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Meschini, Elisa. "Purine-based dual inhibitors of CDK2 and CDK7." Thesis, University of Newcastle Upon Tyne, 2011. http://hdl.handle.net/10443/1363.
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Cyclin-Dependent Kinases (CDKs) play a fundamental role in eukaryotic cell cycle progression, particularly at cell cycle checkpoints, and are therefore important targets for anticancer drug discovery. Activation of CDK2 in complex with Cyclin A regulates entry into S phase of the eukaryotic cell cycle. CDK7, a dual-function enzyme, acts both as a CDK-Activating Kinase (CAK) and as a component of the general transcription factor TFIIH. However, experiments with MAT1-knockdown mice have shown that cell cycle arrest by CAK inhibition would not be detrimental for transcriptional activity in non-dividing cells, as CDK9 in complex with Cyclin T can perform transcriptional duties in the absence of TFIIH. Previous studies have resulted in the identification of NU6102 (1, IC50 mM = 0.005 (CDK2), 4.4 (CDK7)) as a potent and selective CDK2 inhibitor, and NU6247 (2, IC50 mM = 0.12 (CDK2), 0.23 (CDK7)) as an equipotent CDK2/7 inhibitor. It was shown that the sulfonamide group of 1 confers potency and selectivity for CDK2, whereas the pendant piperazinyl substituent of 2 diminishes CDK2 actvity whilst improving activity versus CDK7. S NH N N NH N O O O 2 N N AccordinglyAs part of the work described in the present thesis, sulfonamide 3 (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)) was synthesised and found to be is a potent CDK2 inhibitor, but with some CDK7-inhibitory activity (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)). Further elaboration of the side-chain function has enabled the development of structure-activity relationships (SARs), and the identification of purines (e.g. 4, IC50 mM = 2.6 (CDK2), 0.56 (CDK7)) exhibiting some selectivity for CDK7, albeit with a loss of potency. 4 Subsequent SAR studies conducted on 2 have enabled the following observations to be made: firstly, the purine 6-cyclohexylmethoxy substituent is necessary for activity, with the corresponding 6-unsubstituted purine (5, IC50 mM = 46.9 (CDK2), 20.8 (CDK7)) exhibiting a 100-fold loss of potency against both CDK2 and CDK7. A terminal basic group (e.g. piperazinyl in 2) is required for activity, as replacement by a cyclohexyl substituent results in loss of activity against both kinases (6, 11% inhibition at 10 mM (CDK2), 13% inhibition at 100 mM (CDK7)). The sulfone linker is not a prerequisite for CDK7 activity, with the simple alkylpiperazine derivative (7, IC50 mM = 0.48 (CDK2), 0.51 (CDK7)) exhibiting comparable potency and selectivity. Finally, there appears to be some opportunity for expansion into the gatekeeper pocket of CDK7 by introducing small substituents at the purine C-8 position, with the potential for selectivity over CDK2 (8, 47% inhibition at 100 mM (CDK2), IC50 = 5 mM (CDK7)). Isolation and biological evaluation of the vinyl sulfone 9, an intermediate in the synthesis of 2, indicated a time-dependent inhibition of CDK2, suggesting that 9 is an irreversible inhibitor of CDK2. This would be the first reported irreversible inhibitor of a cyclin-dependent kinase, and therefore the activity of the compound against CDK2 was investigated using protein crystallography and site-directed mutagenesis 5 techniques. From these studies, encouraging evidence has emerged that 9 acts as an irreversible inhibitor of CDK2, covalently binding to a lysine residue within the ATP-binding pocket.
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Costa, Elena. "Design, synthesis and biochemical evaluation of novel CK2 and CDK2 inhibitors." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3423919.
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Kinases are a class of enzymes, which catalyse the transfer of the terminal phosphate of a molecule of ATP, or more rarely of GTP, to a protein that acts as a substrate. This process is reversible and is maintained by the presence of other enzymes, namely the phosphatases, which catalyse the reverse reaction. An imbalance between these enzymes plays a crucial role in the occurrence of diseases, which is attributable to abnormal levels of phosphorylation.
The aim of this research was the design and synthesis of novel potent and selective benzimidazole-based inhibitors of Casein Kinase 2 (CK2) and the design and synthesis of pyrimidine-based inhibitors of Cyclin-dependent Kinase 2 (CDK2). This research was carried out at the University of Padua in collaboration with Newcastle University.
CK2 is a ubiquitous, highly pleiotropic Ser/Thr protein kinase, which has been implicated in every stage of cell cycle progression. DRB was the first CK2 ATP-mimetic inhibitor identified (IC50 = 23 μM) and was also the starting point for a new fruitful class of polyhalogenated benzimidazole-based compounds.
The synthesis of CK2 bifunctional inhibitors, which could simultaneously interact with the ATP pocket and the substrate-binding domain, was proposed. A polyhalogenated benzimidazole scaffold was used as starting point and was then coupled to different peptidic chains.
CDK2, belongs to a family of Ser/Thr kinases. In complex with regulatory subunits, cyclin E or A, CDK2 is involved in driving the cell cycle. The overexpression of CDK2 and the deregulation of cyclins associated with CDK2 are often observed in cancer. NU6300 (2), a purine-based CDK2 inhibitor (IC50 = 63 nM), can be considered the first CDK2 irreversible inhibitor due to a covalent interaction between the vinyl sulfone moiety and Lys89 within the ATP-binding domain of CDK2.
Structure-activity relationship (SAR) studies on the O6-alkylguanine series revealed that the purine pharmacophore is not a prerequisite for CDK2-inhibitory activity. Hence, a series of pyrimidine-based CDK2 inhibitors was synthesised. Structural modifications at the pyrimidine scaffold were then considered in order to investigate the binding affinity, potency and selectivity of the novel CDK2 inhibitors.
All new compounds were evaluated for CK2- and CDK2-inhibitory activity, with promising inhibitors being subjected to more comprehensive biological studies.
Concerning CDK2 inhibitors, docking and co-crystallisation studies were also performed in order to rationalise the results and propose new compounds to be synthesised<br>Le chinasi sono una classe di enzimi in grado di catalizzare il trasferimento di un gruppo fosfato terminale di una molecola di ATP, o più raramente di GTP, ad una proteina che agisce da substrato. Questo processo è reversibile, ed è mantenuto tale dalla presenza di altri enzimi, le fosfatasi, che catalizzano la reazione inversa. Uno squilibrio tra questi enzimi gioca un ruolo rilevante nell’incidenza di diverse patologie, le quali sono attribuibili ad elevati livelli di fosforilazione.
Questo lavoro di ricerca ha avuto lo scopo di progettare e sviluppare nuovi potenti e selettivi inibitori di casein chinasi 2 (CK2) a struttura benzimidazolica, e progettare e sviluppare nuovi inibitori a struttura pirimidinica di chinasi ciclina dipendente 2 (CDK2). La ricerca è stata svolta presso l’Università degli Studi di Padova (IT) in collaborazione con l’Università di Newcastle (UK).
CK2 è una Ser/Thr chinasi ubiquitaria, altamente pleiotropica e coinvolta in ogni fase della progressione del ciclo cellulare. DRB è stato il primo inibitore di CK2 ATP-mimetico ad essere identificato (IC50 = 23 μM), e anche punto di partenza per la sintesi di una nuova classe di inibitori poli-alogenati a struttura benzimidazolica.
E’ stata proposta la sintesi di inibitori bifunzionali di CK2, i quali si pensa vadano ad interagire simultaneamente con la tasca catalitica dell’ATP e con il sito di legame del substrato. Come punto di partenza è stato scelto uno scaffold benzimidazolico poli-alogenato, il quale è stato poi legato ad una serie di sequenze peptidiche.
CDK2 appartiene anch’essa ad una famiglia di chinasi Ser/Thr-dipendenti. CDK2, complessandosi con subunità regolatorie (ciclina A o E), è coinvolta nella progressione del ciclo cellulare. Una caratteristica ricorrente nei tumori sono la sovraespressione di CDK2 e la deregolazione delle cicline ad essa associate. NU6300, un inibitore a scaffold purinico di CDK2 (IC50 = 63 nM), è considerato il primo inibitore di CDK2 irreversibile, questo grazie all’interazione covalente tra il gruppo vinilsulfonico e un residuo di lisina (Lys89), presente all’interno della tasca catalitica dell’enzima CDK2.
Studi di relazione struttura-attività (SAR) riguardanti una serie di O6-alchilguanine hanno rivelato che il farmacoforo di tipo purinico non è un requisito essenziale per l’attività inibitoria su CDK2. Per questo è stata sviluppata una serie di inibitori CDK2 di tipo pirimidinico. Sono state quindi considerate alcune modifiche strutturali allo scaffold pirimidinico, in modo da esaminare l’affinità di binding, la potenza e la selettività dei nuovi inibitori di CDK2.
I nuovi inibitori sono stati valutati per la loro attività inibitoria su CK2 e CDK2 rispettivamente. Gli inibitori più promettenti sono stati poi sottoposti ad ulteriori studi biologici. Per quanto riguarda gli inibitori di CDK2 sono stati effettuati studi di docking molecolare e di co-cristallizzazione per razionalizzare i risultati ottenuti e pianificare nuove vie di sintesi
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Bacevic, Katarina. "Cdk2 as a model for studying evolutionary selection and therapeutic responses in proliferating cancer cells." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT184.
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Les kinases cycline-dépendantes (CDK) sont des protéines régulatrices essentielles du cycle cellulaire. Elles contrôlent la prolifération cellulaire et sont souvent déréglées dans les cancers. De nombreux inhibiteurs de CDKs ont été élaborés et sont actuellement le sujet d'essais cliniques. Bien que Cdk1 soit un régulateur essentiel de cycle cellulaire, Cdk2 n’est pas nécessaire pour la progression du cycle cellulaire, mais favorise la tumorigenèse. Par conséquent, Cdk2 est une cible thérapeutique prometteuse. L’utilisation des inhibiteurs de kinases pour modifier la prolifération cellulaire s’apparente à appliquer une sélection Darwinienne. Cette sélection peut être modélisée mathématiquement. Cette approche a montré que des avantages sélectifs, mêmes marginaux, peuvent être d'une importance majeure dans la compétition inter-cellulaire et la progression du cancer. Selon ce principe, nous avons fait l’hypothèse que le fait que la Cdk2 ait un rôle mineur dans la progression du cycle cellulaire lui confèrerait le statut de cible pertinente pour une thérapie du cancer. Selon cette hypothèse, son inhibition serait bien tolérée, permettant de réduire le niveau d’activité CDK et ainsi agir contre la prolifération déréglée des cellules. Nous avons supposé qu’au lieu d’éliminer entièrement les cellules les plus prolifératives, qui seraient les plus sensibles au traitement, il serait potentiellement intéressant de les exploiter pour concurrencer l’émergence des cellules résistantes, moins prolifératives. L'utilisation d'un traitement continu à faible dose avec les inhibiteurs Cdk2 pourrait permettre de maintenir cet équilibre. L'objectif de la thèse était d'étudier si Cdk2 confère un avantage prolifératif aux cellules cancéreuses, si les cellules peuvent développer une résistance aux inhibiteurs de CDKs, et si oui, déterminer quels étaient les mécanismes de résistance qui permettent de réduire le « fitness » des cellules prolifératives. Pour répondre à ces questions, nous avons généré des lignées cellulaires ayant des degrés variés de résistance à un inhibiteur spécifique de Cdk2 (inhibant également Cdk1 à des concentrations élevées). Nous avons caractérisé leur capacité à proliférer en comparaison avec des cellules parentales et des cellules isogéniques n’exprimant plus Cdk2 en raison d’un « knock-out » du gène. Bien que dans ces premières cellules le gène Cdk2 est retrouvé non muté et que l'expression de la protéine Cdk2 reste inaltérée, l'activité kinase de Cdk2 est diminuée. Les cellules résistantes à l’inhibiteur prolifèrent efficacement in vitro. Cependant, lors des expériences de compétition avec les cellules parentales, sensibles aux inhibiteurs, elles sont perdantes. Ceci montre que le développement d’une résistance à un inhibiteur de kinase entraîne un désavantage sélectif. Malgré une prolifération normale en l’absence de compétiteurs, ce désavantage est mis en évidence dans une population mixte, validant ainsi l’hypothèse de départ. Nous avons constaté que les Cdk2 KO et les cellules résistantes à l’inhibiteur (R50) ont un métabolisme altéré. Ces cellules sont sensibles à l'épuisement des nutriments et du glucose ainsi qu’à l'hypoxie, malgré un taux de consommation d'oxygène normal, ce qui indique une augmentation de la glycolyse aérobique. Les cellules R50 surexpriment la protéine Cdk6, ce qui peut contribuer à la résistance à l'inhibition Cdk2. De plus elles sont sensibles à l’inhibition des Cdk4/6, cibles référencées dans le traitement de certaines classes de cancer du sein. Enfin, les cellules Cdk2 KO présentent un point de contrôle de la phase S perturbé. Ces résultats suggèrent que des inhibiteurs pharmacologiques ciblant Cdk2 pourraient être synergique avec d’autres traitements, par exemple l’inhibition concomitante de la réplication de l'ADN, de la glycolyse, ou de Cdk6. Cela pourrait ainsi diminuer la prolifération des cellules cancéreuses et empêcher l’émergence d'une résistance thérapeutique<br>Cyclin-dependent kinases (Cdk) are essential regulators of the cell cycle that support cell proliferation and are often deregulated in cancer. While Cdk1 is an essential regulator of the cell cycle, Cdk2 is not required for cell cycle progression but promotes tumorigenesis. Therefore, Cdk2 is a promising drug target. Many Cdk inhibitors have been developed and are currently undergoing clinical trials. Darwinian selection can be modelled mathematically, and such studies have shown that even marginal selective advantages can be of great importance in outcomes of cell-cell competition and cancer progression. We hypothesised that the non-essential role of Cdk2 for cell cycle progression may mean that it is a good target for cancer therapy as continual inhibition should be tolerated and should counteract deregulated cell proliferation in cancer. However, as with all chemotherapeutic agents, the development of clinical resistance is likely. We further hypothesized that applying a low-dose treatment with Cdk2 inhibitors should minimize chances of developing resistance, by maintaining competition between robustly proliferating cells that are sensitive to treatment, and resistant cells.The aim of the thesis was to investigate whether Cdk2 confers a proliferative advantage to cancer cells, whether cells can develop resistance to Cdk inhibitors, and if so, whether the mechanisms allowing resistance reduce cellular proliferative fitness.To answer these questions, we have created cell lines with varying degrees of resistance to a selective Cdk2 inhibitor (that at high doses, also inhibits Cdk1) and have characterised their proliferation capacity in comparison with parental cells and isogenic Cdk2 knockout cells. Although in these cells the Cdk2 gene is not mutated and the expression of Cdk2 protein remained unaltered, the kinase activity of Cdk2 is decreased. Similarly, Cdk2 gene knockout (Cdk2 KO) cells have reduced sensitivity to Cdk2 inhibition. Inhibitor-resistant cells proliferate efficiently but are outcompeted by parental, inhibitor-sensitive cells in competition experiments, confirming that inhibitor resistance entails a selective disadvantage. We found that the proliferation of both Cdk2 knockout and inhibitor-resistant (R50) cells is sensitive to nutrient and glucose depletion as well as hypoxia, despite a normal oxygen consumption rate, indicating increased aerobic glycolysis. R50 cells have highly upregulated Cdk6, which may contribute to resistance to Cdk2 inhibition. Moreover, they are sensitised to Cdk4/6 inhibition, which is currently authorised as a treatment for some classes of breast cancer. Finally, Cdk2 knockout cells have an impaired S-phase checkpoint. These results suggest that pharmacological inhibitors targeting Cdk2 might be synthetically lethal with other treatments, eg inhibition of DNA replication, of glycolysis, or of Cdk6. This might diminish cancer cell proliferation and prevent emergence of therapeutic resistance
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Turner, David Michael. "Design and synthesis of purine isosteres as inhibitors of Nek2 and CDK2." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1908.
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Nek2 and CDK2 are serine/threonine protein kinases that are implicated in cell cycle control and cancer. The Nek family of enzymes contains 11 known serine/threonine protein kinases (Nek1-11) and are involved in mitotic cell cycle control. There is evidence that of these 11 kinases, Nek2, Nek6, Nek7 and Nek9 play an important role in the regulation of mitotic events through microtubule control. Particular interest has been placed upon Nek2, which has been shown to have a critical role in mitosis, assisting centrosome disjunction. 6-Ethnyl purine 39, was identified as a submicromolar irreversible inhibitor of Nek2 (IC50 = 150 nM), exhibiting good selectivity over other members of the Nek family. It was believed that this compound formed a triplet of H-bonds with the amino acids of the Nek2 ATP-binding site hinge region via the 2-amino N-H, the purine N3 and the imidazole N9H, allowing an initial noncovalent binding interaction, which facilitates subsequent covalent modification. To validate this binding motif and to act as control compounds, N-methylated purines 52-54 were synthesised, along with the 2-phenoxypurine 55 and the 2-benzylpurine 56. These compounds were all essentially inactive in the Nek2 inhibition assay, which demonstrated that the purine 2-amino N-H group and imidazole N9H were essential for non-covalent binding interactions. 5 To investigate the influence of the purine heterocycle upon Nek2 inhibitory activity and ethynyl group reactivity, heterocyclic derivatives of 39 were synthesised. Deazapurines 87 and 88 were weakly active in the Nek2 inhibition assay (IC50 = 30 μM and 24 μM, respectively), whilst pyrazolopyrimidine 89 and triazolopyrimidine 90 had sub-micromolar activity comparable with the parent purine 39. The 5- formylpyrimidine 101 and triazine 91 were modest inhibitors of Nek2 (IC50 = > 10 μM and 17 μM, respectively). By contrast, pyrimidine 95 (10% inhibition at 100 μM) demonstrated weak Nek2 inhibitory activity. Quantitative 1H NMR (q1H-NMR) kinetic studies were performed to model the reaction of ethynyl-functionalised heterocycles with Cys-22 at the Nek2 ATP-binding site. Compounds 54, 56, and 88-89 were reacted with N-acetylcysteine methyl ester in DMSO-d6 at 24 oC under pseudo first order reaction conditions. The most reactive compounds were triazolopyrimidine 90 and pyrazolopyrimidine 89. The 2- benzylpurine 56 and N-methylanilinopurine 54 had moderate reactivity, comparable with the parent purine 39. The least reactive compound was pyrrolopyrimidine 88, which was approximately 150-fold less reactive than purine 39. 6 CDK2 is a member of the cyclin-dependent kinase (CDK) family of enzymes and a mediator of cell cycle progression. Of the 11 known human CDKs, four (CDK1, CDK2 CDK4 and CDK6) have been directly implicated in cell cycle regulation. Mutation of genes coding for CDKs are common in a variety of cancers, making CDKs attractive chemotherapeutic targets. Purines 249 and 37 were identified as moderate (IC50 = 17 μM) and potent (IC50 = 5 nM) ATP-competitive inhibitors of CDK2, respectively. The focus of this research was to determine whether subtle changes to the core heterocycle would allow retention of CDK-inhibitory activity. CDK2 inhibitors based on the pyrazolopyrimidine and triazolopyrimidine heterocycles (256, 252, 257 and 253) were of comparable potency with the corresponding purines (249 and 37). The most potent compound was triazolopyrimidine 253 with an IC50 value of 3 nM against CDK2. Interestingly, the pyrrolopyrimidine 255 proved only weakly active (IC50 > 100 μM), whereas the functionalised pyrrolopyrimidine 251 had potent CDK2 inhibitory activity (IC50 = 26 nM). Finally, imidazopyridine 254 exhibited activity comparable with purine 249; however, imidazopyridine 250 was found to be approximately 30-fold less potent (IC50 = 140 nM) than the parent 37, for reasons that remain unclear. N.B. diagrams not included in this abstract
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Lebraud, Honorine. "Structure-based design and synthesis of inhibitors of the mitotic kinases Nek2 and CDK2." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2756.
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Cyclin-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.
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Löschmann, Nadine [Verfasser]. "Untersuchung des CDK2, 7 und 9 Inhibitors SNS-032 in chemosensitiven und chemoresistenten Neuroblastomzellen / Nadine Löschmann." Gießen : Universitätsbibliothek, 2015. http://d-nb.info/1071801066/34.
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Ghobadpour, Nazanin. "Role of CDK4/CDK6 inhibitors: Ribociclib, Palbocilcib and Abemacilcib in treatment of metastatic breast cancer." Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-105537.
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Breast cancer is the most common cancer among women in Sweden and globally. Socio-demographic, genetic and productive factors together with some endogenous and exogenous hormones and lifestyle are some of the risk factors that can cause this type of cancer. There is no cure for metastatic breast cancer but the treatment goal is to control the disease and prolong the survival. Treatment options in metastatic breast cancer are exactly like the ones in primary breast cancer. Targeted cancer therapy is a treatment that targets either one or some specific characters of the cancer. Serine-threonine kinases called CDK4/6 inhibitors like Ibrance with chemical name palbociclib, Kisqali with chemical name ribociclib and Verzenios with chemical name abemaciclib are used for target therapy against metastatic breast cancer. CDK4/6 inhibitors in combination with aromatase inhibitors, estrogen receptor down regulators or selective receptor modulators are also used to address the treatment resistant metastatic breast cancers. The aim of this thesis is to investigate the role and the effect of CDK4/6 inhibitors in the treatment of metastatic breast cancer. Six randomized controlled trial studies were selected from the PubMed data base. Results from the trial analysis showed equal effects and relative same adverse event profiles between those three mentioned CDK4/6 inhibitors. The primary endpoint, progression free survival and secondary endpoints including overall survival, clinical benefit rate, overall response rate and safety were investigated in these studies. The duration of progression free survival (PFS) and overall response rate (ORR) was longer and improved in most studies however improvement in overall survival (OS) was not achieved. More research studies are needed to determine optimal treatment for patients with metastatic breast cancer who are medicated by CDK4/6 inhibitors.<br>Bröstcancer är den vanligaste cancerformen bland kvinnor i Sverige och globalt. Sociodemografiska, genetiska och reproduktiva faktorer samt vissa endogena och exogena hormoner och livsstil är några riskfaktorer som kan orsaka denna typ av cancer. Metastatisk bröstcancer innebär att cancer har spridit sig bland andra organ i kroppen bland annat ben, hjärna, lever och lungor. Det finns inget botemedel mot metastatisk bröstcancer men målet är att kontrollera sjukdomen och förlänga överlevnaden och behandlingsalternativ för detta är exakt som den i den primära bröstcancern. Målspecifik cancerterapi är en behandling som riktar sig mot någon specifik karaktär hos cancercellerna. En klass av läkemedel består av en stor familj av serintreoninkinaser som kallas CDK4/6-hämmare: Ibrance (palbocilcib), Kisqali (ribocilcib) och Verzenios (abemaciclib) används mot metastaserad bröstcancer. Syftet med det här examensarbetet var att undersöka rollen samt effekten av CDK4/6 hämmare vid behandling av metastatisk bröstcancer. Sex randomiserade kliniska studier erhölls från artikelsökningar i databasen PubMed. Resultat från artikelanalyser visade likvärdiga effekter samt relativt samma biverkningsprofil mellan de tre ovannämnda CDK4/6 inhibitorerna. Den primära utfallsvariabeln var progressionsfri överlevnad och de sekundära utfallsvariablerna inkluderade total överlevnad, klinisk nytta, den totala svarsfrekvensen och säkerhet undersöktes i dessa studier. Varaktigheten av progressionsfri överlevnad (PFS) samt den totala svarsfrekvensen (ORR) var längre och förbättrades i nästan alla studier medan den totala överlevnaden (OS) kunde inte fastställas då inte tillräckligt med data kunde samlas in. Ytterligare undersökningar behövs för att förstå den fulla mekanismen bakom resistensutveckling samt utöka strategier för att minska negativa biverkningar av CDK4/6 hämmare vid behandling av patienter vid metastatisk bröstcancer.
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Cot, Emilie. "Inhibition chimique des Cdk : mécanisme biochimiques et conséquences cellulaires." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20054.
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Les kinases dépendantes des Cyclines (Cdk) contrôlent le déroulement du cycle cellulaire, mais leur étude est difficile car des mécanismes de compensation se développent lorsqu'une Cdk est absente. Cdk2 est principalement impliquée dans la phase de réplication de l'ADN, cependant l'ablation génétique de Cdk2 chez la souris n'a pas d'effet sur leur développement: les fonctions de Cdk2 sont compensées par d'autres Cdk. L'inhibition chimique permet de bloquer une Cdk et de limiter les compensations. Pour étudier les rôles de Cdk2, nous l'avons inhibé avec NU6102 qui sélectif pour Cdk2 dans le modèle xénope. Nous avons aussi développé des mutants de Cdk2 résistants à NU6102 pour vérifier sa sélectivité et avons cherché à mieux comprendre les paramètres qui déterminent l'affinité entre Cdk2 et un ligand. D'autre part, nous déterminons in vitro que NU6102 serait plus sélectif pour Cdk2 que pour les autres Cdk chez l'humain, et avons décrit les phénotypes induits par cet inhibiteur dans les cellules humaines en cultures. Ces résultats ne permettent pas de confirmer la sélectivité de NU6102 mais montrent que NU6102 a des caractéristiques intéressantes pour être utilisé dans le traitement contre le cancer. L'activité des Cdk est essentielle à l'initiation de la réplication de l'ADN, mais aucun substrat essentiel n'a été identifié chez les métazoaires. Nous avons réalisé un crible des protéines chargées sur la chromatine en présence et en absence d'activité Cdk dans le modèle xénope, afin d'identifier des substrats qui pourraient être impliqués dans la réplication. Ces résultats suggèrent que l'activité Cdk, qui initie la réplication au niveau des origines de réplication de l'ADN, pourrait être impliquée dans d'autres fonctions cellulaires<br>Cycline Dependant Kinases (Cdk) control cell cycle progression. The study of their roles is often difficult because of functional redundancy; when a given Cdk is absent, others may compensate. The main role of Cdk2 in the cell cycle is in the initiation of DNA replication, but absence of Cdk2 is compensated for by Cdk1. For example, mice with a genetic knockout of Cdk2 are viable. The chemical inhibition of Cdks may limit compensation by other Cdks. Therefore, to study Cdk2 roles, we have studied chemical inhibition by NU6102, which seems to be selective for Cdk2 in the Xenopus model. To verify the selectivity and study parameters that determine selectivity, we have designed and produced mutants of Cdk2 which are resistant to NU6102, allowing restoration of function in the presence of inhibitor. Moreover, we demonstrate in vitro that NU6102 is selective for Cdk2 compared to other human Cdks, and we describe phenotypes induced by NU6102 in cultured cells, which are interesting in the light of potential applications of NU6102 in cancer chemotherapy. Cdk activity is essential for initiation of DNA replication, but in metazoans no essential substrates are known. To identify potential Cdk substrates during DNA replication, we have performed a proteomics screen of the proteins loaded onto chromatin in the presence or absence of Cdk activity, in the Xenopus model. The results suggest that Cdk activity is not only required for assembling DNA replication complexes onto origins of replication, but may also be implicated in other cellular functions
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Bondke, Alexander. "Design and synthesis of selective CDK7 inhibitors." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/43965.
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Cyclin-dependent protein kinases (CDKs) have a central role in the regulation of cell proliferation, apoptosis and gene expression. CDK7, in particular, not only regulates the activation of the cell cycle kinases CDK1, CDK2, CDK4 and CDK6, but is also involved in the regulation of transcription as part of the transcription factor TFIIH-complex. While a common feature of cancer is the over-expression of cyclin, there is compelling evidence that CDK2, CDK4 and CDK6 are not essential for the cell cycle, making CDK7 a highly attractive target for anti-cancer drug development. The two pyrazolo[1,5-a]pyrimidine kinase inhibitors BS181 and BS194 were chosen as starting points for the development of a CDK7 selective drug candidate. BS181 is a selective CDK7 inhibitor (IC50: 21 nM) that shows moderate growth inhibition in the MCF7 breast cancer cell line (GI50: 15 μM) as well as in the HCT116 colorectal carcinoma cell line (GI50: 16 μM). The compound suffers from an insufficient oral bioavailability and a poor pharmacokinetic profile. The biological data of the second lead compound BS194 are significantly different: it is a CDK2 pan-inhibitor (IC50: 3 nM) with excellent growth inhibition in MCF7 (GI50: 0.12 μM) and in HCT116 cancer cells (GI50: 0.12 μM). The compound is highly bioavailable and has a good PK profile. The properties of BS181 and BS194 needed to be 'merged' to create a CDK7 selective compound with good overall properties. The multidimensional optimisation of both compounds was driven by iterative circles of computer-aided drug design (CADD), synthesis and biological assessment. Thus, analogues with highly functionalised northern, southern and eastern side chains as well as a novel series of 2,4-diaminopyrimidine kinase inhibitors were designed and subsequently synthesised.
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Shi, Shenhua. "Design and synthesis of novel selective CDK9 inhibitors." Thesis, University of Nottingham, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603594.
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With the findings in anticancer and antiretroviral research, it is strongly believed that CDK9 inhibition would be a useful therapeutic strategy in these fields. A number of pharmacological CDK inhibitors have been previously reported and are undergoing preclinical and clinical evaluation, but none of them selectively targets CDK9. In this project, a number of anilino-4-(thiazol-5-yl)pyrimidine derivatives have been successfully prepared. The most potent compound exhibited best potency with GIso around 10 nM. A set of compounds were tested against CDKl, 2, 7 and 9 and the preliminary structure-activity relationship was established. More than a dozen compounds showed excellent CDK9 inhibitory activity with Ki lower than 10 nM. Lead compound 29, 52 and 67 showed different profile of selectivity towards CDK9, with 21, 6 and 7 nM Ki respectively. Cellular mode of action of lead compound 29 was investigated and the correlation between CDK9 inhibition and anti-tumor activity was confirmed. In the CLL cytotoxicity assay, comparing with clinical compound flavopiridol, compound 52 showed significant selectivity advantage toward norinal B and T-cells against CLL B-cells, which is more than WOO-fold. Several lead compounds, like 52 and 69, are currently scheduled for in vivo assessment. Further research on the basis of this project will result in lead drug candidates for potential cancer therapy.
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Richard, Mathilde. "Les vésicules extracellulaires plasmatiques, de potentiels biomarqueurs de suivi thérapeutique dans le cancer du sein métastatique." Electronic Thesis or Diss., Nantes Université, 2024. http://www.theses.fr/2024NANU1012.
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Les vésicules extracellulaires (VEs), nanoparticules dérivées des membranes cellulaires, sont vectrices de matériels biologiques et circulent via les fluides de l’organisme. Elles se positionnent aujourd’hui comme des biomarqueurs potentiels de la progression du cancer et de la réponse aux thérapies. Le cancer du sein (CS) demeure la première cause de décès par cancer chez la femme, malgré de nombreuses stratégies thérapeutiques innovantes qui permettent d’améliorer la prise en charge et le taux de survie. Ainsi, l’identification de biomarqueurs spécifiques représente un réel espoir pour surveiller l’évolution de la maladie et prédire l’efficacité des traitements. L’objectif principal de ce projet de recherche a été de caractériser les VEs plasmatiques en tant que biomarqueurs potentiels de la réponse au traitement par inhibiteurs de CDK4/6 chez les patientes atteintes d’un cancer du sein métastatique (CSM) positif aux récepteurs hormonaux (RH+). Pour faciliter la recherche translationnelle sur l’utilisation des VEs en tant que biomarqueur circulant, nous avons établi une procédure en trois étapes, applicable en clinique : 1- les VEs ont été isolées du plasma par chromatographie d’exclusion de taille (SEC) semi-automatisée ; 2- la concentration plasmatique des VEs (i.e., vésiculémie) a été déterminée à l’aide du VideoDrop, un microscope utilisant le phénomène d’interférométrie ; et 3- la signature sphingo-lipidique a été analysée par spectrométrie de masse. Nous avons d’abord déterminé la vésiculémie dans une population saine afin d’établir une valeur de référence, en tenant compte des critères de sexe et d’âge. Ensuite, nous avons suivi l’évolution de la vésiculémie physiologique sur une période de 24 mois afin de la comparer avec celle des patientes atteintes d’un CSM RH+ à différents stades de leur prise en charge thérapeutique. Nos résultats révèlent que l’augmentation de la vésiculémie deux mois après le début du traitement est significativement associée à une progression précoce de la maladie. De plus, nous avons analysé la composition lipidique des VEs pour renforcer leur potentiel comme biomarqueurs de progression de la maladie. Une signature spécifique de 16 sphingolipides associés aux VEs nous a permis d’établir un score identifiant avec une précision de 82% les patientes résistantes au traitement par inhibiteurs de CDK4/6. Finalement, cette caractérisation des VEs plasmatiques comme biomarqueurs nous a conduit à explorer leur potentiel chez des patientes atteintes d’un CSM triple négatif et HER2+<br>Extracellular vesicles (EVs), nanoparticle entities derived from cellular membranes, transport biological materials and circulate through body fluids. They are currently positioned as potential biomarkers for cancer progression and therapeutic response. Despite numerous innovative therapeutic strategies improving management and survival rates, breast cancer (BC) remains the leading cause of cancer-related death in women. Thus, the identification of specific biomarkers represents a significant hope for monitoring disease progression and predicting treatment efficacy. The primary objective of this research project was to characterize plasma EVs as potential biomarkers of response to CDK4/6 inhibitors treatment in metastatic breast cancer (MBC) patients with hormone receptor-positive (HR+) status. To facilitate translational research on the use of EVs as circulating biomarkers, we established a three-step procedure clinically applicable: 1- EVs were isolated from plasma using semi-automated size-exclusion chromatography (SEC); 2- plasma EV concentration (i.e., vesiclemia) was determined using the VideoDrop, a microscope utilizing interferometry; and 3- the sphingo-lipidic signature was analyzed by mass spectrometry. We initially measured vesiclemia in a healthy population to establish a reference value, accounting for sex and age. Subsequently, we monitored the physiological vesiclemia evolution over 24 months and compared it with that of MBC HR+ patients at different treatment stages. Our results reveal that an increase in vesiclemia two months after treatment initiation is significantly associated with early disease progression. Furthermore, we analyzed the lipid composition of EVs to reinforce their potential as biomarkers of disease progression. A specific signature of 16 EV-associated sphingolipids enabled us to establish a score identifying CDK4/6 inhibitor-resistant patients with82% accuracy. Finally, this characterization of plasma EVs as biomarkers led us to explore their potential in MBC patients with triple-negative and HER2+ status
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Whittaker, Steven Robert. "The molecular pharmacology of purine CDK inhibitors." Thesis, Institute of Cancer Research (University Of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404908.
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Henderson, Andrew. "Isosteres of sulfonamide inhibitors of cyclin-dependent kinases (CDKs)." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512187.
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Weitensteiner, Sabine. "Cyclin-dependent kinase 5 in endothelial cell migration: Elucidating regulatory mechanisms upstream of Cdk5 and evaluating novel Cdk inhibitors as anti-angiogenic drugs." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-136480.
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Gao, Rui. "Design, synthesis, and optimisation of highly selective macrocyclic CDK9 inhibitors." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/50294/.
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Cyclin dependent kinases (CDKs) belong to a family of serine/threonine protein kinases that play a key role in cell cycle and transcriptional regulation. CDK9, in complex with its regulatory partner cyclin T1, is involved in RNA transcriptional regulation through phosphorylating the RNA polymerase II. Inhibition of CDK9 can target multiple cancer-relevant pathways by downregulating the transcriptionally inducible genes of cancer cells, such as cell cycle regulators and antiapoptotic factors. Although many CDK inhibitors have been in clinical trials, there is only one CDK9-selective compound (BAY1143572) in trials. Therefore, it is still necessary to study other highly selective CDK9 inhibitors. Through the analysis of the cocrystal structure of lead compound 5-(2-((3-(1,4-diazepan-1-yl)phenyl)amino)-pyrimidin-4-yl)-N,4-dimethyl-thiazol-2-amine (5) bound to CDK2 and CDK9, compound (5) favours the “inward” conformation in CDK9, while it adopts both the “inward” and “outward” conformations in CDK2. Aurora A and aurora B (ARKs) inhibitors are also found to adopt the similar “outward” conformation” in ATP binding site of ARKs. Therefore, an “inward” conformation macrocyclic structure was designed to improve the selectivity against CDK9 over CDK2 and ARKs. Three series of macrocyclic compounds were designed, synthesised and tested against CDK9, CDK2, and ARKs. The first series macrocyclic compounds with amide linker shows poor inhibitory activity against CDK9, and the molecular docking study shows these macrocyclic compounds fail to reside in the ATP binding site of CDK9. Following that, ring closing metathesis (RCM) was exploited as an alternative cyclisation strategy. Although the synthesis work was unsuccessful at the first onset, compound 6-methyl-12-oxa-3,6-diaza-2(4,2)-pyrimidina-1,4(1,3)-dibenzenacyclodo-decaphan-8-ene (43) with a similar structure as designed compounds was introduced as the model compound to optimise the RCM reaction. The thiazole group was then demonstrated to be the interfering factor in RCM and compound 11-oxa-3-aza-2(4,2)-pyrimidina-5(1,4)-piperazina-1,4(1,3)-dibenzenacyclo-undecaphan-7-ene (44) with a replacing phenyl group was successfully synthesised and showed poor activity against CDK2 and ARKs (>219 nM for these three kinases). According to the published paper regarding lead compound (5), extensive substituents modification (-CH3, -CN, -F) of compound (44) afforded 11-oxa-3-aza-2(4,2)-pyrimidina-5(1,4)-piperazina-1,4(1,3)-dibenzenacyclo-undecaphan-7-ene-1-carbonitrile (79) as the most selective CDK9 inhibitor. It shows 60-fold selectivity for CDK9 over CDK2 and 90-fold selectivity for CDK9 over ARKs. A further Diversity Kinase Profile Screening demonstrates the high selectivity of compound (79) that only tyrosine-protein kinase Lyn(h) appeared as the off-target. To improve the inhibitory activity against CDK9, alternative macrocyclic pyrimidine systems were designed, and compound 1-methoxy-4-(4-methylpiperazine-1-carbonyl)-3,8-diaza-2(4,2)-pyrimidina-1(1,3),4(1,2)-dibenzenacyclononaphan-7-one (89) showed an attractive Glide score based on molecular docking studies. Because of the time limit, the synthetic work for compound (89) has not been finished.
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Schwermer, Melanie [Verfasser], Alexander [Akademischer Betreuer] Schramm, and Shirley [Akademischer Betreuer] Knauer. "Molekulare Analyse des cdk1/CCNB1 Komplexes und Charakterisierung von cdk-Inhibitoren im Neuroblastom / Melanie Schwermer. Gutachter: Shirley Knauer. Betreuer: Alexander Schramm." Duisburg, 2015. http://d-nb.info/1076980651/34.
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Furnish, Robin. "Evaluating Immune Modulatory Therapeutic Strategies for Diffuse Intrinsic Pontine Glioma." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595849080346532.
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Millan, Laurine. "Caractérisation d’inhibiteurs de complexes CDK‐cycline chez Arabidopsis thaliana." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112149.
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Comme pour tous les organismes pluricellulaires, la croissance et le développement des plantes nécessitent une coordination de la production de cellules via la mitose et la différenciation cellulaire. La progression du cycle cellulaire est contrôlée par les complexes CDK-cycline. Les inhibiteurs de ces complexes, les CKIs, représentent d’excellents candidats pour réguler cet équilibre entre les processus de prolifération et différentiation cellulaires qui ont lieu au cours du développement. Afin de mettre en évidence le rôle d’intégrateurs potentiel des CKIs, le développement floral a été utilisé en tant que modèle.Grâce à l’utilisation de la qRT-PCR, nous avons montré que durant le développement floral d’Arabidopsis thaliana, un groupe restreint de CKIs était exprimé. Nous avons choisi de travailler sur les deux CKIs les plus exprimés, KRP6 et KRP7. Une caractérisation fine de leur profil d’expression durant le développement a été réalisée en utilisant des approches complémentaires telles que l’analyse de l’activité de leur promoteur, de la dynamique de leur transcrit, de leur expression protéique et de leur régulation post-traductionnelle.Jusqu’à présent, seules des approches ‘gain de fonction’ ont été utilisées pour étudier le rôle des CKIs chez les plantes. C’est pour cela que nous avons choisi des approches ‘perte de fonction’ pour analyser le rôle de KRP6 et de KRP7 au cours du développement floral. Ainsi, nous avons généré des doubles mutants d’insertion krp6-krp7, krp3-krp6, krp3-krp7, des triples mutants d’insertion krp3-krp6-krp7 et diverses lignées ARN interférence avec des promoteurs spécifiques. Malgré l’étude de ces nombreuses lignées, nous n’avons pas réussi à mettre en évidence des effets phénotypiques associés à l’absence de la fonction CKI au cours du développement floral. Ces résultats mettent en évidence la redondance fonctionnelle qui semble exister entre les KRPs, ainsi un quadruple mutant pourrait être nécessaire pour entrainer des modifications développementales. Afin de mieux comprendre cette fonction d’intégrateurs des KRPs au cours du développement floral, les partenaires de KRP6 et de KRP7 ont été recherchés. Des criblages double-hybride ont été réalisés afin d’identifier des ADNc, spécifiques du développement floral, codant des protéines capables d’interagir avec KRP6 et KRP7. De façon intéressante, mis à part les cyclines de type D, un nouveau type d’interaction a pu être mis en évidence. Un sous-groupe de la famille des rémorines est capable d’interagir avec KRP6 ou KRP7 en système double-hybride. Les rémorines sont des protéines spécifiques du règne végétal, associées à la membrane plasmique mais dont la fonction reste à clarifier. Une approche BiFC en protoplastes BY-2 a permis de confirmer l’existence de ce type d’interaction. De plus, l’influence des rémorines sur la localisation intracellulaire des KRPs a été étudiée. En présence de ces nouveaux partenaires, KRP7 est capable d’adopter une localisation nucléo-cytoplasmique.Enfin, des résultats récents ont montré que l’AMPK était capable de phosphoryler p27KIP1, l’homologue fonctionnel des KRPs chez les mammifères. Ces évènements de phosphorylation entrainent des modifications de sa localisation intracellulaire et de son activité inhibitrice vis-à-vis des complexes CDK-cycline. Après la réalisation d’analyses in silico ayant permis de prédire des sites putatifs de phosphorylation par SnRK1, l’homologue de l’AMPK chez A. thaliana, pour certains KRPs, la protéine KRP6 sous forme recombinante a été utilisée pour réaliser des essais kinase in vitro. Une phosphorylation de KRP6 est détectée en présence de la sous unité catalytique activée de SnRK1. Contrairement aux mammifères, cet évènement de phosphorylation entraine une altération de l’activité inhibitrice de KRP6 sans modification de sa localisation intracellulaire. Cette abolition de l’activité de KRP6 a été confirmée in planta. En effet, les phénotypes associés à la surexpression de KRP6 peuvent être atténués par la surexpression simultanée de la sous-unité catalytique de SnRK1. L’existence de ce lien entre KRP6 et SnRK1 met en évidence une relation directe entre l’homéostasie énergétique et la prolifération cellulaire<br>As in all multicellular organisms, growth and development in plants require the coordination of cell production by division and cell differentiation. Progression through cell cycle is controlled by the kinase activity of CDK/cyclin complexes. Inhibitors of these complexes, CKIs, represent excellent candidates to regulate the balance between proliferation and differentiation processes during development. To get insight in the potential integrator role of CKIs, floral development was chosen as a developmental model. Using a real time quantitative PCR approach, we bring to light that during floral development of Arabidopsis thaliana, a restricted subset of CKIs was preferentially expressed. It was decided to focus our work on the two major expressed CKIs, KRP6 and KRP7. A better characterization of their expression patterns of during development was undertaken using complementary approaches such as promoter activity analysis, mRNA dynamics, protein expression and post-translational regulation analysis. Because until now ‘gain of function’ approaches have been largely applied to unravel the role of plant CKIs, our challenge was to detect a floral phenotype for KRP6 and KRP7 loss of function mutants, either using knock-out mutants or RNAi lines. We generated krp6-krp7, krp3-krp6, krp3-krp7 double mutants and krp3-krp6-krp7 triple mutant and also several RNAi lines with specifics promoters. Despite the study of these numerous lines, we were not able to highlight phenotypic effects associated with the absence of CKI function during floral development. All these results emphasis functional redundancy which appears to exist between all KRPs, thus quadruple mutant might be needed to provoke some developmental modification.In order to better understand the integrative function of KRPs during floral development, partners of KRP6 and KRP7 were assessed. Two-hybrid screens were performed to identify cDNAs from a “floral-buds-development” library encoding proteins that are able to interact with KRP6 and KRP7. Interestingly, apart from D-type cyclins, we brought to light a new type of interaction. Indeed, a sub-class of the remorin protein family was able to interact with KRP6 or KRP7 in yeast two-hybrid. Remorins are plant specific plasma membrane associated proteins with unknown function. A BiFC approach in BY-2 protoplasts allowed us to confirm remorins/KRP6-7 interactions. Furthermore, the influence of the presence of remorin proteins on KRP6/7 localisation was assessed. KRP7 is able to adopt a nucleo-cytoplasmic localisation in presence of its new partners.Finally, recent results have shown that AMPK is phosphorylating p27KIP1, KRPs functional counterpart in mammals. These phosphorylation events lead to changes in its cellular localisation and its inhibitory activity toward CDK-cyclin complexes. After in silico analysis aiming to predict potential AMPK Arabidopsis homologue SnRK1 phosphorylation sites within some KRPs protein sequences, recombinant KRP6 was used in order to perform in vitro kinase assays. Phosphorylation occurs efficiently on KRP6 when activated SnRK1 catalytic subunit is present. Furthermore, unlike in mammals, this phosphorylation event leads to an alteration of KRP6 inhibitory activity without modification of its cellular localisation. This abolition of KRP6 activity was confirmed by in planta analysis. Indeed, KRP6 overexpression phenotype can be attenuated by simultaneous SnRK1 catalytic subunit overexpression. The existence of this link between KRP6 and SnRK1 underscores a direct relationship between energy homeostasis and cell proliferation
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Grimmler, Matthias. "Identifikation und Charakterisierung von neuen Interaktionspartnern des CDK-Inhibitors p27Kip1." [S.l.] : [s.n.], 2002. http://edoc.ub.uni-muenchen.de/archive/00004707.
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Weiss, Sonja Birgit. "Identifikation und Charakterisierung von neuen Interaktionspartnern des CDK-Inhibitors p57Kip2." [S.l.] : [s.n.], 2003. http://edoc.ub.uni-muenchen.de/archive/00004709.
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Grimmler, Matthias. "Identifikation und Charakterisierung von neuen Interaktionspartnern des CDK-Inhibitors p27Kip1." Diss., lmu, 2003. http://nbn-resolving.de/urn:nbn:de:bvb:19-47077.
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Weiß, Sonja Birgit. "Identifikation und Charakterisierung von neuen Interaktionspartnern des CDK-Inhibitors p57Kip2." Diss., lmu, 2002. http://nbn-resolving.de/urn:nbn:de:bvb:19-47097.
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Kroll, Sebastian Herbert Benjamin. "Towards the synthesis of selective CDK7 inhibitors as potential anti-cancer drugs." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/11657.
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Cyclin-dependent kinase 7 (CDK7) exhibits an interesting target for an anti-cancer therapy approach. CDK7’s triple role in phosphorylation (cell cycle, transcription, estrogen receptor (ER)) in cell regulation makes this kinase interesting. Phosphorylation of cell cycle CDK’s via its CAK-complex, of Ser-5 in RNA-PolII as part of the TFIIH-complex and phosphorylation of Ser-118 in ER all show the importance of this enzyme. Given that CDKs are over-expressed in many cancers, selective inhibition of CDK7 should result in cell cycle arrest and apoptosis predominately in tumour cells. Previously, BS-181 (Figure 0.1) has been reported as the first CDK7 selective inhibitor, which displayed a good in vitro and in vivo profile.1 Based on this initial lead compound, a library of rational-designed analogues was synthesised. Much of this library was based on a computer-aided-drug-design (CADD) approach by docking, which gave valuable insights in possible binding modes and helped to focus targeting the whole active site. [Molecular structure diagrams appear here. To view, please open pdf attachment] Figure 0.1: BS-181 and new analogues. Several of these novel inhibitors showed excellent selectivity versus CDK2 in particular, and potency against CDK7 in the 30 – 60 nM range for their IC50-values. Cellular assays confirmed the growth inhibitory properties of these new compounds, with GI50-values in the low μM range. This work also demonstrates what functional groups were tolerated in the 3-,5- and 7-position.
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Lu, Zhixin. "Investigations on Cancer Cell Biological Effects of CDK8 Inhibitor Q-12." Scholarly Commons, 2018. https://scholarlycommons.pacific.edu/uop_etds/3554.
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Over the past two decades, protein kinases have been intensively investigated as targets to treat neoplastic diseases. Many protein kinase inhibitors not only have therapeutic potential but are becoming invaluable reagents for the study of cell signaling.
We aspired to use our Cyclin-Dependent Kinase 8 inhibitor, Q-12, as a probe for biomarker discovery for CDK8 inhibitor sensitive tumor types. Q-12 shows potent inhibition of cell viability and induction of apoptosis process in some triple-negative breast cancer and colorectal cancer cell lines in vitro. Western blot results indicate that the reduction of STAT1 phosphorylation could be a robust indicator of CDK8 target engagement in all three cancer cell lines used upon Q-12 treatment. Q-12 treatment of triple-negative breast cancer cell line (MDA-MB-468) decreases STAT1 phosphorylation but increases STAT3 phosphorylation. Q-12 activity in MDA-MB-468 cell is dependent
on the activation of STAT3 phosphorylation. All results suggest that there may be a critical STAT1 to STAT3 ratio that may serve as a biomarker for CDK8 inhibitor sensitivity. In this precision medicine era, the discovery of biomarker is urgently needed to minimize the risks of severe side-effects by traditional chemotherapy and improve diagnosis and monitor therapy response across a wide spectrum of disease, especially heterogenous type of disease, like cancer.
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Harlow, Lisa Katherine. "Structure-activity studies of 2-arylaminopurine cyclin-dependant kindase (CDK) inhibitors." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505843.
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Weitensteiner, Sabine [Verfasser], and Stefan [Akademischer Betreuer] Zahler. "Cyclin-dependent kinase 5 in endothelial cell migration : elucidating regulatory mechanisms upstream of Cdk5 and evaluating novel Cdk inhibitors as anti-angiogenic drugs / Sabine Weitensteiner. Betreuer: Stefan Zahler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1017233233/34.
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Shao, Hao. "Design, synthesis and biological evaluation of highly selective CDK9 inhibitors for cancer treatment." Thesis, University of Nottingham, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716491.
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Cyclin-dependent kinases (CDKs) are a family of Ser/Thr kinases involved in cell cycle and transcriptional regulation. CDK9 regulates transcriptional elongation and this unique property has made it a potential target for several diseases, such as cancer, inflammatory and cardiac hypertrophy. Although a number of CDK inhibitors have been identified and taken into clinical trials, all show inhibitory activity against several CDKs and other kinases. The current lack of selective CDK9 inhibitors is limiting efforts to fully understand and validate CDK9 as a drug target. This project aimed to design and synthesize a novel class of 2,4,5-tri-substituted pyrimidine compounds as highly selective CDK9 inhibitors.
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Prevel, Camille. "Développement de biosenseurs fluorescents et d’inhibiteurs pour suivre et cibler CDK4/cycline D dans le mélanome." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONT3505/document.
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Les CDK/cyclines jouent un rôle majeur dans la progression du cycle cellulaire et dans le maintien de la prolifération des cellules cancéreuses, constituant ainsi des biomarqueurs clés et des cibles pharmacologiques attractives. Plus particulièrement, l’activité de CDK4/cycline D, kinase responsable de la progression de la phase G1 et de la transition G1/S, est dérégulée dans de nombreux cancers dont le mélanome. Cette hyperactivation est associée à des mutations, à l’amplification ou à la surexpression de CDK4, cycline D, p16INK4a ou encore pRb.Comme aucune approche sensible et directe n’existe pour évaluer l’activité de CDK4/cycline D dans des conditions physiologiques et pathologiques, le premier objectif de ma thèse a consisté à développer un biosenseur fluorescent permettant d’étudier cette kinase in vitro et in cellulo. Une fois caractérisé et validé in vitro, le biosenseur a été appliqué à la détection d’altérations de CDK4/cycline D dans des biopsies de peau humaine et de xénogreffes de mélanome dans des essais fluorescents d’activité kinase, ainsi que dans des cellules cancéreuses vivantes par microscopie de fluorescence et vidéo microscopie.Par ailleurs, peu d’inhibiteurs sont actuellement disponibles pour inhiber CDK4/cycline D et la plupart d’entre eux ciblent la poche de fixation de l’ATP. C’est pourquoi le second objectif de ma thèse a consisté à identifier des inhibiteurs non compétitifs de l’ATP, soit par élaboration rationnelle de peptides, soit par criblage de petites molécules. A cette fin, deux biosenseurs fluorescents ont été développés qui permettent d’identifier respectivement des composés ciblant l’interface entre CDK4 et cycline D ou des inhibiteurs allostériques capables de perturber la dynamique conformationnelle de CDK4. Des essais de criblage par fluorescence réalisés avec ces biosenseurs ont conduit à l’identification de touches qui ont été validées et caractérisées in vitro et dans des essais de prolifération cellulaire, et qui constituent des candidats prometteurs pour une chimiothérapie sélective du mélanome<br>CDK/cyclins play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting established cancer biomarkers and attractive pharmacological targets. In particular, CDK4/cyclin D, which is responsible for coordinating cell cycle progression through G1 into S phase, is a relevant target in several cancers including melanoma, associated with mutation of CDK4, cyclin D, p16INK4a and pRb.As there are no sensitive and direct approaches to probe CDK4/cyclin D activity in physiological and pathological conditions, the first goal of my thesis has consisted in engineering a fluorescent biosensor to probe this kinase in vitro and in cellulo. Once characterized and validated in vitro, the biosensor was applied to detect CDK4/cyclin D alterations in biopsies from human skin and melanoma xenografts in fluorescence-based activity assays, and in living cancer cells by fluorescence microscopy and timelapse imaging.Moreover, only few inhibitors are currently available to target CDK4/cyclin D and most of them bind the ATP pocket. As such, the second major goal of my thesis project has consisted in identifying non-ATP competitive inhibitors, either through rational design of peptides or by screening small molecule libraries. To this aim, two fluorescent biosensors were engineered which discriminate compounds that target the interface between CDK4 and cyclin D, or that perturb the conformational dynamics of CDK4, respectively, from ATP-pocket binding compounds. Fluorescence-based screening assays performed with these biosensors lead to identification of hits, which were validated and characterized in vitro and in cell proliferation assays, and which constitute promising candidates for selective chemotherapy in melanoma
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Junior, Walter Filgueira de Azevedo. "Introdução à biocristalografia com o estudo estrutural da quinase dependente de ciclina 2 (CDK2) complexada com inibidores." Universidade de São Paulo, 1997. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-17032014-173549/.
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O ciclo celular é controlado pela atividade das quinases dependentes de ciclinas (Ciclin-dependent kinases, CDKs). As CDKs são inativas como monômeros, e a sua ativação necessita da ligação às ciclinas, uma família diversa de proteínas cujos os níveis oscilam durante o ciclo celular, e fosforilação pela CAK (CDK-activating kinase) sobre um resíduo de treonina específico. As CDKs são capazes de fosforilar muitas proteínas que estão envolvidas nos eventos do ciclo celular, incluindo histonas e proteínas supressoras de tumores como pRb. Além da função de regulação positiva das ciclinas e CAK, muitas proteínas inibidoras de CDKs (CDK inhibitors, CKIs) têm sido descobertas, tais como p16, p21 e p28. Visto que, a desregulação das ciclinas e/ou alteração ou ausência de CKIs têm sido associadas com muitos cânceres, há um forte interesse em inibidores químicos de CDKs que possam ter uma função importante na descoberta de novas famílias de agentes anti-tumores. Vistoque, ATP é o autêntico co-fator da CDK2 este pode ser considerado como um \"pseudo-composto líder\" para a descoberta de inibidores de CDK2. Entretanto, há duas preocupações maiores a serem consideradas: composto contendo adenina são ligantes comuns para muitas enzimas nas células, desta forma, qualquer composto altamente carregado como ATP não será absorvido pelas células. Nós descrevemos aqui as estruturas determinadas por difração de raios-X da CDK2 em complexo com dois inibidores diferentes, descloro-flavopiridol (DFP) e Roscovitine. A estrutura do complexo binário CDK2-DFP foi resolvida por substituição molecular e refinada até um Rfactor=20,3% e a estrutura da CDK-2Roscovitine foi refinada até um Rfactor=18%. O descloro-flavopiridol é uma flavona com uma nova estrutura,comparável àquelas de flavonas polihidroxiladas. Estudos prévios mostraram que flavopiridol, um flavonóide, pode inibir cânceres de mama e de pulmão. O Roscovitine é um derivado de adenina e um potente inibidor de CDK2. A comparação das estruturas tridimensionais de CDK2-DFP e CDK2-Roscovitine com a de CDK2-ATP mostraram que o bolsão hidrofóbico de ligação de adenina tem a habilidade surpreendente de acomodar estruturas moleculares diferentes daquelas da ATP<br>Cell cycle progression is tightly controlled by the activity of ciclin-dependent kinases (CDKs). CDKs are inactive as monomers, and activation requires binding to cyclins, a diverse family of proteins whose levels oscillate during cell cycle, and phosphorilation by CDK-activating kinase (CAK) on a specific threonine residue. CDKs are able to phosphorylate many proteins that are in volvedin cell cycle events, including histones and tumor suppressor proteins like the retinoblastoma gene product pRb. In addition to the positive regulatory role of cyclins and CAK, many negative regulatory proteins (CDK Inhibitors, CIGs) have been discovered, such as p16, p21, and p28. Since deregulation of cyclins and/or alteration or absence ofCKIs have been associated with many cancers, there is strong interest in chemical inhibitors of CDKs that could play an important role in the discovery of new family of antitumor agents. Since ATP is the authentic cofactor of CDK2 it can be considered as a \"pseudo-lead compound\" for discovery of CDK2 inhibitors. However there are two major concerns: adenine containing compounds are common ligants for many enzymes in cells, thus, any adenine derivatives may inhibit many enzymes in the cells: second, any highly charged compounds such as ATP will prevent them from uptake by cells. We report here the x-ray structures of CDK2 in complex with two different inhibitors, deschloro-flavopiridol(DFP) and Roscovitine. The structure of the binary complex CDK2-DFP was solved by molecular replacement and refined to Rfactor = 20.3% and the structure ofCDK2-Roscovitine was refined to Rfactor = 18.0 %. The deschloro-flavopiridol(DFP) is a flavone with a novel structure, compared to that of polyhydroxylated flavones. Previous studies have shown that flavopiridol, a flavonoid, can inhibit growth of breast and lung carcinoma cell lines. The Roscovitine is an adenine derivative and a potent CDK2 inhibitor. The two inhibitors are competitive inhibitors for ATP binding to CDK2 and bind to the ATP binding pocket ofCDK2. The comparison of the three-dimensional structures of CDK2-DFP and CDK2-Roscovitine with the CDK2-ATP shows that the hydrophobic adenine-binding pocket has a surprising ability to accommodate molecular structures that are different from ATP.
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Almeida, Buana Carvalho de. "Metabólitos secundários como potenciais inibidores de CDK8 (proteína quinase humana)." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-25042018-142801/.
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Quinases Dependentes de Ciclinas (CDKs) são holoenzimas que possuem uma subunidade catalítica, a CDK, e uma subunidade regulatória, a ciclina. CDKs são Ser/Thr quinases, ou seja, fosforilam resíduos de aminoácidos específicos. A CDK8 fosforila o CTD da RNA Pol II, sendo considerada como regulador transcricional positivo, implicando em efeitos oncogênicos. Em suma, essas descobertas a colocam como alvo de drogas em pesquisas relacionadas ao câncer. Técnicas de biologia molecular foram realizadas a fim de obter o recombinate CDK8::pET28a. A CDK8-HisTag foi expressa em Rosetta(DE3), com cauda de histidina no N-terminal (45 kDa), no entanto a proteína foi expressa como corpos de inclusão. Desta forma, a CDK8-HisTag foi solubilizada com SDS 2%, o detergente foi completamente removido e o refolding da mesma foi obtido com cromatografia líquida de alta eficiência utilizando coluna de Ni2+. A proteína reenovelada e purificada foi analisada por DC e revelou a existência de 13, 6% de hélices α, 34,5 %d e folhas β e 33,1 % de coil. O espectro de emissão de fluorescência da CDK8-HisTag mostrou comprimento de onda máximo em torno de 360 nm. A análise de docking com potenciais inibidores obtidos da espécie W. brachypetala revelou o alcaloide O-metil-hyeronimona como potencial inibidor de CDK8. Este é o primeiro relato de expressão e purificação da proteína CDK8 em E. coli, assim como do screening virtual desta proteína na presença de inibidores naturais obtidos de W. brachypetala.<br>Cyclin Dependent Kinases (CDK) are holoenzyme having a catalytic subunit, CDK, and a regulatory subunit, cyclin. CDKs are Ser / Thr kinases, which phosphorylate specific amino acid residues. The CDK8 phosphorylates the CTD of RNA Pol II. It is considered a positive transcriptional regulator, resulting in oncogenic effects. In short, these findings pose as a drug target in research related to cancer. Molecular biology assays were performed in order to obtain the recombinant CDK8::pET28a. CDK8-HisTag was expressed in Rosetta (DE3), with a N-terminal tail histidine (45 kDa), however, the protein was expressed as inclusion bodies. In this way, a CDK8-HisTag was solubilized with 2% SDS, then the detergent was completely removed and the refolding of the same was obtained with High Performance Liquid Chromatography using Ni2 + column. A refolded and purified protein for DC analysis revealed the existence of 13.6% helix, 34.5% beta and 33.1% coils. The fluorescence emission spectrum of CDK8-HisTag showed the maximum wavelength around 360 nm. An analysis with potential inhibitors obtained from the W. brachypetala species revealed the alkaloid O-metil-hyeronimona as a potential inhibitor of CDK8. This is the first report of expression and purification of the CDK8 protein in E. coli, as well as the virtual screening of this protein in the presence of natural inhibitors obtained from W. brachypetala.
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Parsons, Rachel. "The design and synthesis of pyrimidine based cyclin-dependent kinase (CDK) inhibitors." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408491.
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Northen, Julian S. "Design of novel pyrimido[5,4-d]pyrimidine cyclin dependent kinase (cdk) inhibitors." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391320.
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Hamdi, Amel. "Découverte de petites molécules inhibitrices d’interactions protéiques : développements méthodologiques et application à l’interaction CDK2/CKS1." Rennes 1, 2012. http://www.theses.fr/2012REN1S128.
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Les inhibiteurs d’interactions protéiques constituent des outils de recherche précieux qui permettent de mieux appréhender le fonctionnement des mécanismes de régulation. En outre, les réseaux d’interactions protéiques constituent un immense réservoir de cibles thérapeutiques potentielles, largement sous-exploité. Il est donc probable que de nombreux inhibiteurs d’interactions protéiques puissent constituer des candidats thérapeutiques d’intérêt. Longtemps considérée comme difficile, la découverte de petites molécules inhibitrices d’interactions protéiques connaît aujourd’hui un grand essor, notamment grâce à la disponibilité d’un vaste arsenal de méthodes de criblage. L’un des objectifs de la thèse visait à optimiser un essai BRET (Bioluminescence Resonance Energy Transfer) chez la levure, préalablement développé dans notre unité. Ces travaux n’ont pas abouti à l’optimisation escomptée, mais ils ont permis de mieux comprendre le fonctionnement de l’essai dans les levures et ils suggèrent de nouvelles pistes d’amélioration. L’autre objectif visait à découvrir des petites molécules inhibitrices de l’interaction entre CDK2, protéine kinase jouant un rôle clé dans la division cellulaire, et la protéine CKS1. Les protéines CKS interagissent avec les complexes CDK1/2-Cyclines et jouent de multiples rôles dans la régulation du cycle cellulaire et dans le maintien de l’intégrité du génome mitochondrial. Deux petites molécules inhibitrices de l’interaction CDK2/CKS1 ont été identifiées par criblage de chimiothèque, en utilisant un essai double-hybride en levures. Elles ont été confirmées par des essais d'interaction in vitro et des analogues plus actifs ont été identifiés<br>Small molecule inhibitors of protein interactions are invaluable research tools, enabling a better understanding of regulatory mechanisms. In addition, protein interaction networks represent a relatively untapped, rich source of potential therapeutic targets. Therefore, it is likely that many inhibitors of protein interactions may also represent therapeutic candidates of interest. The discovery of small-molecule inhibitors of protein interactions has been generally considered as an arduous if not impossible task in the past century. However, in the past decade, it has become increasingly popular, partly thanks to the availability of a large arsenal of valuable screening methods. One of the objectives of the PhD work was to optimize a yeast BRET assay (Bioluminescence Resonance Energy Transfer) previously developed in our lab. Although this work has not brought the expected optimization, it has provided a better understanding of the assay in yeast and it has suggested new avenues of optimization. The other objective was to discover small-molecule inhibitors of the interaction between CDK2, a protein kinase that plays a key role in cell division, and the CKS1 regulatory protein. CKS proteins interact with CDK1/2-Cyclin complexes and play multiple roles in regulating the cell cycle and in maintaining mitochondrial genome integrity. Two small-molecule inhibitors of the interaction CDK2/CKS1 were identified by screening a chemical library, using a yeast two-hybrid assay. They have been confirmed by in vitro interaction assays and more active analogues have been identified
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Stott, Francesca Joanne. "Analysis of the INK4 family of cyclin dependent kinase inhibitors, in the mammalian cell cycle." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322001.
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Pellegatti, Laurent. "Méthodologie en chimie hétérocyclique et application à la synthèse d'inhibiteurs de kinases." Thesis, Orléans, 2010. http://www.theses.fr/2010ORLE2046.
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Le cancer constitue l’une des principales causes de mortalité, et représente, de ce fait aujourd’hui un problème de santé publique majeur. Depuis quelques années, les alcaloïdes marins représentent une source d’inspiration importante pour les chimistes en vue d’obtenir de nouveaux médicaments anticancéreux. Dans cette optique, des recherches effectuées au sein de notre laboratoire ont fait état de la synthèse d’analogues de ces alcaloïdes possédant une structure tris aromatique. Nous avons développé des molécules originale analogues de ces alcaloïdes disposant d’un hétérocycle central (1,2,4-triazine et imidazo[1,2-b][1,2,4,5]tétrazine) sur lequel sont greffés deux noyaux phényles diversement substitués. L’obtention de ces composés a également été l’occasion de développer de nouvelles méthodologies de synthèse. Ainsi une nouvelle réaction de type Buchwald-Hartwig sur des méthylsulfanyl-1,2,4-triazines a pu être mise au point ainsi qu’une méthode de CH arylation palladocatalysée sur le noyau imidazo[1,2-b][1,2,4,5]tétrazine. Une partie est aussi consacrée aux réactions multicomposants de type Groebke-Blackburn. Différentes évaluations pharmacologiques ont été réalisées, notamment des tests d’inhibition sur différentes kinases et de cytotoxicité sur diverses lignées cellulaires cancéreuses humaines<br>Cancer, one of the leading causes of death, represents today a major public health problem. Over the last few years, marine alkaloids represent a source of inspiration for chemists in order to obtain new anticancer drugs. For this purpose, as a part of our laboratory researches, analogues of marine alkaloids were synthesized possessing a tris-aromatic structure. We developed originals analogs of these alacaloïds formed by a central heterocycle core (1,2,4-triazine et imidazo[1,2-b][1,2,4,5]tetrazine) on wich is graft two arylic moiety variously substituted. Obtaining these compounds was also an opportunity to develop news synthetic methodologies. So a new Buchwald-Hartwig reaction type based on methylsulfanyl-1,2,4-triazines has been perfect, as palladocatalyzed CH arylation pathway on imidazo[1,2-b][1,2,4,5]tetrazine. A part is devoted to Groebke-Blackburn multicomponant reaction. Various pharmacological analyses were carried out in particular with inhibition of various kinases and cytotoxicity evaluation on various human cancer cell lines
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Knudsen, Erik S., Jack Hutcheson, Paris Vail, and Agnieszka K. Witkiewicz. "Biological specificity of CDK4/6 inhibitors: dose response relationship, in vivo signaling, and composite response signature." IMPACT JOURNALS LLC, 2017. http://hdl.handle.net/10150/625360.
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Recently developed potent and selective CDK4/6 inhibitors fall into two classes based on structure and toxicity profiles in clinical studies. One class, exemplified by palbociclib and ribociclib, exhibits neutropenia as a dose-limiting toxicity and requires discontinuous dosing. In contrast, the structurally distinct CDK4/6 inhibitor abemaciclib is dosed continuously, and has diarrhea and fatigue as dose-limiting toxicities. In preclinical models, palbociclib has been extensively studied and induces cell cycle inhibition in an RB-dependent manner. Thus far, abemaciclib has been less extensively evaluated. We found that abemaciclib cell cycle inhibitory activity is RB-dependent at clinically achievable concentrations. Abemaciclib elicited potent suppression of RB/E2F regulated genes associated with prognosis in ER-positive breast cancer. However, unlike palbociclib, at 250nM-1 mu M doses abemaciclib induced cell death in RB-deficient cell lines. This response was associated with a rapidlyinduced multi-vacuolar phenotype indicative of lysosomal membrane permeabilization that could be ameliorated with chloroquine. This event was not a reflection of inhibition of other CDK family members, but could be recapitulated with CBX4945 that inhibits casein and DYRK/HIPK kinases. To determine if these "off-target" features of abemaciclib were observed in vivo, mice harboring matched RB-positive and negative xenografts were treated with palbociclib and abemaciclib. In vivo, all of the apparent activity of abemaciclib was RB-dependent and strongly elicited suppression of cell cycle regulatory genes in a fashion markedly similar to palbociclib. Using gene expression data from cell lines and tumors treated with abemaciclib and palbociclib a composite signature of response to CDK4/6 inhibition was developed that included many genes that are individually required for tumor cell proliferation or viability. These data indicate that while abemaciclib and palbociclib can exert distinct biological and molecular effects, there are common gene expression features that could be broadly utilized in measuring the response to CDK4/6 inhibition.
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Fredersdorf, Steffen. "A study of cellcycle regulation by cdk-inhibitors of the cip/kip-family." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300159.
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CHIAVETTA, Roberta. "Study of the combined effects of CDK1 inhibitors and senolytic drugs for the clearance of aneuploid-senescent cells." Doctoral thesis, Università degli Studi di Palermo, 2022. http://hdl.handle.net/10447/533837.
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Despite the progresses in discovering new therapeutic drugs and treatments, cancer is still one of the main causes of death. The biggest part of available treatments, moreover, is not always effective against tumour spread and it also has negative effects on the healthy tissues of the individual. For this reason, it is extremely relevant to find new strategies to avoid side effects during the anti-cancer therapies. Aneuploidy, an aberrant number of chromosomes in the cell, is a typical condition of cancer cells caused mainly by segregation errors and chromosomal instability (CIN). CIN is a process by which higher rate of chromosome segregation defects occurs by different mechanisms (chromosome mis-alignments, spindle alterations, mitotic defects cytokinesis failure…) resulting in aneuploidy that, by inducing proteotoxic stress, energy stress and DNA damages, affects proliferation of normal cells. On the other hand, CIN and aneuploidy allow cancer cells to escape pathways leading to cell death (apoptosis), cell cycle arrest and cellular senescence. The reactivation of pathways leading to apoptosis or cellular senescence is a powerful strategy to halt proliferation of cancer cells. In particular, cellular senescence, an irreversible cell cycle arrest, presents itself as an effective mean to stop proliferation of cancer cells that could be then killed specifically, for example by senolytic drugs. Generally, cellular senescence originates from a G1 arrest of the cell cycle caused by DNA damage or other cellular stresses/alterations (such as defects in chromosome segregations).
The cyclin-dependent kinase CDK1 is the most overexpressed kinase in malignant tumours compared to other CDKs. Its function is important for the correct cell cycle progression and its action is highly regulated, in order to preserve the right progression of mitosis. Once CDK1 is degraded, cell can successfully ultimate mitosis, proceeding from anaphase to telophase. Evidence showed how CDK1 and not its partner cyclin B1 is the main responsible of the right progression of mitosis1.
Aneuploidy has been suggested also as a trigger for cellular senescence and aging. Thus, I hypothesized that inducing a cell cycle arrest in the G2/M phase by inhibiting CDK1 could trigger mitotic errors leading to a subsequent G1 arrest and likely senescence of cancer cells. To this aim I have inhibited CDK1 by RNAi and the selective inhibitor RO-3306, an ATP-competitor which interferes with CDK1 activation, in cultured cancer cells and not transformed aneuploid human cells. Moreover, as a positive control of senescence, the flavonoid Curcumin was used due to its known senescence inducer activity in cancer cells. After senescence induction, cells have been treated with the senolytic drugs Fisetin and Quercetin in order to specifically eliminate senescent cells. Globally, my work shows that CDK1 depletion, by RNAi and pharmacological inhibition, leads to G2/M arrest followed by p21waf1/cip1 rise and triggers cellular senescence in cancer cells as shown by SA-βGal positive cells. Accordingly, even though with different mechanisms, Curcumin has induced cell cycle arrest in the G2/M phase, as well as senescence, in tumor cells. Interestingly, the percentage of senescent cells reduces following treatment with the drugs Quercetin and Fisetin, confirming the senolytic action of these compounds. Thus, combining CDK1 inhibition with senolytic drugs can be a powerful strategy for the clearance of aneuploid – senescent cancer cells.
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Bettayeb, Karima. "Optimisation et caractérisation de nouveaux inhibiteurs pharmacologiques de kinases cycline-dépendantes (CDKs)." Rennes 1, 2008. http://www.theses.fr/2008REN1S025.
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Les kinases cycline-dépendantes (CDKs) sont des régulateurs essentiels du cycle de division cellulaire (CDK1, 2, 3, 4, 6, 7), de l’apoptose (CDK1, 5), et de la transcription (CDK7, 9). Les inhibiteurs pharmacologiques de CDKs constituent une nouvelle famille de produits anti-cancéreux potentiels : dix sont actuellement en clinique, dont la roscovitine issue de notre laboratoire. Ma thèse a porté sur l’identification, l’optimisation et la caractérisation détaillée des effets biochimiques et cellulaires de trois nouvelles classes d’inhibiteurs de kinases : indirubines-7-bromées (7BIO), meriolines et nouveaux analogues de la roscovitine (N&N1 et C&R8). Le 7BIO induit une mort cellulaire non-apoptotique par un mécanisme encore inconnu (pas de relargage de cytochrome C, pas d’activation de caspases). En revanche les meriolines et les roscovitine de seconde génération induisent une mort apoptotique classique suite à l’inhibition de CDK9. Qui conduit à une disparition d’un facteur de survie cellulaire essentiel, Mcl-1. Ces trois familles chimiques ont des effets anti-prolifératifs et tumoraux prometteurs<br>Cyclin-dependent kinases (CDKs) are key regulators of cell division cycle (CDK1, 2, 3, 4, 6, 7), apoptosis (CDK1, 5), and transcription (CDK7, 9). Pharmacological inhibitors of CDKs constitute a new family of potential antitumor agents: ten are under clinical trials, among which roscovitine, was discovered in our laboratory. My thesis is about identification, optimisation and characterization of biochemical and cellular effects of three new kinase inhibitory classes: 7-bromo-indirubins (7BIO), meriolin and new analogs of roscovitine (N&N1 and C&R8). 7BIO induces non-apoptotic cell death through an unknown mechanism (no release of cytochrome C, nor caspase activation). On the other hand, meriolins and second generation roscovitine analogs induce a classical apoptosis due to CDK9 inhibition, thus leading to disappearance of the cell survival factor, Mcl-1. These three classes of chemicals display promising anti-proliferative and antitumor properties
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Göpfert, Ulrich. "Untersuchungen zur posttranskriptionellen Regulation der CDK-Inhibitoren p27Kip1 und p21Cip1." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964916177.
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Arnedos, Ballester Monica. "Personalized Medicine and Biomarker Discovery to Targeted Therapies in Breast Cancer : Focus on CDK4/6 Inhibitors." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS181.
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L’avènement du séquençage haut débit a mis en lumière l’hétérogénéité des cancers du sein qui peuvent être groupés en fonction d’altérations moléculaires spécifiques qui sont pour certaines à la base de thérapies ciblées dans le cadre de la médecine personnalisée. Néanmoins de nombreuses complications viennent compromettre le succès thérapeutique de ces approches. En effet, l’une des thérapies ciblées les plus efficaces développées récemment, les inhibiteurs de CDK4/6, sont prescrits chez tous les patients HR+/HER aux stades avancés de la maladie alors même qu’aucun biomarqueur n’a pour l’heure été identifié. Ainsi les données pharmacodynamiques et les marqueurs pronostics font cruellement défaut pour ces patients. Afin d’identifier de tels marqueurs, nous avons conduit une étude clinique « fenêtre d’opportunité » incluant 100 patients à un stade précoce de la maladie. L’analyse en IHC et les études de profilage expression génomique des tumeurs a permis de montrer qu’une courte exposition au palbociclib, un inhibiteur de CDK4/6 induisait un arrêt du cycle cellulaire révélé par une diminution de phospho-Rb et Ki67. Cette corrélation entre diminution du phospho-Rb et la diminution de la prolifération suggère d’ailleurs son utilisation comme biomarqueur de la réponse au palbociclib. Une analyse sur puce à cDNA a permis d’identifier un panel de gènes régulateurs de la prolifération (MKI67, TOP2A, BIRC5) et de la machinerie du cycle cellulaire (PLK1, FOXM1) modulé par le palbociclib. Bien que nous n’ayons pu identifier de marqueurs de résistance au palbociclib en condition basale, nous avons observé des niveaux élevés de CCNE chez les patients traités résistants au palbociclib. Cette donnée a été confirmée chez les patients aux stades avancés de la maladie dans le cadre d’une étude menée en collaboration avec un groupe britannique. D’autres données obtenues en collaboration avec une équipe de l’université Vanderbilt, ont par ailleurs permis de suggérer une contribution des inhibiteurs de CDK4/6 à la réversion de la résistances aux hormonothérapie en inhibant l’expression les gènes cibles du facteur de transcription E2F4. Pour finir, les activités biologiques et cliniques des différents inhibiteurs de CDK4/6 disponibles n’étant pas exactement identiques, un second essai clinique « fenêtre d’opportunité » nous a permis de mettre en évidence un profil de toxicité distinct de l’abemaciclib et de montrer que, contrairement au palbociclib, l’abemaciclib montre une efficacité lorsqu’il est utilisé seul. Une des explications possible de ces différentes activités serait un spectre d’action de l’abemaciclib ciblant plus efficacement la CDK9, même si l’impact clinique associé n’a pas été examiné en détail et qu’une comparaison rigoureuse de l’activité de ces deux inhibiteurs de CDK n’a pas encore été réalisée<br>New sequencing methods have revealed that breast cancer is heterogeneous and characterized by different subgroups harboring specific molecular alterations for which targeted therapies have been developed with the hope of implementing personalized medicine. However, this approach has been proven far too simplistic. Indeed, one of the latest and more efficient targeted therapies to be developed in breast cancer are the CDK4/6 inhibitors, approved for all HR+/HER2- advanced breast cancers. So far, and despite the significant number of patients treated with these drugs, no biomarkers of efficacy have been identified and no clear information about pharmacodynamics have been presented. In order to determine biomarkers of efficacy and pharmacodynamics of palbociclib, the first approved CDK4/6 inhibitor, we conducted a window of opportunity clinical trial in 100 early breast cancer patients. IHC and GE analyses identified that a short period of palbociclib treatment was able to induce cell cycle arrest as determined by decreased phospho-Rb expression and this was accompanied by a profound decrease in proliferation as determined by lnKi67<1 after treatment, with a correlation between changes in proliferation and changes in phospho-Rb, suggesting that early decrease in phospho-Rb could be linked to sensitivity to this drug. Microarray analyses identified that palbociclib modulates genes involved in proliferation (such as MKI67, TOP2A, BIRC5) and cell cycle (such as PLK1, FOXM1). Despite we were not able to identify baseline biomarkers of resistance to this treatment, we observed that levels of CCNE remained high in palbociclib-resistant patients. This finding was further validated in collaboration with an-UK research group who had conducted biomarker research in the advanced setting. Moreover, our data helped also to determine in a different collaboration with Vanderbilt University, that CDK/6 inhibitors might contribute to reverse endocrine resistance generated by activation of genes linked to the E2F4 transcription factor. Finally, as preclinical and clinical data suggest some diversity between different CDK4/6 inhibitors, we decided to conduct a second window of opportunity trial with a second CDK4/6 inhibitor, abemaciclib, who has shown different toxicity profile and, unlike palbociclib, significant efficacy as single-agent. One suggested explanation could be due to a higher impact on CDK9, although its clinical impact has not been determined and no comparison between these two drugs has been performed
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Tikad, Abdellatif. "Développement de nouvelles pyrido[3,2-d]pyrimidines polyfonctionnalisées : application à la synthèse d’inhibiteurs de CDKs." Orléans, 2008. http://www.theses.fr/2008ORLE2079.
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Les pyrido[d]pyrimidines constituent une classe de composés intéressants tant sur le plan chimique que biologique. Une des applications courante pour cette famille concerne leur utilisation dans l’élaboration de nouvelles drogues inhibitrices de kinases. Dans ce travail nous nous sommes intéressés aux pyrido[3,2-d]pyrimidines, les moins décrites dans la littérature, car leurs voies d’accès sont souvent difficiles et les produits de départ sont très onéreux. Dans le cadre de la recherche de nouveaux agents cytotoxiques et d’inhibiteurs de kinases toujours plus sélectifs, nous avons développé de nouvelles méthodologies originales, efficaces et moins coûteuses, pour la synthèse des 2,4-dichloro et 2,4,7-trichloro pyrido[3,2-d]pyrimidines. La réactivité de ces deux synthons clés a été étudiée lors de réactions de substitutions nucléophiles aromatiques et divers couplages pallado-catalysés qui ont permis l’obtention d’un large panel de molécules polyfonctionnalisées en position 2, 4 et 7. Différentes analyses pharmacologiques ont été réalisées. L’inhibition des kinases (DYRK1A, CDK5, GSK3) par nos produits originaux et l’évaluation de leur cytotoxicité sur diverses cellules humaines cancéreuses ont été réalisées. Certaines molécules ont donné des résultats extrêmement prometteurs. Les relations entre structure et activité de ces nouvelles familles de composés sont discutées.
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Ebner, Benedikt [Verfasser]. "Kombinationstherapien mit dem CDK4/6 Inhibitor Palbociclib und weiteren Target-Therapeutika im Urothelkarzinom / Benedikt Ebner." München : Verlag Dr. Hut, 2021. http://d-nb.info/1238422977/34.
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Samuelsson, Magnus. "p57Kip2, a glucocorticoid-induced CDK inhibitor, involved in cell proliferation, apoptosis and differentiation /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-382-1/.
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Thomas, A. J. "The investigation of AT7519 (CDK inhibitor) and AT13387 (HSP90 inhibitor) as novel therapies for the treatment of pancreatic ductal adenocarcinoma." Thesis, University of Liverpool, 2016. http://livrepository.liverpool.ac.uk/3004686/.
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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is usually a systemic disease and so requires chemotherapy even when surgical resection is possible. Current agents such as gemcitabine are only effective in a minority of patients and chemoresistance is an important issue faced in pancreatic cancer. There is an urgent need for more effective therapies; Novel agents may offer an avenue for this. The CDK family of protein kinases are pivotal in cell cycle regulation that is often deranged in cancer. HSP90 is a molecular chaperone affecting multiple key cellular signaling pathways of importance in pancreatic cancer. OBJECTIVES: To determine the efficacy of AT7519, a novel CDK inhibitor, and AT13387, a novel HSP90 inhibitor in pancreatic cancer models in vivo and in vitro both as single agents and in combination with gemcitabine. METHODS: Cell proliferation was measured using the EZ4U assay. Cell cycle analysis was performed with flow cytometry. Apoptosis analysis was using the Caspase-Glo 3/7 luminescent assay. Western blotting assessed expression of client proteins and phosphorylation. A murine xenograft model was employed assessing tumour volumes with external calipers. Experiments were performed using AT7519 and AT13387 as single agents and in combination with gemcitabine. RESULTS: AT7519 inhibited proliferation in cell lines including a gemcitabine resistant line (SUIT-2 GR) with IC50 values ranging 5-2000nM. Cell cycle analysis showed actions in line with CDK inhibition. At 24 hours induction of caspase 3/7 was significantly increased in AT7519 treated cells compared with those in control media (p=0.01). Phosphorylation of client proteins were inhibited. Isobolar analysis of AT7519 combined with gemcitabine suggested an additive effect. AT7519 was tolerated as a single agent and in combination with gemcitabine in a xenograft model and resulted in slowed tumour growth compared to control (p = 0.0446). AT13387 inhibited proliferation of all cell-lines with IC50 values of 29nM-325nM including a cell line with acquired gemcitabine resistance. AT13387 treated cells accumulated in G0/G1 and G2/M phases of the cell cycle (p < 0.05). Late apoptosis was seen after 40 hours post treatment with At13387. AT13387 treatment resulted in down-regulation of HSP90 client proteins and up-regulation of the HSP70 co-chaperone. AT13387 was tolerated and moderately efficicacous as a singe agent in a murine xenograft model but was poorly tolerated in combination with gemcitabine and conferred no benefit over single agents. CONCLUSIONS: AT7519 is a promising agent for combination therapy in pancreatic cancer, as acquired resistance to gemcitabine does not give AT7519 resistance. AT13387 showed promising effects in vitro but was poorly tolerated in an in vivo combination with standard therapy therefore may offer a therapeutic avenue for non-responders to conventional therapies.
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Liu, Wei. "Tetraspanin KAI1/CD82 inhibits cell migration-related cellular events via reorganizing actin network." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/WORLD-ACCESS/Liu/2007-030-Liu.pdf.
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Thesis (M.S. )--University of Tennessee Health Science Center, 2007.<br>Title from title page screen (viewed on July 17, 2008). Research advisor: Xin Zhang, Ph.D. Document formatted into pages (xv, 197 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 159-197).
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Peyressatre, Marion. "Développement de biosenseurs fluorescents et d’inhibiteurs pour suivre et cibler CDK5/p25 dans le glioblastome." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONT3513/document.
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CDK5 est une protéine kinase exprimée de façon ubiquitaire et activée principalement dans le système nerveux central, ou elle joue un rôle important dans la transmission synaptique, la guidance axonale et la migration cellulaire, la plasticité synaptique et le développement neuronal. CDK5 est associée à la protéine p35 au niveau de la membrane cellulaire, et activée par clivage calpaine-dépendant de cette dernière en p25, ce qui conduit à la relocalisation de CDK5/p25 dans le cytoplasme cellulaire. CDK5/p25 phosphoryle de nombreux substrats dont la protéine Tau, contribuant ainsi à l’apparition de plaques neurofibrillaires responsable des pathologies neurodégénératives comme Alzheimer et Parkinson, lorsqu’elle est hyperactivée. Plus récemment, l’expression et l’hyperactivation de CDK5 a été décrite comme impliquée dans le développement de cancers et en particulier de tumeurs cérébrales. Toutefois aucune approche ne permet actuellement de détecter et de mesurer l’activité de CDK5/p25 directement dans des cellules vivantes, au sein des tissus et des tumeurs concernées, dû à un manque d’outils fiables et sensibles pour quantifier les changements dynamiques de son activité kinase. Par ailleurs, peu d’inhibiteurs sont actuellement disponibles pour inhiber CDK5/p25, de manière spécifique, la plupart ciblant la poche de fixation de l’ATP.Le premier objectif de ma thèse a consisté à développer un biosenseur d’activité fluorescent de nature peptidique appelé CDKACT5 qui rapporte l’activité kinase de CDK5/p25 recombinante et dans des extraits cellulaires de manière dynamique et réversible suivant stimulation ou inhibition de cette kinase. Une fois caractérisé et validé in vitro, le biosenseur a été appliqué à la détection d’altérations de CDK5/p25 dans différentes lignées cellulaires de glioblastome dans des essais fluorescents d’activité kinase. Enfin CDKACT5 a été introduit dans des cellules neuronales vivantes afin de suivre les changements dynamiques d’activité de CDK5/p25 par microscopie de fluorescence et vidéo microscopie.Le deuxième objectif de ma thèse a consisté à développer un biosenseur fluorescent conformationnel dans le but d’identifier des inhibiteurs non compétitifs de l’ATP ciblant la boucle d’activation de CDK5. Le biosenseur CDKCONF5 a été exploité pour réaliser un criblage haut débit de trois chimiothèques de petites molécules. Les touches identifiées ont été validées et caractérisées in vitro, pour déterminer leur potentiel inhibiteur dans des tests d’activité kinase et de prolifération cellulaire, ainsi que leur mécanisme d’action. Ces molécules constituent des candidats prometteurs pour une chimiothérapie sélective du glioblastome<br>CDK5 is a protein kinase ubiquitously expressed but mainly activated in the central nervous system, where it plays an important role in neuronal functions such as synaptic transmission, axonal guidance and migration, synaptic plasticity and neuronal development. CDK5 is associated with p35 protein at the cell membrane, then activated by calpain-mediated cleavage of p35 into p25, which promotes relocalization of CDK5/p25 into the cytoplasm. CDK5/p25 phosphorylates a wide variety of substrates including Tau, thereby contributing to appearance of neurofibrillary plaques responsable for neurodegenerative pathologies such as comme Alzheimer’s et Parkinson’s, when hyperactivated. More recent studies suggest that CDK5 expression and hyperactivation are involved in glioblastoma during cell invasion and CDK5 expression has been reported to be correlated with the pathological grade of gliomas. However there are currently no tools available to monitor CDK5/p25 activity in its native cellular environment, in tissues or in tumours, due to an overall lack of reliable tools to quantify dynamic changes in its kinase activity in a sensitive and continuous fashion. Furthermore, few inhibitors are currently available to target CDK5/p25 in a specific fashion and most of them are ATP competitive inhibitors.The first goal of my thesis was to develop a fluorescent peptide biosensor named CDKACT5, that specifically reports on recombinant CDK5/p25 and on endogenous CDK5 activity in cell extracts in a dynamic and reversible fashion following stimulation or inhibition of this kinase. Once validated in vitro, this biosensor was applied to detect alterations in CDK5/p25 activity in different glioblastoma cell lines in fluorescent kinase activity assays. Finally CDKACT5 was introduced into cultured neuronal cells to monitor dynamic changes in CDK5/p25 activity by fluorescence imaging and time-lapse microscopy.The second goal of my thesis project consisted in developing a conformational fluorescent biosensor to identify non-ATP competitive inhibitors targeting the activation loop of CDK5. CDKCONF5 was implemented to perform a high throughput screen of three small molecule libraries. The hits identified were validated and characterized to determine their inhibitory potential in kinase activity and proliferation assays, as well as their mechanism of action. These compounds constitute promising for selective chemotherapy in glioblastoma
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costa, aurora. "INHIBITION OF CYCLIN-DEPENDENT PROTEIN KINASES AS A POTENTIAL NEW THERAPEUTIC STRATEGY FOR THE TREATMENT OF MALIGNANT PLEURAL MESOTHELIOMA." Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227738.
Full textAbstract:
Direct or indirect exposure to asbestos fibers can cause various respiratory pathologies such as asbestosis
or pleural effusion, up to more serious pathologies, even tumors such as malignant pleural mesothelioma
(MPM).
MPM is a highly aggressive tumor for which there is still no effective therapeutic strategy today and, despite
the use of asbestos being banned in many western countries, unfortunately an increase in incidence is still
estimated. This is due both to the long clinical latency that characterizes the development of MPM (about
40 years), and to the environmental persistence of materials containing asbestos. To date, MPM still
represents a therapeutic challenge, and therefore, the identification of new and effective therapies is
urgently needed.
In recent years various approaches have been tested in the preclinical phase by the group where I carried
out my thesis activity, such as the inhibition of the oncogenic kinase SRC, or the reactivation of p53, both
capable of inducing cell death both in MPM lines than in in vivo experiments. More recently they observed
that reactivation of the RBL2/p130 tumor suppressor was also able to induce apoptosis in MPM cell lines.
These data suggested that acting by restoring the function of retinoblastoma family proteins could be an
effective strategy for this tumor.
The aim of my thesis project was to verify whether the inhibition of the cyclin-cycline kinase dependent
complexes, more specifically using the cyclin dependent kinase (CDK) inhibitor abemaciclib, was able to act on the pocket proteins, reactivating their tumor suppressor potential.<br>First of all, we demonstrated that abemaciclib dose-dependently inhibits the cell proliferation in short- and
long-term of MPM cell lines, however, it did not show the same cytotoxic effects on the mesothelium cell
line used as a control. Subsequently, we tested and confirmed that abemaciclib is able to induce apoptosis
in some MPM lines by cytofluorimetric assay of annexin. In Addition, we then evaluated the ability of
abemaciclib on 3D cell cultures, drug showed the capacity inhibiting the formation of 1st and 2nd
generation spheroids but also blocking the proliferation of fully formed spheroids. Again, to investigate the
molecular mechanism through which the inhibitor acts, by western blotting, the phosphorylation levels of
all three pocket proteins (RBL2/p130, RBL1/p107 and RB1/p105) were evaluated, as expected. The
inhibitor induces a reduction in p130 (Ser952) and (Ser941), p107 (Ser975), and p105 (Ser780)
phosphorylation with consequent restoration of protein functionality.
Moreover, since previous studies demonstrate that reactivation of the RBL2/p130 tumor suppressor
mediates apoptosis in MPM cell lines by counteracting the AKT-activated antiapoptotic pathway, we tested
the levels of AKT protein and its phosphorylation, and how we expected this drug induces reduction of
AKT phosphorylation (Ser473).
Finally, we decided to evaluate the sensitivity to abemacilib on ciplatin-resistant cells, and the results show
that the more aggressive mesotlioma cells remained sensitive to this drug even though resistant to cisplatin.
The data obtained are certainly still preliminary, the subsequent studies are necessary above all to
understand what happens to the progression of the cell cycle, but at the same time, they are promising,
urging us to continue and making us hypothesize that this treatment could be effective for patients with
MPM
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Saguner-Imsak, Emine Roze [Verfasser], and Jan [Akademischer Betreuer] Dürig. "Testung eines neuartigen selektiven CDK9-Inhibitors in präklinischen Modellen der chronischen lymphatischen Leukämie / Roze Saguner-Imsak ; Betreuer: Jan Dürig." Duisburg, 2020. http://d-nb.info/1223849503/34.
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Wilson, Anthony James. "Synthesis of novel inhibitors of CDK 4 / Cyclin D1 based on the natural marine sponge pigment fascaplysin." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/4531.
Full textAbstract:
This thesis describes an investigation into the design and synthesis of novel inhibitors of the cell-cycle regulatory enzyme CDK4/Cyclin D1 utilising the structure of the natural product fascaplysin as a lead compound. The structure activity relationship of fascaplysin (i) has been investigated and its structure simplified and rationalised to give novel inhibitor pharmacophores based on structures (ii) and (iii). Inhibitors of structure (ii) were found to possess activity against CDK4/Cyclin D1 of IC50 51-176 μM and to be approximately 15-20 fold selective over CDK2/Cyclin A. The second generation compounds of structure (iii) were synthesised to explore a suspected π-stacking pocket in the active site of CDK4 around the Phe93 residue. These compounds were active against CDK4/Cyclin D1 with IC50 values in the range 7-50 μM and to be 20-100 fold selective over CDK2/Cyclin A. New methodology towards the synthesis of un-natural products bearing a carbohydrate subunit connected via the anomeric 1-position and the 2- and 3- positions of the sugar was also explored. Indole was successfully glycosylated with a glucose subunit at the anomeric position (v) using the anhydrosugar (iv) and indoline was glycosylated with a glucose sugar derivative at the 3-position (viii) using the manno-epoxide (vi).