Structure-based design and synthesis of inhibitors of the mitotic kinases Nek2 and CDK2

PhD ThesisCyclin-dependent kinase 2 (CDK2) and Nek2 (Never-In-Mitosis A related kinases) are cell-cycle associated serine-threonine kinases that play an important role in the regulation of cellular proliferation and mitosis. Aberrant CDK2 and Nek2 activity are strongly associated with cancer, and inhibitors of these protein kinases are of potential therapeutic use as antitumour agents. Inhibitors of Nek2 –Previous studies identified a series of 2-aminoaryl-6-ethynylpurines (e.g. 42), as potent and selective irreversible Nek2 inhibitors with good antitumour activity in vitro and in vivo. 6-Ethynylpurine 42 binds within the ATP domain of Nek2 via a triplet of hydrogen bond interactions with the hinge region, enabling a covalent reaction between Cys-22 and the 6-ethynyl substituent. However, subsequent SAR studies indicated possible off-target activity for this series, which has been investigated through the synthesis and evaluation of control compounds engineered to be inactive against Nek2. With a view to abolishing Nek2-inhibitory activity without imposing dramatic structural changes, the effect of methylation at the purine N-7 (64) and N-9 (63) positions was studied. The corresponding isosteric 6-cyanopurine derivatives (65, 66, and 67) were also synthesised, which were expected to resemble closely 6-ethynylpurines without the capacity to react covalently. Evaluation of these derivatives in cell-based assays confirmed the presence of a growth-inhibitory activity unrelated to Nek2 inhibition. Regioselective N-7 methylation of 65 proved challenging, and a novel approach was developed whereby initial N-9 protection enabled selective methylation at the purine N-7 position, with concomitant loss of the N-9 protecting group giving the target purine 67. Kinetic studies have also been conducted with 42, 63 and 64 to assess the impact of N-7/N-9 methylation on the chemical reactivity of the 6-ethynyl ‘warhead’. A model system was developed to investigate a possible correlation between the chemical and biological reactivity of the 6-ethynyl group of the purine derivatives with thiols. Inhibitors of CDK2 –Previous studies have resulted in the identification of the purine CDK2 inhibitor 60, found to inhibit CDK2 in a time-dependent manner (IC50 = 63 nM) via conjugate addition of a lysine residue (Lys89), located in the ‘hinge region’ of the v ATP-binding domain, to the vinyl sulfone functionality of 60. This is thought to represent the first example of irreversible CDK2 inhibition, and prompted more detailed investigations with 60. Compound 60 exhibited a short half-life in both plasma (44 min) and medium (19 min), ascribed to hydration of the vinyl sulfone. Therefore, a priority was to improve the chemical stability of 60 without compromising inhibitory potency and time-dependent inhibition. Using the crystal structure of 60 in complex with CDK2 to guide inhibitor design, a range of derivatives (162-168) have been synthesised bearing α-substituents (R) on the vinyl sulfone group. In addition, synthetic methodology was developed for the synthesis of the corresponding 2-hydroxyalkyl products which are putative ATPcompetitive inhibitors. From the biological studies conducted on purine derivatives 162- 168, only the α-chlorovinyl sulfone compound 167 (IC50 (4 h) = 14 nM) has emerged as of particular interest, whereas others have shown competitive CDK2 inhibition, confirmed by X-ray crystallography. The β-position of the vinyl sulfone moiety has been briefly explored with purines (323, 333 and 339). In this series, one inhibitor (323) has been shown to bind covalently to CDK2 by structural biology studies. Alternative ‘warheads’ to the vinyl sulfone group have been investigated and exhibited competitive CDK2 inhibitory activity in a time-dependent inhibition assay. Finally, insertion of the vinyl sulfone warhead into known CDK2 inhibitors was investigated in preliminary studies

Provenance, modification and use of manganese-rich rocks at Le Moustier (Dordogne, France)

The use of colouring materials by Neanderthals has attracted a great deal of attention in recent years. Here we present a taphonomic, technological, chemical-mineralogical and functional analysis of fifty-four manganese rich lumps recovered during past and on-going excavations at the lower rockshelter of Le Moustier (Dordogne, France). We compare compositional data for archaeological specimens with the same information for twelve potential geological sources. Morphometric analysis shows that material from Peyrony’s excavations before the First World War provides a highly biased picture of the importance of these materials for Mousterian groups. These early excavations almost exclusively recovered large modified pieces, while Mn-rich lumps from the on-going excavations predominantly consist of small pieces, only half of which bear traces of modification. We estimate that at least 168 pieces were not recovered during early work at the site. Neanderthals developed a dedicated technology for processing Mn-rich fragments, which involved a variety of tools and motions. Processing techniques were adapted to the size and density of the raw material, and evidence exists for the successive or alternating use of different techniques. Morphological, textural and chemical differences between geological and archaeological samples suggest that Neanderthals did not collect Mn-rich lumps at the outcrops we sampled. The association and variability in Mn, Ni, As, Ba content, compared to that observed at the sampled outcrops, suggests that either the Le Moustier lumps come from a unique source with a broad variation in composition, associating Mn, Ni, As, Ba, or that they were collected at different sources, characterized either by Mn-Ni-As or Mn-Ba. In the latter case, changes in raw material composition across the stratigraphy support the idea that Neanderthal populations bearing different stone tool technologies collected Mn fragments from different outcrops. Our results favour a use of these materials for multiple utilitarian and symbolic purposes.publishedVersio

Nouveaux matériaux avancés pour le stockage thermique hautes températures : cas du péritectique Li4Br(OH)3

Ce travail présente le cas d’étude du matériau Li4Br(OH)3comme matériau de stockage thermique pour des applications autour de 300°C. Ce matériau n’est pas un matériau à changement de phaseclassique;catégorie généralement considéréepour les unités de stockage pour une gamme de températures de travail variantentre 300 et 600°C.Il s’agit d’un composé péritectique dont les premières caractérisations thermiques ont montré un réel potentiel en termesde densité énergétiquequi permettrait le développement d’unités de stockage ultra-compactes.This work deals with the case study of Li4Br(OH)3as a thermal storage material for applications around 300°C. This material is not a regular phase change material; a category generally considered for storage units witha working temperature range between 300 and 600°C. It is a peritectic compound whose initial thermal characterizations have shown a real potential in terms of energy densitythat would allow the developmentof ultra-compact storage units

Bis(1,1-dimethyl­guanidinium) tetra­aqua­dimethyl­tin(IV) bis­(sulfate)

Single crystals of the title salt, (C3H10N3)2[Sn(CH3)2(H2O)4](SO4)2, formed concomitantly with the already known [Sn(CH3)3]2SO4·2H2O. In the title structure, the SnIV atom displays a slightly distorted octa­hedral coordination geometry defined by four O water atoms in the equatorial positions and two methyl groups in the axial positions. In the crystal, various O—H⋯O and N—H⋯O hydrogen-bonding inter­actions between the organic cation and the coordinated water mol­ecules as donors and the sulfate O atoms as acceptors result in a three-dimensional structure. The SnIV atom is located on an inversion centre, resulting in half of the complex metal cation being in the asymmetric unit

IDENTIFICATION DE PHASE PAR DIFFRACTION DES RAYONS X Du DIOXYDE DE MANGANESE RECUPERE D’UNE PILE SALINE USAGEE.

Une analyse par diffraction des RX a été réalisée sur la poudre du MnO2recyclé ; Après l’avoir récupérée des piles saline Zn/MnO2 usagées. Le recyclagea été effectué par la méthode d’hydrométallurgie.Dont le but est de voir le processus du changement de phase du MnO2. La comparaison entre le spectre de DRX obtenu du MnO2 recyclé et celui obtenu d’une pille neuve montre un spectre pratiquement similaire avec une différence d’intensité ; ce qui montre la possibilité de recharger le MnO2 pour une nouvelle utilisation. Mots clés :la phaseBirnessite, la phase pyrolusite, DRX sur poudre, identification de phas

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe

The RAS–RAF–MEK–ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels–Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and “on-target” staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus

Tetrazine-mediated bioorthogonal prodrug–prodrug activation

The selective and biocompatible activation of prodrugs within complex biological systems remains a key challenge in medical chemistry and chemical biology. Herein we report, for the first time, a dual prodrug activation strategy that fully satisfies the principle of bioorthogonality by the symbiotic formation of two active drugs. This dual and traceless prodrug activation strategy takes advantage of the INVDA chemistry of tetrazines (here a prodrug), generating a pyridazine-based miR21 inhibitor and the anti-cancer drug camptothecin and offers a new concept in prodrug activation.ISSN:2041-6520ISSN:2041-653

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. © 2016 The Author(s)