Discovery of novel FGF trap small molecules endowed with anti-myeloma activity.

Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.

Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

Mechanistic Modeling of Lys745 Sulfonylation in EGFR C797S Reveals Chemical Determinants for Inhibitor Activity and Discriminates Reversible from Irreversible Agents.

Targeted covalent inhibitors hold promise for drug discovery, particularly for kinases. Targeting the catalytic lysine of epidermal growth factor receptor (EGFR) has attracted attention as a new strategy to overcome resistance due to the emergence of C797S mutation. Sulfonyl fluoride derivatives able to inhibit EGFRL858R/T790M/C797S by sulfonylation of Lys745 have been reported. However, atomistic details of this process are still poorly understood. Here, we describe the mechanism of inhibition of an innovative class of compounds that covalently engage the catalytic lysine of EGFR, through a sulfur(VI) fluoride exchange (SuFEx) process, with the help of hybrid quantum mechanics/molecular mechanics (QM/MM) and path collective variables (PCVs) approaches. Our simulations identify the chemical determinants accounting for the irreversible activity of agents targeting Lys745 and provide hints for the further optimization of sulfonyl fluoride agents.

Unbinding Kinetics of Muscarinic M3 Receptor Antagonists Explained by Metadynamics Simulations.

The residence time (RT), the time for which a drug remains bound to its biological target, is a critical parameter for drug design. The prediction of this key kinetic property has been proven to be challenging and computationally demanding in the framework of atomistic simulations. In the present work, we setup and applied two distinct metadynamics protocols to estimate the RTs of muscarinic M3 receptor antagonists. In the first method, derived from the conformational flooding approach, the kinetics of unbinding is retrieved from a physics-based parameter known as the acceleration factor α (i.e., the running average over time of the potential deposited in the bound state). Such an approach is expected to recover the absolute RT value for a compound of interest. In the second method, known as the tMETA-D approach, a qualitative estimation of the RT is given by the time of simulation required to drive the ligand from the binding site to the solvent bulk. This approach has been developed to reproduce the change of experimental RTs for compounds targeting the same target. Our analysis shows that both computational protocols are able to rank compounds in agreement with their experimental RTs. Quantitative structure-kinetics relationship (SKR) models can be identified and employed to predict the impact of a chemical modification on the experimental RT once a calibration study has been performed.

Molecular Determinants of EphA2 and EphB2 Antagonism Enable the Design of Ligands with Improved Selectivity.

With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.

Free-Energy Simulations Support a Lipophilic Binding Route for Melatonin Receptors.

The effects of the neurohormone melatonin are mediated by the activation of the GPCRs MT1 and MT2 in a variety of tissues. Crystal structures suggest ligand access to the orthosteric binding site of MT1 and MT2 receptors through a lateral channel between transmembrane (TM) helices IV and V. We investigated the feasibility of this lipophilic entry route for 2-iodomelatonin, a nonselective agonist with a slower dissociation rate from the MT2 receptor, applying enhanced sampling simulations and free-energy calculations. 2-Iodomelatonin unbinding was investigated with steered molecular dynamics simulations which revealed different trajectories passing through the gap between TM helices IV and V for both receptors. For one of these unbinding trajectories from the MT1 receptor, an umbrella-sampling protocol with path-collective variables provided a calculated energy barrier consistent with the experimental dissociation rate. The side-chain flexibility of Tyr5.38 was significantly different in the two receptor subtypes, as assessed by metadynamics simulations, and during ligand unbinding it frequently assumes an open conformation in the MT1 but not in the MT2 receptor, favoring 2-iodomelatonin egress. Taken together, our simulations are consistent with the possibility that the gap between TM IV and V is a way of connecting the orthosteric binding site and the membrane core for lipophilic melatonin receptor ligands. Our simulations also suggest that the open state of Tyr5.38 generates a small pocket on the surface of MT1 receptor, which could participate in the recognition of MT1-selective ligands and may be exploited in the design of new selective compounds.

Mechanistic Modeling of Monoglyceride Lipase Covalent Modification Elucidates the Role of Leaving Group Expulsion and Discriminates Inhibitors with High and Low Potency.

Inhibition of monoglyceride lipase (MGL), also known as monoacylglycerol lipase (MAGL), has emerged as a promising approach for treating neurological diseases. To gain useful insights in the design of agents with balanced potency and reactivity, we investigated the mechanism of MGL carbamoylation by the reference triazole urea SAR629 (IC50 = 0.2 nM) and two recently described inhibitors featuring a pyrazole (IC50 = 1800 nM) or a 4-cyanopyrazole (IC50 = 8 nM) leaving group (LG), using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach. Opposite to what was found for substrate 2-arachidonoyl-sn-glycerol (2-AG), covalent modification of MGL by azole ureas is controlled by LG expulsion. Simulations indicated that changes in the electronic structure of the LG greatly affect reaction energetics with triazole and 4-cyanopyrazole inhibitors following a more accessible carbamoylation path compared to the unsubstituted pyrazole derivative. The computational protocol provided reaction barriers able to discriminate between MGL inhibitors with different potencies. These results highlight how QM/MM simulations can contribute to elucidating structure-activity relationships and provide insights for the design of covalent inhibitors.

Different roles for the acyl chain and the amine leaving group in the substrate selectivity of N-Acylethanolamine acid amidase.

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations.

New Coumarin Derivatives as Cholinergic and Cannabinoid System Modulators.

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aß42) was assessed. Compounds 2 and 3, bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Aß42 self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.

New classes of potent heparanase inhibitors from ligand-based virtual screening.

Heparanase is a validated target in cancer therapy and a potential target for several inflammatory pathologies. A ligand-based virtual screening of commercial libraries was performed to expand the chemical space of small-molecule inhibitors. The screening was based on similarity with known inhibitors and was performed in several runs, starting from literature compounds and progressing through newly discovered inhibitors. Among the fifty-five tested compounds, nineteen had IC50 values lower than 5 µM and some showed remarkable potencies. Importantly, tere- and isophthalamides derivatives belong to new structural classes of heparanase inhibitors and some of them showed enzyme affinities (61 and 63, IC50 = 0.32 and 0.12 µM, respectively) similar to those of the most potent small-molecule inhibitors reported so far. Docking studies provided a comprehensive binding hypothesis shared by compounds with significant structural diversity. The most potent inhibitors reduced cell invasiveness and inhibited the expression of proangiogenic factors in tumour cell lines.

Chiral Recognition of Flexible Melatonin Receptor Ligands Induced by Conformational Equilibria.

N-anilinoethylamides are a class of melatoninergic agents with the aniline portion mimicking the indole ring of the natural ligand and the ethylamide chain reproducing that of melatonin. The simplest compound in this class, N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (UCM793), has nanomolar binding affinity for MT1 and MT2 membrane receptors. To explore the effect of chain conformation on receptor binding, a methyl group was inserted on the methylene alpha or beta to the amide nitrogen and conformational equilibria were investigated by NMR spectroscopy and molecular dynamics simulations. Receptor affinity was conserved only for the beta-methyl derivative, which also showed significant stereoselectivity, with the (S) enantiomer being the eutomer. Molecular dynamics simulations, validated by NMR spectroscopy, showed that the beta-methyl group affects the conformational preferences of the ethylamide chain. Docking into the receptor crystal structure provides a rationale for the observed chiral recognition, suggesting that the (S)-beta-methyl group favors the conformation that better fits the receptor binding site.

Comparative Analysis of Virtual Screening Approaches in the Search for Novel EphA2 Receptor Antagonists.

The EphA2 receptor and its ephrin-A1 ligand form a key cell communication system, which has been found overexpressed in many cancer types and involved in tumor growth. Recent medicinal chemistry efforts have identified bile acid derivatives as low micromolar binders of the EphA2 receptor. However, these compounds suffer from poor physicochemical properties, hampering their use in vivo. The identification of compounds able to disrupt the EphA2-ephrin-A1 complex lacking the bile acid scaffold may lead to new pharmacological tools suitable for in vivo studies. To identify the most promising virtual screening (VS) protocol aimed at finding novel EphA2 antagonists, we investigated the ability of both ligand-based and structure-based approaches to retrieve known EphA2 antagonists from libraries of decoys with similar molecular properties. While ligand-based VSs were conducted using UniPR129 and ephrin-A1 ligand as reference structures, structure-based VSs were performed with Glide, using the X-ray structure of the EphA2 receptor/ephrin-A1 complex. A comparison of enrichment factors showed that ligand-based approaches outperformed the structure-based ones, suggesting ligand-based methods using the G-H loop of ephrin-A1 ligand as template as the most promising protocols to search for novel EphA2 antagonists.

Synthesis and characterization of new bivalent agents as melatonin- and histamine H3-ligands.

Melatonin is an endogenous molecule involved in many pathophysiological processes. In addition to the control of circadian rhythms, its antioxidant and neuroprotective properties have been widely described. Thus far, different bivalent compounds composed by a melatonin molecule linked to another neuroprotective agent were synthesized and tested for their ability to block neurodegenerative processes in vitro and in vivo. To identify a novel class of potential neuroprotective compounds, we prepared a series of bivalent ligands, in which a prototypic melatonergic ligand is connected to an imidazole-based H3 receptor antagonist through a flexible linker. Four imidazolyl-alkyloxy-anilinoethylamide derivatives, characterized by linkers of different length, were synthesized and their binding affinity for human MT1, MT2 and H3 receptor subtypes was evaluated. Among the tested compounds, 14c and 14d, bearing a pentyl and a hexyl linker, respectively, were able to bind to all receptor subtypes at micromolar concentrations and represent the first bivalent melatonergic/histaminergic ligands reported so far. These preliminary results, based on binding affinity evaluation, pave the way for the future development of new dual-acting compounds targeting both melatonin and histamine receptors, which could represent promising therapeutic agents for the treatment of neurodegenerative pathologies.

Discovery of a new 1-(phenylsulfonyl)-1H-indole derivative targeting the EphA2 receptor with antiproliferative activity on U251 glioblastoma cell line.

In our continuing effort devoted at developing agents targeting the EphA2 receptor by means of protein-protein interaction (PPI) inhibitors, we report here the design and synthesis of a new class of l-ß-homotryptophan conjugates of 3-ß-hydroxy-Δ5-cholenic acid bearing a set of arylsulfonyl substituents at the indole nitrogen atom. An extensive structure-activity relationship (SAR) analysis indicates that the presence of a bulky lipophilic moiety at the indole nitrogen is fundamental for improving potency on the EphA2 receptor, while abrogating activity on the EphB1-EphB3 receptor subtypes. A rational exploration, guided by the combined application of an experimental design on σp and π physicochemical descriptors and docking simulations, led to the discovery of UniPR1454, a 1-(4-(trifluoromethyl)phenyl)sulfonyl derivative acting as potent and competitive EphA2 antagonist able to inhibit ephrin-A1 dependent signals and to reduce proliferation of glioblastoma (U251) cell line at micromolar concentration.

Pharmacological characterization of second generation FXR agonists as effective EphA2 antagonists: A successful application of target hopping approach.

It is well demonstrated the key role of Eph-ephrin system, specifically of EphA2 receptor, in supporting tumor growth, invasion, metastasis and neovascularization. We previously identified FXR agonists as eligible antagonists of Eph-ephrin system. Herein we characterize new commercially available FXR (Farnesoid X Receptor) agonists as potential Eph ligands including Cilofexor, Nidufexor, Tropifexor, Turofexorate isopropyl and Vonafexor. Our exploration based on molecular modelling investigations and binding assays shows that Cilofexor binds specifically and reversibly to EphA2 receptor with a Ki value in the low micromolar range. Furthermore, Cilofexor interferes with the phosphorylation of EphA2 and the cell retraction and rounding in PC3 prostate cancer cells, both events depending on EphA2 activation. In conclusion, we can confirm that target hopping can be a successful approach to discover new moiety of protein-protein inhibitors.

In silico drug discovery of melatonin receptor ligands with therapeutic potential.

INTRODUCTION: The neurohormone melatonin (N-acetyl-5-methoxytryptamine) regulates circadian rhythms exerting a variety of effects in the central nervous system and in periphery. These activities are mainly mediated by activation of MT1 and MT2 GPCRs. MT1/MT2 agonist compounds are used clinically for insomnia, depression, and circadian rhythm disturbances. AREA COVERED: The following review describes the design strategies that have led to the identification of melatonin receptor ligands, guided by in silico approaches and molecular modeling. Initial ligand-based design, mainly relying on pharmacophore modeling and 3D-QSAR, has been flanked by structure-based virtual screening, given the recent availability of MT1 and MT2 crystal structures. Receptor ligands with different activity profiles, agonist/antagonist and subtype-selective compounds, are available. EXPERT OPINION: An insight on the pharmacological characterization and therapeutic perspectives for relevant ligands is provided. In silico drug discovery has been instrumental in the design of novel ligands targeting melatonin receptors. Ligand-based approaches has led to the construction of a solid framework defining structure-activity relationships to obtain compounds with a tailored pharmacological profile. Structure-based techniques could integrate previous knowledge and provide compounds with novel chemotypes and pharmacological activity as drug candidates for disease conditions in which melatonin receptor ligands are currently being investigated, including cancer and pain.

Protein-Protein Interaction Inhibitors Targeting the Eph-Ephrin System with a Focus on Amino Acid Conjugates of Bile Acids.

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings.

Identification of a Quinone Derivative as a YAP/TEAD Activity Modulator from a Repurposing Library.

The transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are the major downstream effectors in the Hippo pathway and are involved in cancer progression through modulation of the activity of TEAD (transcriptional enhanced associate domain) transcription factors. To exploit the advantages of drug repurposing in the search of new drugs, we developed a similar approach for the identification of new hits interfering with TEAD target gene expression. In our study, a 27-member in-house library was assembled, characterized, and screened for its cancer cell growth inhibition effect. In a secondary luciferase-based assay, only seven compounds confirmed their specific involvement in TEAD activity. IA5 bearing a p-quinoid structure reduced the cytoplasmic level of phosphorylated YAP and the YAP-TEAD complex transcriptional activity and reduced cancer cell growth. IA5 is a promising hit compound for TEAD activity modulator development.

Fighting tertiary mutations in EGFR-driven lung-cancers: Current advances and future perspectives in medicinal chemistry.

Third-generation inhibitors of the epidermal growth factor receptor (EGFR), best exemplified by osimertinib, have been developed to selectively target variants of EGFR bearing activating mutations and the mutation of gatekeeper T790 in patients with EGFR-mutated forms of Non-Small Cell Lung Cancer (NSCLC). While the application of third-generation inhibitors has represented an effective first- and second-line treatment, the efficacy of this class of inhibitors has been hampered by the novel, tertiary mutation C797S, which may occur after the treatment with osimertinib. More recently, other point mutations, including L718Q, G796D, G724S, L792 and G719, have emerged as mutations mediating resistance to third-generation inhibitors. The challenge of overcoming newly developed and recurrent resistances mediated by EGFR-mutations is thus driving the search of alternative strategies in the design of new therapeutic agents able to block EGFR-driven tumor growth. In this manuscript we review the recently emerged EGFR-dependent mechanisms of resistance to third-generation inhibitors, and the achievements lately obtained in the development of next-generation EGFR inhibitors.

Phenotype Screening of an Azole-bisindole Chemical Library Identifies URB1483 as a New Antileishmanial Agent Devoid of Toxicity on Human Cells.

We report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC50 values ranging from 4 to 10 µM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells.