Browsing by Subject "Protein Kinase Inhibitors"
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Item Autoinhibition and Chloride Sensing in the WNK1 Kinase(2012-07-16) Moon, Thomas Matthew; Goldsmith, Elizabeth J.Protein kinases control diverse cellular pathways and have are the subject of intensive study regarding how they maintain specificity toward sub- strates. The research presented here focuses on a 230kDa serine/threonine protein kinase known as WNK1 (with no lysine {k}). The protein was first cloned by Melanie Cobb's laboratory, and its isoforms have been associated with a monogenic form of hypertension as well as with breast and prostate cancer. Recent data have also shown that WNK1 is necessary for maintaining spindle polarity in mitosis and plays a role in post-mitotic abscission. The function of WNK1 is most commonly associated with the regulation of CCCs via the activation of the WNK1 substrates OSR/SPAK. Prior investigation of the system has demonstrated that CCCs are activated by increasing con- centrations of extracellular salt and by intracellular phosphorylation from the OSR/SPAK kinases. Due to it ubiquity in mammalian cell types, a question has arisen as to how the pathway responds to changes in osmolarity. Further, because of the involvement of WNK1 in a diverse set of cellular mechanisms, how is WNK1 activity and substrate specificity controlled? An autoinhibitory domain of WNK1 was characterized by the Cobb Lab regarding its ability to inhibit the kinase in cis and in trans. In this study, we find that the solution structure of the autoinhibitory domain retains a conserved RFXV binding site from the PASK/FRAY homology 2 (PF2) domain present in OSR/SPAK. Titration data shows that incubation with a 5-mer and a 20-mer peptide derived from the WNK1 kinase domain displays extensive chemical shift perturbation as assessed by 1H,15N-HSQC. Expression of this autoinhibitory domain in cis with the WNK1 kinase domain followed by size-exclusion chromatography shows substantial confor- mational changes when dialized from high to low salt. A measurement of the activity of the WNK1 kinase domain in the presence of increasing amounts of sodium chloride indicate an IC50 of 130mM. Further biophysical investigation using differential scanning fluorimetry with the kinase domain shows that the domain undergoes substantial increases in domain stabilization as the concen- tration of salt is increased. Continued analysis of this phenomena has pointed toward evidence of anion sensing by the WNK1 kinase domain. Other protein kinases studied in our lab do not exhibit this salt sensitivity. To determine the binding site of chloride in the WNK1 kinase domain, the inactive WNK1 kinase was cocrystallized in the presence of sodium bro- mide. A dataset was collected using the bromine anomalous edge (0.92 ̊A). The anomalous difference fourier map was calculated and a 5.2 σ peak was observed at the N-terminus of the 3.10 helix present in the DLG motif of the activation loop. To corroborate these data, the structure of the inactive kinase domain previously crystallized in sodium chloride was re-refined. A similar binding site corroborated by a 2mFo − DFc peak of 5.5 σ was observed in subunit A near the N-terminus of the 3.10 helix. When the structure was refined in with a chloride ion placed in the observed density, similar hydrogen bonding interactions between the amide backbone and the chloride ion were observed compared to that in the bromide-soaked structure. The presence of this chloride ion appears to favor sequestration of E268 in αC and R348 in the catalytic loop and promotes an inactive kinase structure. Finally, the crystal structure of the activated WNK1 kinase domain was determined under low-salt conditions. The term 'activated' and not active is used to describe this structure of the WNK1 kinase domain because, although it is phosphorylated at S378 and S382 in the activation-loop during expression, the structure adopts an inactive conformation due to the placement E268 in helix C. The structure displays disorder of many key structural elements such as the N-terminus of αC. A key observation is the lack of a 3.10 helix in the N-terminus of the activation loop and the lack of water or any atom that could be chloride near the amide backbone near the chloride binding site. Based upon the literature surrounding the activation of WNK1 and the data presented in this thesis, we predict a three-tiered regulation of WNK1 driven by a) autophosphorylation b) chloride binding and c) autoinhibitory domain occlusion of the nucleotide and/or docking interfaces present in the WNK1 kinase domain. The coupling of the information that we have gath- ered on the autoinhibitory and kinase domains appear to point to an overall mechanism of salt sensing and self-contained signaling control in the WNK1 kinase cascade.Item One-Pot Measurement of the Kinetic Parameters KI, kinact, and Time-Dependent IC₅₀ for Analysis of Covalent Small Molecule Kinase Inhibitors(2016-01-19) Montalvo, Steven K.; Mondi, Anuja; Westover, Kenneth D.To address the need for the analysis of covalent kinase inhibitors in a high-throughput method, we established a protocol for the facile measurement of kinact and KI utilizing a proprietary off-chip mobility shift assay supplied by PerkinElmer. With this electrophoretic technique, these kinetic parameters, which describe covalent inhibitors better than IC50 measurements, are accurately and reproducibly measured. As a proof of concept measurement, an inhibitor of BMX, compound BMX-IN-1, was shown to have a kinact = 0.0298 ± 0.0024 min-1 and a KI = 0.134 ± 0.021 uM. The industry standard for reporting is kinact/KI and was calculated to be 0.222 ± 0.039 uM-1min-1. To direct medicinal chemists optimizing parameters of covalent inhibitors, simulations of the equations used to fit progress curves were performed in MatLab. These revealed the compelling argument to pursue improved KI before kinact and are discussed further. In addition, some limitations of our assay are presented.Item Studies Toward the Syntheses of Antiarrythmic, Anti-Virulence, and Anticancer Small Molecules(2015-11-23) Adebesin, Adeniyi Michael; Kürti, László; Falck, John R.; Chen, Chuo; Corey, David R.This work is comprised of three projects: a) the development of antiarrythmic analogs of 17(R),18(S)-epoxyeicosatetraenoic acid for the treatment of atrial fibrillation, b) the development of potent inhibitors of QseC mediated virulence gene expression, and c) studies toward a biomimetic total synthesis of nigricanoside A. Antiarrhythmic analogs of 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EEQ): Arrhythmias such as atrial and ventricular fibrillation are a leading cause of death in the United States of America. However, the available drugs for the treatment of these deadly conditions can paradoxically induce proarrhythmic effects, amongst other side effects, and are individually insufficient for treatment. Through a neonatal rat cardiomyocyte assay, 17(R),18(S)-EEQ was found to possess negative chronotropic effects, a characteristic of antiarrhythmic activity. My work on this project led to the development of potent and metabolically robust analogs of 17(R),18(S)-EEQ, which are currently being developed by OMEICOS Therapeutics GmbH, an early stage drug development company, for the treatment of atrial fibrillation. Inhibitors of QseC mediated virulence gene expression: Quorum sensing E. coli regulator C (QseC), a membrane-bound histidine sensor kinase, mediates the expression of various virulence genes in Gram-negative bacteria such as Escherichia coli (EHEC), Salmonella typhimurium, and Francisella tularensis which are pathogenic to humans. Therefore, QseC is a potential target of anti-virulence antibacterial strategies. In collaboration with the Sperandio laboratory, my work on this project led to the development of novel inhibitors of QseC mediated virulence gene expression. Some of the analogs synthesized in this project are currently being investigated by GlaxoSmithKline as anti-virulence agents. Studies toward a biomimetic total synthesis of nigricanoside A: Nigricanoside A, a novel ether-linked glycoglycerolipid with 7 unassigned stereocenters, was reported to possess potent (IC50 ≈ 3 nM) antimitotic activity against MCF-7 and HCT-116 cancer cell lines. However, the rarity of the natural product precluded further structural and biological studies. With the aid of biomimetic hypotheses and literature precedents, the Falck laboratory reduced the stereochemical uncertainty associated with the structure elucidation of the nigricanosides. Furthermore, one of the biomimetic hypotheses inspired the development of a novel stereocontrolled distal epoxidation of conjugated dienols. Armed with this methodology, my work on this project led to the synthesis of the three major fragments of a nigricanoside.Item Targeted therapy: what does the internist really need to know?(2017-09-29) Dowell, Jonathan E.Item Targeting Cyclin Dependent Kinases 4/6 Activity in Pancreatic Ductal Adenocarcinoma(2016-06-06) Franco, Jorge; Brekken, Rolf A.; Burma, Sandeep; Pearson, Gray W.; Knudsen, Erik S.Pancreatic ductal Adenocarcinoma (PDA) is an aggressive and lethal disease that lacks an adequate treatment. Given that patients with PDA only marginally benefit from the current therapies, there is an urgent need to develop more effective approaches that specifically target PDA. Because a significant portion of PDA tumors lose p16 expression and often over-express Cyclin D1, we hypothesized that CDK4/6 activity is deregulated leading to uncontrolled proliferation. Thus, PDA would represent a good candidate for treatment with recently developed CDK4/6 inhibitors. Our study first investigated the sensitivity of PDA cells to CDK4/6 inhibition and found that PDA cells exhibit variable responses to CDK4/6 inhibition, incling models that display significant resistance. Interestingly, these models showed a novel mechanism of resistance to CDK4/6 inhibition. Prior to this study, RB loss was the only mechanism known to circumvent CDK4/6 inhibition. However, herein, we uncovered that CDK4/6 inhibition can lead to aberrant Cyclin E expression, which can compensate for CDK4/6 activity loss and maintain RB in a hyper-phosphorylated state. Subsequent findings demonstrated that this resistance could be blocked by combination therapy with MTOR inhibitors, which prevented aberrant Cyclin E expression and reinforced RB activation. Our second study found that prolonged CDK4/6 inhibition led to an altered metabolic state with an increased in oxidative respiration and glycolysis accompanied by mitochondria accumulation and increase cellular complexity. This heightened metabolic state was mediated by MTOR signaling, which activity was stimulated by amino acid accumulation and an increase in lysosome production in CDK4/6 treated PDA cells. Lastly, we unveiled new combination therapies that targeted the altered metabolism state of CDK4/6 inhibitor treated cells by impinging on antioxidants such as Hemoxygenase 1 (HO-1) and catalase (CAT), whose expression was enhanced post treatment, or by targeting BCL-2/BCL-XL using ABT-737. Taken together our data demonstrate that targeting PDA with CDK4/6 inhibitors can represent an efficacious route for treatment. Activation of MTOR and perhaps other signaling pathways likely contribute to intrinsic and acquired resistance to CDK4/6. These combined data would support the combined use of CDK4/6 with MTOR inhibitors and other agents for the treatment of pancreatic cancer.