Synthesis and Evaluation of Monoaryl Derivatives as Transthyretin Fibril Formation Inhibitors

Transthyretin (TTR) is a homo-tetrameric protein characterized by four identical β-sheet rich monomers assembled together to form a tetramer that is crossed, along the 2-fold symmetry axis, by two similar binding pockets named thyroxin binding sites. Under unknown conditions, TTR can misfold and aggregate triggering the amyloidosis onset. One therapeutic approach consists to stabilize the tetramer with synthetic small molecules that bind TTR binding site hindering the first step of fibril formation. Here, we report the synthesis of new 2-((benzyloxy)imino)acetic, -propanoic and -butanoic acid derivatives, results of their turbidimetric UV assay and the docking study of new monoaryl compounds. The obtained results suggest that, for this class of compounds, (i) the chlorine atom in ortho position on the aromatic ring is the best substituent; (ii) the linker inversion still allows the interaction with thyroxine binding sites; and (iii) the steric hindrance in R1 position is detrimental for the activity

Targeting Different Transthyretin Binding Sites with Unusual Natural Compounds

Misfolding and aggregation of the transthyretin (TTR) protein leads to certain forms of amyloidosis. Some nutraceuticals, such as flavonoids and natural polyphenols, have recently been investigated as modulators of the self-assembly process of TTR, but they generally suffer from limited bioavailability. To discover innovative and more bioavailable natural compounds able to inhibit TTR amyloid formation, a docking study was performed using the crystallographic structure of TTR. This computational strategy was projected as an adhoc inspection of the possible relationship between binding site location and modulation of the assembly process; interactions with the as-yet-unexplored epigallocatechin gallate (EGCG) sites and with the thyroxine (T4) pocket were simultaneously analyzed. All the compounds studied seem to prefer the traditional T4 binding site, but some interesting results emerged from the screening of an in-house database, used for validating the computational protocol, and of the Herbal Ingredients Targets (HIT) catalogue available on the ZINC database

Identification of Human Dihydroorotate Dehydrogenase Inhibitor by a Pharmacophore-Based Virtual Screening Study

Human dihydroorotate dehydrogenase (hDHODH) is an enzyme belonging to a flavin mononucleotide (FMN)-dependent family involved in de novo pyrimidine biosynthesis, a key biological pathway for highly proliferating cancer cells and pathogens. In fact, hDHODH proved to be a promising therapeutic target for the treatment of acute myelogenous leukemia, multiple myeloma, and viral and bacterial infections; therefore, the identification of novel hDHODH ligands represents a hot topic in medicinal chemistry. In this work, we reported a virtual screening study for the identification of new promising hDHODH inhibitors. A pharmacophore-based approach combined with a consensus docking analysis and molecular dynamics simulations was applied to screen a large database of commercial compounds. The whole virtual screening protocol allowed for the identification of a novel compound that is endowed with promising inhibitory activity against hDHODH and is structurally different from known ligands. These results validated the reliability of the in silico workflow and provided a valuable starting point for hit-to-lead and future lead optimization studies aimed at the development of new potent hDHODH inhibitors

Salicylaldoxime derivatives as new leads for the development of carbonic anhydrase inhibitors

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Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor

Design, synthesis and biological evaluation of novel orthosteric-allosteric ligands of the cannabinoid receptor type 2 (CB2R)

It is well known that G protein–coupled receptors (GPCRs) assume multiple active states. Orthosteric ligands and/or allosteric modulators can preferentially stabilize specific conformations, giving rise to pathway-biased signaling. One of the most promising strategies to expand the repertoire of signaling-selective GPCR activators consists of dualsteric agents, which are hybrid compounds consisting of orthosteric and allosteric pharmacophoric units. This approach proved to be very promising showing several advantages over monovalent targeting strategies, including an increased affinity or selectivity, a bias in signaling pathway activation, reduced off-target activity and therapeutic resistance. Our study focused on the cannabinoid receptor type 2 (CB2R), considered a clinically promising target for the control of brain damage in neurodegenerative disorders. Indeed, CB2R was found highly expressed in microglial cells, astrocytes, and even in some neuron subpopulations. Here, we describe the design, synthesis, and biological evaluation of two new classes of potential dualsteric (bitopic) CB2R ligands. The new compounds were obtained by connecting, through different linkers, the pharmacophoric portion of the CB2R positive allosteric modulator (PAM), EC21a, with that of the CB2R selective orthosteric agonist LV62, both developed in our laboratories. A preliminary screening enabled us to identify compound JR64a as the most promising of the series. Indeed, functional examination highlighted a signaling ‘bias’ in favor of G protein activation over βarrestin2 recruitment, combined with high affinity for CB2R and the ability to efficiently prevent inflammation in human microglial cells (HMC3) exposed to LPS/TNFα stimulation, thus demonstrating great promise for the treatment of neurodegenerative diseases

Salicylaldoximes and anthranylaldoximes as alternatives to phenol-based estrogen receptor ligands

Estrogens play a crucial role in the development and function of female reproductive tissues. They have positive effects on the maintenance of bone mineral density, on the liver, and on the cardiovascular and central nervous systems. Selective Estrogen Receptor Modulators (SERMs) are particularly attractive as therapeutic agents because they are able to block estrogen action at those sites where stimulation would be undesirable, such as the breast and uterus, but at the same time stimulate estrogen actions in other tissues where they are desired, such as the bone and liver. Most synthetic estrogen receptor ligands possess a phenolic ring, mimicking the phenolic "Aring" of the natural ligand estradiol. In an attempt to increase the structural diversity of estrogen receptor (ER) ligands, we designed and synthesized molecules containing unprecedented replacements of the prototypical phenolic "A-ring" of estrogens with an oxime and a hydroxy- (salicylaldoximes) or aminomoieties (anthranylaldoximes), forming intramolecularly H-bonded pseudocycles. These new classes of compounds showed interesting ER binding properties on both receptor subtypes (ERα and ERβ). These results proved that the six-membered ring formed by an intramolecular hydrogen bond, and containing an exocyclic oxime OH, is an effective stereoelectronic replacement of the phenolic ring of typical ER ligands

Reversible Monoacylglycerol Lipase Inhibitors: Discovery of a New Class of Benzylpiperidine Derivatives.

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the metabolism of 2-arachidonoylglycerol in the brain and the hydrolysis of peripheral monoacylglycerols. Many studies demonstrated beneficial effects deriving from MAGL inhibition for neurodegenerative diseases, inflammatory pathologies, and cancer. MAGL expression is increased in invasive tumors, furnishing free fatty acids as pro-tumorigenic signals and for tumor cell growth. Here, a new class of benzylpiperidine-based MAGL inhibitors was synthesized, leading to the identification of 13, which showed potent reversible and selective MAGL inhibition. Associated with MAGL overexpression and the prognostic role in pancreatic cancer, derivative 13 showed antiproliferative activity and apoptosis induction, as well as the ability to reduce cell migration in primary pancreatic cancer cultures, and displayed a synergistic interaction with the chemotherapeutic drug gemcitabine. These results suggest that the class of benzylpiperidine-based MAGL inhibitors have potential as a new class of therapeutic agents and MAGL could play a role in pancreatic cancer

Novel Transthyretin Amyloid Fibril Formation Inhibitors: Synthesis, Biological Evaluation, and X-Ray Structural Analysis

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of β-aminoxypropionic acids (compounds 5–21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR∶inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp(2)-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22–32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding

Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands

The design of dualsteric/bitopic agents as single chemical entities able to simultaneously interact with both the orthosteric and an allosteric binding site represents a novel approach in medicinal chemistry. Biased dualsteric/bitopic agents could enhance certain signaling pathways while diminishing the others that cause unwanted side effects. We have designed, synthesized, and functionally characterized the first CB2R heterobivalent bitopic ligands. In contrast to the parent orthosteric compound, our bitopic ligands selectively target CB2R versus CB1R and show a functional selectivity for the cAMP signaling pathway versus βarrestin2 recruitment. Moreover, the most promising bitopic ligand FD-22a displayed anti-inflammatory activity in a human microglial cell inflammatory model and antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Finally, computational studies clarified the binding mode of these compounds inside the CB2R, further confirming their bitopic nature