Elena Petricci

DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

Microwave-assisted carbonylation and cyclocarbonylation of aryl iodides under ligand free heterogeneous catalysis.

Carbonylation reaction is a very effective transformation for the synthesis of esters, amides, and heterocyclic compounds. Heterogeneous catalyzed carbonylation reactions can be carried out using the association of Pd/C and microwave dielectric heating. Alkoxy carbonylation can be performed with stoichiometric amounts of different primary and secondary alcohols in DMF in the presence of DBU as the base. Analogously, iodobenzene, CO, and amines can be transformed into the corresponding amides in good yields after a simple filtration to remove the catalyst. Pd/C was also successfully employed in microwave-assisted cyclocarbonylation of o-iodoaniline with acyl chlorides to give benzoxazinones. Pd/C can be recycled two times without a considerable difference in the reaction yields.

A Multidisciplinary Approach for the Identification of Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: S-DABOCs and DAVPs

Among the FDA approved drugs for the treatment of AIDS, non‐nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of first‐line anti‐HIV‐1 therapy because of the less‐severe adverse effects associated with NNRTIs administration in comparison to therapies based on other anti‐HIV‐1 agents. In this contest, 3,4‐dihydro‐2‐alkoxy‐6‐benzyl‐4‐oxypyrimidines (DABOs) have been the object of many studies aimed at identifying novel analogues endowed with potent inhibitory activity towards HIV‐1 wild type and especially drug‐resistant mutants. Accordingly, based on the encouraging results obtained from the biological screening of our internal collection of S‐DABO derivatives, we started with the systematic functionalization of the pyrimidine scaffold to identify the minimal required structural features for RT inhibition. Herein, we describe how the combination of synthetic, biological, and molecular modeling studies led to the identification of two novel subclasses of S‐DABO analogues: S‐DABO cytosine analogues (S‐DABOCs) and 4‐dimethyamino‐6‐vinylpyrimidines (DAVPs).

Microwave‐Assisted Aminocarbonylation of Ynamides by Using Catalytic [Fe3(CO)12] at Low Pressures of Carbon Monoxide

The microwave-assisted aminocarbonylation of ynamides at low pressures of CO is reported. A new class of (E)-acrylamides that are potentially suitable for several applications has been regioselectively synthesized after microwave irradiation for only 20 min by using eco-friendly [Fe(3)(CO)(12)] as the catalyst precursor and triethylamine as the ligand. This transformation is atom economic as all reactants are used in stoichiometric quantities. Furthermore, the transformation is efficiently applied to the alkoxycarbonylation of alkynes as well. Moreover, running these reactions under microwave irradiation allows the simplification of the reaction conditions with remarkable reductions in time, temperature and gas pressure.

Microwave-Assisted Intramolecular Huisgen Cycloaddition of Azido Alkynes Derived from α-Amino Acids

The intramolecular version of the Huisgen cycloaddition is a potentially useful reaction for the stereocontrolled preparation of 1,5-disubstituted and 1,4,5-trisubstiututed triazoles. When alpha-azido propargyl esters derived from alpha-amino acids are submitted to [3 + 2] cycloaddition, the expected 4H-[1,2,3]triazolo[5,1-c][1,4]oxazin-6-ones are not formed; rather, an oligomeric cyclic polyester is obtained via a prevailing intermolecular cycloaddition. We have discovered that propargyl alpha-azido amides undergo metal-free intramolecular Huisgen cycloaddition in MeCN/H(2)O under microwave dielectric heating. This reaction provides access to new condensed triazoles that can be considered as conformationally constrained peptidomimetics. Moreover, the following microwave-assisted lactam ring opening provides 1,4-disubstituted and 1,4,5-trisubstituted triazole amino acids. The same kind of compounds are obtained from the ester cycloadduct by reaction with primary amines in the presence of AlMe(3). In order to interpretate this unpredictable behavior, an ab initio study of the reaction pathway was undertaken using GAMESS(US) at the B3LYP/6-31G** level of theory. Different relaxed potential energy profiles were obtained for esters and amides, suggesting that the cis-arrangement of the -CO=N- could account for the amide reactivity.

Domino Hydrogenation–Reductive Amination of Phenols, a Simple Process To Access Substituted Cyclohexylamines

Phenols can be efficiently reduced by sodium formate and Pd/C as the catalyst in water and in the presence of amines to give the corresponding cyclohexylamines. This reaction works at rt for 12 h or at 60 °C under microwave dielectric heating for 20 min. With the exception of aniline, primary, secondary amines, amino alcohols, and even amino acids can be used as nucleophiles. The reductive process is based on a sustainable hydrogen source and a catalyst that can be efficiently recovered and reused. The protocol was developed into a continuous-flow production of cyclohexylamines in gram scale achieving very efficient preliminary results (TON 32.7 and TOF 5.45 h(-1)).

Synthesis of 1,2,3-Substituted Pyrroles from Propargylamines via a One-Pot Tandem Enyne Cross Metathesis–Cyclization Reaction

Enyne cross metathesis of propargylamines with ethyl vinyl ether enables the one-pot synthesis of substituted pyrroles. A series of substituted pyrroles, bearing alkyl, aryl, and heteroaryl substituents, has been synthesized in good yields under microwave irradiation. The reactions are rapid and procedurally simple and also represent a facile entry to the synthetically challenging 1,2,3-substituted pyrroles. The value of the methodology is further corroborated by the conversion of pyrroles into 3-methyl-pyrrolines and the derivatization of the 3-methyl-substituent arising from the metathesis reaction.

Discovery, molecular and pharmacological characterization of GSA-10, a novel small-molecule positive modulator of Smoothened.

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer, and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here, we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands, we have identified and characterized a novel quinolinecarboxamide derivative, propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate, (GSA-10), as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological, biochemical, and molecular approaches, we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However, this molecule does not display the hallmarks of reference Smo agonists. Remarkably, GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists, such as MRT-83, SANT-1, LDE225, and M25 in the nanomolar range, by GDC-0449 in the micromolar range, but not by cyclopamine and CUR61414. Thus, GSA-10 allows the pharmacological characterization of a novel Smo active site, which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action, with important implications in physiology and in therapy.

MRT-92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor

The Smoothened (Smo) receptor, a member of class F G protein‐coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7‐transmembrane (7TM) domain. X‐ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM‐directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT‐1). Here we report the development of the acylguanidine MRT‐92, which displays subnanomolar antagonist activity against Smo in various Hh cell‐based assays. MRT‐92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [3H]MRT‐92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site‐directed mutagenesis data, our work convincingly confirms that MRT‐92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmicproximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh‐linked cancers and associated chemoresistance.—Hoch, L., Faure, H., Roudaut, H., Schoenfelder, A., Mann, A., Girard, N., Bihannic, L., Ayrault, O. Petricci, E., Taddei, M., Rognan, D., Ruat, M. MRT‐92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor. FASEB J. 29, 1817‐1829 (2015). www.fasebj.org

An improved synthesis of solid-supported reagents (SSRs) for selective acylation of amines by microwave irradiation

Microwave-assisted acylation of a solid-supported pyrimidine linker with different acyl chlorides gave polymer-bound 4-acyloxypyrimidines, which in turn were used as SSRs for rapid and selective acylation of amines under microwave irradiation.