Novel pyrrole based CB2 agonists: New insights on CB2 receptor role in regulating neurotransmitters' tone.

The cannabinoid system is one of the most investigated neuromodulatory systems because of its involvement in multiple pathologies such as cancer, inflammation, and psychiatric diseases. Recently, the CB2 receptor has gained increased attention considering its crucial role in modulating neuroinflammation in several pathological conditions like neurodegenerative diseases. Here we describe the rational design of pyrrole-based analogues, which led to a potent and pharmacokinetically suitable CB2 full agonist particularly effective in improving cognitive functions in a scopolamine-induced amnesia murine model. Therefore, we extended our study by investigating the interconnection between CB2 activation and neurotransmission in this experimental paradigm. To this purpose, we performed a MALDI imaging analysis on mice brains, observing that the administration of our lead compound was able to revert the effect of scopolamine on different neurotransmitter tones, such as acetylcholine, serotonin, and GABA, shedding light on important networks not fully explored, so far.

Design, Synthesis, and Pharmacological Characterization of a Potent Soluble Epoxide Hydrolase Inhibitor for the Treatment of Acute Pancreatitis.

Acute pancreatitis (AP) is a potentially life-threatening illness characterized by an exacerbated inflammatory response with limited options for pharmacological treatment. Here, we describe the rational development of a library of soluble epoxide hydrolase (sEH) inhibitors for the treatment of AP. Synthesized compounds were screened in vitro for their sEH inhibitory potency and selectivity, and the results were rationalized by means of molecular modeling studies. The most potent compounds were studied in vitro for their pharmacokinetic profile, where compound 28 emerged as a promising lead. In fact, compound 28 demonstrated a remarkable in vivo efficacy in reducing the inflammatory damage in cerulein-induced AP in mice. Targeted metabololipidomic analysis further substantiated sEH inhibition as a molecular mechanism of the compound underlying anti-AP activity in vivo. Finally, pharmacokinetic assessment demonstrated a suitable profile of 28 in vivo. Collectively, compound 28 displays strong effectiveness as sEH inhibitor with potential for pharmacological AP treatment.

Rational design of the zonulin inhibitor AT1001 derivatives as potential anti SARS-CoV-2.

Although vaccines are greatly mitigating the worldwide pandemic diffusion of SARS-Cov-2, therapeutics should provide many distinct advantages as complementary approach to control the viral spreading. Here, we report the development of new tripeptide derivatives of AT1001 against SARS-CoV-2 Mpro. By molecular modeling, a small compound library was rationally designed and filtered for enzymatic inhibition through FRET assay, leading to the identification of compound 4. X-ray crystallography studies provide insights into its binding mode and confirm the formation of a covalent bond with Mpro C145. In vitro antiviral tests indicate the improvement of biological activity of 4 respect to AT1001. In silico and X-ray crystallography analysis led to 58, showing a promising activity against three SARS-CoV-2 variants and a valuable safety in Vero cells and human embryonic lung fibroblasts. The drug tolerance was also confirmed by in vivo studies, along with pharmacokinetics evaluation. In summary, 58 could pave the way to develop a clinical candidate for intranasal administration.

Discovery and Optimization of Indoline-Based Compounds as Dual 5-LOX/sEH Inhibitors: In Vitro and In Vivo Anti-Inflammatory Characterization.

The design of multitarget drugs represents a promising strategy in medicinal chemistry and seems particularly suitable for the discovery of anti-inflammatory drugs. Here, we describe the identification of an indoline-based compound inhibiting both 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH). In silico analysis of an in-house library identified nine compounds as potential 5-LOX inhibitors. Enzymatic and cellular assays revealed the indoline derivative 43 as a notable 5-LOX inhibitor, guiding the design of new analogues. These compounds underwent extensive in vitro investigation revealing dual 5-LOX/sEH inhibitors, with 73 showing the most promising activity (IC50s of 0.41 ± 0.01 and 0.43 ± 0.10 µM for 5-LOX and sEH, respectively). When challenged in vivo in zymosan-induced peritonitis and experimental asthma in mice, compound 73 showed remarkable anti-inflammatory efficacy. These results pave the way for the rational design of 5-LOX/sEH dual inhibitors and for further investigation of their potential use as anti-inflammatory agents.

Extensive Molecular Dynamics Simulations Disclosed the Stability of mPGES-1 Enzyme and the Structural Role of Glutathione (GSH) Cofactor.

A deep in silico investigation of various microsomal prostaglandin E2 synthase-1 (mPGES-1) protein systems is here reported using molecular dynamics (MD) simulations. Firstly, eight different proteins models (Models A-H) were built, starting from the active enzyme trimer system (Model A), namely that bound to three glutathione (GSH) cofactor molecules, and then gradually removing the GSHs (Models B-H), simulating each of them for 100 ns in explicit solvent. The analysis of the obtained data disclosed the structural role of GSH in the chemical architecture of mPGES-1 enzyme, thus suggesting the unlikely displacement of this cofactor, in accordance with experimentally determined protein structures co-complexed with small molecule inhibitors. Afterwards, Model A was submitted to microsecond-scale molecular dynamics simulation (total simulation time=10 µs), in order to shed light about the dynamical behaviour of this enzyme at atomic level and to obtain further structural features and protein function information. We confirmed the structural stability of the enzyme machinery, observing a conformational rigidity of the protein, with a backbone RMSD of ∼3 Šalong the simulation time, and highlighting the strong active contribution of GSH molecules due to their active role in packing the protein chains through a tight binding at monomer interfaces. Furthermore, the focused analysis on R73 residue disclosed its role in solvent exchange events, probably excluding its function as route for GSH to enter towards the endoplasmic reticulum membrane, in line with the recently reported function of cap domain residues F44-D66 as gatekeeper for GSH entrance into catalytic site.

Identification of 2,4-Dinitro-Biphenyl-Based Compounds as MAPEG Inhibitors.

We identified 2,4-dinitro-biphenyl-based compounds as new inhibitors of leukotriene C4 synthase (LTC4 S) and 5-lipoxygenase-activating protein (FLAP), both members of the "Membrane Associated Proteins in Eicosanoid and Glutathione metabolism" (MAPEG) family involved in the biosynthesis of pro-inflammatory eicosanoids. By molecular docking we evaluated the putative binding against the targets of interest, and by applying cell-free and cell-based assays we assessed the inhibition of LTC4 S and FLAP by the small molecules at low micromolar concentrations. The present results integrate the previously observed inhibitory profile of the tested compounds against another MAPEG member, i. e., microsomal prostaglandin E2 synthase (mPGES)-1, suggesting that the 2,4-dinitro-biphenyl scaffold is a suitable molecular platform for a multitargeting approach to modulate pro-inflammatory mediators in inflammation and cancer treatment.

Corrigendum: Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

[This corrects the article DOI: 10.3389/fmolb.2021.715263.].

In Silico Identification and In Vitro Evaluation of New ABCG2 Transporter Inhibitors as Potential Anticancer Agents.

Different molecular mechanisms contribute to the development of multidrug resistance in cancer, including increased drug efflux, enhanced cellular repair mechanisms and alterations of drug metabolism or drug targets. ABCG2 is a member of the ATP-binding cassette superfamily transporters that promotes drug efflux, inducing chemotherapeutic resistance in malignant cells. In this context, the development of selective ABCG2 inhibitors might be a suitable strategy to improve chemotherapy efficacy. Thus, through a multidisciplinary approach, we identified a new ABCG2 selective inhibitor (8), highlighting its ability to increase mitoxantrone cytotoxicity in both hepatocellular carcinoma (EC50from 8.67 ± 2.65 to 1.25 ± 0.80 µM) and transfected breast cancer cell lines (EC50from 9.92 ± 2.32 to 2.45 ± 1.40 µM). Moreover, mitoxantrone co-administration in both transfected and non-transfected HEK293 revealed that compound 8 notably lowered the mitoxantrone EC50, demonstrating its efficacy along with the importance of the ABCG2 extrusion pump overexpression in MDR reversion. These results were corroborated by evaluating the effect of inhibitor 8 on mitoxantrone cell uptake in multicellular tumor spheroids and via proteomic experiments.

Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. On the basis of genetic evidence, it has been postulated that the FHIT protein may function as tumor suppressor, implying a role for the FHIT protein in carcinogenesis. Recently, Gaudio et al. reported that FHIT binds and delocalizes annexin A4 (ANXA4) from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. They also identified the smallest protein sequence of the FHIT still interacting with ANXA4, ranging from position 7 to 13: QHLIKPS. This short sequence of FHIT protein was not only able to bind ANXA4 but also to hold its target in the cytosol during paclitaxel treatment, thus avoiding ANXA4 translocation to the inner side of the cell membrane. Starting from these results, to obtain much information about structure requirements involved in the interaction of the peptide mentioned above, we synthetized a panel of seven peptides through an Ala-scan approach. In detail, to study the binding of FHIT derived peptides with ANXA4, we applied a combination of different biophysical techniques such as differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). Circular dichroism (CD) and nuclear magnetic resonance (NMR) were used to determine the conformational structure of the lead peptide (7-13) and peptides generated from ala-scan technique. The application of different biophysical and structural techniques, integrated by a preliminary biological evaluation, allowed us to build a solid structure activity relationship on the synthesized peptides.

Peptide Derivatives of the Zonulin Inhibitor Larazotide (AT1001) as Potential Anti SARS-CoV-2: Molecular Modelling, Synthesis and Bioactivity Evaluation.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (Mpro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds Mpro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.

Identification of 2-(thiophen-2-yl)acetic Acid-Based Lead Compound for mPGES-1 Inhibition.

We report the implementation of our in silico/synthesis pipeline by targeting the glutathione-dependent enzyme mPGES-1, a valuable macromolecular target in both cancer therapy and inflammation therapy. Specifically, by using a virtual fragment screening approach of aromatic bromides, straightforwardly modifiable by the Suzuki-Miyaura reaction, we identified 3-phenylpropanoic acid and 2-(thiophen-2-yl)acetic acid to be suitable chemical platforms to develop tighter mPGES-1 inhibitors. Among these, compounds 1c and 2c showed selective inhibitory activity against mPGES-1 in the low micromolar range in accordance with molecular modeling calculations. Moreover, 1c and 2c exhibited interesting IC50 values on A549 cell lines compared to CAY10526, selected as reference compound. The most promising compound 2c induced the cycle arrest in the G0/G1 phase at 24 h of exposure, whereas at 48 and 72 h, it caused an increase of subG0/G1 fraction, suggesting an apoptosis/necrosis effect.

The Therapeutic use of the Zonulin Inhibitor AT-1001 (Larazotide) for a Variety of Acute and Chronic Inflammatory Diseases.

BACKGROUND: The involvement of intercellular tight junctions and, in particular, the modulation of their competency by the zonulin pathway with a subsequent increase in epithelial and endothelial permeability, has been described in several chronic and acute inflammatory diseases. In this scenario, Larazotide, a zonulin antagonist, could be employed as a viable therapeutic strategy. OBJECTIVE: The present review aims to describe recent research and current observations about zonulin involvement in several diseases and the use of its inhibitor Larazotide for their treatment. METHODS: A systematic search was conducted on PubMed and Google Scholar, resulting in 209 publications obtained with the following search query: "Larazotide," "Larazotide acetate," "AT-1001," "FZI/0" and "INN-202." After careful examination, some publications were removed from consideration because they were either not in English or were not directly related to Larazotide. RESULTS: The obtained publications were subdivided according to Larazotide's mechanism of action and different diseases: celiac disease, type 1 diabetes, other autoimmune diseases, inflammatory bowel disease, Kawasaki disease, respiratory (infective and/or non-infective) diseases, and other. CONCLUSION: A substantial role of zonulin in many chronic and acute inflammatory diseases has been demonstrated in both in vivo and in vitro, indicating the possible efficacy of a Larazotide treatment. Moreover, new possible molecular targets for this molecule have also been demonstrated.

New Perspectives on Machine Learning in Drug Discovery.

Artificial intelligence methods, in particular, machine learning, has been playing a pivotal role in drug development, from structural design to the clinical trial. This approach is harnessing the impact of computer-aided drug discovery due to large available data sets for drug candidates and its new and complex manner of information interpretation to identify patterns for the study scope. In the present review, recent applications related to drug discovery and therapies are assessed, and limitations and future perspectives are analyzed.

In silico Analysis Revealed Potential Anti-SARS-CoV-2 Main Protease Activity by the Zonulin Inhibitor Larazotide Acetate.

The most severe outcome of COVID-19 infection is the development of interstitial pneumonia causing acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS), both responsible for the infected patients' mortality. ALI and ARDS are characterized by a leakage of plasma components into the lungs, compromising their ability to expand and optimally engage in gas exchange with blood, resulting in respiratory failure. We have previously reported that zonulin, a protein dictating epithelial and endothelial permeability in several districts, including the airways, is involved in ALI pathogenesis in mouse models, and that its peptide inhibitor Larazotide acetate (also called AT1001) ameliorated ALI and subsequent mortality by decreasing mucosal permeability to fluid and extravasation of neutrophils into the lungs. With the recent crystallographic resolution of the SARS-CoV-2 main protease (Mpro), an enzyme fundamental in the viral lifecycle, bound to peptidomimetic inhibitors N3 and 13b, we were able to perform molecular modeling investigation showing that AT1001 presents structural motifs similar to co-crystallized ligands. Specifically, molecular docking, MM-GBSA-based predictions and molecular dynamics showed that AT1001 docks extremely well in the Mpro catalytic domain through a global turn conformational arrangement without any unfavorable steric hindrance. Finally, we have observed that AT1001 can be superimposed onto the crystallized structures of N3 and 13b, establishing a higher number of interactions and accordingly a tighter binding. In vitro studies confirmed AT1001 anti-Mpro and preliminary investigation indicted an anti-viral activity. Combined, these studies suggest that AT1001, besides its well-demonstrated effect in ameliorating mucosal permeability in ALI/ARDS, may also exert a direct anti-SARS-CoV-2 effect by blocking the Mpro. AT1001 has been used extensively in a variety of animal models of ALI demonstrating robust safety and efficacy; it is currently in phase 3 trials in celiac subjects showing strong safety and efficacy profiles. We therefore propose its use as a specific anti-SARS-CoV-2 multitargeting treatment for the current pandemic.

Garcinol and Related Polyisoprenylated Benzophenones as Topoisomerase II Inhibitors: Biochemical and Molecular Modeling Studies.

Garcinol, a polyisoprenylated benzophenone isolated from Garcinia genus, has been reported to inhibit eukaryotic topoisomerase I and topoisomerase II at concentrations comparable to that of etoposide (∼25-100 µM). With the aim to clarify the underlying molecular mechanisms by which garcinol inhibits human topoisomerase IIα and topoisomerase IIß, biochemical assays along with molecular docking and molecular dynamics studies were carried out on garcinol and six congeners. The biochemical results revealed that garcinol derivatives appear to act as catalytic inhibitors of topoisomerase II and to inhibit ATP hydrolysis by topoisomerase II via some form of mixed inhibition. The computational investigation identified the structural elements responsible for binding to the biological target and also provided information for the eventual design of more selective and potent analogues. Collectively, our data suggest that garcinol-type agents may bind to the DNA binding surface and/or ATP domain of type II topoisomerases to antagonize function.

Identification of the 2-Benzoxazol-2-yl-phenol Scaffold as New Hit for JMJD3 Inhibition.

JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors. Compounds were synthesized by a microwave-assisted one-pot reaction under catalyst and solvent-free conditions. Among these, compound 8 presented the highest inhibitory activity (IC50 = 1.22 ± 0.22 µM) in accordance with molecular modeling calculations. Moreover, 8 induced the cycle arrest in S-phase on A375 melanoma cells.

Novel benzoxanthene lignans that favorably modulate lipid mediator biosynthesis: A promising pharmacological strategy for anti-inflammatory therapy.

Lipid mediators (LM) encompass pro-inflammatory prostaglandins (PG) and leukotrienes (LT) but also specialized pro-resolving mediators (SPM) which display pivotal bioactivities in health and disease. Pharmacological intervention with inflammatory disorders such as osteoarthritis and rheumatoid arthritis commonly employs anti-inflammatory drugs that can suppress PG and LT formation, which however, possess limited effectiveness and side effects. Here, we report on the discovery and characterization of the two novel benzoxanthene lignans 1 and 2 that modulate select LM biosynthetic enzymes enabling the switch from pro-inflammatory LT to SPM biosynthesis as potential pharmacological strategy to intervene with inflammation. In cell-free assays, compound 1 and 2 inhibit microsomal prostaglandin E2 synthase-1 and leukotriene C4 synthase (IC50 ∼ 0.6-3.4 µM) and potently interfere with 5-lipoxygenase (5-LOX), the key enzyme in LT biosynthesis (IC50 = 0.04 and 0.09 µM). In human neutrophils, monocytes and M1 and M2 macrophages, compound 1 and 2 efficiently suppress LT biosynthesis (IC50 < 1 µM), accompanied by elevation of 15-LOX-derived LM including SPM. In zymosan-induced murine peritonitis, compound 1 and 2 ameliorated self-limited inflammation along with suppression of early LT formation and elevation of subsequent SPM biosynthesis in vivo. Together, these novel benzoxanthene lignans promote the LM class switch from pro-inflammatory towards pro-resolving LM to terminate inflammation, suggesting their suitability as novel leads for pharmacotherapy of arthritis and related inflammatory disorders.

Chemical shift assignment of mono- and di-bromo triimidazo[1,2-a:1',2'-c:1″,2″-e][1,3,5]triazine derivatives by DFT/NMR integrated approach.

Mono- and di-bromo derivatives of triimidazo[1,2-a:1',2'-c:1″,2″-e][1,3,5]triazine have been proposed as new organic molecules presenting a very rich and complex photophysical behavior. Thus, we afforded the correct chemical shift assignment by integrating the experimental data with DFT calculation of NMR parameters. Our findings lay foundation for a structural reference in the organic synthesis and characterization of new congeners of this intriguing class of molecules.

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.