IntroductionProkineticin 2 (PK2) signaling to gonadotropin-releasing hormone (GnRH) neurons is essential for their embryonic migration from the nasal placode to the brain, and its disruption results in hypogonadotropic hypogonadism. Although PK2 has been implicated in the regulation of reproductive function in adult rodents, its direct cellular targets remain insufficiently defined. Here, we investigated whether GnRH neurons in adult female mice receive and functionally respond to PK2 signals.MethodsWhole-cell patch-clamp recordings were obtained from acute forebrain slices prepared from adult female GnRH-GFP mice. The effects of PK2 and prokineticin receptor antagonists on spontaneous firing and miniature postsynaptic currents (mPSCs) were assessed. Expression of PK2 receptor (PKR2) in GnRH neurons was examined using RNAscope in situ hybridization for simultaneous detection of PKR2 and GnRH mRNAs, followed by confocal laser microscopic analysis.ResultsPK2 increased action potential firing and mPSC frequency in 42.9% (firing) and 42.1% (mPSC) of GnRH neurons. These effects were prevented by intracellular G-protein inhibition with GDP-β-S and by pharmacological blockade of prokineticin receptors using PKRA7 or PC27. Inhibition of nitric oxide synthase by NPLA also eliminated the PK2-induced elevation of mPSC frequency. RNAscope analysis revealed PKR2 mRNA expression in approximately one-third of GnRH neurons.DiscussionThese findings demonstrate that PK2 directly enhances the excitability of a subset of GnRH neurons through PKR2-dependent mechanisms, identifying these neurons as functional targets of PK2 signaling in the mature female mouse brain.ConclusionPK2 serves as a direct modulator of the reproductive neuroendocrine axis in adult female mice by activating PKR2 expressed in a subpopulation of GnRH neurons.

Background: Carbonic anhydrases (CAs) are known to play important roles in several physiological and pathological processes; among them, CAs IX and XII are of particular relevance in cancer therapy due to their involvement in tumor growth and progression. Methods: In this study, a novel series of benzenesulfonamides incorporating a hydrazinocarbonyl-ureido linker alongside a 6-arylpyridine tail was synthesized and evaluated for inhibitory activity through a stopped-flow CO2 hydrase assay on four hCA isoforms. Results: Some of the new compounds exhibited great activity and selectivity toward the tumor-expressed CA XII isoform over the off-target isoforms CA I and CA II. Based on these results, they were selected for ADME prediction studies, showing favorable drug-like properties. To further investigate their binding mode, these compounds were docked into the four hCA isoforms. Conclusions: Overall, the results underscore the potential of compounds bearing a 6-arylpyridine tail along with a hydrazinocarbonyl-ureido linker as a foundation for further inhibitor development.

A straightforward atroposelective access to enantiomerically enriched 2-hydroxy-3-(2-oxochroman-4-yi)naphthalene-1,4-diones (with yields ranging from 32% to 87% and enantiomeric excesses up to 99%) is described. Using an organocatalytic approach, 2-hydroxynaphthoquinone reacts with a 3-coumarin-3-carboxylic acid through a tandem 1,4-addition/decarboxylation process initiated by a thiourea-functionalized cinchona alkaloid, which efficiently controls the stereochemistry of a newly forged stereocenter, while simultaneously directing the formation of a configurationally stable C(sp2)-C(sp3) synclinal atropisomer. The methodology has been explored across a broad substrate scope, and the results are supported by detailed nuclear magnetic resonance (NMR) analyses, single-crystal X-ray diffraction, and density functional theory (DFT) calculations.

The dearomatization of indoles is a powerful strategy for transforming simple substrates into architecturally complex molecular frameworks. This review provides a comprehensive overview of photoinduced methodologies developed for the construction of three‐dimensional polycyclic systems bearing indolyl‐cyclobutane motifs. We summarize classical ultraviolet(‐driven [2 + 2] cycloaddition reactions alongside recent advances in photocatalysis, including sensitizer‐ or photocatalyst‐mediated energy‐transfer and single‐electron transfer processes. Attention is also given to photoinduced hetero‐[2 + 2] cycloadditions, specifically the Büchi and aza‐Büchi reactions, as well as photodimerization pathways of indole derivatives. Together, these approaches provide valuable and versatile tools for the rapid assembly of complex molecular architectures and drug‐like scaffolds.

Background: Carbonic Anhydrases (CAs) represent regulators of cell adaptation to hypoxia, pH regulation, and metabolic fitness. Among cancers, multiple myeloma (MM) is a plasma cell malignancy sustained by hypoxia-driven metabolic adaptation, extracellular acidification, and redox imbalance. Tight regulation of tumor extracellular pH, mediated by Carbonic Anhydrases IX and XII, is crucial for myeloma survival, progression, and stemness, making these isoforms attractive therapeutic targets. Methods: We designed and synthesized a library of terpenoid-based hybrids by derivatizing chlorothymol and 4-isopropyl-3-methylphenol with either the natural coumarin umbelliferon or the 2,2'-dipicolylamine (DPA) scaffold. This chemical strategy aimed to selectively inhibit tumor-associated CAs IX/XII through coumarin- or DPA-mediated recognition, while terpenoid fragments were introduced to enhance lipophilicity, membrane permeability, and potential redox-modulating properties. The compounds were tested by a Stopped-Flow assay for CA inhibition, in cell-based assays for antiproliferative properties and by means of several antioxidant assays. Results: The most active compounds, connecting the coumarin core to a terpenoid tail, inhibited the targeted CAs in the nanomolar range, showing up higher selectivity over off-target isoforms (I and II). In studies performed on MM cell lines, selected derivatives reduced viability (IC50 = 15.8-85.4 µM) and displayed favorable selectivity over normal cells. In silico investigations suggested that the compounds were able to interact selectively with the target enzymes. Conclusions: Collectively, these results support a dual-targeting strategy in which selective inhibition of tumor-associated CAs, combined with redox modulation, interferes with adaptive mechanisms of MM cells, providing a rational framework for the development of multifunctional agents against metabolically resilient hematological malignancies.

ABSTRACT:Neuropathic pain is a major symptom of Fabry-Anderson disease (FD). It develops in childhood, is life-lasting, and resists current therapies; finding new therapeutic strategies is urgently needed. We demonstrate that neuroinflammation control effectively relieves FD pain. We used 2 pharmacological approaches: the microglial inhibitor minocycline and the block of the activity of the chemokine prokineticin-2 with the specific receptor antagonist PC1 (patented compound 1). Ten- and 25-week-old male GLA-/- mice (the FD murine model) were used. These mice were characterized by mechanical allodynia, thermal hyperalgesia, hyposensitivity to cold stimuli, and abdominal pain. Two weeks of treatment with minocycline or PC1 successfully counteracted sensory alterations. A significant inflammatory state, characterized by high levels of prokineticin-2 and proinflammatory cytokines, was present in the FD gut. The sciatic nerve showed initial severe neuroinflammation that attenuated over time. In dorsal root ganglia, neuroinflammation was severe and persistent with prokineticin-2, proinflammatory cytokines, ionized calcium-binding adapter molecule 1, and glial fibrillary acidic protein overexpression; histone demethylases KDM6A and B were also upregulated. We highlighted neuroinflammation in the spinal cord that increased over time. Treatment with minocycline or PC1 significantly counteracted inflammation and neuroinflammation, reducing prokineticin-2 and proinflammatory cytokines levels and increasing anti-inflammatory factor PPARγ expression. Both treatments prevented the onset of micro- and astrogliosis in the spinal cord. We underline the role of neuroinflammation and microglia in FD pain and suggest that treatments that control the activity of the prokineticin system, glial activation, and the production of proinflammatory cytokines and increase anti-inflammatory mediators have a therapeutic effect on pain in FD.

The experience of the SARS-CoV-2 pandemic has highlighted the urgent need to develop broad-spectrum antivirals (BSA) agents capable of targeting viral and/or host factors essential for infection as undeniable weapons against future coronavirus threats with potential pandemic perspectives. In this study, we report the synthesis and biological evaluation of a novel series of indole-based ethyl cinnamate derivatives designed as multitarget inhibitors of both SARS-CoV-2 Main Protease (Mpro) and human cathepsin, namely CatL and CatS, key enzymes involved in viral replication and entry. Enzymatic assays showed that several compounds exert significant inhibition on multiple targets. Structure-activity relationship (SAR) studies highlighted the critical role of the tert-leucine (Tle) residue at P2 position of these (pseudo)dipeptides, which proved to be essential for multitarged enzyme inhibition and antiviral activity. In antiviral assays against representative α- (hCoV-229E) and β-coronavirus (hCoV-OC43), 12 (EC50 = 4.09 µM for hCoV-OC43, 0.77 µM for hCoV-229E), 20 (EC50 = 6.68 µM for hCoV-OC43, 0.62 µM for hCoV-229E), and 3 (EC50 = 2.96 µM for hCoV-OC43, 0.64 µM for hCoV-229E) restricted viral replication with high selectivity values (SI = 98, 56, and 101, respectively). Time-of-drug-addition experiments then revealed that 12 primarily inhibited viral entry by targeting CatL, whereas 20 affected both entry and post-entry stages of hCoV replication cycle likely thanks to its dual-inhibitory activity against both CatL and Mpro. These findings support the development of multitarget inhibitors as promising antivirals able to inhibit both α- and β-coronavirus, so as to reduce the risk of resistance associated with single-target agents. Furthermore, the demonstrated effectiveness of Tle-containing indole-based cinnamates as BSA highlights their potential in the context of the strategic framework for pandemic preparedness.

Background—Aggressive solid tumors are commonly characterized by both basic intracellular pH and acidic extracellular pH, which increase cell survival and proliferation. As carbonic anhydrases IX/XII are involved in this pH regulation, their inhibition is an appealing approach in cancer therapy, avoiding cancer cell survival and proliferation. Substituted coumarins are selective non-classical CA IX and CA XII inhibitors. Methods—In this study, new 7-hydroxycoumarinamides were synthesized and assayed for CA inhibition and antiproliferative activity. Results—All of the coumarinamides showed human CA IX and CA XII selective inhibition over the off-target CA I and CA II isoforms. Coumarin acts as a suicide inhibitor because its heterocyclic ring can be hydrolyzed by CA esterase activity to give the corresponding 2-hydroxycinnamic acid derivative which blocks the entrance of the active site. The 2-hydroxycinnamic acid derivatives deriving from the most potent and selective coumarinamides were docked into CA IX and XII to better understand the activity and selectivity against the two CA isoforms. The most active coumarinamides also produced a decrease of A549 cell proliferation and were able to arrest cells at the G1/S checkpoint. Conclusions—These results may open new perspectives for developing coumarin-based CA IX/XII inhibitors.

HDAC6 and Heat Shock Protein 90 (Hsp90) are key regulators within the androgen response pathway, exhibiting a close interplay and mutual interaction patterns that make their combined inhibition a promising strategy for treating aggressive prostate cancer (PC). Herein, we present the structure-based design of dual inhibitors of Hsp90 and HDAC6 that leveraged the crystal structure requirements of HDAC6 and two distinct Hsp90 binding pockets. The study led to the discovery of compound 17, a potent, nearly balanced, and selective dual inhibitor of HDAC6 and Hsp90 endowed with favorable drug-like properties. The compound demonstrated excellent antiproliferative activity across PC cell lines. In 3D tumor spheroid models, it demonstrated marked anticancer activity and ability to target both established tumor masses and tumor-initiating cell populations. Furthermore, combination studies showed marked synergistic effects that outperformed the coadministration of single-target inhibitors. Overall, compound 17 stands as a promising candidate for further preclinical evaluation against aggressive forms of PC.

The continuous flow telescoped synthesis of 1,1-cyclopropane aminoketones was achieved by optimizing the photocyclization of 1,2-diketones to 2-hydroxycylobutanones (HCBs) and their reaction with aryl- and alkylamines, via tandem condensation C4-C3-ring contraction reaction. With the achieved operational conditions, we were able to obtain a library of cyclopropylamines with good chemical yields, high productivity, and short residence times.

Cathepsins, key members of the papain-like family of cysteine proteases, are crucial for proteolysis processes within human cells, including osteolysis, immunomodulation and apoptosis. Recent research has highlighted the significant role of cathepsins, particularly the L, S, K, and B subtypes, in pancreatic cancer. This has driven the development of novel cathepsin inhibitors as potential treatments to inhibit tumor progression, migration and invasion. Targeting cathepsin S (CatS) has shown promise in reducing tumor progression and enhancing the efficacy of chemotherapeutic agents in preclinical models. Building on our previous work where we employed ethyl p-aminocinnamate ester derivatives for covalent inhibition of cysteine proteases, herein we have designed and synthesized three new derivatives basing on an isosteric replacement (H-F) at the level of cinnamate moiety. These derivatives emerged as potent covalent inhibitors of CatS (1.8-2.6 mu M) with 2F showing also weak inhibition activity against CatL (20 %) and CatB (29 %). In vitro assays of 2F against pancreatic cancer cell lines BXPC3 and CAPAN1 revealed significant antiproliferative activity, with IC50 = 5.79 mu M and 20.75 mu M, respectively. These findings underscore the potential of alpha-fluorocinnamate-based cysteine protease inhibitors as promising candidates for further development in targeting CatS and CatL with the aim to reduce pancreatic cancer cell proliferation.

A photocatalyzed oxidation of functionalized cyclobutanones to access gamma-lactones has been performed in acetonitrile at room temperature, using anthraquinone derivatives as catalysts in the presence of TFA. The best reaction results were obtained by using 3 mol % of 9,10-anthraquinone as a catalyst under 370 nm irradiation in an O2 environment. The scope and the limitations of this reaction have been investigated using both 2- and 3-substituted cyclobutanones, as well as enantiopure ketones, to obtain full preservation of their stereochemical identity after the oxidation. The process furnishes in most cases the desired compounds with high purity after catalyst removal by simple filtration, and reducing the production of solvents waste. A photocatalyzed oxidation of functionalized cyclobutanones to access gamma-lactones has been performed in acetonitrile at room temperature, using 9,10-anthraquinone as catalysts in the presence of TFA. The process furnish good to excellent yields of the desired compounds in 4-16 h reaction and after simple filtration of the recyclable catalyst. image

A series of 1-(4-sulfamoyl-benzoyl)-piperidine-4-carbox-amides deriving from substituted piperazines/ benzyl-amines was designed, synthesized, and tested on human carbonic anhydrase (hCA). The inhibitory activity of the new sulfon-amides was analyzed using acetazol-amide (AAZ) as a standard inhibitor against hCA I, II, IX, and XII. Several sulfon-amides showed both inhibitory activity at low nanomolar concentrations and selectivity against the cytosolic hCA II isoform, and the same trend was observed on the tumor-associated hCA IX and XII. The benzene-sulfon-amido carbox-amides 11 and 15 were the most potent of the piperazino- and benzyl-amino-based series, respectively. Docking and molecular dynamics studies related the high selectivity of compound 11 toward the tumor-associated hCA isoforms to its capability to participate in favorable interactions within hCA IX and hCA XII active sites, whereas no such interactions were detected within both hCA I and hCA II isoforms.

Severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) has infected more than 762 million people to date and has caused approximately 7 million deaths all around the world, involving more than 187 countries. Although currently available vaccines show high efficacy in preventing severe respiratory complications in infected patients, the high number of mutations in the S proteins of the current variants is responsible for the high level of immune evasion and transmissibility of the virus and the reduced effectiveness of acquired immunity. In this scenario, the development of safe and effective drugs of synthetic or natural origin to suppress viral replication and treat acute forms of COVID-19 remains a valid therapeutic challenge. Given the successful history of flavonoids-based drug discovery, we developed esters of substituted cinnamic acids with quercetin to evaluate their in vitro activity against a broad spectrum of Coronaviruses. Interestingly, two derivatives, the 3,4-methylenedioxy 6 and the ester of acid 7, have proved to be effective in reducing OC43-induced cytopathogenicity, showing interesting EC50s profiles. The ester of synaptic acid 7 in particular, which is not endowed with relevant cytotoxicity under any of the tested conditions, turned out to be active against OC43 and SARS-CoV-2, showing a promising EC50. Therefore, said compound was selected as the lead object of further analysis. When tested in a yield reduction, assay 7 produced a significant dose-dependent reduction in viral titer. However, the compound was not virucidal, as exposure to high concentrations of it did not affect viral infectivity, nor did it affect hCoV-OC43 penetration into pre-treated host cells. Additional studies on the action mechanism have suggested that our derivative may inhibit viral endocytosis by reducing viral attachment to host cells.

A small series of hydrazonobenzenesulfonamides was designed, synthesized and studied for their human carbonic anhydrase (hCA) inhibitory activity. The synthesized compounds were evaluated against hCA I, II, IX and XII isoforms using acetazolamide (AAZ) as the standard inhibitor. Various hydrazonosulfonamide derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The most potent and selective hydrazones 8, 9, 10, 11, 19 and 24 were docked into isoforms I, II, IX and XII to better understand their activity and selectivity for the different CA isoforms.

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 Main Protease (Mpro) plays a pivotal role in the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is considered a highly conserved viral target. Disruption of the catalytic activity of Mpro produces a detrimental effect on the course of the infection, making this target one of the most attractive for the treatment of COVID-19. The current success of the SARS-CoV-2 Mpro inhibitor Nirmatrelvir, the first oral drug for the treatment of severe forms of COVID-19, has further focused the attention of researchers on this important viral target, making the search for new Mpro inhibitors a thriving and exciting field for the development of antiviral drugs active against SARS-CoV-2 and related coronaviruses.

The development of drugs for the treatment of advanced prostate cancer (PCA) remains a challenging task. In this study we have designed, synthesized and tested twenty-nine novel HDAC inhibitors based on three different zinc binding groups (trifluoromethyloxadiazole, hydroxamic acid, and 2-mercaptoacetamide). These warheads were conveniently tethered to variously substituted phenyl linkers and decorated with differently substituted pyrrolopyrimidine and purine cap groups. Remarkably, most of the compounds showed nanomolar inhibitory activity against HDAC6. To provide structural insights into the Structure-Activity Relationships (SAR) of the investigated compounds, docking of representative inhibitors and molecular dynamics of HDAC6-inhibitor complexes were performed. Compounds of the trifluoromethyloxadiazole and hydroxamic acid series exhibited promising anti proliferative activities, HDAC6 targeting in PCA cells, and in vitro tumor selectivity. Representative compounds of the two series were tested for solubility, cell permeability and metabolic stability, demonstrating favorable in vitro drug-like properties. The more interesting compounds were subjected to migration assays, which revealed that compound 13 and, to a lesser extent, compound 15 inhibited the invasive behaviour of androgen-sensitive and -insensitive advanced prostate cancer cells. Compound 13 was profiled against all HDACs and found to inhibit all members of class II HDACs (except for HDAC10) and to be selective with respect to class I and class IV HDACs. Overall, compound 13 combines potent inhibitory activity and class II selectivity with favorable drug-like properties, an excellent anti-proliferative activity and marked anti-migration properties on PCA cells, making it an excellent lead candidate for further optimization.

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.