysis: results of a large-scale European multi-instrument interlaboratory study

This dataset contains all the spectra used in "Surface Enhanced Raman Spectroscopy for quantitative analysis: results of a large-scale European multi-instrument interlaboratory study". Data are available in 2 different formats: - a compressed archive with 1 folder ("Dataset”) cointaining all the 3516 TXT files (1 file = 1 spectrum) uploaded by all participants (all spectra of the Interlaboratory study); - 1 single CSV file (“ILSspectra.csv”) with all the 3516 spectra uploaded by all participants in the form of a table. The data are structured as follow, with each row being 1 spectrum, preceded by metadata: "labcode", "substrate", "laser", "method", "sample", "type", "conc", "batch", "replica". Note that for those spectra starting after 400 cm-1 and/or ending before 2000 cm-1 missing values were expressed as NAs

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study

peer reviewedaudience: researcher, professional, studentSurface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community. Copyright © 2020 American Chemical Society