Exploring a New Generation of Pyrimidine and Pyridine Derivatives as Anti-Influenza Agents Targeting the Polymerase PA-PB1 Subunits Interaction.

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.

The Rise of Bacterial G-Quadruplexes in Current Antimicrobial Discovery.

Antimicrobial resistance (AMR) is a silent critical issue that poses several challenges to health systems. While the discovery of novel antibiotics is currently stalled and prevalently focused on chemical variations of the scaffolds of available drugs, novel targets and innovative strategies are urgently needed to face this global threat. In this context, bacterial G-quadruplexes (G4s) are emerging as timely and profitable targets for the design and development of antimicrobial agents. Indeed, they are expressed in regulatory regions of bacterial genomes, and their modulation has been observed to provide antimicrobial effects with translational perspectives in the context of AMR. In this work, we review the current knowledge of bacterial G4s as well as their modulation by small molecules, including tools and techniques suitable for these investigations. Finally, we critically analyze the needs and future directions in the field, with a focus on the development of small molecules as bacterial G4s modulators endowed with remarkable drug-likeness.

Novel 2,4-Dichloro-5-sulfamoylbenzoic Acid Oxime Esters: First Studies as Potential Human Carbonic Anhydrase Inhibitors.

In this study, a focused library of oxime ester derivatives of 2,4-dichloro-5-sulfamoylbenzoic acid (lasamide) containing Schiff bases was synthesized and tested in vitro for their ability to inhibit the cytosolic human carbonic anhydrases (hCAs) I and II, as well as the transmembrane and tumor-associated IX and XII isoforms. As a result, we obtained a first line of knowledge on lasamide derivatives potentially useful for development as CA inhibitors (CAIs). In particular, we focused our attention on the derivative 11, which was selective toward hCAs IX and XII over the cytosolic isoenzymes. An in silico study was conducted to assess the binding mode of 11 within hCAs IX and XII. Also, antiproliferative assays highlighted promising derivatives. The data obtained in this study are currently in use for the development of better-performing compounds on the tumor-associated isoforms.

Exploring the Antiviral Potential of Natural Compounds against Influenza: A Combined Computational and Experimental Approach.

The influenza A virus nonstructural protein 1 (NS1), which is crucial for viral replication and immune evasion, has been identified as a significant drug target with substantial potential to contribute to the fight against influenza. The emergence of drug-resistant influenza A virus strains highlights the urgent need for novel therapeutics. This study proposes a combined theoretical criterion for the virtual screening of molecular libraries to identify candidate NS1 inhibitors. By applying the criterion to the ZINC Natural Product database, followed by ligand-based virtual screening and molecular docking, we proposed the most promising candidate as a potential NS1 inhibitor. Subsequently, the selected natural compound was experimentally evaluated, revealing measurable virus replication inhibition activity in cell culture. This approach offers a promising avenue for developing novel anti-influenza agents targeting the NS1 protein.

Perspectives in the treatment of antibiotic-resistant bacterial infections with active photodynamic partners within the framework of the EURESTOP COST Action (CA21145).

The European Network for diagnosis and treatment of antibiotic-resistant bacterial infections-EURESTOP COST Action CA21145 focuses on tackling the burden of antimicrobial resistance (AMR) and has gathered many members working on photodynamic approaches. This European consortium is presented here in the One Health context, to highlight the potential of antimicrobial photodynamic therapy (aPDT) in the fight against AMR.

Recent developments of agents targeting Vibrio cholerae: patents and literature data.

INTRODUCTION: Vibrio cholerae bacteria cause an infection characterized by acute diarrheal illness in the intestine. Cholera is sustained by people swallowing contaminated food or water. Even though symptoms can be mild, if untreated disease becomes severe and life-threatening, especially in low-income countries. AREAS COVERED: After a description of the most recent literature on the pathophysiology of this infection, we searched for patents and literature articles following the PRISMA guidelines, filtering the results disclosed from 2020 to present. Moreover, some innovative molecular targets (e.g., carbonic anhydrases) and pathways to counteract this rising problem were also discussed in terms of design, structure-activity relationships and structural analyses. EXPERT OPINION: This review aims to cover and analyze the most recent advances on the new druggable targets and bioactive compounds against this fastidious pathogen, overcoming the use of old antibiotics which currently suffer from high resistance rate.

Hedgehog Pathway Inhibition by Novel Small Molecules Impairs Melanoma Cell Migration and Invasion under Hypoxia.

Melanoma is the principal cause of death in skin cancer due to its ability to invade and cause metastasis. Hypoxia, which characterises the tumour microenvironment (TME), plays an important role in melanoma development, as cancer cells can adapt and acquire a more aggressive phenotype. Carbonic anhydrases (CA) activity, involved in pH regulation, is related to melanoma cell migration and invasion. Furthermore, the Hedgehog (Hh) pathway, already known for its role in physiological processes, is a pivotal character in cancer cell growth and can represent a promising pharmacological target. In this study, we targeted Hh pathway components with cyclopamine, glabrescione B and C22 in order to observe their effect on carbonic anhydrase XII (CAXII) expression especially under hypoxia. We then performed a migration and invasion assay on two melanoma cell lines (SK-MEL-28 and A375) where Smoothened, the upstream protein involved in Hh regulation, and GLI1, the main transcription factor that determines Hh pathway activation, were chemically inhibited. Data suggest the existence of a relationship between CAXII, hypoxia and the Hedgehog pathway demonstrating that the chemical inhibition of the Hh pathway and CAXII reduction resulted in melanoma migration and invasion impairment especially under hypoxia. As in recent years drug resistance to small molecules has arisen, the development of new chemical compounds is crucial. The multitarget Hh inhibitor C22 proved to be effective without signs of cytotoxicity and, for this reason, it can represent a promising compound for future studies, with the aim to reach a better melanoma disease management.

The antimicrobial potential of adarotene derivatives against Staphylococcus aureus strains.

Multidrug-resistant (MDR) pathogens are severely impacting our ability to successfully treat common infections. Here we report the synthesis of a panel of adarotene-related retinoids showing potent antimicrobial activity on Staphylococcus aureus strains (including multidrug-resistant ones). Fluorescence and molecular dynamic studies confirmed that the adarotene analogues were able to induce conformational changes and disfunctions to the cell membrane, perturbing the permeability of the phospholipid bilayer. Since the major obstacle for developing retinoids is their potential cytotoxicity, a selected candidate was further investigated to evaluate its activity on a panel of human cell lines. The compound was found to be well tolerated, with IC50 5-15-fold higher than the MIC on S. aureus strains. Furthermore, the adarotene analogue had a good pharmacokinetic profile, reaching a plasma concentration of about 6 µM after 0.5 h after administration (150 mg/kg), at least twice the MIC observed against various bacterial strains. Moreover, it was demonstrated that the compound potentiated the growth-inhibitory effect of the poorly bioavailable rifaximin, when used in combination. Overall, the collected data pave the way for the development of synthetic retinoids as potential therapeutics for hard-to-treat infectious diseases caused by antibiotic-resistant Gram-positive pathogens.

Selection of Natural Compounds with HMGA-Interfering Activities and Cancer Cell Cytotoxicity.

HMGA proteins are intrinsically disordered (ID) chromatin architectural factors characterized by three DNA binding domains (AT-hooks) that allow them to bind into the DNA minor groove of AT-rich stretches. HMGA are functionally involved in regulating transcription, RNA processing, DNA repair, and chromatin remodeling and dynamics. These proteins are highly expressed and play essential functions during embryonic development. They are almost undetectable in adult tissues but are re-expressed at high levels in all cancers where they are involved in neoplastic transformation and cancer progression. We focused on identifying new small molecules capable of binding into the minor groove of AT-rich DNA sequences that could compete with HMGA for DNA binding and, thus, potentially interfere with their activities. Here, a docking-based virtual screening of a unique high diversity in-house library composed of around 1000 individual natural products identified 16 natural compounds as potential minor groove binders that could inhibit the interaction between HMGA and DNA. To verify the ability of these selected compounds to compete with HMGA proteins, we screened them using electrophoretic mobility shift assays. We identified Sorocein C, a Diels-Alder (D-A)-type adducts, isolated from Sorocea ilicifolia and Sorocea bonplandii with an HMGA/DNA-displacing activity and compared its activity with that of two structurally related compounds, Sorocein A and Sorocein B. All these compounds showed a cytotoxicity effect on cancer cells, suggesting that the Sorocein-structural family may provide new and yet unexplored chemotypes for the development of minor groove binders to be evaluated as anticancer agents.

Exploring the Potential of Anthraquinone-Based Hybrids for Identifying a Novel Generation of Antagonists for the Smoothened Receptor in HH-Dependent Tumour.

Natural products (NPs) are highly profitable pharmacological tools due to their chemical diversity and ability to modulate biological systems. Accessing new chemical entities while retaining the biological relevance of natural chemotypes is a fundamental goal in the design of novel bioactive compounds. Notably, NPs have played a crucial role in understanding Hedgehog (HH) signalling and its pharmacological modulation in anticancer therapy. However, HH antagonists developed so far have shown several limitations, thus growing interest in the design of second-generation HH inhibitors. Through smart manipulation of the NPs core-scaffold, unprecedented and intriguing architectures have been achieved following different design strategies. This study reports the rational design and synthesis of a first and second generation of anthraquinone-based hybrids by combining the rhein scaffold with variously substituted piperazine nuclei that are structurally similar to the active portion of known SMO antagonists, the main transducer of the HH pathway. A thorough functional and biological investigation identified RH2_2 and RH2_6 rhein-based hybrids as valuable candidates for HH inhibition through SMO antagonism, with the consequent suppression of HH-dependent tumour growth. These findings also corroborated the successful application of the NPs-based hybrid design strategy in the development of novel NP-based SMO antagonists.

Inhibitors of UHRF1 base flipping activity showing cytotoxicity against cancer cells.

Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1.

Antimalarial drugs: what's new in the patents?

INTRODUCTION: The efficacy of current therapeutic warheads in preventing malaria transmission or treating the disease is often hampered by the emergence of drug-resistance. No effective vaccines are available to date, and novel drugs able to counteract drug-resistant forms of malaria and/or to target multiple stages of the parasite's lifecycle are urgently needed. AREAS COVERED: This review covers patents that protect antimalarial small molecules bearing the artemisinin or other chemical scaffolds, as well as vaccines, that have been published in the period 2015-2022. Literature was searched in public databases of articles and patents. Patents protecting small molecules that prevent malaria transmission are not discussed herein. EXPERT OPINION: Significant progress has been made in the design of antimalarial agents. Most of these candidates have been tested in standardized strains, with the use of Plasmodium clinical isolates for testing still underdeveloped. Several compounds have been profiled in in vivo mouse models of malaria, including humanized mice. Despite having different efficacy, these new molecules might further progress the field and hopefully will advance to clinical development soon.

Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity.

The SARS-CoV-2 protease (3CLpro) is one of the key targets for the development of efficacious drugs for COVID-19 treatment due to its essential role in the life cycle of the virus and exhibits high conservation among coronaviruses. Recent studies have shown that flavonoids, which are small natural molecules, have antiviral activity against coronaviruses (CoVs), including SARS-CoV-2. In this study, we identified the docking sites and binding affinity of several natural compounds, similar to flavonoids, and investigated their inhibitory activity towards 3CLpro enzymatic activity. The selected compounds were then tested in vitro for their cytotoxicity, for antiviral activity against SARS-CoV-2, and the replication of other coronaviruses in different cell lines. Our results showed that Baicalein (100 µg/mL) exerted strong 3CLpro activity inhibition (>90%), whereas Hispidulin and Morin displayed partial inhibition. Moreover, Baicalein, up to 25 µg/mL, hindered >50% of SARS-CoV-2 replication in Vero E6 cultures. Lastly, Baicalein displayed antiviral activity against alphacoronavirus (Feline-CoV) and betacoronavirus (Bovine-CoV and HCoV-OC43) in the cell lines. Our study confirmed the antiviral activity of Baicalein against SARS-CoV-2 and demonstrated clear evidence of its pan-coronaviral activity.

Selective Targeting of Cancer-Related G-Quadruplex Structures by the Natural Compound Dicentrine.

Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (Kb~106 vs. 105 M-1), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.

Inhibition of adenovirus transport from the endosome to the cell nucleus by rotenone.

Regardless of the clinical impact of human adenovirus (HAdV) infections in the healthy population and its high morbidity in immunosuppressed patients, a specific treatment is still not yet available. In this study, we screened the CM1407 COST Action's chemical library, comprising 1,233 natural products to identify compounds that restrict HAdV infection. Among them, we identified rotenolone, a compound that significantly inhibited HAdV infection. Next, we selected four isoflavonoid-type compounds (e.g., rotenone, deguelin, millettone, and tephrosin), namely rotenoids, structurally related to rotenolone in order to evaluate and characterized in vitro their antiviral activities against HAdV and human cytomegalovirus (HCMV). Their IC50 values for HAdV ranged from 0.0039 µM for rotenone to 0.07 µM for tephrosin, with selective indices ranging from 164.1 for rotenone to 2,429.3 for deguelin. In addition, the inhibition of HCMV replication ranged from 50% to 92.1% at twice the IC50 concentrations obtained in the plaque assay for each compound against HAdV. Our results indicated that the mechanisms of action of rotenolone, deguelin, and tephrosin involve the late stages of the HAdV replication cycle. However, the antiviral mechanism of action of rotenone appears to involve the alteration of the microtubular polymerization, which prevents HAdV particles from reaching the nuclear membrane of the cell. These isoflavonoid-type compounds exert high antiviral activity against HAdV at nanomolar concentrations, and can be considered strong hit candidates for the development of a new class of broad-spectrum antiviral drugs.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

Targeting the tubulin C-terminal tail by charged small molecules.

The disordered tubulin C-terminal tail (CTT), which possesses a higher degree of heterogeneity, is the target for the interaction of many proteins and cellular components. Compared to the seven well-described binding sites of microtubule-targeting agents (MTAs) that localize on the globular tubulin core, tubulin CTT is far less explored. Therefore, tubulin CTT can be regarded as a novel site for the development of MTAs with distinct biochemical and cell biological properties. Here, we designed and synthesized linear and cyclic peptides containing multiple arginines (RRR), which are complementary to multiple acidic residues in tubulin CTT. Some of them showed moderate induction and promotion of tubulin polymerization. The most potent macrocyclic compound 1f was found to bind to tubulin CTT and thus exert its bioactivity. Such RRR containing compounds represent a starting point for the discovery of tubulin CTT-targeting agents with therapeutic potential.

Identification of Small Molecular Chaperones Binding P23H Mutant Opsin through an In Silico Structure-Based Approach.

N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.