Cancer-Stem-Cell Phenotype-Guided Discovery of a Microbiota-Inspired Synthetic Compound Targeting NPM1 for Leukemia.

The human microbiota plays an important role in human health and disease, through the secretion of metabolites that regulate key biological functions. We propose that microbiota metabolites represent an unexplored chemical space of small drug-like molecules in the search of new hits for drug discovery. Here, we describe the generation of a set of complex chemotypes inspired on selected microbiota metabolites, which have been synthesized using asymmetric organocatalytic reactions. Following a primary screening in CSC models, we identified the novel compound UCM-13369 (4b) whose cytotoxicity was mediated by NPM1. This protein is one of the most frequent mutations of AML, and NPM1-mutated AML is recognized by the WHO as a distinct hematopoietic malignancy. UCM-13369 inhibits NPM1 expression, downregulates the pathway associated with mutant NPM1 C+, and specifically recognizes the C-end DNA-binding domain of NPM1 C+, avoiding the nucleus-cytoplasm translocation involved in the AML tumorological process. The new NPM1 inhibitor triggers apoptosis in AML cell lines and primary cells from AML patients and reduces tumor infiltration in a mouse model of AML with NPM1 C+ mutation. The disclosed phenotype-guided discovery of UCM-13369, a novel small molecule inspired on microbiota metabolites, confirms that CSC death induced by NPM1 inhibition represents a promising therapeutic opportunity for NPM1-mutated AML, a high-mortality disease.

New Trends in Aging Drug Discovery.

Aging is considered the main risk factor for many chronic diseases that frequently appear at advanced ages. However, the inevitability of this process is being questioned by recent research that suggests that senescent cells have specific features that differentiate them from younger cells and that removal of these cells ameliorates senescent phenotype and associated diseases. This opens the door to the design of tailored therapeutic interventions aimed at reducing and delaying the impact of senescence in life, that is, extending healthspan and treating aging as another chronic disease. Although these ideas are still far from reaching the bedside, it is conceivable that they will revolutionize the way we understand aging in the next decades. In this review, we analyze the main and well-validated cellular pathways and targets related to senescence as well as their implication in aging-associated diseases. In addition, the most relevant small molecules with senotherapeutic potential, with a special emphasis on their mechanism of action, ongoing clinical trials, and potential limitations, are discussed. Finally, a brief overview of alternative strategies that go beyond the small molecule field, together with our perspectives for the future of the field, is provided.

2-(Fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl (UCM-1306), an Orally Bioavailable Positive Allosteric Modulator of the Human Dopamine D1 Receptor for Parkinson's Disease.

Tolerance development caused by dopamine replacement with l-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson's disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances l-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson's disease.

Discovery of V-0219: A Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor toward Oral Treatment for "Diabesity".

Peptidic agonists of the glucagon-like peptide-1 receptor (GLP-1R) have gained a prominent role in the therapy of type-2 diabetes and are being considered for reducing food intake in obesity. Potential advantages of small molecules acting as positive allosteric modulators (PAMs) of GLP-1R, including oral administration and reduced unwanted effects, could improve the utility of this class of drugs. Here, we describe the discovery of compound 9 (4-{[1-({3-[4-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-yl}methyl)piperidin-3-yl]methyl}morpholine, V-0219) that exhibits enhanced efficacy of GLP-1R stimulation, subnanomolar potency in the potentiation of insulin secretion, and no significant off-target activities. The identified GLP-1R PAM shows a remarkable in vivo activity, reducing food intake and improving glucose handling in normal and diabetic rodents. Enantioselective synthesis revealed oral efficacy for (S)-9 in animal models. Compound 9 behavior bolsters the interest of a small-molecule PAM of GLP-1R as a promising therapeutic approach for the increasingly prevalent obesity-associated diabetes.

A Positive Allosteric Modulator of the Serotonin 5-HT2C Receptor for Obesity.

The 5-HT2CR agonist lorcaserin, clinically approved for the treatment of obesity, causes important side effects mainly related to subtype selectivity. In the search for 5-HT2CR allosteric modulators as safer antiobesity drugs, a chemical library from Vivia Biotech was screened using ExviTech platform. Structural modifications of identified hit VA240 in synthesized analogues 6-41 afforded compound 11 (N-[(1-benzyl-1H-indol-3-yl)methyl]pyridin-3-amine, VA012), which exhibited dose-dependent enhancement of serotonin efficacy, no significant off-target activities, and low binding competition with serotonin or other orthosteric ligands. PAM 11 was very active in feeding inhibition in rodents, an effect that was not related to the activation of 5-HT2AR. A combination of 11 with the SSRI sertraline increased the anorectic effect. Subchronic administration of 11 reduced food intake and body weight gain without causing CNS-related malaise. The behavior of compound 11 identified in this work supports the interest of a serotonin 5-HT2CR PAM as a promising therapeutic approach for obesity.

A new serotonin 5-HT6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding.

Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells.

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

Chemoproteomic Approach to Explore the Target Profile of GPCR ligands: Application to 5-HT1A and 5-HT6 Receptors.

Determination of the targets of a compound remains an essential aspect in drug discovery. A complete understanding of all binding interactions is critical to recognize in advance both therapeutic effects and undesired consequences. However, the complete polypharmacology of many drugs currently in clinical development is still unknown, especially in the case of G protein-coupled receptor (GPCR) ligands. In this work we have developed a chemoproteomic platform based on the use of chemical probes to explore the target profile of a compound in biological systems. As proof of concept, this methodology has been applied to selected ligands of the therapeutically relevant serotonin 5-HT1A and 5-HT6 receptors, and we have identified and validated some of their off-targets. This approach could be extended to other drugs of interest to study the targeted proteome in disease-relevant systems.

Fatty acid synthase is a metabolic marker of cell proliferation rather than malignancy in ovarian cancer and its precursor cells.

Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitor-resistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated ß-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell lines­although resisting senescence­reveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth-inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug development.

The extracellular entrance provides selectivity to serotonin 5-HT7 receptor antagonists with antidepressant-like behavior in vivo.

The finding that ergotamine binds serotonin receptors in a less conserved extended binding pocket close to the extracellular entrance, in addition to the orthosteric site, allowed us to obtain 5-HT7R antagonist 6 endowed with high affinity (Ki=0.7 nM) and significant 5-HT1AR selectivity (ratio>1428). Compound 6 exhibits in vivo antidepressant-like effect (1 mg/kg, ip) mediated by the 5-HT7R, which reveals its interest as a putative research tool or pharmaceutical in depression disorders.

Serotonin 5-HT6 receptor antagonists for the treatment of cognitive deficiency in Alzheimer's disease.

Alzheimer's disease (AD) is one of the most frequent causes of death and disability worldwide and has a significant clinical and socioeconomic impact. In the search for novel therapeutic strategies, serotonin 5-HT6 receptor (5-HT6R) has been proposed as a promising drug target for cognition enhancement in AD. This manuscript reviews the compelling evidence for the implication of this receptor in learning and memory processes. We have summarized the current status of the medicinal chemistry of 5-HT6R antagonists and the encouraging preclinical findings that demonstrate their significant procognitive behavioral effects in a number of learning paradigms, probably acting through modulation of multiple neurotransmitter systems and signaling pathways. The results of the ongoing clinical trials are eagerly awaited to shed some light on the validation of 5-HT6R antagonists as a new drug class for the treatment of symptomatic cognitive impairment in AD, either as stand-alone therapy or in combination with established agents.

New serotonin 5-HT1A receptor agonists endowed with antinociceptive activity in vivo.

We report the synthesis of new compounds 4-35 based on two different openings (A and B) of the chromane ring present in the previously identified 5-HT1A receptor (5-HT1AR) ligand 3. The synthesized compounds were assessed for binding affinity, selectivity, and functional activity at the 5-HT1AR. Selected candidates resulting from B opening were also evaluated for their potential antinociceptive effect in vivo and pharmacokinetic properties in vitro. Analogue 19 [2-(4-{[2-(2-ethoxyphenoxy)ethyl]amino}butyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione] has been characterized as a high-affinity and potent 5-HT1AR agonist (Ki = 2.3 nM; EC50 = 19 nM). Pharmacokinetic studies indicated that compound 19 displays a good metabolic stability in human liver microsomes (t1/2 ∼ 3 h and CLint = 3.5 mL/min/kg, at 5 µM), and a low level of protein binding (25%, at 5 µM). Interestingly, 19 (3 mg/kg, ip, and 30 mg/kg, po) caused significant attenuation of formalin-induced behavior in early and late phases of the mouse intradermal formalin test of pain, and this in vivo effect was reversed by the selective 5-HT1AR antagonist WAY-100635. Thus, the new 5-HT1AR agonist identified in this work, 19, exhibits oral analgesic activity, and the results herein represent a step toward identifying new therapeutics for the control of pain.

New synthetic inhibitors of fatty acid synthase with anticancer activity.

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds 5-30 that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC(50) < 50 µM have been tested for their ability to inhibit FASN activity. Among them, derivative 30 blocks the 90% of FASN activity at low concentration (4 µM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK pathways. Remarkably, 30 does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor 30 may aid the validation of this enzyme as a therapeutic target for the treatment of cancer.

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines.

INTRODUCTION: Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. METHODS: In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. RESULTS: In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. CONCLUSIONS: G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development.

New serotonin 5-HT(1A) receptor agonists with neuroprotective effect against ischemic cell damage.

We report the synthesis of new compounds 4-35 based on structural modifications of different moieties of previously described lead UCM-2550. The new nonpiperazine derivatives, representing second-generation agonists, were assessed for binding affinity, selectivity, and functional activity at the 5-HT(1A) receptor (5-HT(1A)R). Computational ß(2)-based homology models of the ligand-receptor complexes were used to explain the observed structure-affinity relationships. Selected candidates were also evaluated for their potential in vitro and in vivo neuroprotective properties. Interestingly, compound 26 (2-{6-[(3,4-dihydro-2H-chromen-2-ylmethyl)amino]hexyl}tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione) has been characterized as a high-affinity and potent 5-HT(1A)R agonist (K(i) = 5.9 nM, EC(50) = 21.8 nM) and exhibits neuroprotective effect in neurotoxicity assays in primary cell cultures from rat hippocampus and in the MCAO model of focal cerebral ischemia in rats.

Development of non-peptide ligands of growth factor receptor-bound protein 2-SRC homology 2 domain using molecular modeling and NMR spectroscopy.

We report a novel series of non-peptide ligands that inhibit the growth factor receptor-bound protein 2 (Grb2)-Src homology 2 (SH2) domain binding, designed using a combined computational and NMR-driven approach. We have identified a new lead compound, 1n (IC(50) = 56 µM), which is cytotoxic in HER2-positive breast cancer cells and disrupts the interaction between HER2 and Grb2. Thus, 1n can be used as a scaffold for the development of efficient Grb2-SH2 domain binding inhibitors.

Development of molecular probes for the human 5-HT(6) receptor.

In this work we report the synthesis of a set of labeled ligands targeting the human 5-HT(6) receptor (h5-HT(6)R). Among the synthesized compounds, fluorescent probe 10 (K(i) = 175 nM and Φ(f) = 0.21) and biotinylated derivative 15 (K(i) = 90 nM) deserve special attention because they enable direct observation of the h5-HT(6)R in cells. Thus, they represent the first molecular probes for 5-HT(6)R visualization. These results are the starting point for introducing a variety of tags in these or other 5-HT(6)R ligand scaffolds aimed at the development of optimized probes with tailored profiles in terms of fluorescence, affinity, or selectivity.

Benzimidazole derivatives as new serotonin 5-HT6 receptor antagonists. Molecular mechanisms of receptor inactivation.

On the basis of our previously described pharmacophore model for serotonin 5-HT(6) receptor (5-HT(6)R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT(6)R. Site-directed mutagenesis and a beta(2)-adrenoceptor-based homology model of the 5-HT(6)R were used to predict the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in CH(2)-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined experimental and computational study has permitted to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT(6)R antagonists.

Development of Fluorescent Ligands for the Human 5-HT1A Receptor.

In this work, we report the design and synthesis of a set of fluorescent probes targeting the human 5-HT1A receptor (h5-HT1AR). Among the synthesized compounds, derivative 4 deserves special attention as being a high-affinity ligand (K i = 2 nM) with good fluorescent properties (I em > 1000 au and a fluorescence quantum yield, Φf, of 0.26), which enables direct observation of the h5-HT1AR in cells. Thus, it represents the first efficacious fluorescent probe for the specific labeling of h5-HT1AR in cells. Our results provide the basis for the introduction of a variety of tags in scaffolds of G protein-coupled receptor (GPCR) ligands that enable visualization, covalent binding, or affinity pull-down of receptors. These strategies should contribute to the optimization of the therapeutic exploitation of known or new members of the GPCR superfamily by providing valuable information about their location or level of expression.

Novel Inhibitors of Fatty Acid Synthase with Anticancer Activity.

PURPOSE: Fatty acid synthase (FASN) is overexpressed in human breast carcinoma. The natural polyphenol (-)-epigallocatechin-3-gallate blocks in vitro FASN activity and leads to apoptosis in breast cancer cells without any effects on carnitine palmitoyltransferase-1 (CPT-1) activity, and in vivo, does not decrease body weight. We synthesized a panel of new polyphenolic compounds and tested their effects on breast cancer models. EXPERIMENTAL DESIGN: We evaluated the in vitro effects of the compounds on breast cancer cell growth (SK-Br3, MCF-7, and MDA-MB-231), apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase], cell signaling (HER2, ERK1/2, and AKT), and fatty acid metabolism enzymes (FASN and CPT-1). In vivo, we have evaluated their antitumor activity and their effect on body weight in a mice model of BT474 breast cancer cells. RESULTS: Two compounds potently inhibited FASN activity and showed high cytotoxicity. Moreover, the compounds induced apoptosis and caused a marked decrease in the active forms of HER2, AKT, and ERK1/2 proteins. Interestingly, the compounds did not stimulate CPT-1 activity in vitro. We show evidence that one of the FASN inhibitors blocked the growth of BT474 breast cancer xenografts and did not induce weight loss in vivo. CONCLUSIONS: The synthesized polyphenolic compounds represent a novel class of FASN inhibitors, with in vitro and in vivo anticancer activity, that do not exhibit cross-activation of beta-oxidation and do not induce weight loss in animals. One of the compounds blocked the growth of breast cancer xenografts. These FASN inhibitors may represent new agents for breast cancer treatment. (Clin Cancer Res 2009;15(24):7608-15).