Tezacaftor is a direct inhibitor of sphingolipid delta-4 desaturase enzyme (DEGS).

BACKGROUND: We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. RESULTS: We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. CONCLUSIONS: We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance.

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

An extra virgin olive oil-enriched chocolate spread positively modulates insulin-resistance markers compared with a palm oil-enriched one in healthy young adults: A double-blind, cross-over, randomised controlled trial.

AIMS: To investigate if extra virgin olive oil (EVOO) or palm oil enriched chocolate spreads consumption leads to different results in terms of plasma ceramides concentration, glucose and lipid metabolism, inflammatory markers and appetite regulation in young healthy subjects. METHODS: In a 2-week, double-blind, cross-over, randomised controlled trial, 20 healthy, normal-weight subjects with a mean age of 24.2 years (SD: 1.2), consumed chocolate spread snacks (73% of energy [%E] from fat, 20% from carbohydrates and 7% from proteins), providing 570 Kcal/day added to an isocaloric diet. The chocolate spreads were identical, except for the type of fat: EVOO oil, rich in monounsaturated fatty acids (MUFAs), or palm oil, rich in Saturated Fatty Acids (SFAs). RESULTS: EVOO-enriched chocolate spread consumption led to better circulating sphingolipids and glucose profile, with reduced plasma ceramide C16:0, ceramide C16:0/ceramide C22:0-ceramide C24:0 ratio and sphingomyelin C18:0 (P = 0.030, P= 0.032 and P = 0.042, respectively) compared to the palm oil-enriched chocolate spread diet. HOMA-IR and plasma insulin were lower, while the Quicki and the McAuley Index were higher after the EVOO diet compared to the palm oil diet (P = 0.046, P = 0.045, P = 0.018 and P = 0.039 respectively). Subjects maintained a stable weight throughout the study. No major significant changes in total cholesterol, triglycerides, HDL, inflammatory markers, and appetite-regulating hormones/visual analogue scale were observed between the groups. CONCLUSIONS: Partially replacing SFAs with MUFAs in a chocolate-based snack as part of a short-term isocaloric diet in healthy individuals may limit SFAs detrimental effects on insulin sensitivity and decrease circulating harmful sphingolipids in young adults.

Searching for New Microbiome-Targeted Therapeutics through a Drug Repurposing Approach.

Commonly used non-antibiotic drugs have been associated with changes in gut microbiome composition, paving the way for the possibility of repurposing FDA-approved molecules as next-generation microbiome therapeutics. Herein, we developed and validated an ex vivo high-throughput screening platform─the mini gut model─to underpin human gut microbiome response to molecular modulators. Ten FDA-approved compounds, selected based on maximum structural diversity of molecular fingerprints, were screened against the gut microbiome of five healthy subjects to characterize the ability of human-targeted drugs to modulate the human gut microbiome network. Three compounds, THIP hydrochloride, methenamine, and mesna, have shown promise as novel gut microbiome therapeutics in light of their capability of promoting health-associated features of the gut microbiome. Our findings provide a resource for future research on drug-microbiome interactions and lay the foundation for a new era of more precise gut microbiome modulation through drug repurposing, aimed at targeting specific dysbiotic events.

Discovery and SAR Evolution of Pyrazole Azabicyclo[3.2.1]octane Sulfonamides as a Novel Class of Non-Covalent N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA) Inhibitors for Oral Administration.

Inhibition of intracellular N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity is a promising approach to manage the inflammatory response under disabling conditions. In fact, NAAA inhibition preserves endogenous palmitoylethanolamide (PEA) from degradation, thus increasing and prolonging its anti-inflammatory and analgesic efficacy at the inflamed site. In the present work, we report the identification of a potent, systemically available, novel class of NAAA inhibitors, featuring a pyrazole azabicyclo[3.2.1]octane structural core. After an initial screening campaign, a careful structure-activity relationship study led to the discovery of endo-ethoxymethyl-pyrazinyloxy-8-azabicyclo[3.2.1]octane-pyrazole sulfonamide 50 (ARN19689), which was found to inhibit human NAAA in the low nanomolar range (IC50 = 0.042 µM) with a non-covalent mechanism of action. In light of its favorable biochemical, in vitro and in vivo drug-like profile, sulfonamide 50 could be regarded as a promising pharmacological tool to be further investigated in the field of inflammatory conditions.

Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome.

Intracellular chloride concentration [Cl-]i is defective in several neurological disorders. In neurons, [Cl-]i is mainly regulated by the action of the Na+-K+-Cl- importer NKCC1 and the K+-Cl- exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Modulation of Efficient Diiodo-BODIPY in vitro Phototoxicity to Cancer Cells by Carbon Nano-Onions.

Photodynamic therapy is currently one of the most promising approaches for targeted cancer treatment. It is based on responses of vital physiological signals, namely, reactive oxygen species (ROS), which are associated with diseased condition development, such as tumors. This study presents the synthesis, incorporation, and application of a diiodo-BODIPY-based photosensitizer, based on a non-covalent functionalization of carbon nano-onions (CNOs). In vitro assays demonstrate that HeLa cells internalize the diiodo-BODIPY molecules and their CNO nanohybrids. Upon cell internalization and light exposure, the pyrene-diiodo-BODIPY molecules induce an increase of the ROS level of HeLa cells, resulting in remarkable photomediated cytotoxicity and apoptosis. Conversely, when HeLa cells internalize the diiodo-BODIPY/CNO nanohybrids, no significant cytotoxicity or ROS basal level increase can be detected. These results define a first step toward the understanding of carbon nanomaterials that function as molecular shuttles for photodynamic therapeutics, boosting the modulation of the photosensitizer.

Novel, Potent, and Druglike Tetrahydroquinazoline Inhibitor That Is Highly Selective for Human Topoisomerase II α over ß.

We disclose a novel class of 6-amino-tetrahydroquinazoline derivatives that inhibit human topoisomerase II (topoII), a validated target of anticancer drugs. In contrast to topoII-targeted drugs currently in clinical use, these compounds do not act as topoII poisons that enhance enzyme-mediated DNA cleavage, a mechanism that is linked to the development of secondary leukemias. Instead, these tetrahydroquinazolines block the topoII function with no evidence of DNA intercalation. We identified a potent lead compound [compound 14 (ARN-21934) IC50 = 2 µM for inhibition of DNA relaxation, as compared to an IC50 = 120 µM for the anticancer drug etoposide] with excellent metabolic stability and solubility. This new compound also shows ~100-fold selectivity for topoIIα over topoß, a broad antiproliferative activity toward cultured human cancer cells, a favorable in vivo pharmacokinetic profile, and the ability to penetrate the blood-brain barrier. Thus, ARN-21934 is a highly promising lead for the development of novel and potentially safer topoII-targeted anticancer drugs.

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Distinctive lipid signatures of bronchial epithelial cells associated with cystic fibrosis drugs, including Trikafta.

In recent years, a number of drugs have been approved for the treatment of cystic fibrosis (CF). Among them, newly released Trikafta, a combination of 3 drugs (VX-661/VX-445/VX-770), holds great promise to radically improve the quality of life for a large portion of patients with CF carrying 1 copy of F508del, the most frequent CF transmembrane conductance regulator (CFTR) mutation. Currently available disease-modifying CF drugs work by rescuing the function of the mutated CFTR anion channel. Recent research has shown that membrane lipids, and the cell lipidome in general, play a significant role in the mechanism of CFTR-defective trafficking and, on the other hand, its rescue. In this paper, by using untargeted lipidomics on CFBE41o- cells, we identified distinctive changes in the bronchial epithelial cell lipidome associated with treatment with Trikafta and other CF drugs. Particularly interesting was the reduction of levels of ceramide, a known molecular player in the induction of apoptosis, which appeared to be associated with a decrease in the susceptibility of cells to undergo apoptosis. This evidence could account for additional beneficial roles of the triple combination of drugs on CF phenotypes.

Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign.

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives.

Brain-wide Mapping of Endogenous Serotonergic Transmission via Chemogenetic fMRI.

Serotonin-producing neurons profusely innervate brain regions via long-range projections. However, it remains unclear whether and how endogenous serotonergic transmission specifically influences regional or global functional activity. We combined designed receptors exclusively activated by designed drugs (DREADD)-based chemogenetics and functional magnetic resonance imaging (fMRI), an approach we term "chemo-fMRI," to causally probe the brain-wide substrates modulated by endogenous serotonergic activity. We describe the generation of a conditional knockin mouse line that, crossed with serotonin-specific Cre-recombinase mice, allowed us to remotely stimulate serotonergic neurons during fMRI scans. We show that endogenous stimulation of serotonin-producing neurons does not affect global brain activity but results in region-specific activation of a set of primary target regions encompassing corticohippocampal and ventrostriatal areas. By contrast, pharmacological boosting of serotonin levels produced widespread fMRI deactivation, plausibly reflecting the mixed contribution of central and perivascular constrictive effects. Our results identify the primary functional targets of endogenous serotonergic stimulation and establish causation between activation of serotonergic neurons and regional fMRI signals.

Kernel-Based, Partial Least Squares Quantitative Structure-Retention Relationship Model for UPLC Retention Time Prediction: A Useful Tool for Metabolite Identification.

We propose a new QSRR model based on a Kernel-based partial least-squares method for predicting UPLC retention times in reversed phase mode. The model was built using a combination of classical (physicochemical and topological) and nonclassical (fingerprints) molecular descriptors of 1383 compounds, encompassing different chemical classes and structures and their accurately measured retention time values. Following a random splitting of the data set into a training and a test set, we tested the ability of the model to predict the retention time of all the compounds. The best predicted/experimental R2 value was higher than 0.86, while the best Q2 value we observed was close to 0.84. A comparison of our model with traditional and simpler MLR and PLS regression models shows that KPLS better performs in term of correlation (R2), prediction (Q2), and support to MetID peak assignment. The KPLS model succeeded in two real-life MetID tasks by correctly predicting elution order of Phase I metabolites, including isomeric monohydroxylated compounds. We also show in this paper that the model's predictive power can be extended to different gradient profiles, by simple mathematical extrapolation using a known equation, thus offering very broad flexibility. Moreover, the current study includes a deep investigation of different types of chemical descriptors used to build the structure-retention relationship.

Potent α-amino-ß-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies.

4-Cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in animal models. In the present work, we describe structure-activity relationship (SAR) studies on 3-aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to elucidate the principal structural and stereochemical features needed to achieve effective NAAA inhibition. Investigations on the influence of the substitution at the ß-position of the 2-oxo-3-azetidinyl ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA inhibitors as therapeutic agents for the treatment of pain and inflammation.