Aminopyrazine Inhibitors Binding to an Unusual Inactive Conformation of the Mitotic Kinase Nek2: SAR and Structural Characterization†

We report herein the first systematic exploration of inhibitors of the mitotic kinase Nek2. Starting from HTS hit aminopyrazine 2, compounds with improved activity were identified using structure-based design. Our structural biology investigations reveal two notable observations. First, 2 and related compounds bind to an unusual, inactive conformation of the kinase which to the best of our knowledge has not been reported for other types of kinase inhibitors. Second, a phenylalanine residue at the center of the ATP pocket strongly affects the ability of the inhibitor to bind to the protein. The implications of these observations are discussed, and the work described here defines key features for potent and selective Nek2 inhibition, which will aid the identification of more advanced inhibitors of Nek2

Thréonine synthase et aspartate kinase d'A. thaliana (études structurales de deux enzymes allostériques contrôlées par la S-adénosylméthionine)

La Thréonine synthase (TS) et l'Aspartate kinase (AK) sont deux enzymes aIlostériques impliquées dans la voie de synthèse des acides aminés dérivés de l'aspartaie. Deux isoformes de TS et trois isoformes d'AK, prèsentes chez Arabidopsis thaliana, ont été clonées et étudiées au laboratoire. La caractérisation de ces enzymes a mis en évidence, pour les deux TS et pour une isoforme d'AK (AK 1), une sensibilité à la S-adénosylméthionine spécifique aux plantes. Alors que l'activité TS est stimulée par la SAM, l'inhibition de l'activité AKI est inhibée par la lysine et la SAM en synergie. Afin d'observer au niveau moléculaire le contrôle de ces activités enzymatiques et de mieux comprendre les différences de sensibilité entre les plantes et les microorganismes, une étude structurale de ces deux enzymes a été entreprise. La résolution de la structure de la TS en présence de son cofacteur, le pyridoxal phosphate (PLP), a permis de mettre en évidence une position non classique du PLP pour une enzyme dépendante du PLP de type II, rendant compte de la faible activité et de la faible affinité de la TS pour son substrat en absence d'activateur. La résolution de la structure de la TS en présence de PLP et de SAM a montré que la fixation de l'activateur induit des changements de conformation asymétriques du dimère de TS conduisant à l'activation d'un seul monomère. La structure de l'AK 1 révèle pour la première fois l'organisation structurale d'une Aspartate kinase et présente la forme inhibée de l'enzyme. Cette enzyme composée d'un domaine catalytique et d'un domaine régulateur de type ACT, présente un mode d'organisation nouveau pour une enzyme possédant des motifs ACT.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Allosteric threonine synthase: reorganization of the PLP site upon asymmetric activation through SAM binding to a novel site

French abstract: La thréonine synthase est une enzyme à pyridoxal-phosphate qui catalyse la dernière étape de la synthèse de la thréonine chez les plantes et les microorganismes. Chez les plantes, cette enzyme est activée par le produit terminal de la synthèse de la méthionine, la S-Adénosylméthionine (SAM). Des chercheurs du laboratoire de Physiologie Cellulaire végétale ont obtenu les structures cristallographiques de la thréonine synthase de plante en absence et en présence de SAM. La comparaison de ces structures montre que la SAM active l'enzyme en induisant d'importants changements de conformation conduisant à une réorientation du pyridoxal-phosphate du site actif

Amino acid biosynthesis: New architectures in allosteric enzymes.

This review focuses on the allosteric controls in the Aspartate-derived and the branched-chain amino acid biosynthetic pathways examined both from kinetic and structural points of view. The objective is to show the differences that exist among the plant and microbial worlds concerning the allosteric regulation of these pathways and to unveil the structural bases of this diversity. Indeed, crystallographic structures of enzymes from these pathways have been determined in bacteria, fungi and plants, providing a wonderful opportunity to obtain insight into the acquisition and modulation of allosteric controls in the course of evolution. This will be examined using two enzymes, threonine synthase and the ACT domain containing enzyme aspartate kinase. In a last part, as many enzymes in these pathways display regulatory domains containing the conserved ACT module, the organization of ACT domains in this kind of allosteric enzymes will be reviewed, providing explanations for the variety of allosteric effectors and type of controls observed

A Novel Organization of ACT Domains in Allosteric Enzymes Revealed by the Crystal Structure of Arabidopsis Aspartate Kinase

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine–sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III

Structural and functional integration of the PLCγ interaction domains critical for regulatory mechanisms and signaling deregulation

Multidomain proteins incorporating interaction domains are central to regulation of cellular processes. The elucidation of structural organization and mechanistic insights into many of these proteins, however, remain challenging due to their inherent flexibility. Here, we describe the organization and function of four interaction domains in PLCγ1 using a combination of structural biology and biochemical approaches. Intramolecular interactions within the regulatory region center on the cSH2 domain, the only domain that also interacts with the PLC-core. In the context of fibroblast growth-factor receptor signaling, the coordinated involvement of nSH2 and cSH2 domains mediates efficient phosphorylation of PLCγ1 resulting in the interruption of an autoinhibitory interface by direct competition and, independently, dissociation of PLCγ1 from the receptor. Further structural insights into the autoinhibitory surfaces provide a framework to interpret gain-of-function mutations in PLCγ isoforms linked to immune disorders and illustrate a distinct mechanism for regulation of PLC activity by common interaction domains

Design of potent and selective hybrid inhibitors of the mitotic kinase Nek2 : structure−activity relationship, structural biology, and cellular activity

We report herein a series of Nek2 inhibitors based on an aminopyridine scaffold. These compounds have been designed by combining key elements of two previously discovered chemical series. Structure based design led to aminopyridine (R)-21, a potent and selective inhibitor able to modulate Nek2 activity in cells

Design of Potent and Selective Hybrid Inhibitors of the Mitotic Kinase Nek2: Structure–Activity Relationship, Structural Biology, and Cellular Activity

We report herein a series of Nek2 inhibitors based on an aminopyridine scaffold. These compounds have been designed by combining key elements of two previously discovered chemical series. Structure based design led to aminopyridine <b>(</b><i><b>R</b></i><b>)-21</b>, a potent and selective inhibitor able to modulate Nek2 activity in cells