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.

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.

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.

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.

A sulfonyl fluoride derivative inhibits EGFRL858R/T790M/C797S by covalent modification of the catalytic lysine.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.

Chemical modification of NSC12 leads to a specific FGF-trap with antitumor activity in multiple myeloma.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity in vivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells in vitro by inhibiting FGF-dependent receptor activation and slowing MM growth in vivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

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.

Design and SAR Analysis of Covalent Inhibitors Driven by Hybrid QM/MM Simulations.

Quantum mechanics/molecular mechanics (QM/MM) hybrid technique is emerging as a reliable computational method to investigate and characterize chemical reactions occurring in enzymes. From a drug discovery perspective, a thorough understanding of enzyme catalysis appears pivotal to assist the design of inhibitors able to covalently bind one of the residues belonging to the enzyme catalytic machinery. Thanks to the current advances in computer power, and the availability of more efficient algorithms for QM-based simulations, the use of QM/MM methodology is becoming a viable option in the field of covalent inhibitor design. In the present review, we summarized our experience in the field of QM/MM simulations applied to drug design problems which involved the optimization of agents working on two well-known drug targets, namely fatty acid amide hydrolase (FAAH) and epidermal growth factor receptor (EGFR). In this context, QM/MM simulations gave valuable information in terms of geometry (i.e., of transition states and metastable intermediates) and reaction energetics that allowed to correctly predict inhibitor binding orientation and substituent effect on enzyme inhibition. What is more, enzyme reaction modelling with QM/MM provided insights that were translated into the synthesis of new covalent inhibitor featured by a unique combination of intrinsic reactivity, on-target activity, and selectivity.

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines.

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.

Balancing reactivity and antitumor activity: heteroarylthioacetamide derivatives as potent and time-dependent inhibitors of EGFR.

Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 lung cancer cells (expressing EGFR L858R/T790M mutant) at low micromolar concentration.

Novel Symmetrical Benzazolyl Derivatives Endowed with Potent Anti-Heparanase Activity.

Heparanase is the only mammalian endo-ß-d-glucuronidase involved in a variety of major diseases. The up-regulation of heparanase expression increases tumor size, angiogenesis, and metastasis, representing a validated target in the anti-cancer field. To date, only a few small-molecule inhibitors have been described, but none have gotten through pre-clinical development. Previously, we explored 2-(4-(4-(bromo-methoxybenzamido)benzylamino)phenyl) benzazole derivatives as anti-heparanase agents, proposing this scaffold for development of broadly effective heparanase inhibitors. Herein, we report an extended investigation of new symmetrical 2-aminophenyl-benzazolyl-5-acetate derivatives, proving that symmetrical compounds are more effective than asymmetrical analogues, with the most-potent compound, 7g, being active at nanomolar concentration against heparanase. Molecular docking studies were performed on the best-acting compounds 5c and 7g to rationalize their interaction with the enzyme. Moreover, invasion assay confirmed the anti-metastatic potential of compounds 5c, 7a, and 7g, proving the inhibition of the expression of proangiogenic factors in tumor cells.

Novel Benzazole Derivatives Endowed with Potent Antiheparanase Activity.

Heparanase is the sole mammalian enzyme capable of cleaving glycosaminoglycan heparan sulfate side chains of heparan sulfate proteoglycans. Its altered activity is intimately associated with tumor growth, angiogenesis, and metastasis. Thus, its implication in cancer progression makes it an attractive target in anticancer therapy. Herein, we describe the design, synthesis, and biological evaluation of new benzazoles as heparanase inhibitors. Most of the designed derivatives were active at micromolar or submicromolar concentration, and the most promising compounds are fluorinated and/or amino acids derivatives 13a, 14d, and 15 that showed IC50 0.16-0.82 µM. Molecular docking studies were performed to rationalize their interaction with the enzyme catalytic site. Importantly, invasion assay confirmed the antimetastatic potential of compounds 14d and 15. Consistently with its ability to inhibit heparanase, compound 15 proved to decrease expression of genes encoding for proangiogenic factors such as MMP-9, VEGF, and FGFs in tumor cells.

Antiproliferative and pro-apoptotic activity of melatonin analogues on melanoma and breast cancer cells.

Melatonin plays different physiological functions ranging from the regulation of circadian rhythms to tumor inhibition, owing to its antioxidant, immunomodulatory and anti-aging properties. Due to its pleiotropic functions, melatonin has been shown to elicit cytoprotective processes in normal cells and trigger pro-apoptotic signals in cancer cells. The therapeutic potential of melatonin analogues prompted us to investigate the in vitro and in vivo antitumor activity of new melatonin derivatives and explore the underlying molecular mechanisms. The experiments revealed that the new melatonin analogues inhibited the growth of melanoma and breast cancer cells in a dose- and time-dependent manner. In addition, our results indicated that melatonin derivative UCM 1037 could induce apoptosis in melanoma and breast cancer cells, as well as cell necrosis, in MCF-7. Together, apoptosis and necrosis could be two possible mechanisms to explain the cytotoxic effect of the melatonin analogue against cancer cells. The suppression of tumor growth by the melatonin analogues was further demonstrated in vivo in a xenograft mice model. A decrease in the activation of MAPK pathway was observed in all cancer cells following UCM 1037 treatment. Overall, this study describes a promising antitumor compound showing antiproliferative and cytotoxic activity in melanoma and breast cancer cells.

Atropisomerism and Conformational Equilibria: Impact on PI3Kδ Inhibition of 2-((6-Amino-9H-purin-9-yl)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one (IC87114) and Its Conformationally Restricted Analogs.

IC87114 [compound 1, (2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4(3H)-one nucleus to the o-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the aS and aR isomers of compound 1 do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The aS and aR atropisomers of 1 were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9H-purin-9-yl pendant to the quinazolin-4(3H)-one nucleus of both aS and aR isomers of 1 had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound 1 analogues to PI3Kδ enzyme.

Free-energy studies reveal a possible mechanism for oxidation-dependent inhibition of MGL.

The function of monoacylglycerol lipase (MGL), a key actor in the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2AG), is tightly controlled by the cell's redox state: oxidative signals such as hydrogen peroxide suppress MGL activity in a reversible manner through sulfenylation of the peroxidatic cysteines, C201 and C208. Here, using as a starting point the crystal structures of human MGL (hMGL), we present evidence from molecular dynamics and metadynamics simulations along with high-resolution mass spectrometry studies indicating that sulfenylation of C201 and C208 alters the conformational equilibrium of the membrane-associated lid domain of MGL to favour closed conformations of the enzyme that do not permit the entry of substrate into the active site.

Fatty Acid Amide Hydrolase (FAAH), Acetylcholinesterase (AChE), and Butyrylcholinesterase (BuChE): Networked Targets for the Development of Carbamates as Potential Anti-Alzheimer's Disease Agents.

The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

Synthesis, Structural Elucidation, and Biological Evaluation of NSC12, an Orally Available Fibroblast Growth Factor (FGF) Ligand Trap for the Treatment of FGF-Dependent Lung Tumors.

NSC12 is an orally available pan-FGF trap able to inhibit FGF2/FGFR interaction and endowed with promising antitumor activity. It was identified by virtual screening from a NCI small molecule library, but no data were available about its synthesis, stereochemistry, and physicochemical properties. We report here a synthetic route that allowed us to characterize and unambiguously identify the structure of the active compound by a combination of NMR spectroscopy and in silico conformational analysis. The synthetic protocol allowed us to sustain experiments aimed at assessing its therapeutic potential for the treatment of FGF-dependent lung cancers. A crucial step in the synthesis generated a couple of diastereoisomers, with only one able to act as a FGF trap molecule and to inhibit FGF-dependent receptor activation, cell proliferation, and tumor growth when tested in vitro and in vivo on murine and human lung cancer cells.

Heparanase: a rainbow pharmacological target associated to multiple pathologies including rare diseases.

In recent years, heparanase has attracted considerable attention as a promising target for innovative pharmacological applications. Heparanase is a multifaceted protein endowed with enzymatic activity, as an endo-ß-D-glucuronidase, and nonenzymatic functions. It is responsible for the cleavage of heparan sulfate side chains of proteoglycans, resulting in structural alterations of the extracellular matrix. Heparanase appears to be involved in major human diseases, from the most studied tumors to chronic inflammation, diabetic nephropathy, bone osteolysis, thrombosis and atherosclerosis, in addition to more recent investigation in various rare diseases. The present review provides an overview on heparanase, its biological role, inhibitors and possible clinical applications, covering the latest findings in these areas.

Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase.

Heparanase is a ß-d-glucuronidase which cleaves heparan sulfate chains in the extracellular matrix and on cellular membranes. A dysregulated heparanase activity is intimately associated with cell invasion, tumor metastasis and angiogenesis, making heparanase an attractive target for the development of anticancer therapies. SST0001 (roneparstat; Sigma-Tau Research Switzerland S.A.) is a non-anticoagulant 100% N-acetylated and glycol-split heparin acting as a potent heparanase inhibitor, currently in phase I in advanced multiple myeloma. Herein, the kinetics of heparanase inhibition by roneparstat is reported. The analysis of dose-inhibition curves confirmed the high potency of roneparstat (IC50 ≈ 3 nM) and showed, at higher concentrations, a Hill coefficient consistent with the engagement of two molecules of inhibitor. A homology model of human heparanase GS3 construct was built and used for docking experiments with inhibitor fragments. The model has high structural similarity with the recently reported crystal structure of human heparanase. Different interaction schemes are proposed, which support the hypothesis of a complex binding mechanism involving the recruitment of one or multiple roneparstat chains, depending on its concentration. In particular, docking solutions were obtained in which (i) a single roneparstat molecule interacts with both heparin-binding domains (HBDs) of heparanase or (ii) two fragments of roneparstat interact with either HBD-1 or HBD-2, consistent with the possibility of different inhibitor:enzyme binding stoichiometries. This study provides unique insights into the mode of action of roneparstat as well as clues of its interaction with heparanase at a molecular level, which could be exploited to design novel potential inhibitor molecules.

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.