Luigi Scipione

Given its pivotal role in the viral life cycle, blocking integrase (IN) through IN strand transfer inhibitors (INSTIs) represented a breakthrough in the treatment of HIV infection, establishing these antiretroviral regimens as first‐line options against AIDS. However, the onset of drug‐resistant strains has challenged the efficacy of INSTIs, demanding new efforts in the search for therapeutic tools that work through alternative modes of action. In this context, the discovery of allosteric IN inhibitors (ALLINIs) has highlighted new opportunities to target IN beyond its active site, enhancing antiviral efficacy and the genetic barrier to resistance. Extensive drug discovery campaigns have resulted in the development of effective ALLINIs with both in vitro and in vivo efficacy, two of which are advancing in clinical trials as next‐generation therapeutic tools. Nevertheless, advancements in structural biology have critically aided in elucidating the underlying effects of ALLINIs, highlighting a complex, multimodal mode of action that ultimately yields defects in virion maturation. This review focuses on ALLINIs, providing a comprehensive overview of major drug discovery efforts devoted to this field, with an emphasis on the medicinal chemistry and structural biology findings that have revealed unprecedented opportunities for developing innovative and effective anti‐HIV drugs.

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.

Terminal deoxynucleotidyl transferase (TdT) is overexpressed in some cancer types, where it drives the mutagenic repair of double strand breaks through non canonical non-homologous end joining pathway. The TdT enzyme belongs to the X family of polymerases, together with the DNA polymerase λ (pol λ) and β (pol β). However, TdT exclusively displays template-independent nucleotide polymerisation. Pursuing our studies in developing TdT inhibitors, herein we deepened the structure-activity relationships of new structural analogues of our previously identified hit compounds. The diketo hexenoic acid derivatives here analysed showed high selectivity towards TdT and inhibition potencies spanning from the low micromolar range to the nanomolar. Docking studies highlighted the chemical features involved in the TdT binding, well contributing to the rationalisation of the structural requirements needed for the enzymatic inhibition.

The continuation of the phytochemical analysis to determine the alkaloid content of Vinca difformis subsp. sardoa Stearn, led to the isolation of the further compound vincamajine (1) which was reported from the species for the first time during this study. Its structure was established by means of extensive NMR, IR and MS analyses which are completely reported in this paper for the first time. In addition, the first chemophenetic evaluation about this compound was also carried out evidencing some important results which were fully described and discussed in this paper.

The HIV-1 integrase (IN) plays a critical role in the viral lifecycle by integrating the viral DNA into the host chromosome. The catalytic function of IN has been exploited as a target, with five drugs acting as active site binders (IN strand transfer inhibitors, INSTIs). However, IN mutations conferring low-level resistance to INSTIs have been reported. Therefore, new IN inhibitors with different mechanisms of action are needed. The allosteric inhibition of IN, exerted by allosteric IN inhibitors (ALLINIs), is gaining interest. ALLINIs inhibit IN by inducing aberrant IN multimerization with different mechanisms. Furthermore, recent discoveries unveiled that IN has an under-studied yet equally vital second function. This involves IN binding to the RNA genome in virions, necessary for proper virion maturation. In this work, we describe a series of quinolinonyl derivatives as inhibitors of both the IN catalytic functions and IN-RNA interactions, which impair both early and late steps of viral replication.

In this work, for the first time, different parts of the Finger Lime (Citrus australasica F. Muell.), such as pulp, peel and seeds, were analyzed by HS-SPME-GC/MS, and NMR techniques in order to describe its volatile and non-volatile chemical profile. The results highlighted the presence of a high number of terpenes with limonene as principal component in all investigated parts (ranging from 40.4% to 62.6%) and molecules belonging to the classes of amino acids, organic acids, carbohydrates, fatty acids, phenols and miscellaneous compounds that followed a different trend between the investigated different parts. In this study, the inhibition of ChEs (AChE and BChE) was evaluated using the spectrophotometric method of Ellman. The results showed that only peel extract weakly inhibited AChE (14%). Based on these data, this extract was further investigated by GC/MS after derivatization. Furthermore, peel extract was chosen to evaluate the in vitro effects on two human glioblastoma cells lines (U87 and LN18). Flow cytometry results showed that citrus extract was more effective in down-regulating the expression of the adhesion molecule CD44. In fact, after 72 h with 400 µg/mL of citrus extract, CD44 expression levels were reduced in both U87 and LN18 glioblastoma cell lines. This was confirmed by immunofluorescence analysis, which also showed a modification of CD44 antigen localization in both U87 and LN18 cell lines. Moreover, wound assay data supported its ability to reduce glioblastoma cell’s motility. The migration ability of U87 cells decreased (85% control vs. 50% at 400 μg/mL), while it was even more pronounced in resistant LN18 cells (93% control vs. 15% at 400 μg/mL). The findings highlighted that citrus peel extract could have an anti-invasive activity for glioma management.

It has been more than four years since the first report of SARS-CoV-2, and humankind has experienced a pandemic with an unprecedented impact. Moreover, the new variants have made the situation even worse. Among viral enzymes, the SARS-CoV-2 main protease (M^pro) has been deemed a promising drug target vs. COVID-19. Indeed, M^pro is a pivotal enzyme for viral replication, and it is highly conserved within coronaviruses. It showed a high extent of conservation of the protease residues essential to the enzymatic activity, emphasizing its potential as a drug target to develop wide-spectrum antiviral agents effective not only vs. SARS-CoV-2 variants but also against other coronaviruses. Even though the FDA-approved drug nirmatrelvir, a M^pro inhibitor, has boosted the antiviral therapy for the treatment of COVID-19, the drug shows several drawbacks that hinder its clinical application. Herein, we report the synthesis of new thiazolidine-4-one derivatives endowed with inhibitory potencies in the micromolar range against SARS-CoV-2 M^pro. In silico studies shed light on the key structural requirements responsible for binding to highly conserved enzymatic residues, showing that the thiazolidinone core acts as a mimetic of the Gln amino acid of the natural substrate and the central role of the nitro-substituted aromatic portion in establishing π-π stacking interactions with the catalytic His-41 residue.

In this work, phytochemical analysis on different extracts of Roccella tinctoria DC. was reported using different techniques with respect to the past. Twenty volatile and three non-volatile compounds were identified, some of which were found in this species for the first time. The methanolic extracts and their non-volatile components were then evaluated for their antitumor effects in cancerous A549 and Mz-ChA-1 cells and for their tolerability in non-cancerous BEAS-2B and H69 cells, showing IC₅₀ values from 94.6 µg/mL to 416.4 µg/mL, in general. The same extracts and compounds were also tested for their antifungal effects in Candida albicans, with only compound 2 being active, with an MIC₅₀ value of 87 µg/mL. In addition, they were tested for their anti-Candida adhesion activity, anti-Candida biofilm formation, and anti-Candida mature biofilm inhibition, with efficacy percentages generally above 50% but not for all of them. Lastly, the DF3 extract and compounds 1–2 were tested in vivo according to the Galleria mellonella survival assay, showing positive mortality rates above 50% at different concentrations. All these biological assays were conducted on this species for the first time. Comparisons with other lichens and compounds were also presented and discussed.

Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC₅₀ ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC₅₀ and K_D compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.