It has been more than four years since the first report of SARS‐CoV‐2, the virus responsible for the coronavirus disease 2019 (COVID‐19) pandemic, the scientific community is focused on vaccine development in an exceptionally rapid time frame, as well as the evaluation of a wide range of potential treatments in clinical trials, a few of which have also reached the market. However, these drugs are characterized by several limits (including low response to treatment in some patients, low effectiveness against the new variants, severe side effects, etc.), thus underscoring the need to speed up the research. Among potential antiviral targets, the SARS‐CoV‐2 nonstructural protein 13 (nsp13) is highly promising thanks to its pivotal role in viral replication. Pursuing the studies on the development of nsp13 inhibitors, herein, the design, synthesis, and biological evaluation of new SARS‐CoV‐2 nsp13 inhibitors are reported. In general, the newly designed dikehexenoic derivatives are proven active against both the enzymatic activities showing measurable IC₅₀ under 30 μM concentration, while the diketobutanoic series shows less promising results. Moreover, the tested compounds are capable of blocking viral replication without exerting cytotoxicity. Docking studies predict their binding into an allosteric pocket within the RecA2 domain.

A small library of novel thiazolidinone-based sulfonamide derivatives was designed, synthesized and evaluated for their ability to target human carbonic anhydrase (hCA) isoforms IX and XII, which are overexpressed in malignant cells and play a key role in metastasis and therapeutic response of cancer cells. A molecular hybridization approach was employed to design the molecules by combining different moieties identified as having antitumor activity. The thiazolidinone core was functionalized with benzenesulfonamide as a zinc-binding group and different isatin derivatives to enhance the chemical profile and optimize the hydrophilic/lipophilic balance. Biological evaluation against hCA I, II, IX and XII isoforms showed promising inhibitory activities, and some compounds exhibited selectivity and high inhibitory activity against hCA IX and hCA XII while not affecting off-target hCA I and hCA II. In particular, compound 3h demonstrated high selectivity with K_i values of 57.8 nM for hCA IX and 44.3 nM for hCA XII.

Since its emergence in late 2019, SARS-CoV-2, the causative agent of COVID-19, has continued to spread globally, with more than 7 million reported deaths as of March 2025. Among the viral nonstructural proteins, nsp12 serves as the RNA-dependent RNA polymerase (RdRp), mediating viral genome replication and transcription in concert with its cofactors nsp7 and nsp8. To date, only two nucleoside analogs specifically targeting SARS-CoV-2 nsp12, remdesivir and molnupiravir, have been authorized by the FDA for COVID-19 treatment. In response to the need for additional safe and effective antiviral agents, we screened two extensive in silico libraries of safe-in-man compounds (>9,000) and natural compounds (>249,000), against the SARS-CoV-2 nsp12/7/8 complex, targeting the orthosteric and two allosteric nsp12 sites, using the EXSCALATE (EXaSCale smArt pLatform Against paThogEns) platform. Compounds were then selected based on docking score significance, novelty for the target, and clinical safety profiles. The top 119 candidates were subsequently evaluated in a biochemical assay to assess their potential to inhibit SARS-CoV-2 nsp12/7/8 polymerase activity, identifying 42 compounds able to block it, among which four showed IC₅₀ and EC₅₀ values in the nanomolar or low micromolar range. When tested in cell-based assays to evaluate their efficacy on SARS-CoV-2 replication, they proved to inhibit it in the same concentration ranges. Mechanism of action studies revealed different modalities of inhibition. These results provide the basis for the development of novel antiviral compounds against SARS-CoV-2, targeting both the RdRp active site and an allosteric site, further suggesting that the Computer-Aided Drug Discovery (CADD) approach, together with experimental validation, can provide the basis for accelerated antiviral drug development.

COVID-19 pandemic stimulated tremendous efforts to develop therapeutic strategies targeting SARS-CoV-2, leading to the evaluation of a wide range of potential treatments in clinical trials. However, effective therapeutics remain elusive when the development of new variants and the limits of antiviral drugs is considered. Therefore, the development of antiviral drugs against SARS-CoV-2 is of paramount importance. Among potential drug targets, the SARS-CoV-2 nsp13 is highly attractive thanks to its pivotal role in viral replication. Pursuing our studies on the development of nsp13 inhibitors, in this work we describe the design, synthesis, and biological evaluation of novel inhibitors targeting SARS-CoV-2 nsp13. The newly designed N-benzyl indole derivatives were active against both enzymatic activities showing measurable IC₅₀ under 30 μM concentration, while N-alkyl derivatives showed less promising results. Interestingly, the tested compounds blocked viral replication with no cytotoxicity. Docking studies predicted their binding into an allosteric conserved site located in the RecA2 domain.

The SARS-CoV-2 pandemic has posed a tremendous burden globally, highlighting the urgent need for new effective antivirals that are possibly useful against future emerging Coronaviruses (hCoVs). In this context, major efforts were focused on the inhibition of highly conserved and essential targets playing a pivotal role in viral replication. Among them, SARS-CoV-2 nsp13 stands out, being the most conserved enzyme within hCoVs. Following our previous reports describing the identification of indole-based diketo acid (DKA) derivatives as SARS-CoV-2 nsp13 inhibitors endowed with antiviral activity, we applied a scaffold hopping strategy to identify new nsp13 inhibitors. Therefore, we investigated a series of 4-phenyl pyrrolyl DKAs and their structural analogs characterized by molecular simplification or DKA isosteric replacement. The derivatives showed potency against both nsp13-associated activities exhibiting measurable IC₅₀s in the low micromolar/submicromolar range, highlighting a promising dual inhibitory profile accordingly. Structure–activity relationship (SAR) studies were performed, highlighting the main structural features increasing the activity of the different compound classes. Interestingly, SAR trends were confirmed in the presence of the BSA/TCEP system despite variations in potency. To shed light on the interaction of the best acting compounds 13b, 15a, and 17d, docking studies were performed, suggesting a putative binding mode in agreement with our previous findings.

In the search for novel antiviral agents targeting SARS-CoV-2 non-structural protein 13 (Nsp13), we applied an integrated in silico drug design workflow combining ADME profiling, molecular interaction field analysis, molecular docking and thermodynamic evaluations to identify thiazolo-pyridine derivatives as promising scaffolds. This computational strategy enabled the rational prioritization of compounds and guided the design and synthesis of a novel series of [1,3]thiazolo[5,4-b]pyridine-6-carboxamides (19 and 20) and [1,3]thiazolo[5,4-h][1,6]naphthyridines (21 and 22). Among the synthesized compounds, 19h and 19n emerged as the most potent candidates, demonstrating dual inhibitory activity with IC₅₀ values for unwinding and ATPase activities in the low micromolar range. Kinetic analyses confirmed a competitive inhibition mechanism, as these compounds successfully compete with the natural substrate ATP for the enzyme's active site. Overall, our findings highlight the effectiveness of integrated computational approaches for accelerating antiviral lead optimization and establish the thiazolo-pyridine core as viable chemotype for further Structure-Activity Relationship (SAR) studies and preclinical development.