Crystal structures of Lymphocytic choriomeningitis virus endonuclease domain complexed with diketo-acid ligands

International audienceThe Arenaviridae family, together with the Bunyaviridae and Orthomyxoviridae families, is one of the three negative-stranded RNA viral families that encode an endonuclease in their genome. The endonuclease domain is at the N-terminus of the L protein, a multifunctional protein that includes the RNA-dependent RNA polymerase. The synthesis of mRNA in arenaviruses is a process that is primed by capped nucleotides that are 'stolen' from the cellular mRNA by the endonuclease domain in cooperation with other domains of the L protein. This molecular mechanism has been demonstrated previously for the endonuclease of the prototype Lymphocytic choriomeningitis virus (LCMV). However, the mode of action of this enzyme is not fully understood as the original structure did not contain catalytic metal ions. The pivotal role played by the cap-snatching process in the life cycle of the virus and the highly conserved nature of the endonuclease domain make it a target of choice for the development of novel antiviral therapies. Here, the binding affinities of two diketo-acid (DKA) compounds (DPBA and L-742,001) for the endonuclease domain of LCMV were evaluated using biophysical methods. X-ray structures of the LCMV endonuclease domain with catalytic ions in complex with these two compounds were determined, and their efficacies were assessed in an in vitro endonuclease-activity assay. Based on these data and computational simulation, two new DKAs were synthesized. The LCMV endonuclease domain exhibits a good affinity for these DKAs, making them a good starting point for the design of arenavirus endonuclease inhibitors. In addition to providing the first example of an X-ray structure of an arenavirus endonuclease incorporating a ligand, this study provides a proof of concept that the design of optimized inhibitors against the arenavirus endonuclease is possible

Cellular and Molecular Mechanisms of Methotrexate Resistance in Melanoma

Endocrinolog

Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: Effect of the alpha-phosphorus configuration on HIV-1 RT activity

International audienceno abstrac

CovaDOTS: In Silico Chemistry-Driven Tool to Design Covalent Inhibitors Using a Linking Strategy.

International audienceWe recently reported an integrated fragment-based optimization strategy called DOTS (Diversity Oriented Target-focused Synthesis) that combines automated virtual screening (VS) with semirobotized organic synthesis coupled to in vitro evaluation. The molecular modeling part consists of hit-to-lead chemistry, based on the growing paradigm. Here, we have extended the applicability of the DOTS strategy by adding new functionalities, allowing a generic chemistry-driven linking approach with a particular emphasis on covalent drugs. Indeed, the covalent mode of action can be described as a specific case of linking, where suitable linkers are sought to fuse a bound organic compound with a nucleophilic protein side chain. The proof of concept is established using three retrospective study cases in which known noncovalent inhibitors have been converted to covalent inhibitors. Our method is able to automatically design reference covalent inhibitors (and/or analogs) from an initial activated substructure and predict their binding mode. More importantly, the reference compounds are ranked high among several hundred putative adducts, demonstrating the utility of the approach to design covalent inhibitors

Reinforced cementitious panels evaluated

Aussi disponible en fran\ue7ais: \uc9valuation des panneaux cimentaires renforc\ue9sPeer reviewed: NoNRC publication: Ye

CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Modulation

International audienceDifferentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors

Structural characterisation and inhibition of Arenavirus replication complex elements : assembly, function and inhibition of embedded nucleases

Arenaviruses, belongs to a family of emerging enveloped segmented and ambisens RNA viruses associated withneurological and hemorrhagic diseases in humans. Arenavirus transcription and genome replication are cytoplasmic ensured by aribonucleoproteine replicase complex NP-L. After penetration, L protein initiates transcription to produce NP and L mRNAs[ 1].The priming of transcription is the result of a cap-snatching mechanism ensured by an endonuclease domain associated to the Lpolymerase. As the concentration of NP in the cell increases, genome segments are replicated, to produce full-length copies(cRNA). cRNAs are now templates for transcription of GPC mRNA (from the S segment) and Z mRNA (from the L segment).The NP caries an exonuclease in charge of clearing out from the cytoplasm dsRNA triggering innate immunity response. Bothnucleases have a similar two metal ion catalytic mechanism, with the particularity of transitioning ion brought by the RNAsubstrate. Any alteration of the remaining ion impairs greatly theses activities[2]. We present a global study aiming to characterizethe assembly of the NP[3], through flexible domains[4], a step critical for vRNApackaging and the polsitioning of L for vRNAreplication, as well as using a combined approach of biophysical screening, crystallography and in silico docking, identifyingactive compounds against both nucleases[5]. Crystal structures of the nucleases domain complexed with several compounds wereobtained[67]. By developing specific compounds to alter both transcription and innate immunity shadowing, our strategy is togive the cell a fighting chance to clear the infection. Combining structure, enzymology, rational synthesis, hit-To-leadoptimization, in cellula evaluation, and screening methods, we are presenting the results of a 2nd generation of molecules pavingthe way to the design of a 3rd generation increasing specificity towards Arenaviral nucleases in the context of the replicationcomplex[8]

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology

Masitinib is a protein kinase inhibitor that sensitises refractory pancreatic adenocarcinoma cells to treatment with the nucleoside analog gemcitabine. Here the authors show that Masitinib activates deoxycytidine kinase to enhance phosphorylation of nucleoside analogue pro-drugs, increasing their potency