Universiẗat, Diss., 2002--Würzburg.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

Over the last twenty years, fragment-based drug discovery (FBDD) has emerged as a highly successful way to provide lead compounds for subsequent optimisation into drug candidates. Initial hits usually exhibit lower potency than those identified by more traditional techniques, such as High-Throughput Screening (HTS), but the optimisation phase of FBDD is highly efficient, thus providing superior lead-like compounds. The recent application of FBDD in a variety of protein kinase campaigns has successfully led to the identification of novel binding sites and highly efficient chemical ligands. This demonstrates the utility of the FBDD strategy against well-established kinase targets, where selectivity is otherwise challenging due to significant conservation of the ATP-binding site. Protein kinase CK2 is a ubiquitously expressed and constitutively active regulator of cell growth, proliferation and apoptosis. Elevated levels of CK2 protein and activity have historically been involved in human cancer, including lung, cervical and head and neck cancer types, and its overexpression is associated with worse prognosis. A number of CK2 inhibitors are currently available displaying activity against multiple cancers in vitro and in the clinic, however the majority of these candidates target the ATP-binding site and thus display poor selectivity in kinase panel assays. Here we explore the application of FBDD towards the development of potent and selective inhibitors of the catalytic α-subunit of CK2. This project exploits a novel, conserved binding site, named the αD pocket, for the generation of allosteric inhibitor molecules. Following structure-based optimisation of a potent inhibitor series, and characterisation of a previously unreported binding mode, a fragment linking strategy between the lead αD-site fragment and a low-affinity pseudosubstrate peptide is investigated. This work validates the utility of FBDD towards the discovery of new binding modes, presents a first in class CK2α allosteric inhibitor series and provides the first X-ray crystal structure of protein kinase CK2 in complex with a ligand binding in the substrate-binding channel.

The combination of bone morphogenetic protein 7 (BMP7) and neurotrophins (e.g. brain-derived neurotrophic factor, BDNF) protects septal neurons during hypoglycemic stress. I investigated the signaling mechanisms underlying this synergistic protection. BMP7 (5 nM) increased phosphorylation and nuclear translocation of BMP-responsive Smads 1/5/8 within 30 min in cultures of rat embryonic septal neurons. BDNF (100 ng/ml) enhanced the BMP7-induced increase in phospho-Smad levels in both nucleus and cytoplasm; this effect was more pronounced after a hypoglycemic stress. BDNF increased both Akt and Erk phosphorylation, but pharmacological blockade of these kinase pathways (with wortmannin and U0126, respectively) did not reduce the Smad phosphorylation produced by the BMP7+BDNF combination. Inhibitors of casein kinase II (CK2) activity reduced the (BMP7 + BDNF)-induced Smad phosphorylation, and this trophic factor combination increased CK2 activity in hypoglycemic cultures. These findings suggest that BDNF can increase BMP-dependent Smad phosphorylation via a mechanism requiring CK2. Preliminary results indicate that a cytoplasmic component robustly inhibits CK2. Protection of septal cholinergic neurons during a hypoglycemic stress is inhibited by a CK2 inhibitor and by a Phosphatidylinositol 3-kinase inhibitor, indicating that increases in CK2 activity and in Smad phosphorylation are only part on the protective mechanisms.

In the current thesis we analyse protein phosphorylation pathways in P. falciparum, the protozoa responsible for the most virulent form of malaria in order to both understand the role and scope of this protein modification in the parasite, and to explore its feasibility as a new drug target. With the aim to map phosphorylation pathways controlled by P. falciparum Casein Kinase 2 (PfCK2), we developed a new chemical-biological approach based on γ-modified ATP analogues bearing reporting groups on the transferred phosphate in order to selectively tag CK2 substrates. Despite being able to efficiently synthesise a small set of analogues, the data presented here shows that the P-N linkage bond between the nucleotide and the tag is stable during the assay conditions but not during the product analysis due to its acidic liability (e.g. with HPLC, MALDI); suggesting that a different type of linkage should be chosen in the future. Detailed characterisation studies of the parasite PfCK2 presented here showed a number of important features differing from human CK2. Docking analyses with a CK2 inhibitor showed that the PfCK2 ATP binding pocket is smaller than human CK2 due to the presence of Val116 and Leu45 which in the human kinase are replaced by more bulky isoleucine residues: Ile120 and Ile49. The difference between the human and parasite CK2 orthologues extends further to mechanisms of activation and regulation. Shown here is the autophosphorylation of PfCK2 that, unlike the human orthologue, occurs within subdomain I at Thr63. This autophosphorylation is essential for full catalytic activity. In addition we also showed that Thr63 phosphorylation regulates the interaction between the calalytic α-subunit and the regulatory β2-subunit. Here, we also presented evidence for tyrosine phosphorylated proteins in parasite infected red blood cells. PfCK2 can act as a dual specificity kinase phosphorylating P. falciparum Minichromosome Maintenance protein 2 (PfMCM2) on Tyr16 in vitro. It is therefore possible that PfCK2 may contribute to tyrosine phosphorylation within the parasite. Finally, we also reported a study regarding MCM2-Ser13 phosphorylation which successfully identified PfCK1 as the kinase responsible for this event.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Oct. 8, 2008). Includes bibliographical references. Available online via the OhioLINK ETD Center. Also available in print.

Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 86-100).

Universiẗat, Diss., 2005--Köln.

En 2015, on estime le nombre de nouveaux cas de cancer à plus de 385000 et le nombre de décès à 149500. Ces chiffres, plus élevés chez l'homme que chez la femme, connaissent ces dernières années une nette recrudescence chez les patients de sexe féminin. Globalement, la principale difficulté pour lutter contre les cancers se situe dans la capacité à les détecter avant qu'ils ne métastasent et dans les nombreux phénomènes de résistance aux traitements chimiothérapeutiques.De par son implication dans la croissance des cellules cancéreuses, le réseau vasculaire tumorale représente une cible intéressante ainsi qu'une alternative prometteuse aux traitements actuels. Ainsi, la classe de composés qualifiés de VDAs (pour « Vascular Disrupting Agents ») visent les cellules endothéliales ainsi que les péricytes, provoquant ainsi des phénomènes d'ischémie et de nécrose cellulaire. Un exemple de ce type de composés n'est autre que la Combrétastatine A-4 (ou CA-4), qui est un composé naturel extrait d'un arbuste sud-africain, le Combretum caffrum. Cette molécule a été pour la première fois étudiée par G.R. Pettit en 1989 qui a alors démontré qu'elle pouvait inhiber très efficacement la polymérisation des dimères α et β de tubuline en microtubules, résultant alors en la non-formation du cytosquelette, et l'apoptose de la cellule. Le développement de nouveaux analogues de la CA-4 s'avère crucial car ce composé dispose non seulement d'une solubilité réduite mais peut aussi être instable lorsqu'il est administré. Le travail effectué lors de cette thèse a donc consisté dans un premier temps en la synthèse de nouveaux dérivés de la CA-4 en remplaçant le noyau B par différents hétérocycles et en effectuant plusieurs pharmacomodulations ; ces derniers étant alors évalués pour leurs propriétés inhibitrices de la polymérisation de la tubuline et antiprolifératives. Avec plus de 24 millions de personnes touchées dans le monde, les pathologies neurodégénératives représentent un problème majeur de santé publique. Il est également très important de considérer l'incidence future de ces pathologies, et on estime à plus de 42 millions le nombre de personnes qui seraient atteintes par ce fléau d'ici à 2020. La famille des protéines kinases DYRK a fait l'objet ces dernières années d'une attention toute particulière de par son implication dans de nombreux phénomènes physiologiques et notamment au niveau des phases primaires du développement du système nerveux central. Plus spécifiquement, les kinases DYRK1A et DYRK1B ont été étudiées de par leurs implications dans de nombreux cancers (notamment le glioblastome) et certaines pathologies neurocérébrales. Le dérèglement de l'expression de ces protéines pourrait être la cause de retards mentaux, du développement de la maladie d'Alzheimer, de la trisomie 21, ainsi que de phénomènes de résistance. Nous avons identifié au sein de notre laboratoire une molécule inhibant ces 2 kinases. Initialement développé dans le but d'inhiber la protéine kinase CK2 (caséine kinase 2), notre composé a montré une activité inhibitrice et une sélectivité sur DYRK1A et DYRK1B. De par son activité, cette molécule est aujourd'hui notre composé « hit » et nous visons, à travers les travaux de cette thèse, la synthèse de multiples analogues dans le but d'améliorer l'activité initiale mais aussi sa spécificité sur DYRK1A ou sur DYRKB. Dans cette optique, nous avons réalisé différentes pharmacomodulations de nos composés dits « indénobenzo[b]thiophènes » et étudier les effets de tels composés sur DYRK1A, DYRK1B, DYRK2 mais aussi sur CK2. Ceci nous a permis d'affiner nos connaissances vis-à-vis des kinases de type DYRK et de sélectionner les molécules les plus prometteuses afin de (i) réaliser une étude autour de leurs caractéristiques physico-chimiques (Log P, solubilités dans différents solvants), (ii) d'analyser leurs comportements au niveau de la barrière hémato-encéphalique (BHE) et (iii) de réaliser des nano-encapsulations / Up to now, cancer is the second deadliest pathology in the World and is still considered as one of the most challenging public health issue. Globally, it has been assessed to be the main pathologic cause of death by the World Health Organization. This bad prognosis is partly due to the ability of cancer cells to give metastases but also to resistances phenomenon impeding drastically the effect of chemotherapeutic and radiotherapeutic treatments. As a consequence, there is currently a critical lack of effective treatments which would completely eradicate tumor cells, with minimal side effects. In spite of some difficulties in this competitive research area, the discovery of cancer therapeutics remains stimulating and we aim to achieve the synthesis of novel anticancer agents.Given its pivotal role in tumor growth and survival, the tumor vasculature represents an attractive target for anticancer therapy. Apart from angiogenesis inhibitors that compromise the formation of new blood vessels, the class of vascular disrupting agents (VDAs) targets endothelial cells and pericytes of the already established tumor vasculature, resulting in tumor ischemia and necrosis. A striking example of VDA is the combretastatin A-4, also known as CA-4, which was originally isolated from the bark of the South African willow tree Combretum caffrum by the American scientist G.R. Pettit in 1989. These products demonstrated to be efficient against a wide array of cancers such as breast, colon, lung or ovarian cell lines. New CA-4 analogs containing different heterocycles instead of the hydroxymethoxy substituted pharmacomodulable B ring were prepared and evaluated for their in cellulo tubulin polymerization inhibition and antiproliferative activities. In the other hand, tumor cell survival is a complex process which remains poorly understood. As part of the survival machinery, chemoresistances and DNA repairs are central elements regulated by a prosurvival/proapoptotic signal balance. Protein kinases are known to be directly involved in this signal transmission through molecular interactions. As such, DYRK kinases and most specifically DYRK1A/1B, were part of numerous recent studies due to their involvement in cancer and other pathologies. DYRK1B (also called Mirk kinase) is an ubiquitous kinase which was proved to be over-expressed in many cancers such as pancreatic, ovarian or colon. Its involvement as a regulator of DNA repair and tumor cell survival was assessed, phosphorylating specifically serine, threonine and tyrosine residues. Also, another closely related isoform known as DYRK1A, was mapped in the Down syndrome critical region located itself on chromosome 21. Interestingly, this kinase was not only uncovered to play a fundamental role in glioblastomas survival but was also associated with abnormal brain development in early stages and mental retardations. Particularly, DYRK1A was found to hyperphosphorylate microtubule-associated tau protein, resulting into genesis of neurofibrillary tangles. As a consequence, DYRK1A has become one of the most targeted proteins in order to improve cognitive impairment of patients suffering from Down syndrome or Alzheimer’s disease. Initially designed to target protein kinase CK2, one of our molecules was also tested on DYRK kinases. This compound exhibited a strong activity against DYRK1A/DYRK1B whilst being inactive on other protein kinases. Consequently, it was considered as our hit compound and (i) we synthetized derivatives as dual or single inhibitors of DYRK1A/DYRK1B, (ii) evaluated their biological activities (with emphasis on the blood brain barrier), and (iii) finally synthesized nanoparticles loaded with our inhibitors