The increasing emergence of viral infections highlights the urgent need for new, broad-spectrum antiviral drugs. Peptide-based therapeutics, such as mucroporin-M1 derived from scorpion venom, offer promising antiviral potential due to their specificity and low toxicity. Here, we report on the design, synthesis, and structural and biological characterization of mucroporin-M1 analogues incorporating α-aminoisobutyric acid (Aib) residues and N-terminal lipidation to enhance helical stability and proteolytic resistance. Structural analyses via circular dichroism and NMR confirmed amphipathic helical conformations for some of the analogues. These modifications significantly improved resistance to enzymatic degradation and enhanced membranolytic activity. Biological evaluation showed that some peptides exert potent virucidal effects against enveloped RNA and DNA viruses belonging to the Flaviviridae or Herpesviridae family, with minimal cytotoxicity, while they are inactive against non-enveloped viruses. These findings suggest that chemically optimized mucroporin-M1 analogues can represent promising candidates for developing peptide-based antivirals endowed with broad-spectrum virucidal activity.

Influenza viruses remain a major global health threat due to their rapid evolution and ability to evade current therapies. Among viral targets, the PA-PB1 interface of the RNA polymerase complex has emerged as an attractive site for small-molecule inhibition. Based on compound 1, a previously identified PA-PB1 interaction inhibitor featuring a cycloheptathiophene-3-carboxamide scaffold, we designed and synthesized a new series of derivatives to investigate the role of the cycloheptyl moiety in antiviral activity and water solubility. In parallel, we developed an improved three-step synthetic route to access 2-amidothiophene-3-carboxamide analogs more efficiently. The new derivatives (2-16) provided valuable structure-activity relationship insights, highlighting how modifications at C-5 influence both anti-influenza potency and solubility. Among them, the C-5 phenyl analog 9 displayed the strongest antiviral activity, achieving sub-micromolar EC50 values (0.19-1.11 µM) across a panel of influenza strains, along with a CC50 value > 100 µM. Notably, the C-5 methyl analog 5 showed the greatest enhancement in aqueous solubility (75.2 µM) while maintaining low-micromolar potency (EC50 of 2 µM) and no significant toxicity (CC50 > 100 µM). Despite structural divergence from the starting hit 1, both compounds preserved the PA-PB1 interaction inhibition mechanism, as demonstrated by enzyme-linked immunosorbent assay (ELISA) and supported by docking studies within the PAC cavity.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects. The triggers of viral neuropathogenesis during congenital infection (cCMV) are still unclear, and treatment options are limited. We used both a two-dimensional model of dynamic neurogenesis and cerebral organoids (COs), recapitulating the developing brain in the first trimester of gestation, to investigate the neuropathogenesis induced by HCMV. We also evaluated antiviral and neuroprotective effects of different compounds, both approved, direct-acting drugs and investigational, host-directed antivirals. In differentiating neurons, treatment with direct-acting antivirals blocked HCMV active replication and provided some protection from virus-induced defects. COs exposed to two different strains of HCMV showed viral spread throughout the organoids, dysregulation of key players of neurogenesis, alteration of the tissue cytoarchitecture, and triggering of innate antiviral and pro-inflammatory responses. Inter-strain differences in virus release and growth attenuation were detected in infected COs. Regardless of the strain, treatment with direct-acting antivirals, particularly letermovir, completely abolished HCMV replication, protected COs from virus-induced disorganization of tissue architecture, and dampened innate immune and pro-inflammatory response activation. Importantly, we also demonstrated the efficacy of the antiviral treatment in HCMV-infected COs in blocking an already established infection. This study contributes to shed light on HCMV-induced neuropathogenesis that occurs during congenital infection. Importantly, we demonstrated the neuroprotective effects of letermovir in models of human developing brain, holding promise for its evaluation as a candidate therapeutic agent to ameliorate cCMV-associated neurodevelopmental defects.

Targeted protein degradation has emerged as a new strategy in drug development, particularly in the anti-cancer field. Although still limited, in recent years, the application of Proteolysis Targeting Chimera (PROTAC) technology against viral infections has been investigated, establishing its broad therapeutic potential. Here, we present the design, synthesis, and biological characterization of a series of PROTACs targeting SARS-CoV-2 Main Protease (MPro) based on a previously identified MPro inhibitor linked to ligands of either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligase. Among the synthesized compounds, the VHL-addressing PROTAC 6 emerged as the most potent Mpro degrader, effectively reducing SARS-CoV-2 Mpro protein levels in infected 293T-hACE2 cells with a DC50 value of 0.9 μM and a Dmax of almost 90% at 25 μM. In parallel, it displayed antiviral activity against SARS-CoV-2 in the low micromolar range, also confirmed in the disease-relevant model of human lung Calu-3 cells, and retained activity against the human endemic coronavirus HCoV-OC43. PROTAC 6 resulted inactive in inhibiting SARS-CoV-2 Mpro catalytic activity in vitro and mechanistic studies confirmed that its antiviral activity is due to the binding and successive induction of degradation of SARS-CoV-2 Mpro. Kinetic solubility experiments showed that PROTAC 6 exhibits very low solubility in phosphate-buffered saline solution but significant higher solubility in two biorelevant dissolution media mimicking intestinal conditions under fasted and fed states, as FaSSIF and FeSSIF. In conclusion, we developed SARS-CoV-2 Mpro degraders based on a novel warhead and confirmed the significant role of targeted protein degradation technology in advancing the identification of new, effective anti-coronavirus PROTACs.

Flaviviruses constitute a global health threat, mainly because of the increasing geographical spread of vectors and the suboptimal efficacy of existing prophylactic vaccines. Despite the substantial clinical burden imposed by these pathogens, a critical gap remains in the approved specific antiviral therapies. This Perspective critically evaluates recent advances in antiflaviviral drug discovery, focusing on viral nonstructural proteins (NSs), host-targeted therapeutic targets, and emerging targeted protein degradation (TPD) technologies. We highlight direct-acting antivirals (DAAs) against NS2B-NS3, NS5, and NS4B, host-targeted antivirals (HTAs) that modulate virus-dependent cellular pathways, and the transformative potential of TPD, thereby filling the gap of insufficient systematic summaries in this field. By integrating rational drug design strategies and dissecting the pharmacological profiles of agents with different scaffolds, this Perspective aims to provide a panoramic snapshot of the current landscape and a conceptual roadmap for accelerating the development of next-generation, broad-spectrum antiflaviviral drugs.

Emerging viral threats highlight the urgent need for antivirals targeting viral replication mechanisms. Based on previous research, we expanded a series of asymmetric benzene-1,4-disulfonamides. As these proved to be synthetically challenging and generally more toxic than expected, we optimized a hit structure by omitting one sulfonamide group. The resulting compound combines micromolar antiviral efficacy with low toxicity and straightforward synthesis. Molecular docking and enzyme-linked immunosorbent assay (ELISA) assays confirmed that the compound targets the NS5 RNA polymerase domain of dengue virus-2, blocking its interaction with NS3 and thereby inhibiting viral replication. A focused series of structural analogs further demonstrated the essential contribution of each individual part of the hit molecule.

Targeted degradation is emerging as a new therapeutic approach in the treatment of different diseases. It allows hijacking the cellular pathways deputed to protein or nucleic acid homeostasis to degrade a target macromolecule of interest involved in a pathogenic process. In the last decades, targeted protein degradation has been widely applied for the treatment of cancer or neurodegenerative disorders and some of such therapies are already in clinical use. More recently, therapeutic degraders such as PROTACs, LYTACs, HyTs, BacPROTACs, and others have also been explored in the field of antimicrobial and antiviral drug discovery. The peculiar mechanism of action, along with the opportunity to degrade both microbial and host targets, holds great promise for overcoming some limitations of classic antimicrobials, e.g. drug resistance, as well as for increasing the potency of current therapies. With a focus on the antimicrobial field, this Review aims at providing a comprehensive, state-of-the-art description of targeted degradation mechanisms and strategies developed so far, as well as to discuss advantages, disadvantages, and caveats of this innovative approach for combating infectious diseases.

Influenza viruses still represent a great concern for Public Health by causing yearly seasonal epidemics and occasionally worldwide pandemics. Moreover, spillover events at the animal-human interface are becoming more frequent nowadays, also involving animal species not previously found as reservoirs. To restrict the effects of influenza virus epidemics, especially in at-risk population, and to prepare a drug arsenal for possible future pandemics, researchers worldwide have been working on the development of antiviral strategies since the 80's of the last century. One of the main obstacles is the considerable genomic variability of influenza viruses, which constantly poses the issues of drug-resistance emergence and immune evasion. This review summarizes the approved therapeutics for clinical management of influenza, promising new anti-flu compounds and monoclonal antibodies currently undergoing clinical evaluation, and molecules with efficacy against influenza virus in preclinical studies. Moreover, we discuss some innovative anti-influenza therapeutic approaches such as combination therapies and targeted protein degradation. Given the limited number of drugs approved for influenza treatment, there is a still strong need for novel potent anti-influenza drugs endowed with a high barrier to drug resistance and broad-spectrum activity against influenza viruses of animal origin that may be responsible of future large outbreaks and pandemics.

Dengue virus (DENV) poses a major public health concern as it is responsible for approximately 100 million human infections annually. Since no antiviral drugs are currently available to treat DENV infection, the development of effective therapeutic strategies is urgently needed. For anti-DENV drug discovery, the interaction between DENV NS3 and NS5 proteins represents an attractive target, as it is essential for viral replication and is highly conserved across all DENV serotypes. In this study, we report two distinct virtual screenings of commercially available drug-like compounds, which were performed to identify inhibitors of the NS3-NS5 interaction. Both screening approaches led to the identification of hit compounds that were able to reduce NS3-NS5 binding in vitro in a dose-dependent manner, as measured by an ELISA-based assay. Moreover, the hits inhibited the replication of DENV-2 at low micromolar and non-cytotoxic concentrations. Among these, hit 3 exhibited the highest selectivity index and showed antiviral activity against all four DENV serotypes. Biophysical studies indicated that hit 3 exerts its antiviral activity by directly binding to NS5. Hit 3 was then selected for structure-activity relationship studies, leading to the identification of structural analogues that retained anti-DENV activity through the disruption of NS3-NS5 interaction. Overall, this study reports the identification of a series of novel chemical scaffolds endowed with pan-dengue antiviral activity, representing a promising foundation for the development of new anti-DENV agents.

SARS-CoV-2 Main protease (Mpro) is the most explored coronavirus antiviral target, being most antivirals approved or under development protease inhibitors. Mpro is active as a dimer and the molecular details of its maturation are poorly understood. Some compounds that crystallize at the dimerization interface rather than at the catalytic pocket have been proposed as allosteric inhibitors. Here, we characterize a series of novel compounds starting from a scaffold identified by an in silico screening for Mpro catalytic pocket. Several compounds showed anti-SARS-CoV-2 activity in infected cells, but they did not inhibit Mproin vitro. Time-of-addition studies pointed to a stage compatible with Mpro targeting. Molecular modelling studies suggested that compounds 1 and 11 bind Mpro similarly to the allosteric inhibitor AT7519. Small-angle X-ray scattering studies revealed that 1 and 11 strongly shift Mpro equilibrium to the monomeric form, while the allosteric inhibitor pelitinib and the catalytic inhibitors nirmatrelvir and GC376 stabilize the dimer. Compounds 1 and 11 inhibited Mpro proteolytic activity in SARS-CoV-2 infected cells acting as allosteric inhibitors that stabilize the monomeric form. In conclusion, we validated an allosteric site in Mpro that could be exploited for the development of effective anti-SARS-CoV-2 antivirals targeting Mpro with a novel mechanism.

Keratoacanthoma (KA) is a rapidly growing epithelial neoplasm characterized by clinical and histopathological features that often overlap with well-differentiated squamous cell carcinoma (SCC), posing diagnostic challenges. This review provides a comprehensive overview of KA, emphasizing advances in non-invasive diagnostic techniques such as dermoscopy, reflectance confocal microscopy (RCM), and line-field confocal optical coherence tomography (LC-OCT), which improve lesion characterization and differentiation from SCC. We discuss the histopathological phases of KA and highlight key features aiding in diagnosis. Furthermore, we explore the emerging role of human papillomavirus (HPV), particularly β-genus types, as a cofactor in KA carcinogenesis through modulation of apoptosis and DNA damage response pathways, especially under ultraviolet (UV) radiation exposure. Therapeutic strategies remain centered on complete surgical excision; however, alternative treatments, including radiotherapy, cryotherapy, topical agents, and systemic retinoids, are discussed with their respective benefits and limitations. Finally, we review current HPV vaccines and novel vaccine candidates targeting a broad spectrum of mucosal and cutaneous HPV types. This review underscores the importance of integrated diagnostic and therapeutic approaches to optimize KA management and highlights future directions in understanding its pathogenesis and treatment.

In recent years, the emergence of new viruses and the re-emergence of old ones have posed a significant challenge to global Public Health. Viruses characterised by high morbidity and mortality rates have the potential to spread rapidly, causing large epidemic outbreaks and even pandemics. In this context, viral infections still lacking effective treatments represent a serious threat to human health. For this reason, sustained development and implementation of countermeasures are urgently needed against these infections, as they are for diseases for which the emergence of drug resistance is rapidly increasing. In this regard, compared to de novo drug discovery, drug repurposing could represent a highly efficient, faster, and more affordable strategy to develop new drugs. Here, we provide a comprehensive review of the different experimental and computational approaches used for drug repurposing and discuss their advantages and limitations in comparison with other drug discovery strategies. In addition, as an example of the successful application of drug repurposing, we present the case of approved antifungal drugs that could be repurposed to counteract viral infections.

Influenza viruses (IV) are single-stranded RNA viruses with a negative-sense genome and have the potential to cause pandemics. While vaccines exist for influenza, their protection is only partial. Additionally, there is only a limited number of approved anti-IV drugs, which are associated to emergence of drug resistance. To address these issues, for years we have focused on the development of small-molecules that can interfere with the heterodimerization of PA and PB1 subunits of the IV RNA-dependent RNA polymerase (RdRP). In this study, starting from a cycloheptathiophene-3-carboxamide compound that we recently identified, we performed iterative cycles of medicinal chemistry optimization that led to the identification of compounds 43 and 45 with activity in the nanomolar range against circulating A and B strains of IV. Mechanistic studies demonstrated the ability of 43 and 45 to interfere with viral RdRP activity by disrupting PA-PB1 subunits heterodimerization and to bind to the PA C-terminal domain through biophysical assays. Most important, ADME studies of 45 also showed an improvement in the pharmacokinetic profile with respect to the starting hit.

Inspired by our previous finding that targeting the 150-cavity with a multisite-binding strategy emerged as an effective approach to obtain more potent and selective neuraminidase (NA) inhibitors against influenza virus, we present here the design, synthesis, and optimization of novel boron-containing N-substituted oseltamivir (OSC) derivatives. Exploratory structure-activity relationship (SAR) studies led to the identification of compounds 27c and 33c as the most potent NA inhibitors, surpassing OSC in potency against both wild-type group-1 NAs and oseltamivir-resistant NAs. These compounds demonstrated significant antiviral activity against several wild-type strains and H1N1pdm09 strains (EC50 = 0.03 ± 0.005 and 0.03 ± 0.0008 μM, respectively). Additionally, these compounds did not exhibit significant toxicity (CC50 > 200 μM in CEF cells; CC50 > 250 μM in MDCK cells). These findings highlight 27c and 33c as promising next-generation anti-influenza agents.

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.

Human cytomegalovirus (HCMV) is the viral leading cause of congenital defects in newborns worldwide. Many aspects of congenital CMV (cCMV) infection, which currently lacks a specific treatment, as well as the main determinants of neuropathogenesis in the developing brain during HCMV infection are unclear. In this study, we modeled HCMV infection at different stages of neural development. Moreover, we evaluated the effects of both approved and investigational anti-HCMV drugs on viral replication and gene expression in two different neural progenitor cell lines, i.e., human embryonic stem cells-derived neural stem cells (NSCs) and fetus-derived neuroepithelial stem (NES) cells. Ganciclovir, letermovir, nitazoxanide, and the ozonide OZ418 reduced viral DNA synthesis and the production of infectious virus in both lines of neural progenitors. HCMV infection dysregulated the expression of genes that either are markers of neural progenitors, such as SOX2, NESTIN, PAX-6, or play a role in neurogenesis, such as Doublecortin. Treatment with antiviral drugs had different effects on HCMV-induced dysregulation of the genes under investigation. This study contributes to the understanding of the molecular mechanisms of cCMV neuropathogenesis and paves the way for further consideration of anti-HCMV drugs as candidate therapeutic agents for the amelioration of cCMV-associated neurological manifestations.

The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein–protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA–PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a–n) or several amino acid groups (compounds 3a–n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 μM in PA–PB1 ELISA, an EC50 value of 2.8 μM in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds.

To date, Proteolysis Targeting Chimera (PROTAC) technology has been successfully applied to mediate proteasomal-induced degradation of several pharmaceutical targets mainly related to oncology, immune disorders, and neurodegenerative diseases. On the other hand, its exploitation in the field of antiviral drug discovery is still in its infancy. Recently, we described two indomethacin (INM)-based PROTACs displaying broad-spectrum antiviral activity against coronaviruses. Here, we report the design, synthesis, and characterization of a novel series of INM-based PROTACs that recruit either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. The panel of INM-based PROTACs was also enlarged by varying the linker moiety. The antiviral activity resulted very susceptible to this modification, particularly for PROTACs hijacking VHL as E3 ligase, with one piperazine-based compound (PROTAC 6) showing potent anti-SARS-CoV-2 activity in infected human lung cells. Interestingly, degradation assays in both uninfected and virus-infected cells with the most promising PROTACs emerged so far (PROTACs 5 and 6) demonstrated that INM-PROTACs do not degrade human PGES-2 protein, as initially hypothesized, but induce the concentration-dependent degradation of SARS-CoV-2 main protease (Mpro) both in Mpro-transfected and in SARS-CoV-2-infected cells. Importantly, thanks to the target degradation, INM-PROTACs exhibited a considerable enhancement in antiviral activity with respect to indomethacin, with EC50 values in the low-micromolar/nanomolar range. Finally, kinetic solubility as well as metabolic and chemical stability were measured for PROTACs 5 and 6. Altogether, the identification of INM-based PROTACs as the first class of SARS-CoV-2 Mpro degraders demonstrating activity also in SARS-CoV-2-infected cells represents a significant advance in the development of effective, broad-spectrum anti-coronavirus strategies.

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections.

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.