ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Bioisosterism is a field that is widely applied to biological molecules, including drugs and agrochemicals. Bioisosterism is the replacement of an active fragment in a molecule with another fragment similar in activity. The replacement is designed to alter the behavior of the molecule in its target environment. In previous work a bioisostere database called the Quantum Isostere Database (QID) was built out of descriptors derived from the theory of Quantum Chemical Topology (QCT). The current work aims to expand the existing QID to include ring fragments. A series of rings were characterised by QCT properties taken from the ring. It was found that four features of a ring each independently have a systematic effect on the ring’s properties. In other words, each of the characteristics of a ring can be changed and have the same effect on the ring’s properties irrespective of the other ring features. The rings were also characterised using the three QCT properties taken from a point within the ring. The three properties established a space where rings were positioned based on their respective three properties. The positions of the rings showed that the space was able to discern between ring types, and that the features of a ring could be predicted if only its three properties were known. To improve the QID the alignment method and scoring were tested. The alignment procedure is unable to correctly align collinear fragments. Therefore, a principal axis alignment procedure was successfully employed to align collinear fragments. For terminal fragments an alternative alignment procedure was proposed to account for the increased rotational freedom. A global axis system meant that the direction dependent properties for all fragments were expressed in this new axis system. This idea was extended further and it was found that the geometry of a molecule was imprinted in the electrostatics when they were expressed in the global axis system. Finally, a pKa prediction method which correlates a single ab initio bond length was tested against two data sets (enols and guanidines). The method relies on subsets to form, where molecules within a subset share a chemical or structural commonality. These subsets were able to distinguish between the five tautomeric forms for the guanidines and different conformations for the enols. All predictions were within 1.0 pKa units of experimental values.

Cyclic-AMP dependent protein kinase (PKA) is a key intracellular signal transduction kinase that is modulated by Gs- and Gi-coupled GPCRs. Under normal physiological conditions, PKA exists as an inactive holoenzyme made up of two catalytic subunits and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits (PKACa) are released to perform various downstream phosphorylation events. However, aberrant PKA activation can cause various diseases including Cushing’s Syndrome, which is an endocrine disorder caused by the overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system. This disorder can be caused by pituitary adenomas that release unregulated amounts of ACTH, adrenal adenomas that release unregulated amounts of cortisol without ACTH stimulation, and ectopic tumors outside the hypothalamus-pituitary-adrenal axis that produce ACTH. In recent genomic studies of patients with ACTH-independent Cushing’s Syndrome, the L205R-PKACamutant has been discovered. Through various studies on the mutant enzyme multiple research groups learned that the single point mutation causes a loss in sensitivity to cAMP signaling, a loss in binding to PKA regulatory subunits, and unregulated phosphorylation of PKACasubstrates, which ultimately leads to the increased cortisol biosynthesis in these patients. The first part of this work describes the enzymology and inhibition studies of known inhibitors against both wt- and L205R-PKACa. Early in the enzymology studies we developed at medium throughput endpoint assay that used Rhodamine-kemptide as the substrate and as a chromophore separating substrate and phosphorylated product using a reverse-phase HPLC method. The analysis of the substrate peptide against both wild-type and mutant enzyme showed a 6-fold decrease in the KMand a 2-fold decrease in kcat, and a similar but lower order of magnitude effect was observed for the studies with ATP. The inhibition studies were performed using the substrate competitive inhibitor PKI(5-24), which showed a 253-fold higher potency towards the wild-type enzyme over the mutant while the ATP-competitive inhibitor was determined to be equipotent. Using this information we used modeling studies to aid in the development of mutant selective functional inhibitors for the substrate-binding pocket. Additionally, we begun to explore the use of Proteolysis Targeting Chimeras, or PROTACs, as another means for targeting the L205R mutant enzyme.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13085号 / 論医博第2126号 / 新制||医||1021(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 渡邊 直樹, 教授 岩田 想, 教授 富樫 かおり / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The LKB1/STRAD/MO25 complex (LSMK) has been identified as the major upstream kinase for AMP-activated protein kinase (AMPK). PKA phosphorylates LKB1 at the Ser428 residue in humans and Ser431 residue in mice. We investigated PKA as an upstream kinase for LSMK. LKB1 that had been incubated with PKA prior to incubation with AMPK experienced up to a 51% increase in AMPK Kinase activity compared to LKB1 alone (p < 0.05). When blocked with a PKA Inhibitor, the kinase effect of PKA on LKB1 was eliminated. Rat epitrochlearis muscle tissue incubated with epinephrine experienced no increase in AMPK activity compared with controls indicating that epinephrine does not cause AMPK activity in this type of tissue. In conclusion, phosphorylation by PKA can increase the AMPKK activity of LKB1-STRAD-MO25 in vitro. Because LKB1 has been found to be constitutively active, it is postulated that phosphorylation by PKA may act to enhance LKB1-AMPK interaction and thus achieve its effect.

cAMP receptors contain highly conserved cAMP binding pockets, in part responsible for allosteric activation, yet CBDs exhibit a wide array of cAMP binding affinities. While several cAMP:CBD crystallographic structures have been solved, they are insufficient to explain differences in cAMP:CBD affinities. We hypothesize that it is the position of the apo autoinhibitory equilibrium and/or a change in the state-specific association constants of the active and inactive CBD forms that are primarily responsible for modulating ~1000-fold difference in cAMP affinities. Interestingly, we discovered that PKARIα and HCN2 have comparable state-specific association constants, suggesting that the position of the apo autoinhibitory equilibrium is primarily responsible for the large difference in observed cAMP affinities in these systems. In addition, the individual components of the cAMP binding pocket (i.e. BBR, PBC, and lid) show functional variability across different CBDs. In RIα, both the BBR and lid are dispensable for high affinity cAMP binding, leaving the PBC as the key determinant of cAMP affinity. Interestingly, in addition the PBC:cAMP contact side-chains, non-contact side-chains are also important in modulating cAMP affinity (ie. L201 and Y205). Further dissection of the contributions arising from the apo pre-equilibrium and the cAMP binding pockets is required to better understand cAMP affinity and selectivity. / Thesis / Master of Science (MSc)

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