Discovery of first novel sigma/HDACi dual-ligands with a potent in vitro antiproliferative activity.

Designing and discovering compounds for dual-target inhibitors is challenging to synthesize new, safer, and more efficient drugs than single-target drugs, especially to treat multifactorial diseases such as cancer. The simultaneous regulation of multiple targets might represent an alternative synthetic approach to optimize patient compliance and tolerance, minimizing the risk of target-based drug resistance due to the modulation of a few targets. To this end, we conceived for the first time the design and synthesis of dual-ligands σR/HDACi to evaluate possible employment as innovative candidates to address this complex disease. Among all synthesized compounds screened for several tumoral cell lines, compound 6 (Kiσ1R = 38 ± 3.7; Kiσ2R = 2917 ± 769 and HDACs IC50 = 0.59 µM) is the most promising candidate as an antiproliferative agent with an IC50 of 0.9 µM on the HCT116 cell line and no significant toxicity to normal cells. Studies of molecular docking, which confirmed the affinity over σ1R and a pan-HDACs inhibitory behavior, support a possible balanced affinity and activity between both targets.

Hit identification of novel small molecules interfering with MALAT1 triplex by a structure-based virtual screening.

Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.

Uncovering Novel Capsaicin Inhibitory Activity towards Human Carbonic Anhydrase Isoforms IX and XII by Combining In Silico and In Vitro Studies.

Hot pepper (Capsicum annuum) represents one of the most widespread functional foods of the Mediterranean diet, and is associated with a reduced risk of developing cardiovascular disease, cancer, and mental disorders. In particular, its bioactive spicy molecules, named Capsaicinoids, exhibit polypharmacological properties. Among them, Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the most studied and reported in variegated scientific contributions for its beneficial effects, often linked to mechanisms of action unrelated to the activation of Transient Receptor Potential Vanilloid 1 (TRPV1). In this study, we present the application of in silico methods to Capsaicin for evaluating its inhibitory activity against the tumor-associated human (h) expressed CA IX and XII. In vitro assays confirmed Capsaicin inhibitory activity towards the most relevant tumor-related hCA isoforms. In particular, the hCAs IX and XII showed an experimental KI value of 0.28 µM and 0.064 µM, respectively. Then, an A549 model of non-small cell lung cancer, typically characterized by an elevated expression of hCA IX and XII, was employed to test the inhibitory effects of Capsaicin in vitro under both normoxic and hypoxic conditions. Finally, the migration assay revealed that Capsaicin [10 µM] inhibits cells from moving in the A549 cells model.

Development of [1,2]oxazoloisoindoles tubulin polymerization inhibitors: Further chemical modifications and potential therapeutic effects against lymphomas.

Lymphomas are among the ten most common cancers, and, although progress has been achieved in increasing survival, there is still an unmet need for more effective therapeutic approaches, including better options for patients with refractory tumors that initially respond but then relapse. The lack of effective alternative treatment options highlights the need to develop new therapeutic strategies capable of improving survival prospects for lymphoma patients. Herein, we describe the identification and exploration of the SAR of a series of [1,2]oxazolo[5,4-e]isoindoles as potent small molecules that bind to the colchicine site of tubulin and that have promise for the treatment of refractory lymphomas. Exploration of the chemical space of this class of compounds at the pyrrole moiety and at the [1,2]oxazole ring highlighted two compounds bearing a 3,5-dimethoxybenzyl and a 3,4,5-trimethoxybenzyl group as potent candidates and showed that structural modifications at the isoxazole moiety are generally not favorable for activity. The two best candidates showed efficacy against different lymphoma histotypes and displayed 88 and 80% inhibition of colchicine binding fitting well into the colchicine pocket, as demonstrated by X-ray crystallography T2R-TTL-complexes, docking and thermodynamic analysis of the tubulin-colchicine complex structure. These results were confirmed by transcriptome data, thus indicating [1,2]oxazolo[5,4-e]isoindoles are promising candidates as antitubulin agents for the treatment of refractory lymphomas.

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma.

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.

Riluzole-Rasagiline Hybrids: Toward the Development of Multi-Target-Directed Ligands for Amyotrophic Lateral Sclerosis.

Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole (1)-rasagiline (2) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3-8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6. It showed monoamine oxidase A (MAO-A) inhibitory activity (IC50 = 6.9 µM) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC50 = 8.7 µM) might add a piece to the puzzle of its anti-ALS molecular profile.

Chromene Derivatives as Selective TERRA G-Quadruplex RNA Binders with Antiproliferative Properties.

In mammalian cells, telomerase transcribes telomeres in large G-rich non-coding RNA, known as telomeric repeat-containing RNA (TERRA), which folds into noncanonical nucleic acid secondary structures called G-quadruplexes (G4s). Since TERRA G4 has been shown to be involved in telomere length and translation regulation, it could provide valuable insight into fundamental biological processes, such as cancer growth, and TERRA G4 binders could represent an innovative strategy for cancer treatment. In this work, the three best candidates identified in our previous virtual screening campaign on bimolecular DNA/RNA G4s were investigated on the monomolecular Tel DNA and TERRA G4s by means of molecular modelling simulations and in vitro and in cell analysis. The results obtained in this work highlighted the stabilizing power of all the three candidates on TERRA G4. In particular, the two compounds characterized by a chromene scaffold were selective TERRA G4 binders, while the compound with a naphthyridine core acted as a dual Tel/TERRA G4-binder. A biophysical investigation by circular dichroism confirmed the relative stabilization efficiency of the compounds towards TERRA and Tel G4s. The TERRA G4 stabilizing hits showed good antiproliferative activity against colorectal and lung adenocarcinoma cell lines. Lead optimization to increase TERRA G4 stabilization may provide new powerful tools against cancer.

Synthesis of 2H-Imidazo[2',1':2,3] [1,3]thiazolo[4,5-e]isoindol-8-yl-phenylureas with promising therapeutic features for the treatment of acute myeloid leukemia (AML) with FLT3/ITD mutations.

Despite progressive advances in understanding the molecular biology of acute myeloid leukemia (AML), the conventional therapeutic approach has not changed substantially, and the outcome for most patients is poor. Thus, continuous efforts on the discovery of new compounds with improved features are required. Following a multistep sequence, we have identified a new tetracyclic ring system with strong antiproliferative activity towards several haematological cell lines. The new compounds possess structural properties typical of inactive-state-binding kinase inhibitors and are structurally related to quizartinib which is known as type-II tyrosine kinase inhibitor. In particular, the high activity found in two cell lines MOLM-13 and MV4-11, expressing the constitutively activated mutant FLT3/ITD, indicates inhibition of FLT3 kinase and on the basis of structure-activity relationship (SAR) the presence of an ureido moiety demonstrates to play a key role in driving the antiproliferative activity towards these cell lines. Molecular modelling studies supported the mechanism of recognition of the most active compounds within the FLT3 pocket where quizartinib binds. Moreover, Molecular Dynamics simulation (MDs) revealed the formation of a recurrent H-bond with Asp829, which more stabilizes the complex of 9c and the FLT3 inactive state. In MV4-11 cell line compound 9c reduces the phosphorylation of FLT3 (Y591) and some of its downstream targets leading to cell cycle arrest at G1 phase and induction of apoptosis. In an MV4-11 xenograft mouse model, 9c significantly reduces the tumor growth at the dose of 1-3 mg/kg without apparent toxicity.

Identification of SET/EED dual binders as innovative PRC2 inhibitors.

Background: The inhibition of PRC2, implicated in the pathogenesis of several tumors, can be a useful therapeutic strategy for cancer treatment. In the literature, two types of PRC2 modulators are reported: competitive inhibitors of S-adenosyl methionine binding to the catalytic subunit EZH2; and allosteric ligands that prevent the interaction of the trimethylated H3K27 lysine in histone 3 to the EED subunit. The lack of dual EZH2/EED modulators drove us to search for compounds capable of recognizing both domains. Materials & methods: This goal was pursued by combining pharmacophore- and docking-based virtual screening of the Multi-Target Ligand Chemotheca database. Prediction tools for absorption, distribution, metabolism and excretion and pan-assay interference compounds were also applied. Results: Finally, five 1,2,3-triazole derivatives were identified as promising dual EZH2/EED modulators. Conclusion: Our multistage screening protocol highlighted the great potential of Chemotheca for identifying polypharmacological agents.

Novel propargylamine-based inhibitors of cholinesterases and monoamine oxidases: Synthesis, biological evaluation and docking study.

A combination of several pharmacophores in one molecule has been successfully used for multi-target-directed ligands (MTDL) design. New propargylamine substituted derivatives combined with salicylic and cinnamic scaffolds were designed and synthesized as potential cholinesterases and monoamine oxidases (MAOs) inhibitors. They were evaluated invitro for inhibition of acetyl- (AChE) and butyrylcholinesterase (BuChE) using Ellman's method. All the compounds act as dual inhibitors. Most of the derivatives are stronger inhibitors of AChE, the best activity showed 5-bromo-N-(prop-2-yn-1-yl)salicylamide 1e (IC50 = 8.05 µM). Carbamates (4-bromo-2-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2d and 2,4-dibromo-6-[(prop-2-yn-1-yl)carbamoyl]phenyl ethyl(methyl)carbamate 2e were selective and the most active for BuChE (25.10 and 26.09 µM). 4-Bromo-2-[(prop-2-yn-1-ylimino)methyl]phenol 4a was the most potent inhibitor of MAOs (IC50 of 3.95 and ≈10 µM for MAO-B and MAO-A, respectively) along with a balanced inhibition of both cholinesterases being a real MTDL. The mechanism of action was proposed, and binding modes of the hits were studied by molecular docking on human enzymes. Some of the derivatives also exhibited antioxidant properties. Insilico prediction of physicochemical parameters affirm that the molecules would be active after oral administration and able to reach brain tissue.

Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid Est Veritas?

Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.

4-Oxoquinolines and monoamine oxidase: When tautomerism matters.

4-Oxoquinoline derivatives have been often used in drug discovery programs due to their pharmacological properties. Inspired on chromone and 4-oxoquinoline chemical structure similarity, a small series of quinoline-based compounds was obtained and screened, for the first time, toward human monoamine oxidases isoforms. The data showed the N-(3,4-dichlorophenyl)-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide 10 was the most potent and selective MAO-B inhibitor (IC50 = 5.30 ± 0.74 nM and SI: ≥1887). The data analysis showed that prototropic tautomerism markedly influences the biological activity. The unequivocal characterisation of the quinoline tautomers was performed to understand the attained data. To our knowledge, there have been no prior reports on the characterisation of quinolone tautomers by 2D NMR techniques, namely by 1H-15N HSQC and 1H-15N HMBC, which are proposed as expedite tools for medicinal chemistry campaigns. Computational studies on enzyme-ligand complexes, obtained after MM-GBSA calculations and molecular dynamics simulations, supported the experimental data.

In Silico Food-Drug Interaction: A Case Study of Eluxadoline and Fatty Meal.

Food-drug interaction is an infrequently considered aspect in clinical practice. Usually, drugs are taken together with meals and what follows may adversely affect pharmacokinetic and pharmacodynamic properties, and hence, the therapeutic effects. In this study, a computational protocol was proposed to explain the different assimilations of two µ-receptors agonists, eluxadoline and loperamide, with a peculiar pharmacokinetic profile. Compared to loperamide, eluxadoline is absorbed less after the intake of a fatty meal, and the LogP values do not explain this event. Firstly, keeping in mind the different pH in the intestinal tract, the protonation states of both compounds were calculated. Then, all structures were subjected to a conformational search by using MonteCarlo and Molecular Dynamics methods, with solvation terms mimicking the water and weak polar solvent (octanol). Both computational results showed that eluxadoline has less conformational freedom in octanol, unlike loperamide, which exhibits constant behavior in both solvents. Therefore, we hypothesize that fatty meal causes the "closure" of the eluxadoline molecule to prevent the exposure of the polar groups and their interaction with water, necessary for the drug absorption. Based on our results, this work could be a reasonable "case study", useful for future investigation of the drug pharmacokinetic profile.

Natural Products Extracted from Fungal Species as New Potential Anti-Cancer Drugs: A Structure-Based Drug Repurposing Approach Targeting HDAC7.

Mushrooms can be considered a valuable source of natural bioactive compounds with potential polypharmacological effects due to their proven antimicrobial, antiviral, antitumor, and antioxidant activities. In order to identify new potential anticancer compounds, an in-house chemical database of molecules extracted from both edible and non-edible fungal species was employed in a virtual screening against the isoform 7 of the Histone deacetylase (HDAC). This target is known to be implicated in different cancer processes, and in particular in both breast and ovarian tumors. In this work, we proposed the ibotenic acid as lead compound for the development of novel HDAC7 inhibitors, due to its antiproliferative activity in human breast cancer cells (MCF-7). These promising results represent the starting point for the discovery and the optimization of new HDAC7 inhibitors and highlight the interesting opportunity to apply the "drug repositioning" paradigm also to natural compounds deriving from mushrooms.

In Silico Identification and Biological Evaluation of Antioxidant Food Components Endowed with IX and XII hCA Inhibition.

The tumor-associated isoenzymes hCA IX and hCA XII catalyze the hydration of carbon dioxide to bicarbonate and protons. These isoforms are highly overexpressed in many types of cancer, where they contribute to the acidification of the tumor environment, promoting tumor cell invasion and metastasis. In this work, in order to identify novel dual hCA IX and XII inhibitors, virtual screening techniques and biological assays were combined. A structure-based virtual screening towards hCA IX and XII was performed using a database of approximately 26,000 natural compounds. The best shared hits were submitted to a thermodynamic analysis and three promising best hits were identified and evaluated in terms of their hCA IX and XII inhibitor activity. In vitro biological assays were in line with the theoretical studies and revealed that syringin, lithospermic acid, and (-)-dehydrodiconiferyl alcohol behave as good hCA IX and hCA XII dual inhibitors.

Inside Perspective of the Synthetic and Computational Toolbox of JAK Inhibitors: Recent Updates.

The mechanisms of inflammation and cancer are intertwined by complex networks of signaling pathways. Dysregulations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway underlie several pathogenic conditions related to chronic inflammatory states, autoimmune diseases and cancer. Historically, the potential application of JAK inhibition has been thoroughly explored, thus triggering an escalation of favorable results in this field. So far, five JAK inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of different diseases. Considering the complexity of JAK-depending processes and their involvement in multiple disorders, JAK inhibitors are the perfect candidates for drug repurposing and for the assessment of multitarget strategies. Herein we reviewed the recent progress concerning JAK inhibition, including the innovations provided by the release of JAKs crystal structures and the improvement of synthetic strategies aimed to simplify of the industrial scale-up.

Multi-Targeting Bioactive Compounds Extracted from Essential Oils as Kinase Inhibitors.

Essential oils (EOs) are popular in aromatherapy, a branch of alternative medicine that claims their curative effects. Moreover, several studies reported EOs as potential anti-cancer agents by inducing apoptosis in different cancer cell models. In this study, we have considered EOs as a potential resource of new kinase inhibitors with a polypharmacological profile. On the other hand, computational methods offer the possibility to predict the theoretical activity profile of ligands, discovering dangerous off-targets and/or synergistic effects due to the potential multi-target action. With this aim, we performed a Structure-Based Virtual Screening (SBVS) against X-ray models of several protein kinases selected from the Protein Data Bank (PDB) by using a chemoinformatics database of EOs. By evaluating theoretical binding affinity, 13 molecules were detected among EOs as new potential kinase inhibitors with a multi-target profile. The two compounds with higher percentages in the EOs were studied more in depth by means Induced Fit Docking (IFD) protocol, in order to better predict their binding modes taking into account also structural changes in the receptor. Finally, given its good binding affinity towards five different kinases, cinnamyl cinnamate was biologically tested on different cell lines with the aim to verify the antiproliferative activity. Thus, this work represents a starting point for the optimization of the most promising EOs structure as kinase inhibitors with multi-target features.

Mediterranean products as promising source of multi-target agents in the treatment of metabolic syndrome.

Alteration of nutritional habits play an essential role on the risk of developing Metabolic Syndrome (MetS). Several epidemiological studies have shown that assuming diets rich of foods included in the Mediterranean diet (MetDiet) pattern like, such as olive oil, nuts, fruit, fiber, vegetables, wine and grain cereals has protective effects on the different risk factors characterizing the MetS. The beneficial effects of the MetDiet in the MetS are mainly due to the antioxidant and anti-inflammatory properties of the most abundant phytochemical components of such foods as polyphenols like resveratrol and oleuropein, allyl sulfides, ellagic acid, mono- and poly-unsaturated fatty acids (MUFA and PUFA), tocopherols and flavonoids like quercetin, which have shown positive results in the prevention of cardiovascular diseases (CVDs), with related risk factors, like hypertension, hypercholesterolemia and obesity. In this review, we highlighted the multi-target activities of the bioactive components contained in some foods typical of the Mediterranean area like olive oil, onion, liquorice, rosemary, oregano, hazelnut, pistachio, "Melannurca" apple, red wine, hot pepper, Citrus sp. fruits, saffron and garlic, with particular focus on their impact on health outcomes in relation to MetS main key factors, such as insulin resistance (IR) and type 2 diabetes mellitus (T2DM), endothelial dysfunctions, inflammatory response, oxidative stress and dyslipidaemic and hypercholesterolemic effects.

A drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine.

Multiple myeloma (MM) is an incurable hematological malignancy driven by several genetic and epigenetic alterations. The hyperactivation of the Polycomb repressive complex 2 (PRC2), a multi-subunit oncogenic histone methyltransferase, has been implicated in the pathogenesis of this malignancy. Upon protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED, PRC2 primarily methylates lysine 27 of histone H3 (H3K27me3), thus modulating the chromatin structure and inducing transcriptional repression. Herein, we highlight a new mechanism of action that can contribute to explain the anti-tumor activity of hydroxychloroquine (HCQ), an anti-malaric agent also known as autophagy inhibitor. By structural studies, we demonstrate that HCQ inhibits the allosteric binding of PRC2 to EED within the H3K27me3-binding pocket, thus antagonizing the PRC2 catalytic activity. In silico results are compatible with the significant reduction of the H3K27me3 levels in MM cells exerted by HCQ. Overall, these findings disclose a novel epigenetic activity of HCQ with potential implications for its clinical repositioning.

Targeting multiple G-quadruplex-forming DNA sequences: Design, biophysical and biological evaluations of indolo-naphthyridine scaffold derivatives.

It is well recognized that the non-canonical DNA structures known as G-quadruplexes (G4s) have a potential anticancer significance and several compounds have been discovered and evaluated as promising G4 binders with anticancer activity. Here, starting from a promising hit with an indolo-naphthyridine scaffold, a small series of five indolo-naphthyridine based derivatives have been designed and evaluated as G4-targeting compounds. FRET biophysical studies were performed on multiple DNA G4 structures, leading to the identification of a multi-target G4 stabilizer with a slight preference for the c-KIT1 and a good G4 over duplex selectivity. The good affinity of this compound against c-KIT1 G4 was also confirmed by SPR and MST experiments, while biological assays revealed its cytotoxic activity on tumour cells. Finally, Molecular Dynamics simulations helped to elucidate the stabilization effect of the selected compound against the c-KIT1 G4.