Chemistry and Modelling for Protein Recognition

Team Publications

Year of publication 2018

Luis M.G. Abegão, Ruben D Fonseca, Tárcius N Ramos, Florence Mahuteau-Betzer, Sandrine PIGUEL, José J. Rodrigues Jr, Cleber R. Mendonca, Sylvio Canuto, Daniel Luis Silva, and Leonardo De Boni (2018 Apr 12)

Oxazole dyes with potential for photoluminescence bioprobes: A two-Photon absorption study

The Journal of Physical Chemistry C : 122 : 10526-10534 : DOI : 10.1021/acs.jpcc.8b01904 Learn more
Summary

In this work, six π-conjugated oxazole compounds dissolved in dichloromethane (DCM) were characterized with linear and nonlinear optical measurements. Z-Scan with femtosecond laser pulses was employed to determine the two-photon absorption (TPA) spectra. Other photophysical parameters, such as: absorbance, solvatochromism, lifetime fluorescence and fluorescence anisotropy were evaluated with linear optical techniques. The experimental TPA cross-section spectra were adjusted by Sum-Over-States (SOS) model, in which important parameters such as transition dipole moments and broadening parameters were determined. In order to better understand the TPA spectra of the oxazole compounds, quantum-chemical calculations using the response function formalism and the DFT level of theory were performed. Using the results provided by the quantum-chemical calculations and the broadening parameters estimated through the application of the SOS model, the TPA spectra were simulated by the superposition (summation) of individual homogeneous Lorentzian absorption profiles.

jp-2018-01904m_0006

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Marius Mamone, Jessy Aziz, Julie Le Bescont, Sandrine Piguel (2018 Jan 18)

Aminocarbonylation of N-Containing Heterocycles with Aromatic Amines Using Mo(CO)6

Synthesis : 50 : 1521-1526 : DOI : 10.1055/s-0037-1609152 Learn more
Summary

We describe herein the palladium-catalyzed aminocarbonylation of nitrogen-containing heterocycles with aniline derivatives using molybdenum hexacarbonyl as a CO solid source, expanding the scope of the limited examples. This method is compatible with a variety of substitutions on the aniline moiety. The simple reaction conditions include easily available Pd(dppf)Cl2 catalyst, DBU as base in DMF at 120 °C for 3 hours in sealed tube thereby leading to the isolation of 21 compounds with yields ranging from 18 to 82%. We also show that double aminocarbonylation reactions are possible in satisfactory yields regarding both coupling partners.

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Hammerer F., Poyer F., Fourmois L., Chen S., Garcia G., Teulade-Fichou M.P., Maillard P., Mahuteau-Betzer F. (2018 Jan 1)

Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy

Bioorganic & Medicinal Chemistry : 26 : 107-118 : DOI : 10.1016/j.bmc.2017.11.024 Learn more
Summary

The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency.

Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy

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Year of publication 2017

Pauline Gilson, Fernando Josa-Prado, Claire Beauvineau, Delphine Naud-Martin, Laetitia Vanwonterghem, Florence Mahuteau-Betzer, Alexis Moreno, Pierre Falson, Laurence Lafanechère, Véronique Frachet, Jean-Luc Coll, Jose Fernando Díaz, Amandine Hurbin, Benoit Busser (2017 Sep 2)

Identification of pyrrolopyrimidine derivative PP-13 as a novel microtubule-destabilizing agent with promising anticancer properties.

Scientific reports : 7 : 10209 : DOI : 10.1038/s41598-017-09491-9 Learn more
Summary

Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in β-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones.

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Aziz Jessy, Piguel Sandrine (2017 Aug 25)

An update on direct C–H bond functionalization of nitrogen-containing fused heterocycles

Synthesis : 49 : 4562-4585 : DOI : 10.1055/s-0036-1590859 Learn more
Summary

This report highlights the recent advances in direct C–H bond functionalization of 5,5- and 6,5-fused heterocycles containing at least two nitrogen atoms. Besides C–C bond formation, C–N, C–S, C–P, and C–Si bonds can also be created via a metal-catalyzed process. Some examples, where a C–H functionalization approach was applied for the synthesis of drug candidates, will be presented as well.

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Claire Beauvineau, Corinne Guetta, Marie-Paule Teulade-Fichou, Florence Mahuteau-Betzer (2017 Aug 14)

PhenDV, a turn-off fluorescent quadruplex DNA probe for improving the sensitivity of drug screening assays

Organic & Biomolecular Chemistry : 15 : 7117-7121 : DOI : 10.1039/c7ob01705g Learn more
Summary

We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push–pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes

PhenDV-Mahuteau-Betzer

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Marie-Paule Teulade-Fichou, Joël Lefebvre, Corinne Guetta, Florent Poyer, Florence Mahuteau-Betzer (2017 Jul 13)

Copper-alkyne complexation is responsible for the Nucleolar Localisation of Quadruplex Nucleic Acid Drugs Labelled by Click Chemistry.

Angewandte Chemie (International ed. in English) : 56 : 11365-11369 : DOI : 10.1002/anie.201703783 Learn more
Summary

G-quadruplex(es) (G4) are non-canonical nucleic acid structures found in guanine-rich sequences. They can be targeted with small molecules (G4-ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localisation of PhenDC3, one of the most powerful G4 ligands, by synthesising two clickable azide and alkyne derivatives (PhenDC3-alk, PhenDC3-az) and labelling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper-based CuAAC and copper-free SPAAC methodologies in fixed cells implicated Cu(I) /alkyne intermediates in the non-specific localisation of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labelling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localise drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.

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Chennoufi R., Mahuteau-Betzer F., Tauc P., Teulade-Fichou M.P., Deprez E. (2017 Jun 18)

Triphenylamines Induce Cell Death Upon 2-Photon Excitation

Molecular Imaging : 16 : 1-4 : DOI : 10.1177/1536012117714164 Learn more
Summary

Photodynamic therapy (PDT) is a promising therapeutic method for several diseases, in particular for cancer. This approach uses a photosensitizer, oxygen, and an external light source to produce reactive oxygen species (ROS) at lethal doses to induce cell death. One drawback of current PDT is the use of visible light which has poor penetration in tissues. Such a limitation could be overcome by the use of novel organic compounds compatible with photoactivation under near-infrared light excitation. Triphenylamines (TPAs) are highly fluorescent compounds that are efficient to induce cell death upon visible light excitation (458 nm), but outside the biological spectral window. Interestingly, we recently showed that TPAs target cytoplasmic organelles of living cells, mainly mitochondria, and induce a high ROS production upon 2-photon excitation (in the 760-860 nm range), leading to a fast apoptosis process. However, we observed significant differences among the tested TPA compounds in terms of cell distribution and time courses of cell death-related events (apoptosis vs necrosis). In summary, TPAs represent serious candidates as photosensitizers that are compatible with 2-photon excitation to simultaneously trigger and imaging cell death although the relationship between their subcellular localization and the cell death mechanism involved is still a matter of debate.

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Chaput Ludovic, Mouawad Liliane (2017 Jun 12)

Efficient conformational sampling and weak scoring in docking programs? Strategy of the wisdom of crowds

Journal of Cheminformatics : 9 : 37 : DOI : 10.1186/s13321-017-0227-x Learn more
Summary

Background: In drug design, an efficient structure-based optimization of a ligand needs the precise knowledge of the protein-ligand interactions. In the absence of experimental information, docking programs are necessary for ligand positioning, and the choice of a reliable program is essential for the success of such an optimization. The performances of four popular docking programs, Gold, Glide, Surflex and FlexX, were investigated using 100 crystal structures of complexes taken from the Directory of Useful Decoys-Enhanced database.

Results: The ligand conformational sampling was rather efficient, with a correct pose found for a maximum of 84 complexes, obtained by Surflex. However, the ranking of the correct poses was not as efficient, with a maximum of 68 top-rank or 75 top-4 rank correct poses given by Glidescore. No relationship was found between either the sampling or the scoring performance of the four programs and the properties of either the targets or the small molecules, except for the number of ligand rotatable bonds. As well, no exploitable relationship was found between each program performance in docking and in virtual screening; a wrong top-rank pose may obtain a good score that allows it to be ranked among the most active compounds and vice versa. Also, to improve the results of docking, the strengths of the programs were combined either by using a rescoring procedure or the United Subset Consensus (USC). Oddly, positioning with Surflex and rescoring with Glidescore did not improve the results. However, USC based on docking allowed us to obtain a correct pose in the top-4 rank for 87 complexes. Finally, nine complexes were scrutinized, because a correct pose was found by at least one program but poorly ranked by all four programs. Contrarily to what was expected, except for one case, this was not due to weaknesses of the scoring functions.

Conclusions: We conclude that the scoring functions should be improved to detect the correct poses, but sometimes their failure may be due to other varied considerations. To increase the chances of success, we recommend to use several programs and combine their results.

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Morgan Pellerano, Sergey Tcherniuk, Corine Perals, Thi Nhu Ngoc Van, Elsa Garcin, Florence Mahuteau-Betzer, Marie-Paule Teulade-Fichou, May C Morris (2017 Apr 22)

Targeting Conformational Activation of CDK2 Kinase.

Biotechnology journal : 12 : 1600531 : DOI : 10.1002/biot.201600531 Learn more
Summary

Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP-competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T-loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T-loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.

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Gilles Galvani, K Harsha Vardhan Reddy, Claire Beauvineau, NourEddine Ghermani, Florence Mahuteau-Betzer, Mouad Alami, Samir Messaoudi (2017 Feb 17)

Conversion of 3-Bromo-2H-coumarins to 3-(Benzofuran-2-yl)-2H-coumarins under Palladium Catalysis: Synthesis and Photophysical Properties Study.

Organic letters : 19 : 910-913 : DOI : 10.1021/acs.orglett.7b00069 Learn more
Summary

An intriguing conversion of 3-bromo-2H-coumarins to 3-(benzofuran-2-yl)-2H-coumarins under palladium catalysis is reported. The process involves, from only one single starting material, three transformations and two bond formations in one pot: C-C bond formation via C-H activation and C-O bond formation through 2H-coumarin-to-benzofuran ring contraction under palladium catalysis. Moreover, the photophysical properties of all synthesized compounds were studied.

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J Santo, C Lopez-Herrera, C Apolit, Y Bareche, L Lapasset, C Chavey, S Capozi, F Mahuteau-Betzer, R Najman, P Fornarelli, I C Lopez-Mejía, G Béranger, F Casas, E-Z Amri, B Pau, D Scherrer, J Tazi (2017 Feb 14)

Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

International journal of obesity : 41 : 390-401 : DOI : 10.1038/ijo.2016.220 Learn more
Summary

Bakground/Objectives:

Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug.

Subjects/Methods:

Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet.

Results:

We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids.

Conclusions:

Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

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Year of publication 2016

Chaput L., Martinez-Sanz J., Saettel N., Mouawad L. (2016 Oct 17)

Benchmark of four popular virtual screening programs: construction of the active/decoy dataset remains a major determinant of measured performance

Journal of Cheminformatics : 8:56 : DOI : 10.1186/s13321-016-0167-x Learn more
Summary

Abstract
In a structure-based virtual screening, the choice of the docking program is essential for the success of a hit identification. Benchmarks are meant to help in guiding this choice, especially when undertaken on a large variety of protein targets. Here, the performance of four popular virtual screening programs, Gold, Glide, Surflex and FlexX, is compared using the Directory of Useful Decoys-Enhanced database (DUD-E), which includes 102 targets with an average of 224 ligands per target and 50 decoys per ligand, generated to avoid biases in the benchmarking. Then, a relationship between these program performances and the properties of the targets or the small molecules was investigated.
Results
The comparison was based on two metrics, with three different parameters each. The BEDROC scores with α = 80.5, indicated that, on the overall database, Glide succeeded (score > 0.5) for 30 targets, Gold for 27, FlexX for 14 and Surflex for 11. The performance did not depend on the hydrophobicity nor the openness of the protein cavities, neither on the families to which the proteins belong. However, despite the care in the construction of the DUD-E database, the small differences that remain between the actives and the decoys likely explain the successes of Gold, Surflex and FlexX. Moreover, the similarity between the actives of a target and its crystal structure ligand seems to be at the basis of the good performance of Glide. When all targets with significant biases are removed from the benchmarking, a subset of 47 targets remains, for which Glide succeeded for only 5 targets, Gold for 4 and FlexX and Surflex for 2.
Conclusion
The performance dramatic drop of all four programs when the biases are removed shows that we should beware of virtual screening benchmarks, because good performances may be due to wrong reasons. Therefore, benchmarking would hardly provide guidelines for virtual screening experiments, despite the tendency that is maintained, i.e., Glide and Gold display better performance than FlexX and Surflex. We recommend to always use several programs and combine their results.

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Baladi T., Granzhan A., Piguel S. (2016 May 1)

Microwave-Assisted C-2 Direct Alkenylation of Imidazo[4,5-b]pyridines: Access to Fluorescent Purine Isosteres with Remarkably Large Stokes Shifts

European Journal of Organic Chemistry : 2016 : 2421-2434 : DOI : 10.1002/ejoc.201600166 Learn more
Summary

We describe herein the first C-2 direct alkenylation of the valuable 3H-imidazo[4,5-b]pyridine promoted by microwave-assisted Pd/Cu co-catalysis. The reaction is rapid and compatible with a wide range of functional groups either on the imidazo[4,5-b]pyridine ring or on the styryl bromides thereby leading to the isolation of 23 compounds with moderate to good yields. The relevance of this method is demonstrated by its application to the synthesis of new cross-conjuguated push-pull 2-vinyl- and 2-alkynylimidazo[4,5-b]pyridines characterized by satisfactory fluorescence quantum yields and remarkable solvatofluorochromic properties.

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Jessy Aziz, Tom Baladi, Sandrine Piguel (2016 Apr 26)

Direct Alkynylation of 3H-Imidazo[4,5-b]pyridines Using gem-Dibromoalkenes as Alkynes Source.

The Journal of organic chemistry : 81 : 4122-4133 : DOI : 10.1021/acs.joc.6b00406 Learn more
Summary

C2 direct alkynylation of 3H-imidazo[4,5-b]pyridine derivatives is explored for the first time. Stable and readily available 1,1-dibromo-1-alkenes, electrophilic alkyne precursors, are used as coupling partners. The simple reaction conditions include an inexpensive copper catalyst (CuBr·SMe2 or Cu(OAc)2), a phosphine ligand (DPEphos) and a base (LiOtBu) in 1,4-dioxane at 120 °C. This C-H alkynylation method revealed to be compatible with a variety of substitutions on both coupling partners: heteroarenes and gem-dibromoalkenes. This protocol allows the straightforward synthesis of various 2-alkynyl-3H-imidazo[4,5-b]pyridines, a valuable scaffold in drug design.

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