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

Metadynamics for perspective drug design: Computationally driven synthesis of new protein-protein interaction inhibitors targeting the EphA2 receptor

Metadynamics (META-D) is emerging as a powerful method for the computation of the multidimensional freeenergy surface (FES) describing the protein-ligand binding process. Herein, the FES of unbinding of the antagonist N-(3α-hydroxy-5β-cholan-24-oyl)-L-β-homotryptophan (UniPR129) from its EphA2 receptor was reconstructed by META-D simulations. The characterization of the free-energy minima identified on this FES proposes a binding mode fully consistent with previously reported and new structure-activity relationship data. To validate this binding mode, new N-(3α-hydroxy-5β-cholan-24-oyl)-L-β-homotryptophan derivatives were designed, synthesized, and tested for their ability to displace ephrin-A1 from the EphA2 receptor. Among them, two antagonists, namely compounds 21 and 22, displayed high affinity versus the EphA2 receptor and resulted endowed with better physicochemical and pharmacokinetic properties than the parent compound. These findings highlight the importance of free-energy calculations in drug design, confirming that META-D simulations can be used to successfully design novel bioactive compounds

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

UniPR1331, a small molecule targeting Eph/ephrin interaction, prolongs survival in glioblastoma and potentiates the effect of antiangiogenic therapy in mice

Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to standard therapeutic approaches that combine surgery, radiotherapy, and chemotherapy. As opposed to healthy tissues, EphA2 has been found highly expressed in specimens of glioblastoma, and increased expression of EphA2 has been shown to correlate with poor survival rates. Accordingly, agents blocking Eph receptor activity could represent a new therapeutic approach. Herein, we demonstrate that UniPR1331, a pan Eph receptor antagonist, possesses significant in vivo anti-angiogenic and anti-vasculogenic properties which lead to a significant anti-tumor activity in xenograft and orthotopic models of GBM. UniPR1331 halved the final volume of tumors when tested in xenografts (p<0.01) and enhanced the disease-free survival of treated animals in the orthotopic models of GBM both by using U87MG cells (40 vs 24 days of control, p<0.05) or TPC8 cells (52 vs 16 days, p<0.01). Further, the association of UniPR1331 with the anti-VEGF antibody Bevacizumab significantly increased the efficacy of both monotherapies in all tested models. Overall, our data promote UniPR1331 as a novel tool for tackling GBM

Mannich base derivatives as novel EGFR irreversible inhibitors

The epidermal growth factor receptor (EGFR), a tyrosine kinase receptor involved in oncogenic phenomena, is a validated target for cancer therapy.1 Gefitinib and erlotinib are the earliest small molecule EGFR tyrosine kinase inhibitors (TKIs) approved for non-small cell lung cancer (NSCLC) treatment. These compounds are able to establish a reversible interaction with the ATP binding pocket of the kinase domain. A single point mutation in the ATP binding site involving the gatekeeper residue Thr790 (T790M) prevents the ATP-competitive reversible inhibitors from binding and leads to the development of drug resistance.2 A second generation of EGFR-targeted agents allowed to overcame this resistance by irreversible interaction with the target: these compounds irreversibly alkylate a cysteine (Cys797) within the catalytic site of EGFR.4 The main limit of this class of compounds is the intrinsic reactivity due to the presence of a Michael acceptor group, responsible for the covalent bond formation with the cysteine residue. This high reactivity may give rise to unexpected toxicity and lack of target specificity.2 With the aim to reduce the undesired interactions with non-target related proteins, we substituted the highly electrophilic Michael acceptor group with a low reactive group that preserve the ability to react with the nucleophile within the target protein. For this purpose, we explored a series of β-aminocarbonyl groups, inserted on a traditional quinazoline-based driver portion, synthesizing a series of Mannich base derivatives, which had shown to be chemically stable under physiologic conditions, while retaining an irreversible inhibition of the target

Novel cycloAmpRGD-Sunitinib Dual Conjugates as Potent Targeted Anti-angiogenic Tools

In recent years, targeted therapies that selectively address receptors and pathways involved in tumor genesis and progression have attracted growing interest. Some integrin subfamilies (e.g. αVβ3, αVβ5 and α5β1) have shown to be involved, even by close cooperation with other cell receptors (e.g. vascular endothelial growth factor receptors, VEGFRs) in tumor angiogenesis, which plays crucial role in tumor development and dissemination. Recent studies clearly demonstrated a complex in vivo regulation of tumor angiogenesis events; in particular, the αVβ3 integrin receptor is physically and functionally correlated with the VEGFR2 receptor within endothelial cells (ECs), suggesting that dual specific agents capable of inhibiting them would have a great anti-angiogenesis potential. Our research group has recently introduced a new class of cyclic semipeptide ligands, cycloAmpRGD, containing the Arg-Gly-Asp (RGD) sequence and 4-aminoproline scaffolds. These ligands demonstrated to efficiently and selectively bind to the αVβ3 integrin and their binding capability is preserved even in the presence of covalently conjugated “bulky loads” (cytotoxic and chelating agents). Here we report the synthesis, characterization and biological evaluation of a series of dual conjugates of type I, wherein the ligand cycloAmpRGD is covalently associated to a Sunitinib-derived moiety, a clinically approved anti-angiogenic multikinase inhibitor. The binding competence of these candidates toward the αVβ3 integrin in EC lines, their kinase inhibitory activity toward VEGFR2, and their ability to block endothelial cell capillary formation in vitro are described, in comparison with the single agents and related combinations. Encouraging results point to the notion that the covalent conjugation of cycloAmpRGD and Sunitinib may be of high therapeutic potential for tumor angiogenesis inhibition

Predicting the reactivity of nitrile-carrying compounds with cysteine: a combined computational and experimental study.

Here, we report on a mechanistic investigation based on DFT calculations and kinetic measures aimed at determining the energetics related to the cysteine nucleophilic attack on nitrile-carrying compounds. Activation energies were found to correlate well with experimental kinetic measures of reactivity with cysteine in phosphate buffer. The agreement between computations and experiments points to this DFT-based approach as a tool for predicting both nitrile reactivity toward cysteines and the toxicity of nitriles as electrophile agents

Predicting the Reactivity of Nitrile-Carrying Compounds with Cysteine: A Combined Computational and Experimental Study

Here, we report on a mechanistic investigation based on DFT calculations and kinetic measures aimed at determining the energetics related to the cysteine nucleophilic attack on nitrile-carrying compounds. Activation energies were found to correlate well with experimental kinetic measures of reactivity with cysteine in phosphate buffer. The agreement between computations and experiments points to this DFTbased approach as a tool for predicting both nitrile reactivity toward cysteines and the toxicity of nitriles as electrophile agents