The Novel A-Type Proanthocyanidin-Rich Phytocomplex SP4™ Acts as a Broad-Spectrum Antiviral Agent against Human Respiratory Viruses.

The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections.

Mechanisms of antiviral activity of the new hDHODH inhibitor MEDS433 against respiratory syncytial virus replication.

Human respiratory syncytial virus (RSV) is an important cause of acute lower respiratory infections, for which no effective drugs are currently available. The development of new effective anti-RSV agents is therefore an urgent priority, and Host-Targeting Antivirals (HTAs) can be considered to target RSV infections. As a contribution to this antiviral avenue, we have characterized the molecular mechanisms of the anti-RSV activity of MEDS433, a new inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of de novo pyrimidine biosynthesis. MEDS433 was found to exert a potent antiviral activity against RSV-A and RSV-B in the one-digit nanomolar range. Analysis of the RSV replication cycle in MEDS433-treated cells, revealed that the hDHODH inhibitor suppressed the synthesis of viral genome, consistently with its ability to specifically target hDHODH enzymatic activity. Then, the capability of MEDS433 to induce the expression of antiviral proteins encoded by Interferon-Stimulated Genes (ISGs) was identified as a second mechanism of its antiviral activity against RSV. Indeed, MEDS433 stimulated secretion of IFN-ß and IFN-λ1 that, in turn, induced the expression of some ISG antiviral proteins, such as IFI6, IFITM1 and IRF7. Singly expression of these ISG proteins reduced RSV-A replication, thus likely contributing to the overall anti-RSV activity of MEDS433. Lastly, MEDS433 proved to be effective against RSV-A replication even in a primary human small airway epithelial cell model. Taken as a whole, these observations provide new insights for further development of MEDS433, as a promising candidate to develop new strategies for treatment of RSV infections.

Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 µM and 10.1 µM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 µM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses.

Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

The Novel hDHODH Inhibitor MEDS433 Prevents Influenza Virus Replication by Blocking Pyrimidine Biosynthesis.

The pharmacological management of influenza virus (IV) infections still poses a series of challenges due to the limited anti-IV drug arsenal. Therefore, the development of new anti-influenza agents effective against antigenically different IVs is therefore an urgent priority. To meet this need, host-targeting antivirals (HTAs) can be evaluated as an alternative or complementary approach to current direct-acting agents (DAAs) for the therapy of IV infections. As a contribution to this antiviral strategy, in this study, we characterized the anti-IV activity of MEDS433, a novel small molecule inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 exhibited a potent antiviral activity against IAV and IBV replication, which was reversed by the addition of exogenous uridine and cytidine or the hDHODH product orotate, thus indicating that MEDS433 targets notably hDHODH activity in IV-infected cells. When MEDS433 was used in combination either with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, or with an anti-IV DAA, such as N4-hydroxycytidine (NHC), synergistic anti-IV activities were observed. As a whole, these results indicate MEDS433 as a potential HTA candidate to develop novel anti-IV intervention approaches, either as a single agent or in combination regimens with DAAs.

The New Generation hDHODH Inhibitor MEDS433 Hinders the In Vitro Replication of SARS-CoV-2 and Other Human Coronaviruses.

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 inhibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

Effective deploying of a novel DHODH inhibitor against herpes simplex type 1 and type 2 replication.

Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.