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116 results on '"Isabelle Krimm"'

1. Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket

Author

Alexandre Bancet, Rita Frem, Florian Jeanneret, Angélique Mularoni, Pauline Bazelle, Caroline Roelants, Jean-Guy Delcros, Jean-François Guichou, Catherine Pillet, Isabelle Coste, Toufic Renno, Christophe Battail, Claude Cochet, Thierry Lomberget, Odile Filhol, and Isabelle Krimm

Subjects
Molecular biology, Cell biology, Structural biology, Cancer, Science
Abstract

Summary: Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

Published
2024
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2. Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

Author

Hubert Gorny, Angélique Mularoni, Jean-Guy Delcros, Céline Freton, Jordane Preto, and Isabelle Krimm

Subjects
VDAC1, microscale thermophoresis, nano-differential scanning fluorimetry, Therapeutics. Pharmacology, RM1-950
Abstract

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.

Published
2023
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3. The Journey towards Solubility Assessment of Small Molecules Using HPLC-DAD

Author

Nada Hemmani, Anaïs Cacilie Martin, Andy Larivière, Mathis Tresallet, Kensa Tchang, Isabelle Krimm, and Marc Le Borgne

Subjects
HPLC, in silico prediction, shake-flask, small molecules, solubility, Medicine
Abstract

In the process of developing bioactive small molecules, solubility determination is a crucial step. Many research papers treating problems related to solubility are published, but none of them fully describes the methods and steps for solubility assessment. In addition, in silico prediction tools and databases such as SwissADME, ACD/Percepta, DrugBank and many others offer the possibility to have approximative solubility values based on the structure of the molecule. Although significant differences can be observed depending on the database and the conditions of the experiment such as solvent, pH, temperature… etc. The lack of data can be a barrier to obtaining details on solubility measurement methods. This presentation aims to describe, step by step, the journey of tryptophan solubility determination using high-performance liquid chromatography-diode array detector (HPLC-DAD).

Published
2022
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4. The intrinsically disordered N-terminus of the voltage-dependent anion channel.

Author

Jordane Preto and Isabelle Krimm

Subjects
Biology (General), QH301-705.5
Abstract

The voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.

Published
2021
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5. 1D NMR WaterLOGSY as an efficient method for fragment-based lead discovery

Author

Claire Raingeval, Olivier Cala, Béatrice Brion, Marc Le Borgne, Roderick Eliot Hubbard, and Isabelle Krimm

Subjects
waterlogsy, solvent-exposed, saturation transfer difference, binding mode, fragment-based lead discovery, Therapeutics. Pharmacology, RM1-950
Abstract

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein.

Published
2019
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6. Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

Author

Abdelhakim Ahmed-Belkacem, Lionel Colliandre, Nazim Ahnou, Quentin Nevers, Muriel Gelin, Yannick Bessin, Rozenn Brillet, Olivier Cala, Dominique Douguet, William Bourguet, Isabelle Krimm, Jean-Michel Pawlotsky, and Jean- François Guichou

Subjects
Science
Abstract

Cyclophilins play a key role in the life cycle of many viruses and represent important drug targets for broad-spectrum antiviral therapies. Here, the authors use fragment-based drug discovery to develop non-peptidic inhibitors of human cyclophilins with high activity against replication of a number of viral families.

Published
2016
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7. Synthesis, Optimization, Antifungal Activity, Selectivity, and CYP51 Binding of New 2-Aryl-3-azolyl-1-indolyl-propan-2-ols

Author

Nicolas Lebouvier, Fabrice Pagniez, Young Min Na, Da Shi, Patricia Pinson, Mathieu Marchivie, Jean Guillon, Tarek Hakki, Rita Bernhardt, Sook Wah Yee, Claire Simons, Marie-Pierre Lézé, Rolf W. Hartmann, Angélique Mularoni, Guillaume Le Baut, Isabelle Krimm, Ruben Abagyan, Patrice Le Pape, and Marc Le Borgne

Subjects
azoles, antifungal agents, indole, microwave irradiation, X-ray crystallography, Candida species, Medicine, Pharmacy and materia medica, RS1-441
Abstract

A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different chemical approaches were then developed. The first one, in seven steps, involved the synthesis of the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole and the final opening of oxiranes by imidazole or 1H-1,2,4-triazole. The second route allowed access to the target compounds in only three steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested against Candida albicans and other Candida species. The enantiomers of the best anti-Candida compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g), were analyzed by X-ray diffraction to determine their absolute configuration. The (−)-8g enantiomer (Minimum inhibitory concentration (MIC) = IC80 = 0.000256 µg/mL on C. albicans CA98001) was found with the S-absolute configuration. In contrast the (+)-8g enantiomer was found with the R-absolute configuration (MIC = 0.023 µg/mL on C. albicans CA98001). By comparison, the MIC value for FLC was determined as 0.020 µg/mL for the same clinical isolate. Additionally, molecular docking calculations and molecular dynamics simulations were carried out using a crystal structure of Candida albicans lanosterol 14α-demethylase (CaCYP51). The (−)-(S)-8g enantiomer aligned with the positioning of posaconazole within both the heme and access channel binding sites, which was consistent with its biological results. All target compounds have been also studied against human fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.

Published
2020
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8. Comparing binding modes of analogous fragments using NMR in fragment-based drug design: application to PRDX5.

Author

Clémentine Aguirre, Tim ten Brink, Jean-François Guichou, Olivier Cala, and Isabelle Krimm

Subjects
Medicine, Science
Abstract

Fragment-based drug design is one of the most promising approaches for discovering novel and potent inhibitors against therapeutic targets. The first step of the process consists of identifying fragments that bind the protein target. The determination of the fragment binding mode plays a major role in the selection of the fragment hits that will be processed into drug-like compounds. Comparing the binding modes of analogous fragments is a critical task, not only to identify specific interactions between the protein target and the fragment, but also to verify whether the binding mode is conserved or differs according to the fragment modification. While X-ray crystallography is the technique of choice, NMR methods are helpful when this fails. We show here how the ligand-observed saturation transfer difference (STD) experiment and the protein-observed 15N-HSQC experiment, two popular NMR screening experiments, can be used to compare the binding modes of analogous fragments. We discuss the application and limitations of these approaches based on STD-epitope mapping, chemical shift perturbation (CSP) calculation and comparative CSP sign analysis, using the human peroxiredoxin 5 as a protein model.

Published
2014
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9. BcL-xL conformational changes upon fragment binding revealed by NMR.

Author

Clémentine Aguirre, Tim Ten Brink, Olivier Walker, Florence Guillière, Dany Davesne, and Isabelle Krimm

Subjects
Medicine, Science
Abstract

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4'-fluoro-[1,1'-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices [Formula: see text]2, [Formula: see text]3 and the very beginning of [Formula: see text]5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.

Published
2013
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10. Discovery of fragment molecules that bind the human peroxiredoxin 5 active site.

Author

Sarah Barelier, Dominique Linard, Julien Pons, André Clippe, Bernard Knoops, Jean-Marc Lancelin, and Isabelle Krimm

Subjects
Medicine, Science
Abstract

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.

Published
2010
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11. Biophysical Dissection of Isolated GPCRs: The Adenosine A2A Receptor under the Bistouries

Author

Jean-Louis Banères, Thomas Botzanowski, Jean A. Boutin, Barbara Calamini, Jérôme Castel, Laurent J. Catoire, Sarah Cianférani, Claire Demesmay, Gavin Ferguson, Gilles Ferry, Julie Kniazeff, Isabelle Krimm, Thierry Langer, Guillaume Lebon, Marie Ley, Miklos Nyerges, Magali Schwob, Catherine Venien-Bryan, Renaud Wagner, Gabrielle Zeder-Lutz, Claudia Zilian-Stohrer, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Pharmacochimie et Biologie pour le Développement (PHARMA-DEV), Institut de Recherche pour le Développement (IRD)-Institut de Chimie de Toulouse (ICT), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT), Sanofi-Aventis R&D, SANOFI Recherche, Food and Agriculture Organization, Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Infrastructure Nationale de Protéomique, FR2048 ProFI, Separative Methods - Techniques séparatives (TechSep), Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherches SERVIER (IRS), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Vienna [Vienna], Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), Biotechnologie et signalisation cellulaire (BSC), and Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
purification, ligands, adenosine receptor A 2A, [SDV]Life Sciences [q-bio], biophysical approach, expression, adenosine receptor A 2A biophysical approach NMR mass spectrometry molecular pharmacology expression purification reconstitution ligands techniques review, reconstitution, review, molecular pharmacology, techniques, NMR, mass spectrometry
Abstract

International audience; In an effort to provide an overview of the biophysical approaches used to study G-protein-coupled receptors, we chose to consider the adenosine A2A receptor as a model, as it is widely reported in the literature to explore the way GPCRs are studied nowadays. After a brief introduction of the receptor, we gathered descriptions of the various tools used to investigate the pharmacology and structure of the A2A receptor. We began by describing the key developments which have led to successful studies of GPCRs including the cloning, expression and purification of A2A, and the subsequent characterizations including quality control, binding and functional studies that have been necessary for the further understanding of the receptor. Then, we reviewed the reconstitution of A2A into nanodiscs as well as the use of this biological material in structural mass spectrometry, NMR, calorimetry and various other approaches to gain not only information about the structure and function of A2A, but also the dynamics of the receptor and the tools necessary to pursue such investigations. The body of techniques presented herein are applicable to all GPCRs amenable to purification.

Published
2023
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13. AB668, a novel highly selective protein kinase CK2 inhibitor with a distinct anti-tumor mechanism as compared to CX-4945 and SGC-CK2-1

Author

Alexandre Bancet, Rita Frem, Florian Jeanneret, Angélique Mularoni, Pauline Bazelle, Caroline Roelants, Jean-Guy Delcros, Jean-François Guichou, Catherine Pillet, Isabelle Coste, Toufic Renno, Christophe Battail, Claude Cochet, Thierry Lomberget, Odile Filhol, and Isabelle Krimm

Abstract

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further explore its therapeutic potential. Here we have discovered AB668, a new bivalent inhibitor that binds both at the ATP site and an allosteric αD pocket unique to CK2. The molecule inhibits CK2 activity with an outstanding selectivity over other kinases. Using caspase activation assay, live-cell imaging and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to the non-selective ATP-competitive inhibitor CX-4945 that reached clinic and to the selective ATP-competitive SGC-CK2-1 molecule. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668 has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death and stimulating distinct biological pathways in several cancer cell lines while sparing healthy cells. Our data suggest that targeting a cryptic CK2 αD pocket validates an allosteric approach to targeting CK2 and provides a starting point for creating drug-like CK2 inhibitors for aggressive cancers.

Published
2022
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14. A fragment-based drug discovery strategy applied to the identification of NDM-1 β-lactamase inhibitors

Author

Jérémy Caburet, Benjamin Boucherle, Sofiane Bourdillon, Giorgia Simoncelli, Federica Verdirosa, Jean-Denis Docquier, Yohann Moreau, Isabelle Krimm, Serge Crouzy, Marine Peuchmaur, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département de pharmacochimie moléculaire (DPM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Università degli Studi di Siena = University of Siena (UNISI), Université de Liège, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Krimm, Isabelle, and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
Pharmacology, [CHIM.ANAL] Chemical Sciences/Analytical chemistry, High throughput virtual screening, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], metallo-β-lactamase, [CHIM.ORGA]Chemical Sciences/Organic chemistry, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Organic Chemistry, General Medicine, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, NDM-1, [CHIM.ORGA] Chemical Sciences/Organic chemistry, beta-Lactams, beta-Lactamases, Anti-Bacterial Agents, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [CHIM] Chemical Sciences, Drug Discovery, [CHIM]Chemical Sciences, beta-Lactamase Inhibitors, STD NMR screening, Fragment-Based Drug Discovery
Abstract

International audience; Hydrolysis of β-lactam drugs, a major class of antibiotics, by serine or metallo-β-lactamases (SBL or MBL) is one of the main mechanisms for antibiotic resistance. New Delhi Metallo-β-lactamase-1 (NDM-1), an acquired metallo-carbapenemase first reported in 2009, is currently considered one of the most clinically relevant targets for the development of β-lactam-β-lactamase inhibitor combinations active on NDM-producing clinical isolates. Identification of scaffolds that could be further rationally pharmacomodulated to design new and efficient NDM-1 inhibitors is thus urgently needed. Fragment-based drug discovery (FBDD) has become of great interest for the development of new drugs for the past few years and combination of several FBDD strategies, such as virtual and NMR screening, can reduce the drawbacks of each of them independently. Our methodology starting from a high throughput virtual screening on NDM-1 of a large library (more than 700,000 compounds) allowed, after slicing the hit molecules into fragments, to build a targeted library. These hit fragments were included in an in-house untargeted library fragments that was screened by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR). 37 fragments were finally identified and used to establish a pharmacophore. 10 molecules based on these hit fragments were synthesized to validate our strategy. Indenone 89 that combined two identified fragments shows an inhibitory activity on NDM-1 with a K i value of 4 µM.

Published
2022
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16. A Deep Dive into VDAC1 Conformational Diversity Using All-Atom Simulations Provides New Insights into the Structural Origin of the Closed States

Author

Jordane Preto, Hubert Gorny, Isabelle Krimm, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Krimm, Isabelle

Subjects
Models, Molecular, Protein Conformation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], QH301-705.5, beta-barrel transporter, voltage-dependent anion channel, [SDV.CAN]Life Sciences [q-bio]/Cancer, Molecular Dynamics Simulation, Catalysis, Inorganic Chemistry, Mice, [SDV.CAN] Life Sciences [q-bio]/Cancer, Animals, Physical and Theoretical Chemistry, Biology (General), Molecular Biology, QD1-999, Spectroscopy, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Voltage-Dependent Anion Channel 1, Organic Chemistry, General Medicine, molecular dynamics, Computer Science Applications, Chemistry, Ion Channel Gating
Abstract

The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter which controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high conducting “open” state and low conducting “closed” states emerging at high transmembrane potential. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltage known to promote closure. Conformers exhibiting stable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our low-conducting models suggest that closed states predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed in experiments and corroborates a recent study describing entropy as a key factor for VDAC gating.

Published
2022
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17. NMR investigation of protein–ligand interactions for G-protein coupled receptors

Author

Claire Raingeval, Isabelle Krimm, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, and Centre National de la Recherche Scientifique (CNRS)

Subjects
Magnetic Resonance Spectroscopy, [SDV]Life Sciences [q-bio], Allosteric regulation, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Computational biology, Fluorine-19 NMR, Ligands, 01 natural sciences, Receptors, G-Protein-Coupled, 03 medical and health sciences, Drug Discovery, [CHIM]Chemical Sciences, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Receptor, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, G protein-coupled receptor, Pharmacology, 0303 health sciences, 010405 organic chemistry, Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 3. Good health, 0104 chemical sciences, Molecular Medicine, Signal transduction, Two-dimensional nuclear magnetic resonance spectroscopy, Function (biology), Protein ligand
Abstract

In this review, we report NMR studies of ligand–GPCR interactions, including both ligand-observed and protein-observed NMR experiments. Published studies exemplify how NMR can be used as a powerful tool to design novel GPCR ligands and investigate the ligand-induced conformational changes of GPCRs. The strength of NMR also lies in its capability to explore the diverse signaling pathways and probe the allosteric modulation of these highly dynamic receptors. By offering unique opportunities for the identification, structural and functional characterization of GPCR ligands, NMR will likely play a major role for the generation of novel molecules both as new tools for the understanding of the GPCR function and as therapeutic compounds for a large diversity of pathologies.

Published
2019
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18. 1D NMR WaterLOGSY as an efficient method for fragment-based lead discovery

Author

Béatrice Brion, Marc Le Borgne, Roderick E. Hubbard, Claire Raingeval, Isabelle Krimm, Olivier Cala, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre National de la Recherche Scientifique (CNRS), Molécules bioactives et chimie médicinale (B2MC), and Université de Lyon-Université de Lyon

Subjects
solvent-exposed, Magnetic Resonance Spectroscopy, Stereochemistry, Fragment-based lead discovery, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, binding mode, 01 natural sciences, Glycogen phosphorylase, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, saturation transfer difference, Drug Discovery, medicine, Pharmacology, Binding Sites, 010405 organic chemistry, Chemistry, Ligand, WaterLOGSY, lcsh:RM1-950, A protein, fragment-based lead discovery, Proteins, General Medicine, Human serum albumin, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Epitope mapping, lcsh:Therapeutics. Pharmacology, Saturation transfer, medicine.drug, Research Paper
Abstract

International audience; WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-x L , Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein. ARTICLE HISTORY

Published
2019
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19. Modification and Biological Evaluation of a Series of 1,5-Diaryl-1,2,4-triazole Compounds as Novel Agents against Pancreatic Cancer Metastasis through Targeting Myoferlin

Author

Dazhao Mi, Peili Wang, Shao Ting, Mingyao Liu, Zhengfang Yi, Yuanjin Zhang, Xin Wang, Haixiang Pei, Weikai Guo, Yihua Chen, Yunqi Li, Yuan He, Isabelle Krimm, Technicolor R & I [Cesson Sévigné], Technicolor, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre National de la Recherche Scientifique (CNRS), Shihezi University, and Iowa State University (ISU)

Subjects
Epithelial-Mesenchymal Transition, Mice, Nude, Muscle Proteins, Antineoplastic Agents, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Matrix metalloproteinase, 01 natural sciences, Receptor tyrosine kinase, Metastasis, Structure-Activity Relationship, 03 medical and health sciences, Cell Movement, In vivo, Cell Line, Tumor, Pancreatic cancer, Drug Discovery, medicine, [CHIM]Chemical Sciences, Animals, Humans, Neoplasm Invasiveness, Molecular Targeted Therapy, Epithelial–mesenchymal transition, Neoplasm Metastasis, Survival rate, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Mice, Inbred BALB C, 0303 health sciences, biology, Chemistry, Calcium-Binding Proteins, Membrane Proteins, Triazoles, medicine.disease, Xenograft Model Antitumor Assays, In vitro, 3. Good health, 0104 chemical sciences, Pancreatic Neoplasms, 010404 medicinal & biomolecular chemistry, Drug Design, Cancer research, biology.protein, Molecular Medicine
Abstract

Pancreatic cancer is one of the most common cancers with an extremely low survival rate. Metastasis, as one of the key reasons of cancer-related death, is found in more than 50% pancreatic cancer patients at diagnosis. Novel therapeutic targets and drugs blocking cancer metastasis are urgently needed. Herein, we report a series of 1,5-diaryl-1,2,4-triazole derivatives as potent antimetastatic agents. Lead compound 6y displayed effective antimetastatic activities in pancreatic cancer in vitro and in vivo. Concomitant studies indicated that 6y probably binds with myoferlin (MYOF), a novel potential antitumor metastasis target, which regulates vesicle trafficking and metastasis-related proteins. Subsequent biophysical and biochemical methods verified that 6y bound to MYOF. Mechanism studies revealed that 6y inhibited pancreatic cancer metastasis through reversing the epithelial mesenchymal transition, inhibiting the secretions of matrix metalloproteinase and blocking the receptor tyrosine kinases. Our findings suggest that targeting MYOF with 6y may be a promising therapeutic strategy to prevent pancreatic cancer metastasis.

Published
2019
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20. Fragment Linking Strategies for Structure-Based Drug Design

Author

Alexandre Bancet, Claire Raingeval, Thierry Lomberget, Jean-François Guichou, Isabelle Krimm, Marc Le Borgne, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Molécules bioactives et chimie médicinale (B2MC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Université de Lyon, Biomolécules Cancer et Chimiorésistances (B2C), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Le Borgne, Marc, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Superadditivity, [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Computational biology, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Ligands, 01 natural sciences, Small Molecule Libraries, Structure-Activity Relationship, 03 medical and health sciences, chemistry.chemical_compound, Fragment (logic), [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Drug Discovery, Drug approval, [CHIM]Chemical Sciences, Drug Approval, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Clinical Trials as Topic, 0303 health sciences, Binding Sites, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Drug discovery, Inhibitors, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Proteins, X ray crystallography, Screening assays, 0104 chemical sciences, 3. Good health, Pharmaceutical Preparations, chemistry, Drug Design, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Molecular Medicine, Structure based, Protein identification, Linkers, Linker, Lead compound, Protein Binding
Abstract

International audience; Fragment-based drug discovery is a strategy widely used in both academia and pharmaceutical companies to generate small-molecule protein inhibitors and drug candidates. Among the approaches reported in the literature (growing, linking, and merging), the linking approach theoretically offers the opportunity to rapidly gain in binding energy. Nevertheless, this approach is poorly represented when considering the compounds currently in clinical trials. Here, we report an exhaustive view of the cases published so far in the literature, together with the methods used to identify the two initial fragments either simultaneously or successively. We review the different types of linkers published and discuss how these linkers are designed to obtain the lead compound. Mixing merging and linking methods, where the linker is duplicated from a known inhibitor, appears as an interesting strategy. To reach superadditivity, we propose to grow one of the fragments in order to minimize the distance between the two binders and then link the resulting compounds using flexible alkyl-derived linkers.

Published
2020
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21. The intrinsically disordered N-terminus of the voltage-dependent anion channel

Author

Jordane Preto, Isabelle Krimm, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Krimm, Isabelle

Subjects
0301 basic medicine, Models, Molecular, Cytoplasm, Protein Denaturation, Physiology, Protein Conformation, Lipid Bilayers, Apoptosis, Protein Structure Prediction, Gating, Mitochondrion, Crystallography, X-Ray, Biochemistry, Physical Chemistry, 01 natural sciences, Molecular dynamics, Mice, Neoplasms, Biochemical Simulations, Macromolecular Structure Analysis, Biology (General), Membrane potential, 0303 health sciences, Crystallography, Ecology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010304 chemical physics, biology, Chemistry, Physics, Condensed Matter Physics, Mitochondria, Electrophysiology, Computational Theory and Mathematics, Modeling and Simulation, Physical Sciences, Mitochondrial Membranes, Crystal Structure, Cellular Structures and Organelles, Bacterial outer membrane, Algorithms, Research Article, Anions, Steric effects, Protein Structure, Voltage-dependent anion channel, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], QH301-705.5, Molecular Dynamics Simulation, Membrane Potential, Cellular and Molecular Neuroscience, 03 medical and health sciences, Protein Domains, Cations, 0103 physical sciences, Genetics, Solid State Physics, Animals, Computer Simulation, Cysteine, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Ions, Force field (physics), Voltage-Dependent Anion Channel 1, Biology and Life Sciences, Computational Biology, Proteins, Cell Biology, Intrinsically Disordered Proteins, 030104 developmental biology, Membrane protein, Mutation, biology.protein, Biophysics, Peptides, Software
Abstract

The voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC., Author summary The voltage-dependent anion channel (VDAC) is a membrane protein playing a pivotal role in the transport of ions or ATP across the mitochondrial outer membrane as well as in the induction of apoptosis. At high enough membrane potential, VDAC is known to transition from an open state to multiple closed states, reducing the flow of ions through the channel and blocking the passage of large metabolites. While the structure of the open state was resolved more than a decade ago, a molecular description of the gating mechanism of the channel is still missing. Here we show that the N-terminus of VDAC is an intrinsically disordered region and that such a property has a profound impact on its dynamics either as a free peptide or as part of the channel. By taking disordered properties of the N-terminus into account, we managed to generate long-lived closed conformations of the channel at experimental values of the membrane potential. Our results provide new insights into the molecular mechanism driving the gating of VDAC.

Published
2020
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22. Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

Author

Claire Raingeval, Claire Demesmay, Renaud Wagner, Lucie Hartmann, Lydia N. Caro, Vincent Dugas, Sarah Mohamed-Bouteben, Lucile Lecas, Isabelle Krimm, Separative Methods - Techniques séparatives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS), Separative Methods - Techniques séparatives (TechSep), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
Low protein, Receptor, Adenosine A2A, 02 engineering and technology, Protein-ligand interaction, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Ligands, 01 natural sciences, Biochemistry, Proof of Concept Study, Chromatography, Affinity, Analytical Chemistry, Affinity chromatography, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Drug Discovery, Weak affinity chromatography, Environmental Chemistry, Humans, [CHIM]Chemical Sciences, Binding site, Organic Chemicals, Integral membrane protein, Spectroscopy, ComputingMilieux_MISCELLANEOUS, G protein-coupled receptor, Miniaturization, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, 010401 analytical chemistry, Membranes, Artificial, 021001 nanoscience & nanotechnology, Small molecule, 0104 chemical sciences, Immobilized Proteins, Membrane protein, Biophysics, Nanoparticles, Target protein, Nanodisc, 0210 nano-technology, Protein Binding
Abstract

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 μg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

Published
2020
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23. Identifying Protein Allosteric Transitions for Drug Discovery with 1D NMR

Author

Isabelle Krimm

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Allosteric modulator, Stereochemistry, Allosteric regulation, Plasma protein binding, Ligands, Benzoates, Biochemistry, 03 medical and health sciences, Glycogen phosphorylase, 0302 clinical medicine, Allosteric Regulation, Drug Discovery, Humans, General Pharmacology, Toxicology and Pharmaceutics, Pharmacology, biology, Drug discovery, Chemistry, Phenyl Ethers, Protein dynamics, Glycogen Phosphorylase, Organic Chemistry, Nuclear magnetic resonance spectroscopy, Deuterium, 030104 developmental biology, Allosteric enzyme, biology.protein, Molecular Medicine, Allosteric Site, 030217 neurology & neurosurgery
Abstract

Allosteric drugs present many advantages over orthosteric drugs and are therefore an attractive approach in drug discovery, despite being highly challenging. First, the binding of ligands in protein allosteric pockets do not ensure an allosteric effect, and second, allosteric ligands can possess diverse modes of pharmacology even within a compound family. Herein we report a new method to: 1) detect allosteric communication between protein binding sites, and 2) compare the effect of allosteric ligands on the allosteric transitions of the protein target. The method, illustrated with glycogen phosphorylase, consists of comparing 1D saturation transfer difference (STD) NMR spectra of a molecular spy (here fragments) in the absence and presence of allosteric ligands. The modification of the STD NMR spectrum of the fragment indicates whether the protein dynamics/conformations have been changed in the presence of the allosteric modulator, thereby highlighting allosteric coupling between the binding pocket of the reference compound (in this case the fragment) and the allosteric pocket.

Published
2017
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24. Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors

Author

Ruben Abagyan, Renaud Prudent, Thierry Lomberget, Céline F. Sautel, Béatrice Laudet, Matthias Engel, Isabelle Krimm, Odile Filhol, Marc Le Borgne, Irina Kufareva, Mohamed Ettaoussi, Claude Cochet, Paul Brear, Benoit Bestgen, Virginie Moucadel, Skaggs School of Pharmacy and Pharmaceutical Sciences [San Diego], University of California [San Diego] (UC San Diego), University of California-University of California, Molécules bioactives et chimie médicinale (B2MC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, University of Cambridge [UK] (CAM), Invasion mechanisms in angiogenesis and cancer (IMAC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U1172 Inserm - U837 (JPArc), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE)-Université de Lille, Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre de Résonance Magnetique Nucleaire (CRMN), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Saarland University [Saarbrücken], Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), University of California (UC)-University of California (UC), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 (JPArc), Université Lille Nord de France (COMUE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Kufareva, Irina [0000-0001-9083-7039], Filhol, Odile [0000-0003-1964-7958], Borgne, Marc Le [0000-0003-1398-075X], Apollo - University of Cambridge Repository, and Le Borgne, Marc

Subjects
0301 basic medicine, genetic structures, lcsh:Medicine, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Crystallography, X-Ray, 01 natural sciences, Substrate Specificity, Adenosine Triphosphate, Cell Movement, Catalytic Domain, Phosphorylation, lcsh:Science, Casein Kinase II, ComputingMilieux_MISCELLANEOUS, Cancer, Tumor, Crystallography, Multidisciplinary, Molecular medicine, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Small molecule, 3. Good health, Molecular Docking Simulation, 5.1 Pharmaceuticals, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Development of treatments and therapeutic interventions, [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Protein subunit, Kinases, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Article, Target validation, Cell Line, 03 medical and health sciences, Targeted therapies, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Target identification, Cell Line, Tumor, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Protein kinase A, Protein Kinase Inhibitors, Cell Proliferation, Binding Sites, 010405 organic chemistry, Cell growth, lcsh:R, Surface Plasmon Resonance, In vitro, 0104 chemical sciences, Protein Subunits, Kinetics, 030104 developmental biology, Cell culture, X-Ray, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Biophysics, lcsh:Q, Holoenzymes, Peptides
Abstract

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

Published
2019
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26. 2-Aminothiazole derivatives as selective allosteric modulators of the protein kinase CK2. Part 1: Identification of an allosteric binding site

Author

Isabelle Krimm, Claude Cochet, Irina Kufareva, Ahmed A. M. Kamal, Thierry Lomberget, Benoit Bestgen, Rolf W. Hartmann, Ruben Abagyan, Matthias Engel, Wei-Guang Seetoh, Chris Abell, Marc Le Borgne, Molécules bioactives et chimie médicinale (B2MC), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Skaggs School of Pharmacy and Pharmaceutical Sciences [San Diego], University of California [San Diego] (UC San Diego), University of California-University of California, Univ Cambridge, Dept Chem, Cambridge CB2 1EW, England, affiliation inconnue, Saarland University [Saarbrücken], Transduction du signal : signalisation calcium, phosphorylation et inflammation, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and University of California (UC)-University of California (UC)

Subjects
Circular dichroism, Stereochemistry, Allosteric regulation, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, 01 natural sciences, Molecular Docking Simulation, Article, 03 medical and health sciences, Structure-Activity Relationship, Allosteric Regulation, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Drug Discovery, Structure–activity relationship, Humans, Enzyme kinetics, Binding site, Naphthyridines, Protein kinase A, Casein Kinase II, Protein Kinase Inhibitors, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Molecular Structure, Chemistry, Protein Stability, Temperature, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Kinetics, Thiazoles, Mutation, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Molecular Medicine, Phenazines, Signal transduction, Allosteric Site, [CHIM.CHEM]Chemical Sciences/Cheminformatics, Protein Binding
Abstract

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD-NMR, circular dichroism spectroscopy and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC(50) of 3.4 μM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anti-cancer drugs.

Published
2019
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27. Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Author

Anass Jawhari, Maciej Baranowski, Olivier Cala, Javier Pérez, Isabelle Krimm, Erika Cecon, Ralf Jockers, Maja Pučić-Baković, Claire Raingeval, Gordan Lauc, Sébastien Igonet, CALIXAR, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), GENOS, Synchrotron SOLEIL, M.B. was supported by the Horizon 2020 Program of the European Union (iNEXT grant, project 653706)., European Project: 653706,H2020,H2020-INFRAIA-2014-2015,iNEXT(2015), Méthodes et criblage RMN pour les molécules bioactives, Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École normale supérieure - Lyon (ENS Lyon), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, 0301 basic medicine, Magnetic Resonance Spectroscopy, Receptor, Adenosine A2A, Protein Conformation, Drug Evaluation, Preclinical, lcsh:Medicine, Adenosine A2A receptor, Ligands, Article, 03 medical and health sciences, Protein structure, Cyclic AMP, medicine, Humans, [CHIM]Chemical Sciences, lcsh:Science, Integral membrane protein, G protein-coupled receptor, Multidisciplinary, Protein Stability, Chemistry, lcsh:R, Protein engineering, Adenosine receptor, Adenosine, 030104 developmental biology, Solubility, Structural biology, Biophysics, lcsh:Q, medicine.drug
Abstract

We thank Emmanuel DEJEAN for continuous support, Kelly GARNIER for help with thermal-shift assay and the CALIXAR team for helpful discussions. We would like to thank Patrick SCHULTZ for initial electron microscopy work and discussions and Alice ROTHNIE for critical reading of the manuscript. We thank Dr. Francisco CIRUELA ALFEREZ (University of Barcelona) for kindly providing HEK-293 cells stably expressing the A2AR-Nanoluc fusion protein and Ivan GUDELJ for help with mass spectrometry measurements.; International audience; Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A(2A) receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A(2A)R that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A(2A) receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A(2A)R in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Published
2018
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28. Ligand-Orientation Based Fragment Selection in STD NMR Screening

Author

Olivier Cala, Isabelle Krimm, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Binding Sites, Orientation (computer vision), Chemistry, Ligand, Proteins, Small Molecule Libraries, Computational biology, Plasma protein binding, Ligands, Combinatorial chemistry, Fragment (logic), [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Saturation transfer, Drug Design, Drug Discovery, Animals, Humans, Molecular Medicine, Binding site, Nuclear Magnetic Resonance, Biomolecular, Selection (genetic algorithm), Protein Binding
Abstract

International audience; While saturation transfer difference (STD) is a widely used NMR method for ligand screening, the selection of specific binders requires the validation of the hits through competition experiments or orthogonal biophysical techniques. We show here that the quantitative STD analysis is a reliable and robust approach to discriminate between specific and nonspecific ligands, allowing selection of fragments that bind proteins with a privileged binding mode, in the absence of any structural data for the protein.

Published
2015
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29. Le criblage de fragments

Author

Isabelle Krimm

Subjects
Drug, Fragment (logic), business.industry, media_common.quotation_subject, Therapeutic protein, Medicine, General Medicine, Computational biology, business, General Biochemistry, Genetics and Molecular Biology, 3. Good health, media_common
Abstract

Fragment-based screening is now recognised as a powerful method for the design of novel potent molecules against therapeutic protein targets including challenging targets. Here, the main concepts used in the fragment-based drug design approach are reviewed, together with the reasons for its success. Methods and strategies used to identify, validate and select fragments are discussed. Future challenges and developments that are expected for the next decade are presented.

Published
2015
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31. The Dynamics of Copper Uptake in Human Cu,Zn-Superoxide Dismutase: a Solid-State NMR Study

Author

Claire Raingeval, Olivier Cala, Béatrice Brion, Isabelle Krimm, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Groupement d'études de résonance magnétique, and Bussy, Agnès

Subjects
[CHIM] Chemical Sciences, [CHIM]Chemical Sciences
Abstract

http://thcgerm.free.fr/spip.php?article123; National audience; Les expériences de saturation transfer difference (STD) et de Water–ligand observed via gradient spectroscopy (WaterLOGSY) sont des techniques simples et rapides de criblage, largement utilisées en RMN (interactions protéine-ligand, criblage de fragments). Adaptées à la détection de ligands faible affnité (Kd ~ mM-uM), ces méthodes présentent l’avantage d’être exclusivement centrées sur l’analyse des signaux 1D 1H des ligands et ne nécessitent pas de posséder des informations sur la cible protéique elle-même. Nous montrons ici l’intérêt particulier de ces deux expériences pour étudier l’orientation du ligand, renseignement crucial pour la validation et la sélection de ligands de protéine, notamment dans le cadre du criblage de fragments.

Published
2017

32. Découverte d’une nouvelle famille de petites molécules non-peptidiques inhibitrices des Cyclophilines ayant une probable activité antivirale

Author

Olivier Cala, Abdelhakim Ahmed-Belkacem, Lionel Colliandre, Nazim Ahnou, Quentin Nevers, Muriel Gelin, Yannick Bessin, Rozenn Brillet, Dominique Douguet, William Bourguet, Isabelle Krimm, Jean -Michel Pawlotsky, Jean-François Guichou, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Groupement d'études de résonance magnétique, Bussy, Agnès, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[CHIM] Chemical Sciences, [CHIM]Chemical Sciences
Abstract

http://thcgerm.free.fr/spip.php?article123; National audience

Published
2017

33. Protein–ligand structure guided by backbone and side-chain proton chemical shift perturbations

Author

Clémentine Aguirre, Isabelle Krimm, Olivier Cala, Tim ten Brink, Jean-François Guichou, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Proton, Stereochemistry, Ligands, 010402 general chemistry, 01 natural sciences, Biochemistry, chemistry.chemical_compound, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Amide, Side chain, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Nuclear Magnetic Resonance, Biomolecular, Spectroscopy, Carbon Isotopes, 010405 organic chemistry, Ligand, Peroxiredoxins, 0104 chemical sciences, Molecular Docking Simulation, NMR spectra database, Crystallography, chemistry, Docking (molecular), Protons, Protein target, Protein ligand
Abstract

The fragment-based drug design approach consists of screening libraries of fragment-like ligands, to identify hits that typically bind the protein target with weak affinity ( $$100\,\upmu \hbox {M}$$ –5 mM). The determination of the protein–fragment complex 3D structure constitutes a crucial step for uncovering the key interactions responsible for the protein–ligand recognition, and for growing the initial fragment into potent active compounds. The vast majority of fragments are aromatic compounds that induce chemical shift perturbations (CSP) on protein NMR spectra. These experimental CSPs can be quantitatively used to guide the ligand docking, through the comparison between experimental CSPs and CSP back-calculation based on the ring current effect. Here we implemented the CSP back-calculation into the scoring function of the program PLANTS. We compare the results obtained with CSPs measured either on amide or aliphatic protons of the human peroxiredoxin 5. We show that the different kinds of protons lead to different results for resolving the 3D structures of protein–fragment complexes, with the best results obtained with the $$\hbox {H}_{\alpha }$$ protons.

Published
2014
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34. NMR for Chemical Biology and Drug Discovery

Author

Isabelle Krimm, Bussy, Agnès, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
TheoryofComputation_MISCELLANEOUS, [CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, GeneralLiterature_INTRODUCTORYANDSURVEY, ComputerApplications_MISCELLANEOUS, ComputingMilieux_MISCELLANEOUS, GeneralLiterature_MISCELLANEOUS
Abstract

Invitation Keynote Conférence internationale; International audience

Published
2016

35. Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach

Author

Rahila Rahimova, Isabelle Krimm, Suzanne Peyrottes, Preeti, Rémi Guillon, Corinne Lionne, Christian Périgaud, David Egron, Nushin Aghajari, Laurent Chaloin, Zsuzsanna Marton, Charles Dumontet, Lars Petter Jordheim, Institut de Recherche en Infectiologie de Montpellier ( IRIM ), Centre National de la Recherche Scientifique ( CNRS ) -Université de Montpellier ( UM ), Archéologie, Terre, Histoire, Sociétés [Dijon] ( ARTeHiS ), Ministère de la Culture et de la Communication ( MCC ) -Université de Bourgogne ( UB ) -Centre National de la Recherche Scientifique ( CNRS ), Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques ( ISA ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ) -École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] ( IBMM ), Ecole Nationale Supérieure de Chimie de Montpellier ( ENSCM ) -Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre de Recherche en Cancérologie de Lyon ( CRCL ), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Bases moléculaires et structurales des systèmes infectieux ( BMSSI ), Université Claude Bernard Lyon 1 ( UCBL ), Institut de Recherche en Infectiologie de Montpellier (IRIM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Archéologie, Terre, Histoire, Sociétés [Dijon] (ARTeHiS), Ministère de la Culture et de la Communication (MCC)-Université de Bourgogne (UB)-Centre National de la Recherche Scientifique (CNRS), ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Bases moléculaires et structurales des systèmes infectieux (BMSSI), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Nucleotidase activity, In silico, Antineoplastic Agents, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Naphthalenes, 01 natural sciences, Molecular Docking Simulation, Benzoates, 03 medical and health sciences, Structure-Activity Relationship, Non-competitive inhibition, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Nucleotidase, Cell Line, Tumor, Drug Discovery, Structure–activity relationship, Humans, Computer Simulation, Pyrroles, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, 5'-Nucleotidase, 030304 developmental biology, 0303 health sciences, Binding Sites, 010405 organic chemistry, Chemistry, Imidazoles, [ CHIM.THER ] Chemical Sciences/Medicinal Chemistry, Drug Synergism, In vitro, 0104 chemical sciences, 3. Good health, Biochemistry, Purines, Benzamides, Molecular Medicine, [ CHIM.ANAL ] Chemical Sciences/Analytical chemistry, Drug Screening Assays, Antitumor, Databases, Chemical
Abstract

International audience; We used a combined approach based on fragment-based drug design (FBDD) and in silico methods to design potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), which has been recognized as an important therapeutic target in hematological cancers. Two subgroups of small compounds (including adenine and biaryl moieties) were identified as cN-II binders and a fragment growing strategy guided by molecular docking was considered. Five compounds induced a strong inhibition of the 5'-nucleotidase activity in vitro, and the most potent ones were characterized as noncompetitive inhibitors. Biological evaluation in cancer cell lines showed synergic effect with selected anticancer drugs. Structural studies using X-ray crystallography lead to the identification of new binding sites for two derivatives and of a new crystal form showing important domain swapping. Altogether, the strategy developed herein allowed identifying new original noncompetitive inhibitors against cN-II that act in a synergistic manner with well-known antitumoral agents.

Published
2015
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36. Overview of liquid-NMR approaches for fragment-based lead discovery

Author

Isabelle Krimm, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Marina Veronesi, Stefania Girotto and Luca Mollica - Istituto Italiano di Tecnologia, and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2015

37. Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

Author

Lionel Colliandre, Abdelhakim Ahmed-Belkacem, Dominique Douguet, Olivier Cala, William Bourguet, Quentin Nevers, Muriel Gelin, Isabelle Krimm, Rozenn Brillet, Yannick Bessin, Nazim Ahnou, Jean-Michel Pawlotsky, Jean-François Guichou, Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de Référence Virus des hépatites B, C et Delta, Institut National de la Transfusion Sanguine [Paris] (INTS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01, the Fonds Europeen de Developpement Economique et Regional (FEDER) Number 48748, the Agence Nationale de Recherche sur le SIDA et les Hepatites Virales (ANRS) and the Fondation pour la Recherche Medicale (FRM)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Bussy, Agnès, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [CHIM.ANAL] Chemical Sciences/Analytical chemistry, CYCLOSPORINE-A, Hepatitis C virus, Science, General Physics and Astronomy, Isomerase, Biology, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Article, MECHANISMS, T-CELL-ACTIVATION, 03 medical and health sciences, DESIGN, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, medicine, polycyclic compounds, Peptide bond, Cyclophilin, Alisporivir, Multidisciplinary, Drug discovery, General Chemistry, ALISPORIVIR, Antivirals, Small molecule, Virology, 3. Good health, APOPTOSIS, GENOTYPE, DRUG DISCOVERY, MITOCHONDRIAL PERMEABILITY TRANSITION, 030104 developmental biology, Biochemistry, REPLICATION, Nucleic acid, Structure-based drug design, Pathogens
Abstract

Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections., Cyclophilins play a key role in the life cycle of many viruses and represent important drug targets for broad-spectrum antiviral therapies. Here, the authors use fragment-based drug discovery to develop non-peptidic inhibitors of human cyclophilins with high activity against replication of a number of viral families.

Published
2015
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38. NMR for fragment-based drug design

Author

Isabelle Krimm, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2015

39. Overview of Probing Protein‐Ligand Interactions Using NMR

Author

Isabelle Krimm, Clémentine Aguirre, and Olivier Cala

Subjects
Chemistry, Proteins, Nuclear magnetic resonance spectroscopy of nucleic acids, General Medicine, Fluorine-19 NMR, Nuclear Overhauser effect, Nuclear magnetic resonance spectroscopy, Nuclear magnetic resonance crystallography, Ligands, Biochemistry, Structural Biology, Computational chemistry, Triple-resonance nuclear magnetic resonance spectroscopy, Transverse relaxation-optimized spectroscopy, Nuclear Magnetic Resonance, Biomolecular, Two-dimensional nuclear magnetic resonance spectroscopy
Abstract

Nuclear magnetic resonance (NMR) is a powerful technique for the study and characterization of protein-ligand interactions. In this unit we review both experiments where the NMR spectrum of the protein is observed (protein-observed NMR experiments) and those where the NMR spectra of the ligand is observed (ligand-observed NMR experiments) for the identification of binding partners, the measurement of protein-ligand affinity, the design of molecules that are active against biological targets such as proteins, and the assessment of the binding modes of the ligands. Ligand-observed methods discussed in this unit are Nuclear Overhauser Effect (NOE)-based approaches, with well-known experiments such as the Saturation Transfer Difference, Water-Ligand Observed via Gradient Spectroscopy (WaterLOGSY), and transferred-Nuclear Overhauser Effect Spectroscopy (tr-NOESY) experiments, and also the INPHARMA experiment. Regarding protein-observed experiments, this unit focuses on the use of chemical shift perturbations observed in protein-NMR spectra upon ligand binding. Also discussed is how these chemical shift perturbations can be used for the analysis of protein-ligand complexes, including fast structure determination when combined with docking.

Published
2015
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40. Applications of NMR in fragment-based drug design

Author

Isabelle Krimm, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Howard S. and Abell C.

Subjects
Drug, 3D structures, Chemistry, Drug discovery, media_common.quotation_subject, screening, Combinatorial chemistry, fragment-based drug design, NMR, 3. Good health, Fragment (logic), [CHIM.ANAL]Chemical Sciences/Analytical chemistry, False positive paradox, protein–fragment complexes, media_common
Abstract

International audience; The role of NMR in drug discovery has been significantly reinforced over recent years, particularly in the field of fragment-based drug design. NMR is one of the most robust methods for screening fragment libraries against therapeutic targets, generating few false positives. The application of ligand-observed and protein-observed experiments (STD, WaterLOGSY, transferred-NOESY and 2D-HSQC) is reviewed in this chapter, showing that NMR is also a powerful method for analysing the 3D structures of protein–fragment complexes.

Published
2015
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41. A Combination of Spin Diffusion Methods for the Determination of Protein-Ligand Complex Structural Ensembles

Author

Bettina Elshorst, Gerhard Hessler, Alexander Schiffer, Isabelle Krimm, Thomas Langer, Donghan Lee, Stefan Bartoschek, Adam Mazur, Peter Monecke, Christian Griesinger, Jens Pilger, Herman Schreuder, K-Ulrich Wendt, Stefan Becker, Melanie Wegstroth, Department of NMR-based Structural Biology [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Sanofi-Aventis Deutschland GmbH, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and This project was performed under the auspices of the BMBF project number 1615870A: 'Neue Verfahren der Bio-NMR zur Optimierung und Beschleunigung strukturbasierter Wirkstoff-entwicklung'.

Subjects
Epoxide hydrolase 2, Magnetic Resonance Spectroscopy, Stereochemistry, drug design, 010402 general chemistry, Crystallography, X-Ray, Ligands, 01 natural sciences, Catalysis, Diffusion, Glycogen phosphorylase, NMR spectroscopy, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Hydrolase, PLANTS, DOCKING, Binding site, Epoxide Hydrolases, Binding Sites, RECEPTOR, Ligand, Chemistry, 010405 organic chemistry, Proteins, General Chemistry, Nuclear magnetic resonance spectroscopy, General Medicine, Small molecule, Cyclic AMP-Dependent Protein Kinases, SOLUBLE EPOXIDE HYDROLASE, NMR, 0104 chemical sciences, Molecular Docking Simulation, kinases, ANT COLONY OPTIMIZATION, Docking (molecular), glycogen phosphorylase, Protein Binding
Abstract

We thank Sandra Schimanski-Breves and Alexander Liesum for crystallization, Dirk Bossemeyer and Michael Gassel for protein production of PKA.; International audience; Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinaseA and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.

Published
2015
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42. Protein–protein interactions within peroxiredoxin systems

Author

Isabelle Krimm, Olivier Walker, Valérie Noguera-Mazon, and Jean-Marc Lancelin

Subjects
Models, Molecular, Cell signaling, Protein Conformation, Peroxiredoxins, Cell Biology, Plant Science, General Medicine, Plasma protein binding, respiratory system, Biology, Biochemistry, Protein–protein interaction, Protein structure, Peroxidases, Glutaredoxin, Thioredoxin, Peroxiredoxin, Function (biology), Plant Proteins, Protein Binding
Abstract

Peroxiredoxin systems in plants were demonstrated involved in crucial roles related to reactive oxygenated species (ROS) metabolism and the linked cell signalling to ROS. Peroxiredoxins function as peroxidasic systems that combine at least a reactivating reductant agent like thioredoxins, and sometimes glutaredoxins and glutathion. In the past three years a number of peroxiredoxin structures were solved by crystallography in different experimental crystallisation conditions. The structures have revealed a significant propensity of peroxiredoxins for oligomerism that was confirmed by biophysical studies in solution using NMR and other methods as analytical ultra-centrifugation. These studies showed that quaternary structures of peroxiredoxins involve specific protein-protein interaction interfaces that rely upon the peroxiredoxin types and/or their redox conditions. The protein-protein interactions with the reactivating redoxins essentially lead to transient unstable complexes. We review herein the different protein-protein interactions characterized or deduced from those reports.

Published
2006
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43. F-ara-AMP is a substrate of cytoplasmic 5′-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation

Author

Charles Dumontet, Lars Petter Jordheim, Marie-Claude Gagnieu, Carlos M. Galmarini, Emeline Cros, Jean-Marc Lancelin, Isabelle Krimm, and Anne-Sophie Bretonnet

Subjects
Cytoplasm, Magnetic Resonance Spectroscopy, Stereochemistry, Biochemistry, 5'-nucleotidase, Dephosphorylation, Genetics, medicine, Humans, 5'-Nucleotidase, Chromatography, High Pressure Liquid, chemistry.chemical_classification, Nucleoside analogue, Chemistry, food and beverages, General Medicine, Kinetics, Enzyme, Molecular Medicine, Phosphorylation, Vidarabine Phosphate, Nucleoside, Vidarabine, Intracellular, medicine.drug
Abstract

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5′-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a K m in the millimolar range and V max = 35 nmol/min/mg, with both methods. Cytoplasmic 5′-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

Published
2006
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44. Performance of Protein–Ligand Docking with Simulated Chemical Shift Perturbations

Author

Clémentine Aguirre, Thomas E. Exner, Isabelle Krimm, Tim ten Brink, ISA - Méthodes et criblage RMN pour les molécules bioactives, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Theoretical Medicinal Chemistry, Eberhard Karls Universität Tübingen, Financial support was provided by the Agence National de la Recherche (Project No. ANR-11-JS07-0008), and ANR-11-JS07-0008,PLUS,Simulation des interactions protéine-ligand dans le contexte du Fragment-Based Drug-Design(2011)

Subjects
Models, Molecular, Conformational change, COSTRUCTURES, Protein Conformation, General Chemical Engineering, Library and Information Sciences, Crystallography, X-Ray, Ligands, chemistry.chemical_compound, Computational chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Amide, BINDING, Computer Simulation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Nuclear Magnetic Resonance, Biomolecular, Binding Sites, fungi, Proteins, General Chemistry, COMPLEXATION-INDUCED CHANGES, Amides, NMR, Computer Science Applications, High-Throughput Screening Assays, Molecular Docking Simulation, Crystallography, Protein–ligand docking, chemistry, Searching the conformational space for docking, Docking (molecular), Protons, Algorithms, Databases, Chemical
Abstract

International audience; Protein chemical shift perturbations (CSPs) that result from the binding of a ligand to the protein contain structural information about the complex. Therefore, the CSP data, typically obtained during library screening from two-dimensional (2D) nuclear magnetic resonance (NMR) spectra, are often available before attempts to solve the experimental structure of the complex are started, and can be used to solve the complex structure with CSP-based docking. Here, we compare the performance of the post-docking filter and the guided-docking approaches using either amide or α-proton CSPs with 10 protein–ligand complexes. We show that the comparison of experimental CSPs with CSPs simulated for virtual ligand positions can be used to evidence protein conformational change upon binding and possibly improve the CSP-based docking

Published
2015
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45. Structural Implications on the Interaction of Scorpion α-like Toxins with the Sodium Channel Receptor Site Inferred from Toxin Iodination and pH-Dependent Binding

Author

Michael Gurevitz, Isabelle Krimm, Francoise Bouet, Oren Froy, Nicolas Gilles, Dalia Gordon, and Jean-Marc Lancelin

Subjects
Models, Molecular, animal structures, Leiurus, Sodium, Neurotoxins, Scorpion Venoms, chemistry.chemical_element, Grasshoppers, Binding, Competitive, Biochemistry, Sodium Channels, Cellular and Molecular Neuroscience, biology.animal, Animals, Periplaneta, Binding site, Receptor, Cockroach, Scorpion toxin, biology, Sodium channel, Hydrogen-Ion Concentration, biology.organism_classification, Protein Structure, Tertiary, Amino Acid Substitution, chemistry, Mutagenesis, Site-Directed, Tyrosine, Locust, Iodine, Synaptosomes
Abstract

The alpha-like toxin from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity to receptor site 3 on insect sodium channels but does not bind to rat brain synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase in the electropositive charge on the toxin surface resulting from protonation of its four histidines. Radioiodination of Tyr(14) of Lqh III abolished its binding to locust but not cockroach sodium channels, whereas the noniodinated toxin bound equally well to both neuronal preparations. Radioiodination of Tyr(10) or Tyr(21) of the structurally similar alpha-toxin from L. quinquestriatus hebraeus (LqhalphaIT), as well as their substitution by phenylalanine, had only minor effects on binding to cockroach neuronal membranes. However, substitution of Tyr(21), but not Tyr(14), by leucine decreased the binding affinity of LqhalphaIT approximately 87-fold. Thus, Tyr(14) is involved in the bioactivity of Lqh III to locust receptor site 3 and is not crucial for the binding of LqhalphaIT to this site. In turn, the aromatic ring of Tyr(21) takes part in the bioactivity of LqhalphaIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.

Published
2002
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46. NMR as a screening and structural method for protein-ligand interactions

Author

Isabelle Krimm, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

National audience

Published
2014

47. Targeting protein-protein interactions using fragment-like molecules

Author

Olivier Cala, Clémentine Aguirre, Tim Ten Brink, Béatrice Brion, Jean François Guichou, Isabelle Krimm, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

communication par affiche; International audience

Published
2014

48. NMR applications in FBDD regarding fragment binding mode and framgent-induced protein conformational change

Author

Isabelle Krimm, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2014

49. Applications of NMR for fragment-based drug design

Author

Isabelle Krimm, Olivier Cala, Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

National audience

Published
2014

50. Studies of interaction Cyclophilin D - fragments molecules

Author

Pierre Bais, Jean-François Guichou, Olivier Cala, Isabelle Krimm, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Criblage de fragment, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Bussy, Agnès

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

communication par affiche; National audience

Published
2014

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