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The design, synthesis, and screening of dual PI3K/mTOR inhibitors that gave nanomolar enzymatic and cellular activities on both targets with an acceptable kinase selectivity profile are described. A docking study was performed to understand the binding mode of the compounds and to explain the differences in biological activity. In addition, cellular effects of the best dual inhibitors were determined on six cancer cell lines and compared to those on a healthy diploid cell line for cellular cytotoxicity. Two compounds are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. A more detailed analysis of the cellular effect of these PI3K/mTOR dual inhibitors demonstrated that they induce GI-phase cell cycle arrest in breast cancer cells and trigger apoptosis. These compounds show an interesting kinase profile as dual PI3K/mTOR tool compounds or as a chemical series for further optimization to progress into in vivo experiments.
To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4 (CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and olefinic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular activities. The diarylcyclopropyl compounds with SR-configuration (−)-1b, (−)-2b and the cyclopropylvinyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enantiomerically pure forms for the three series of compounds. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for potential antivascular activities.
2’-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA-DNA and DNA-RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA-RNA duplexes similar to the one of the parent DNA-RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed [capital Delta]G values (37°C) that are in line with the measured Tm values for both the DNA-DNA and DNA-RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA-DNA and DNA-RNA duplexes involving Un are similar to those of the dT-rA and dU-rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA-DNA and RNA-DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2’-exo sugar pucker conformation close to an A-helix type without perturbing the C2’-endo sugar pucker conformations of their 2’-deoxynucleoside neighbours. These results confirm the potential of 2’-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.
High-throughput screening assays have become the starting point of many drug discovery programs for large pharmaceutical companies as well as academic organisations. Despite the increasing throughput of screening technologies, the almost infinite chemical space remains out of reach, calling for tools dedicated to the analysis and selection of the compound collections intended to be screened.
We present Screening Assistant 2 (SA2), an open-source JAVA software dedicated to the storage and analysis of small to very large chemical libraries. SA2 stores unique molecules in a MySQL database, and encapsulates several chemoinformatics methods, among which : providers management, interactive visualisation, scaffold analysis, diverse subset creation, descriptors calculation, sub-structure / SMART search, similarity search and filtering. We illustrate the use of SA2 by analysing the composition of a database of 15 million compounds collected from 73 providers, in terms of scaffolds, frameworks, and undesired properties as defined by recently proposed HTS SMARTS filters. We also show how the software can be used to create diverse libraries based on existing ones.
Screening Assistant 2 is a user-friendly, open-source software that can be used to manage collections of compounds and perform simple to advanced chemoinformatics analyses. Its modular design and growing documentation facilitate the addition of new functionalities, calling for contributions from the community. The software can be downloaded at http://sa2.sourceforge.net/.
Highly regioselective : An efficient synthesis of the imidazo[1,2-b]pyrazole core has been developed, and the first regioselective palladium-catalyzed direct arylation of the C-3 position is described (see scheme). Good to excellent yields were obtained for a wide range of aryl partners with electron-rich and electron-poor substituents. This methodology allows rapid access to a large variety of imidazo[1,2-b]pyrazole products and could open the way to the design of new biologically active compounds.
To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs were prepared from 1,3-diaryl-2-nitroprop-1-enes (6-12) obtained in a two-step synthesis from appropriate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6-12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological activities involved in antivascular action. It was found that several new compounds exhibited interesting biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells, inhibition of tubulin polymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity.
High Throughput Screening (HTS) is a standard technique widely used to find hit compounds in drug discovery projects. The high costs associated with such experiments have highlighted the need to carefully design screening libraries in order to avoid wasting resources. Molecular diversity is an established concept that has been used to this end for many years. In this article, a new approach to quantify the molecular diversity of screening libraries is presented. The approach is based on the Delimited Reference Chemical Subspace (DRCS) methodology, a new method that can be used to delimit the densest subspace spanned by a reference library in a reduced 2D continuous space. A total of 22 diversity indices were implemented or adapted to this methodology, which is used here to remove outliers and obtain a relevant cell-based partition of the subspace. The behavior of these indices was assessed and compared in various extreme situations and with respect to a set of theoretical rules that a diversity function should satisfy when libraries of different sizes have to be compared. Some gold standard indices are found inappropriate in such a context, while none of the tested indices behave perfectly in all cases. Five DRCS-based indices accounting for different aspects of diversity were finally selected, and a simple framework is proposed to use them effectively. Various libraries have been profiled with respect to more specific subspaces, which further illustrate the interest of the method.
A library of substituted chromeno[3,4-b]indoles was developed as Lamellarin isosters. Synthesis was achieved from indoles after a four-step pathway sequence involving C-3 iodination, a Suzuki cross-coupling reaction, and a one pot deprotection/lactonisation step. Twenty final compounds were tested in order to determine their activity against topoisomerase I and kinases, the two major biological activities of Lamellarins. One newly synthesized derivative exhibited a strong topoisomerase activity comparable to reference compounds such as campthotecin and Lamellarin with only a weak kinase inhibition. Two other lead compounds were identified as new nanomolar DYRK1A inhibitors and several other drugs affected the kinases in the sub-micromolar range. These results will enable us to use the chromeno[3,4-b]indole as a pharmacophore to develop potent treatments for neurological or oncological disorders in which DYRK1A is fully involved.
The synthesis of several novel 4-azaindoles was carried out by novel Fischer reaction which offers as a main advantage, the synthesis of the bisfunctionalized 4-azaindolic building block in one step. The final compounds were evaluated on a panel of 5 kinases in order to evaluate their selectivity and on 7 cancer cell lines to determine their cytotoxic effects. RAF-1 and DYRK1A inhibitions were found, docking studies explain fully the results.
A series of diversely substituted biarylolefins based on carbohydrate and dihydroxyethylene scaffolds were synthesized and evaluated for antiproliferative activity against a panel of human tumor cell lines. Among the thirty-five yet unknown biarylolefins prepared, six displayed potent antiproliferative activities with IC(50) values in the micromolar and submicromolar range. As a new type of antiproliferative agent, the most potent compound 26 showed an IC(50) value of 70 nM against SK-OV3 cell line (ovarian cancer). All the synthesized compounds exhibited a poor or modest tubulin polymerization inhibitory activity suggesting another mode of action for these compounds. Molecular docking simulations to the colchicine binding site of tubulin of representative compounds have been used to explain the lack of activity as inhibitors of tubulin polymerization.
High-throughput screening (HTS) is a well-established technology which can test up to several million compounds in a few weeks. Despite these appealing capabilities, available resources and high costs may limit the number of molecules screened, making diversity analysis a method of choice to design and prioritize screening libraries. With a constantly increasing number of molecules available for screening, chemical space has become a key concept for visualizing, analyzing, and comparing chemical libraries. In this first article, we present a new method to build delimited reference chemical subspaces (DRCS). A set of 16 million screening compounds from 73 chemical providers has been gathered, resulting in a database of 6.63 million standardized and unique molecules. These molecules have been used to create three DRCS using three different sets of chemical descriptors. A robust principal component analysis model for each space has been obtained, whereby molecules are projected in a reduced two-dimensional viewable space. The specificity of our approach is that each reduced space has been delimited by a representative contour encompassing a very large proportion of molecules and reflecting its overall shape. The methodology is illustrated by mapping and comparing various chemical libraries. Several tools used in these studies are made freely available, thus enabling any user to compute DRCS matching specific requirements.
We here report the synthesis and biological evaluation of new 3-[(2-indolyl)]-5-phenyl-3,5-pyridine, 3-[(2-indolyl)]-5-phenyl-2,4-pyridine and 3-[(2-indolyl)]-5-phenyl-2,6-pyrazine derivatives designed as potential CDK inhibitors. Indoles and phenyls were used to generate several substitutions of the pyridine and pyrazine rings. The synthesis included Stille or Suzuki type reactions, which were carried out on the 3,5-dibromopyridine, 2,4-dichloropyridine and 2,6-dichloro-1-4-pyrazine moieties. Cell effects of the V-shaped family were in the micromolar range. Kinase assays were conducted and showed that compound 11 inhibited CDK5 with an inhibitory concentration of 160 nM with a moderate selectivity over GSK3 compared to the reference C which exhibited a slightly lower activity on CDK5 (1.5 mu M). Compound 11 was also found to be the most potent compound in the series and was identified as a new lead for DYRK1A inhibitor discovery (IC(50) = 60 nM). Docking studies were carried out in order to investigate the inhibition of DYRK1A. (C) 2011 Elsevier Masson SAS. All rights reserved.
A series of combretastatin A4 (CA4) analogues with a lactam or lactone ring fused to the trimethoxyphenyl or the B-phenyl moiety were synthesized in an efficient and stereoselective manner by using a domino Heck-Suzuki-Miyaura coupling reaction. The vascular-disrupting potential of these conformationally restricted CA4 analogues was assessed by various in vitro assays : inhibition of tubulin polymerization, modification of endothelial cell morphology, and disruption of endothelial cell cords. Compounds were also evaluated for their growth inhibitory effects against murine and human tumor cells. B-ring-constrained derivatives that contain an oxindole ring (in contrast to compounds with a benzofuranone ring) as well as analogues bearing a six-membered lactone core fused to the trimethoxyphenyl ring are endowed with significant biological activity. The most potent compound of this series (oxindole 9b) is of particular interest, as it combines chemical stability and a biological activity profile characteristic of a vascular-disrupting agent.
Multiple protein structure methods have been proposed for incorporating protein flexibility in molecular docking. One approach for docking ligands onto a rigid receptor is to use an ensemble of multiple rigid structures determined experimentally by X-ray or NMR spectroscopy or generated by numerical simulations. In this work we present all empirical method for generating a wide range of conformational states of a wobbling receptor using restrained Molecular Dynamics simulations (MD) and we propose a partitioning protocol for selecting a few representative conformations of the binding site from restrained MID sampling.
Defining a large number of protein structures is computationally expensive when the MD simulations use an explicit solvent representation. For computational efficiency, solvent effect is therefore represented by an ensemble of restraints applied on a subset of specific atoms, using a distance-dependent permittivity function. The parameters used for the restraints and the permittivity are described. Several 100 ns restrained MD simulations are performed using different sets of parameters. In order to optimize the parameters, the results are compared to a 30 ns MD simulation in explicit solvent. Conformational sampling is speeded up by a factor of around 10-20 when performing restrained MD simulations. A partitioning k-means algorithm is applied to select representative structures of the receptor binding site. The methodology was evaluated on the ligand binding domain of the flexible Peroxysome Proliferator-Activated Receptor-gamma (PPAR gamma).
Chemical libraries or databases are collections of compounds which can be screened (virtually or experimentally) in order to discover drug candidates. These libraries are very variable in their content (description of structures, molecular descriptors, literature links...) and their size (number of compounds). Over the last decade, a large number of papers have been published on the subject. In this review, we summarize these studies by introducing different types of compound collections and reviewing the main kinds of software used to manipulate them. We present the descriptors which have a fundamental role in the characterisation of the molecules, and describe how they are used to define the molecular filters applied before screening, in order to obtain both a representation of chemical spaces and selections of subsets by diversity or similarity.
We report the synthesis of an adenosyl-derived indolylphosphonamide analogue of salicyladenosylmonophosphate involved in the plague and tuberculosis siderophore biosyntheses. The compound proved to be a potent inhibitor of the Yersinia pestis salicylate adenylation domain YbtE catalyzing the initial step of yersiniabactin biosynthesis.
The extraction of coupling constant values from NMR spectra is an important step in assigning the configuration of organic molecules. A method for the automatic multiplet analysis in weakly coupled spin systems is described here. It relies on a multi-step procedure that forms the AUJ (AUtomatic J) algorithm. Tolerance to low-magnitude second-order effects is achieved by an efficient multiplet centering and symmetrization step. Time-domain signal analysis through a linear model produces a raw evaluation of coupling constants and their associated multiplicities. The final result is obtained through numerical optimization of the multiplet parameters. Two examples are presented, one from an experimental spectrum, and the other one, of higher complexity, from a computer-simulated multiplet.
Electrophile-induced cyclization of (E)- and (Z)-2-ene-1,5-diols to tetrahydrofurans and oxetanes is described. Significant differences between the present report and previous work have been noted. A tentative model is proposed.