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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.

One current approach in the treatment of cancer is the inhibition of cyclin dependent kinase (CDK) enzymes with small molecules. Here the discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors is described, including details of the design and successful synthesis of novel ring-constrained thiazolylpyrimidines. The structure-activity relationship (SAR) trends exhibited by this constrained thiazolylpyrimidine family of CDK inhibitors are presented and compared with those from an unconstrained series of analogues. One significant finding from this aspect of the project was that ring-constrained thiazolylpyrimidines in general inhibit CDK2-cyclin E with greater potency than the corresponding unconstrained forms. Furthermore, an X-ray crystal structure of 2-methyl-N-[3-nitrophenyl]-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine, a representative from the constrained thiazolylpyrimidine series, in complex with CDK2-cyclin A is reported; confirming the binding mode within the CDK2 ATP binding pocket. A further assessment of SARs through the synthesis of control compounds and an extended study into the synthesis of N-substituted derivatives is described. The identification of CDK inhibitors that possess a strong selectivity profile across the CDK family is important. For example, the identification of highly CDK4-selective inhibitors should enable researchers to study the biological role of this important enzyme and to enable a block of cell division in the G1 phase. Here synthetic attempts to prepare a potentially CDK4 selective inhibitor compound, namely 5-methyl-N8-[4-(piperazin-1-yl)phenyl]thiazolo[4,5-h]quinazoline-2,8-diamine, are described. This approach was inspired by SAR data published on a structurally related inhibitor, 8-cyclopentyl-5-methyl-2-[4-(piperazin-1-yl)phenylamino]pyrido[2,3-d]pyrimidin-7(8H)-one.

The tuberous sclerosis-2 (Tsc-2) gene product, tuberin, functions as a renal tumor suppressor. Treatment of Eker (Tsc-2 EK/+) rats and primary renal epithelial cells derived from Tsc-2 EK/+ rats (QTRRE cells) with 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of heterozygosity at the Tsc-2 locus in kidney tumors and QTRRE cells. QTRRE cells are carcinogenic in athymic nude mice. Analysis of kidney tumors formed in Tsc-2 EK/+ + rats following 8-months of TGHQ treatment reveals increases in B-Raf, Raf-1, pERK, cyclin D1, p27Kip1, 4EBP1, p-4EBP1(Thr70), p-4EBP1(Ser65), and p-4EBP1(Thr37/46) protein expression. These data establish the involvement of mTOR and MAPK signaling cascades in tuberin null tumors. Similar increases in 4EBP1 and p4EBP1 are observed in renal tumor QTRRE-xenografts in nude mice. Concomitant with increases in expression of these proteins in TGHQ-induced renal tumors, similar changes are observed in QTRRE cells, which also exhibit high ERK, B-Raf and Raf-1 kinase activity; and increased expression of cyclin D1, p27, p-4EBP1 (Thr70), p-4EBP1 (Ser65), and p-4EBP1 (Thr37/46). Manipulation of the Raf/MEK/ERK kinase cascade in QTRRE cells, with kinase inhibitors and siRNA, indicates that Raf-1/MEK/ERK participates in crosstalk with 4EBP1 to regulate translation of cyclin D1.Cyclin D1 and p27 protein levels are increased in the cytoplasm in our RCC models. In normal HK-2 cells, p27 and cyclin D1 are localized to the nucleus. Due to the instability of the cyclin D1-CDK4 complex, p27 interaction is necessary for cyclin D1-CDK4 complex assembly and stabilization in the nucleus. Manipulation of p27 protein levels in QTRRE cells with phosphodiesterase inhibitors, dibutyryl cAMP, and the proteosome inhibitor MG132, all result in a parallel increase in p27 and cyclin D1. Furthermore, p27 siRNA and sorafenib treatment both cause a decrease in p27 and cyclin D1. Further manipulation of cAMP, Rap1B, and B-Raf proteins, revealed that cAMP/PKA/Rap1B/B-Raf activation and B-Raf//ERK MAPK inhibition both modulate p27 expression and compartmental localization in tuberous sclerosis renal cancer. Phosphodiesterase inhibitors play a role in regulating the expression, degradation, and cytoplasmic localization of p27. Therefore, cytoplasmic p27-cyclin D1 mislocalization and stabilization may have an oncogenic role in the cytosol and play a crucial role in tumor formation.

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Protein-protein interactions (PPIs) are key drug targets and recent breakthroughs in this area are providing insight into the types of molecules needed to selectively and potently inhibit a target traditionally seen as untractable. The rules that have been used to design classic substratecompetitive drugs (for example Lipinski's rule of five) may not apply in this new field in the same way. Here I present work performed in three systems that are well-validated drug targets for oncogenesis: the CDK2/cyclin A complex, the PLK1 Polobox domain and MDM2. In each case the site of the protein-protein interaction is defined and understood and the rationale for pharmaceutical intervention is clear. I use these as a model system to evaluate the characteristics of drugs that target protein-protein interaction sites and present work on the development of inhibitors as potential leads for subsequent drug development. In Chapter 1 I introduce the problems, challenges and rewards of PPI drug development; in Chapter 2 I present co-crystal structures of MDM2 with isoindolinone inhibitors; in Chapter 3 I detail attempts to co-crystallise the Plk1 Polobox with inhibitors and screen potential inhibitors; in Chapter 4 I present the results of screening to identify inhibitors of Cyclin A recruitment; and in Chapter 5 I discuss other strategies for inhibition of the CDK2/cyclin A complex, including results with a covalent inhibitor. Through these projects I have been able to demonstrate the wide applicability of the PPI inhibition approach, identify key features of drugs able to inhibit PPIs and contribute to drug design in each system.

Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy. In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors. In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.

Cardiac hypertrophy leading to heart failure remains a leading cause of morbidity and mortality in the 21st century despite major therapeutic advances. Improved understanding of novel molecular and cellular processes contributing to cardiac hypertrophy therefore continues to be important. Cyclin-dependent Kinase 9 (CDK9), part of a family of proteins controlling cell cycle and growth, has emerged as one such potential candidate over the last 5 years. CDK9 is the catalytic subunit of the CDK9/CyclinT complex and acts by phosphorylating the carboxy-terminal domain of RNA polymerase II. Hypertrophic signals, such as Endothelin-1 (ET-1) and phenylephrine, have been shown to cause CDK9 activation leading to a hypertrophic response in cultured mouse cardiomyocytes associated with reactivation of the foetal gene program. CDK9 also forms a complex with GATA4 to play a role in differentiation of mouse ES cells into cardiomyocytes. These findings suggest a specific role for CDK9 in controlling growth and differentiation of cardiomyocytes and merits further study in models where cardiomyocyte differentiation and proliferation are key contributors. In contrast to mammals, zebrafish retain a high cardiomyocyte proliferative capacity throughout their life span and can readily repair following injury. I have examined the role of CDK9 on global and cardiac development in the zebrafish embryo. I have also assessed the impact of CDK9 manipulation on response to ventricle injury using a laser-induced injury model developed and validated as part of my thesis. My findings confirm that normal growth of the embryonic ventricle is associated with a rapid increase in cardiomyocyte number, that was of 50% in the period 96-120 hpf, accompanied by increasing chamber trabeculation. This is also characterized by an increase in the gene expression of most of cardiac development relevant transcription factors, i.e. GATA4, 5 and 6, and MEF2c. The significant reduced cardiovascular function (14% of Ejection Fraction compared to 20% in controls) at 2 h post laser injury in the zebrafish embryonic heart promptly recovers at 24 hour post-laser, accompanied by acceleration of cardiomyocyte proliferation, that increased of 49% in injured ventricles compared to 20% in controls in the period 2-24 h post-laser. Pharmacological and genetic inhibition of CDK9 activity also significantly reduced cardiac growth, cardiomyocyte number, ventricle function and impairs functional recovery following laser injury. Conversely, genetic inhibition of LARP7, a CDK9 repressor, resulted in increased cardiomyocyte number and was associated with full functional and cellular recovery following laser-injury. In conclusion, I have provided evidence, in the zebrafish embryonic heart, that CDK9 plays an important role in cardiac growth and development and impacts significantly on cardiomyocyte proliferation. I have also shown that CDK9 manipulation significantly affects cellular and functional recovery following laser-induced injury. Further studies are required to further define the role of CDK9 and LARP7 in the heart and develop therapeutic strategies using this pathway that could contribute to cellular repair mechanisms in the adult mammalian heart.

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