Chemistry and Modelling for Protein Recognition

Team Publications

Year of publication 2016

Morgan Pellerano, Delphine Naud-Martin, Marion Peyressatre, Camille Prével, Marie-Paule Teulade-Fichou, May Morris, Florence Mahuteau-Betzer (2016 Mar 17)

TP-2Rho Is a Sensitive Solvatochromic Red-Shifted Probe for Monitoring the Interactions between CDK4 and Cyclin D.

Chembiochem : a European journal of chemical biology : 17 : 737-744 : DOI : 10.1002/cbic.201500641 Learn more
Summary

Understanding the intricate steps of protein kinase regulation requires characterization of protein-protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP-2Rho, to peptide and protein derivatives of cyclin-dependent kinase 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclin D1 and a peptide substrate. We demonstrate the sensitivity of TP-2Rho in relation to of those other dyes used for monitoring peptide-protein and protein-protein interactions. Moreover, we show that TP-Rho-labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclin D.

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Rahima Chennoufi, Houcine Bougherara, Nathalie Gagey-Eilstein, Blaise Dumat, Etienne Henry, Frédéric Subra, Stéphanie Bury-Moné, Florence Mahuteau-Betzer, Patrick Tauc, Marie-Paule Teulade-Fichou, Eric Deprez (2016 Mar 8)

Mitochondria-targeted Triphenylamine Derivatives Activatable by Two-Photon Excitation for Triggering and Imaging Cell Apoptosis.

Scientific reports : 6 : 21458 : DOI : 10.1038/srep21458 Learn more
Summary

Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760-860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.

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Chaput L., Martinez-Sanz J., Quiniou E., Rigolet P., Saettel N., Mouawad L. (2016 Jan 18)

vSDC: a method to improve early recognition in virtual screening when limited experimental resources are available

Journal of Cheminformatics : 8:1 : DOI : 10.1186/s13321-016-0112-z Learn more
Summary

Background: In drug design, one may be confronted to the problem of finding hits for targets for which no small inhibiting molecules are known and only low-throughput experiments are available (like ITC or NMR studies), two common difficulties encountered in a typical academic setting. Using a virtual screening strategy like docking can alleviate some of the problems and save a considerable amount of time by selecting only top-ranking molecules, but only if the method is very efficient, i.e. when a good proportion of actives are found in the 1-10 % best ranked molecules.

Results: The use of several programs (in our study, Gold, Surflex, FlexX and Glide were considered) shows a divergence of the results, which presents a difficulty in guiding the experiments. To overcome this divergence and increase the yield of the virtual screening, we created the standard deviation consensus (SDC) and variable SDC (vSDC) methods, consisting of the intersection of molecule sets from several virtual screening programs, based on the standard deviations of their ranking distributions.

Conclusions: SDC allowed us to find hits for two new protein targets by testing only 9 and 11 small molecules from a chemical library of circa 15,000 compounds. Furthermore, vSDC, when applied to the 102 proteins of the DUD-E benchmarking database, succeeded in finding more hits than any of the four isolated programs for 13-60 % of the targets. In addition, when only 10 molecules of each of the 102 chemical libraries were considered, vSDC performed better in the number of hits found, with an improvement of 6-24 % over the 10 best-ranked molecules given by the individual docking programs.

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Blaise Dumat, Elodie Faurel-Paul, Pauline Fornarelli, Nicolas Saettel, Germain Metgé, Céline Fiorini-Debuisschert, Fabrice Charra, Florence Mahuteau-Betzer, Marie-Paule Teulade-Fichou (2016 Jan 7)

Influence of the oxazole ring connection on the fluorescence of oxazoyl-triphenylamine biphotonic DNA probes.

Organic & biomolecular chemistry : 14 : 358-370 : DOI : 10.1039/c5ob02225h Learn more
Summary

On the basis of our previous work on DNA fluorophores derived from vinylpyridinium-triphenylamine, we explored the structure space around the electron-rich triphenylamine (TP) core by changing the vinyl bond to an oxazole ring. As 2,5-diaryloxazoles are known to be highly fluorescent and efficient two photon absorbers, we synthesized analogues with two different connections of the oxazole to the triphenylamine core: TP-Ox2Py and TP-Ox5Py sets. Since the benzimidazolium group was proven to be more effective in the TP series than the pyridinium, we also synthesized a TP-Ox5Bzim set. The TP-Ox5Py series retains the TP-Py properties: on/off behavior on DNA, good two-photon cross-section and bright staining of nuclear DNA by microscopy under both one or two-photon excitation. On the other hand, the TP-Ox2Py series does not display fluorescence upon binding to DNA. The TP-Ox5Bzim set is fluorescent even in the absence of DNA and displays lower affinity than the corresponding TP-Ox5Py. CD experiments and docking were performed to understand these different behaviors.

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Year of publication 2015

Tom Baladi, Valentina Abet, Sandrine Piguel (2015 Nov 13)

State-of-the-art of small molecule inhibitors of the TAM family: the point of view of the chemist.

European journal of medicinal chemistry : 105 : 220-37 : DOI : 10.1016/j.ejmech.2015.10.003 Learn more
Summary

The TAM family of tyrosine kinases receptors (Tyro3, Axl and Mer) is implicated in cancer development, autoimmune reactions and viral infection and is therefore emerging as an effective and attractive therapeutic target. To date, only a few small molecules have been intentionally designed to block the TAM kinases, while most of the inhibitors were developed for blocking different protein kinases and then identified through selectivity profile studies. This minireview will examine in terms of chemical structure the different compounds able to act on either one, two or three TAM kinases with details about structure-activity relationships, drug-metabolism and pharmacokinetics properties where they exist.

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Guillaume Kellermann, Markus Kaiser, Florent Dingli, Olivier Lahuna, Delphine Naud-Martin, Florence Mahuteau-Betzer, Damarys Loew, Evelyne Ségal-Bendirdjian, Marie-Paule Teulade-Fichou, Sophie Bombard (2015 Sep 3)

Identification of human telomerase assembly inhibitors enabled by a novel method to produce hTERT.

Nucleic acids research : 43 : e99 : DOI : 10.1093/nar/gkv425 Learn more
Summary

Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors.

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Kathleen Solmont, Hamza Boufroura, Amel Souibgui, Pauline Fornarelli, Anne Gaucher, Florence Mahuteau-Betzer, Béchir Ben Hassine, Damien Prim (2015 May 13)

Divergent strategy for the synthesis of original dihydrobenzo- and dihydronaphtho-acridines.

Organic & biomolecular chemistry : 13 : 6269-6277 : DOI : 10.1039/c5ob00456j Learn more
Summary

A straightforward access to numerous novel substituted dihydrobenzo- and dihydronaphthoacridines is described using a unique molecular platform in two key steps. A large range of carbon-based substituents such as aromatic, vinyl, alkynyl fragments through Pd-catalysed couplings has been installed. The molecular diversity is extended to the introduction of aza-heterocycles and further authorizes the installation of alkylamino chains by means of Cu-promoted C-N bond formation. Possible access to quinolinium salts is also described. The methodology revealed convenient preparation of a wide panel of molecules that display various rigidity/flexibility and lipophilic/hydrophilic balances. Finally, the influence of structural modulations on the photophysical properties of these novel architectures is also studied. It is noteworthy that styryl and alkynyl derivatives are emissive in water (ΦF up to 12%).


Divergent_strategy

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Florence Mahuteau-Betzer (2015 May 12)

The French National Compound Library: advances and future prospects.

Médecine sciences : M/S : 31 : 417-422 : DOI : 10.1051/medsci/20153104016 Learn more
Summary

The French National Compound Library (Chimiothèque Nationale) has been created in 2003 and is the federation of local collections. It contains more than 56 000 small molecules and natural compounds synthesised or isolated in different laboratories over the past years. This explains the diversity of the collection. The strength of this initiative is the ability to connect chemists and biologists for the development of hits. This development involves the synthesis of analogues or/and chemical tools to find new targets. These collaborations lead to the identification of new chemical probes. These probes able to modulate a biological function are essential to study biological pathways. They can also be useful for therapeutic applications. This article will describe the major achievements and perspectives of the French Chemical Library.

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Noëlie Campos, Renier Myburgh, Aude Garcel, Audrey Vautrin, Laure Lapasset, Erika Schläpfer Nadal, Florence Mahuteau-Betzer, Romain Najman, Pauline Fornarelli, Katjana Tantale, Eugénia Basyuk, Martial Séveno, Julian P Venables, Bernard Pau, Edouard Bertrand, Mark A Wainberg, Roberto F Speck, Didier Scherrer, Jamal Tazi (2015 Apr 19)

Long lasting control of viral rebound with a new drug ABX464 targeting Rev – mediated viral RNA biogenesis.

Retrovirology : 12 : 30 : DOI : 10.1186/s12977-015-0159-3 Learn more
Summary

Background: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis.

Results: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies.

Conclusions: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.

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Year of publication 2014

Vabre R., Legraverend M., Piguel S. (2014 Jun 1)

Synthesis and evaluation of spectroscopic properties of newly synthesized push–pull 6-amino-8-styryl purines

Dyes and Pigments : 105 : 145-151 : DOI : 10.1016/j.dyepig.2014.01.025 Learn more
Summary

New 6-amino-8-styryl purines were synthesized using direct C–H bond functionalization. These push–pull compounds showed strong fluorescence, high quantum yields and a noteworthy fluorosolvatochromism. Deprotected purines 7a–c are promising targets for incorporation into nucleic acids as they are still fluorescent in aqueous media.

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Delphine Naud-Martin, Xavier Martin-Benlloch, Florent Poyer, Florence Mahuteau-Betzer, Marie-Paule Teulade-Fichou (2014 Feb 1)

Acri-2,7-Py, a bright red-emitting DNA probe identified through screening of a distyryl dye library.

Biotechnology journal : 9 : 301-310 : DOI : 10.1002/biot.201300197 Learn more
Summary

The identification of DNA sensors is still a challenge since no DNA probe possesses all the photophysical properties required for live-cell imaging: high fluorescence yield, red emission, permeability, no photobleaching and no cytotoxicity. We describe the preparation of a distyryl dye library and its evaluation on a panel of nucleic acids with various structures (duplex DNA, quadruplex DNA and RNA). The screening involved measuring the modification of the fluorescence properties of the dyes with or without nucleic acids on a microplate reader, and allowed the identification of selective quadruplex DNA ligands with good affinities. Using this screening method we discovered a new bright red-emitting DNA stain, Acri-2,7-Py, for fixed cells. In living cells, the staining was not nuclear and photodamage generated through illumination induced cellular death. These processes require further studies to determine the relevance of Acri-2,7-Py in photodynamic therapy.

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Vabre R., Couradin J., Naud-Martin D., Legraverend M., Piguel S. (2014 Jan 1)

Palladium-Catalyzed Cross-Coupling between 8-Substituted 6-Thiophenyl­purines and Boronic Acids

Synthesis : 46 : 933-942 : DOI : 10.1055/s-0033-1340734 Learn more
Summary

A palladium(II)-catalyzed, copper(I)-mediated ­Liebeskind–Srogl cross-coupling for the C-6 arylation/alkenylation on purine scaffold is reported. Various boronic acids reacted readily with 8-substituted 6-thiophenylpurines.

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Year of publication 2013

Ténin Traoré, Andrea Cavagnino, Nicolas Saettel, François Radvanyi, Sandrine Piguel, Isabelle Bernard-Pierrot, Véronique Stoven, Michel Legraverend (2013 Nov 19)

New aminopyrimidine derivatives as inhibitors of the TAM family.

European journal of medicinal chemistry : 70 : 789-801 : DOI : 10.1016/j.ejmech.2013.10.037 Learn more
Summary

In this study, we describe the synthesis of new pyrimidine analogs of BMS-777607, a potent and selective inhibitor of Met kinase. Inhibition of Met and Axl remained high whereas inhibition of Tyro3 and Mer decreased to some extend. The preferential moderate inhibition of the non-phosphorylated form of Abl1 of some derivatives suggests that they behave as type II inhibitors. This hypothesis was confirmed by docking studies into the structure of Met (3F82) and in a Tyro3 model where key interactions with the hinge region, the DFG-out motif and the allosteric pocket explain this inhibition.

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Blaise Dumat, Guillaume Bordeau, Elodie Faurel-Paul, Florence Mahuteau-Betzer, Nicolas Saettel, Germain Metge, Céline Fiorini-Debuisschert, Fabrice Charra, Marie-Paule Teulade-Fichou (2013 Aug 20)

DNA switches on the two-photon efficiency of an ultrabright triphenylamine fluorescent probe specific of AT regions.

Journal of the American Chemical Society : 135 : 12697-12706 : DOI : 10.1021/ja404422z Learn more
Summary

We report on the design and synthesis of two-photon fluorescent triphenylamines bearing two or three vinyl branches terminated by a N-methyl benzimidazolium moiety. The new compounds (TP-2Bzim, TP-3Bzim) are light-up fluorescent DNA probes with a long wavelength emission (>580 nm). Compared to their pyridinium models, the TP-Bzim dyes exhibit a remarkable improvement of both their DNA affinity and fluorescence quantum yield, especially for the two-branch derivative (TP-2Bzim: ΦF = 0.54, Ka = 10(7) M(-1)), resulting in a large fluorescence emission turn-on ratio of up to 140. Concomitantly, the two-photon absorption cross-section of TP-2Bzim is dramatically enhanced upon DNA binding (δ = 1080 vs 110 GM for the free form). This effect of the DNA matrix on the nonlinear absorption is uncovered for the first time. This is attributed to a tight fit of the molecule inside the minor groove of AT-rich DNA which induces geometrical rearrangements in the dye ground state as supported by circular dichroism and molecular modeling data. Consequently, TP-2bzim displays an exceptional two-photon molecular brightness (δ×ΦF = 583 GM), a value unrivalled for a small biofluorophore. These properties enable to image nuclear DNA in fixed cells at submicromolar concentration ([TP-2Bzim] = 100 nM) and to visualize ultrabright foci of centromeric AT-rich chromatin. Finally TP-2Bzim exhibits a high photostability, is live-cell permeant, and does not require RNase treatment. This outstanding combination of optical and biological properties makes TP-2Bzim a bioprobe surpassing the best DNA stainers and paves the way for studying further nonlinear optical processes in DNA.

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Souibgui A., Gaucher A., Marrot J., Aloui F., Mahuteau-Betzer F., Ben Hassine B., Prim D. (2013 Jul 1)

A Flexible Strategy Towards Thienyl-, Oxazolyl- and Pyridyl-Fused Fluorenones

European Journal of Organic Chemistry : 2013 : 4515-4522 : DOI : 10.1002/ejoc.201300233 Learn more
Summary

Abstract The preparation of new thienyl-, oxazolyl-, and pyridyl-fused benzo[c]fluorenones has been developed from a unique and easily available common precursor. Starting from bromotetralone, a short sequence allowed the construction of various heterocycles followed by Suzuki coupling and final acid-promoted formation of the central cyclopentanone unit. The flexible strategy allowed the selective preparation of fused penta- to hepta-cyclic architectures. Fully aromatic targets have been obtained through the application of an oxidation process. The influence of structural modulations on the photophysical properties are described.

Flexible-strategy-2013

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