Continuous Flow Biocatalytic Reductive Amination by Co-Entrapping Dehydrogenases with Agarose Gel in a 3D-Printed Mould Reactor.

Herein, we show how the merge of biocatalysis with flow chemistry aided by 3D-printing technologies can facilitate organic synthesis. This concept was exemplified for the reductive amination of benzaldehyde catalysed by co-immobilised amine dehydrogenase and formate dehydrogenase in a continuous flow micro-reactor. For this purpose, we investigated enzyme co-immobilisation by covalent binding, or ion-affinity binding, or entrapment. Entrapment in an agarose hydrogel turned out to be the most promising solution for this biocatalytic reaction. Therefore, we developed a scalable and customisable approach whereby an agarose hydrogel containing the co-entrapped dehydrogenases was cast in a 3D-printed mould. The reactor was applied to the reductive amination of benzaldehyde in continuous flow over 120 h and afforded 47 % analytical yield and a space-time yield of 7.4 g L day-1 using 0.03 mol% biocatalysts loading. This work also exemplifies how rapid prototyping of enzymatic reactions in flow can be achieved through 3D-printing technology.

PROTACs bearing piperazine-containing linkers: what effect on their protonation state?

Proteolysis targeting chimeras (PROTACs) represent an emerging class of compounds for innovative therapeutic application. Their bifunctional nature induces the formation of a ternary complex (target protein/PROTAC/E3 ligase) which allows target protein ubiquitination and subsequent proteasomal-dependent degradation. To date, despite great efforts being made to improve their biological efficacy PROTACs rational design still represents a challenging task, above all for the modulation of their physicochemical and pharmacokinetics properties. Considering the pivotal role played by the linker moiety, recently the insertion of a piperazine moiety into the PROTAC linker has been widely used, as this ring can in principle improve rigidity and increase solubility upon protonation. Nevertheless, the pK a of the piperazine ring is significantly affected by the chemical groups located nearby, and slight modifications in the linker could eliminate the desired effect. In the present study, the pK a values of a dataset of synthesized small molecule compounds including PROTACs and their precursors have been evaluated in order to highlight how a fine modulation of piperazine-containing linkers can impact the protonation state of these molecules or similar heterobifunctional ones. Finally, the possibility of predicting the trend through in silico approaches was also evaluated.

Discovery of novel SARS-CoV-2 inhibitors targeting the main protease Mpro by virtual screenings and hit optimization.

Two years after its emergence, SARS-CoV-2 still represents a serious and global threat to human health. Antiviral drug development usually takes a long time and, to increase the chances of success, chemical variability of hit compounds represents a valuable source for the discovery of new antivirals. In this work, we applied a platform of variably oriented virtual screening campaigns to seek for novel chemical scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors. The study on the resulting 30 best hits led to the identification of a series of structurally unrelated Mpro inhibitors. Some of them exhibited antiviral activity in the low micromolar range against SARS-CoV-2 and other human coronaviruses (HCoVs) in different cell lines. Time-of-addition experiments demonstrated an antiviral effect during the viral replication cycle at a time frame consistent with the inhibition of SARS-CoV-2 Mpro activity. As a proof-of-concept, to validate the pharmaceutical potential of the selected hits against SARS-CoV-2, we rationally optimized one of the hit compounds and obtained two potent SARS-CoV-2 inhibitors with increased activity against Mpro both in vitro and in a cellular context, as well as against SARS-CoV-2 replication in infected cells. This study significantly contributes to the expansion of the chemical variability of SARS-CoV-2 Mpro inhibitors and provides new scaffolds to be exploited for pan-coronavirus antiviral drug development.

Indomethacin-based PROTACs as pan-coronavirus antiviral agents.

Indomethacin (INM), a well-known non-steroidal anti-inflammatory drug, has recently gained attention for its antiviral activity demonstrated in drug repurposing studies against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Although the mechanism of action of INM is not yet fully understood, recent studies have indicated that it acts at an early stage of the coronaviruses (CoVs) replication cycle. In addition, a proteomic study reported that the anti-SARS-CoV-2 activity of INM could be also ascribed to its ability to inhibit human prostaglandin E synthase type 2 (PGES-2), a host protein which interacts with the SARS-CoV-2 NSP7 protein. Although INM does not potently inhibit SARS-CoV-2 replication in infected Vero E6 cells, here we have explored for the first time the application of the Proteolysis Targeting Chimeras (PROTACs) technology in order to develop more potent INM-derived PROTACs with anti-CoV activity. In this study, we report the design, synthesis, and biological evaluation of a series of INM-based PROTACs endowed with antiviral activity against a panel of human CoVs, including different SARS-CoV-2 strains. Two PROTACs showed a strong improvement in antiviral potency compared to INM. Molecular modelling studies support human PGES-2 as a potential target of INM-based antiviral PROTACs, thus paving the way toward the development of host-directed anti-CoVs strategies. To the best of our knowledge, these PROTACs represent the first-in-class INM-based PROTACs with antiviral activity and also the first example of the application of PROTACs to develop pan-coronavirus agents.

Studiare le migrazioni dal basso?: appunti per un dibattito tra Italia e Brasile / Studying migration from below?: Discussion notes between Italy and Brazil

L´obiettivo centrale di questo articulo è segnalare alcuni spunti di ricerca e porre alcune questioni metodologiche che collocano al centro del dibattito internazionele il fenomeno migratorio. La prospettiva adottata è quella dello sguardo comparato tra Italia e Brasile, si propone un approccio che riesca a tenere conto del ruolo svolto dai protagonisti delle migrazioni.(AU)

This articles´main point is to indicate some research ideas and to raise some methodological questions that are in centre of internacional debate on migrations. Tthe adopted perpective is the comparative look between Italy and Brazil and we propose an approach capable of taking into account the role played by the protagonists of migrations.(AU)