Adrenergic receptors (AR) are G protein-coupled receptors (GPCRs) responsible for regulating physiological processes including the fight-or-flight response, muscle contraction, blood flow, and energy release. Numerous protein kinases play key roles in AR signaling cascades such as GPCR kinases (GRKs) and inflammatory kinases whose aberrant activity contributes to cardiovascular disease, hypertension, type 2 diabetes, and obesity. Thus, a detailed molecular understanding of the structure, function, and regulation of these kinases will therefore facilitate the development of novel therapeutic interventions.Obesity results in chronic inflammation of adipocytes through increased expression of inflammatory kinases, namely, IKKe and TBK1, which indirectly attenuate B3 adrenergic receptor signaling to decrease energy expenditure and disrupt glucose homeostasis. Together, excessive activity of IKKε and TBK1 exacerbates the obese phenotype and leads to the development of type 2 diabetes. The drug amlexanox is a modest potency inhibitor of both kinases that produces weight loss and improves insulin sensitivity when administered to obese mice. Herein, I report the co-crystal structure of TBK1 in complex with amlexanox and a comprehensive profiling of the structure-activity relationships (SAR) of amlexanox analogs. Through the determination of seven co-crystal structures of TBK1 in complex with amlexanox and amlexanox analogs inhibitors, we uncovered mechanisms for improving potency, cellular efficacy, and selectivity against IKKe and TBK1. Future efforts on targeting IKKe and TBK1 should carefully consider pharmacokinetics, as we observe a stark disconnect between in vitro potency and efficacy in cells and animals. Taken together, amlexanox undoubtedly represents a pharmacophore amenable to therapeutic development whose analogs may have clinical value in the treatment of diabetes and obesity.Heart contractility is carefully regulated through the activation of the a1 adrenergic receptor and subsequent desensitization by GRKs. Excessive activation of a1AR, such as during cardiac arrest, leads to abnormally high levels of intracellular Ca2+ and activation of calmodulin (Ca2+CaM) which inhibits GRK5 and promotes nuclear translocation of the kinase, leading to maladaptive ventricular hypertrophy. I determined the architecture of the Ca2+CaM–GRK5 complex through small X-ray scattering and electron microscopy, which show that Ca2+CaM inhibits GRK5 via bitopic bridging to two different regions of GRK5. The natural product calmodulin inhibitor malbrancheamide, which I showed binds exclusively to the C-terminal lobe of Ca2+CaM in a co-crystal structure, was used to probe the Ca2+CaM–GRK5 interaction. I establish malbrancheamide as a tool for probing the regulatory effects of half the Ca2+CaM interaction. The bitopic nature of Ca2+CaM binding to GRK5 produces different modes of regulation which may therefore be exploited for therapeutic development. In particular, disrupting the C-terminal interaction may retain GRK5 at the cell membrane and block cardiac hypertrophy. Together, my work suggests that natural products may serve as useful probes for studying Ca2+CaM protein-protein interactions. Future efforts should consider the utility of screening natural product compound libraries to identify additional molecules that may be of use in studying maladaptive cardiac pathologies.
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Structural Studies and Pharmacological Targeting of Protein Kinases in Obesity and Heart Disease
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