Howard Schulman - US grants

Studies in a variety of tissues have rekindled interest in the possible second messenger role of calcium in mediating the action of numerous hormones, neurotransmitters, growth factors and other regulatory agents. Stimulation of neural tissue by membrane depolarization as well as hormonal regulation of liver cell function may involve calcium-dependent phosphorylation. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, I propose to study the possibility that certain of the biochemical and physiological effects of calcium may be mediated by phosphorylation of a diverse array of substrate proteins catalyzed by a calcium-dependent kinase with broad substrate specificity. The major calcium/calmoldulin-dependent protein kinase phospharylating microtubule-associated protein-2 (MAP-2) in rat brain has been purified to homogeneity. The enzyme has a broad substrate specificity characteristic of 'general' protein kinase, such as the cAMP-dependent protein kinase. The possibility that this protein kinase is a mediator of calcium action in diverse tissues will be examined. The physical characterization of the kinase will be extended and the role of autophosphorylation tested. The tissue and subcellular localization of the kinase will be determined by biochemical and immunological assays. The ability of this kinase to phosphorylate numerous potential substrates will be investigated. Differences in the specificity of the calcium kinase and the cAMP kinase will be analyzed by sequencing the phosphorylation sites of selected substrates and by testing synthetic peptide substrates. Activation of the calcium/calmodulin kinase in vivo will be examined in PC12 cells which contain three putative substrates - tyrosine hydroxylase and two groups of microtubule-associated proteins, MAP-2 and tau. Calcium influx into PC12 will be stimulated by depolarization of the cell membrane or by activation of the acetylcholine receptor. 32P incorporation into the target proteins will be analyzed and sites of phosphorylation compared with in vitro phosphorylation by the calcium kinase. Functional changes in the three substrates will be correlated with phosphorylation. Numerous biochemical effects of calcium may be mediated in a variety of tissues via activation of this calcium/calmodulin-dependent protein kinase.

Intracellular calcium serves as a second messenger to mediate the effects of many hormones, growth, factors, neurotransmitters and other extracellular signals. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, this proposal aims to test the possibility that certain of the biochemical and physiological effects of calcium may be mediated by phosphorylation of a diverse array of substrate proteins catalyzed by the multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase). The proposal will examine its regulation by calcium can by autophosphorylation, will determine which type of receptor is subserved by the kinase and will expand on the putative role of the kinase in the regulation of carbohydrate and amino acid metabolism and in nuclear membrane breakdown. Site directed mutagenesis will be used to either block or mimic autophosphorylation sites that are involved in activating or inhibiting the CaM kinase. The mechanism for regulation by autophosphorylation will be investigated. The inhibitory "pseudosubstrate" hypothesis of kinase regulation will be tested with truncation mutants lacking the inhibitory domain. These experiments will generate a calcium-independent form of the kinase to be used in examining two of its many cellular functions. One such function, the calcium-dependent regulation of carbohydrate and amino acid metabolism will be examined in Reuber H35 hepatoma cells. Inhibitory antibodies to the kinase or calcium-independent kinase preparations will be microinjected into the hepatoma cells and the ability of hormonal stimulation to regulate enzymes such as pyruvate kinase and phenylalanine hydroxylase examined. Similar studies are proposed to examine the role of the kinase in the calcium-triggered breakdown of nuclear membrane in sea urchin eggs, another of its postulated functions. Finally, involvement of the CaM kinase in mediating the effect of receptors that evaluate calcium via the phosphatidylinositol and other signalling systems will be examined. To achieve this, a model substrate of the kinase, tyrosine hydroxylase, will be transfected into Swiss 3T3 cells which have numerous receptors with diverse mechanisms for elevating calcium. Cells will be stimulated by signals such as vasopressin, bombesin and platelet-derived growth factor and the activation of the kinase monitored by its phosphorylation of tyrosine hydroxylase.

enzyme activity; calmodulin; calmodulin dependent protein kinase; neurotransmitters; isozymes; neurotransmitter transport; neuroanatomy; enzyme inhibitors; hippocampus; RNA splicing; synapsins; phosphorylation; calcineurin; potassium channel; in situ hybridization; Chelonia; laboratory rat; laboratory mouse;

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is a major protein kinase orchestrating the physiological effects of hormones, neurotransmitters and growth factors in numerous tissues. It responds to stimuli that elevate intracellular Ca2+ and phosphorylates substrates which are localized in the cytosol, membrane and cytoskeletal compartments. Its designation as a general protein kinase brings us to an important question--how does a cell utilize CaM kinase as a mediator of multiple Ca2+-linked hormones, yet still achieve specificity in its cellular responses? We propose to test the hypothesis that the autoregulatory properties of CaM kinase enable it to decode the frequency of Ca2+ oscillations and spikes, thus adding a novel form of temporal response specificity. Furthermore, we propose to test the hypothesis that spatial targeting of CaM kinase isoforms to distinct intracellular sites helps to determine site-specific effects of hormones. Temporal regulation of this multimeric enzyme occurs by its unique autophosphorylation properties. We will extend our studies demonstrating that autophosphorylation occurs when an activated subunit phosphorylates a proximate neighbor in the holoenzyme which also has calmodulin bound. These cooperative properties will be elaborated, since they form the basis for a model by which the kinase may be selectively activated at appropriate Ca2+ spike frequencies. The model will be tested in vitro using immobilized kinase exposed to rapid fluctuations in Ca2+ and in situ by testing frequency dependent activation of the kinase and of the Cl-channel that it regulates in epithelial cells. One of our newly cloned CaM kinase isoforms is targeted to the nucleus and others to the cytoskeleton suggesting a spatial basis for response specificity. We will identify the position of catalytic/regulatory and targeting domains within the structure by electron microscopy. We will identify the nuclear localization sequence and determine whether the enzyme enters the nucleus as a monomeric intermediate or as a multimer. Targeting of the cytoskeletal isoform and its translocation by activation will also be examined. With this basic knowledge, we will target engineered constructs to the nucleus and to the cytoskeleton and use these as reporters of functional Ca2+/calmodulin elevated at these sites by several distinct Ca2-linked signal transduction pathways.

The overall goal of the conference is to bring together a diverse group of scientists working on all phases of calcium and its role in cell function. In addition to its role as an essential mineral for bone growth, regulation of calcium levels inside cells and the action of calcium on intracellular targets impinges on virtually every primary biological activity in cells from both prokaryotes and eukaryotes. The action of calcium in cells is critical for regulation of cardiac and skeletal muscle function, neuronal communication in the brain, the response of immune cells to infection and in most other critical cellular functions. The field provides the intersection of well established physiology and pathophysiology with new findings at the molecular level that provide insights into calcium and cell function. In addition to the formal sessions, informal workshops will be arranged during the meeting to discuss current questions in calcium research. Poster sessions will be held to allow all attendees to exchange new information and participate in the conference. Speakers will be from various fields including physiology, cell biology, biochemistry, pharmacology, molecular biology and genetics. This meeting is an important mechanism by which scientists funded by government grants exchange information that reduces redundancy in research and increases the speed by which new discoveries are incorporated by other scientists across the calcium field. The topics to be covered are: Structure and function of calcium-binding proteins--these are the calcium sensors that indicate to cells when they have been stimulated; Molecular properties of calcium channels--these proteins regulate entry of calcium into cells that trigger cell excitation; Calcium signaling in the cell and the nucleus--how changes in calcium levels in cells is used to provide information within the cell and how this information is transmitted throughout the cell to regulate cell division and the produc tion of protein products from specific genes; Calcium in human diseases--findings from the studies on cell biology above have enabled muscular dystrophy, stroke, Alzheimer's and other diseases to be better understood at a molecular and cellular level and allows for design of new therapeutics which will be discussed.

The hippocampus in intact animals, in slice preparation, and as isolated neurons in culture offers an experimental opportunity to study both long- term depression (LTD) and long-term potentiation (LTD), forms of synaptic plasticity utilized in learning and memory. Ca2+ is a key signaling molecular regulating synaptic plasticity in the hippocampus and we will focus on several aspects of Ca2+ action including the activation of protein kinases/phosphatases, nitric oxide synthase, and transcription of genes that underlie changes in synaptic function during LTD and LTP. Nitric oxide (no) is a retrograde messenger that we have found to stimulate synaptic release in a Ca2+-independent manner. We have collaborated with Dr. Richard Scheller to demonstrate NO alters protein-protein interactions among the synaptic proteins VAMP, syntaxin, n-secl, and SNAP-25 that may be responsible for such release. We will define, quantitate, and identify the sites of these changes. We will determine which neurotransmitter classes are affected and whether NO alters stimulated release. We have demonstrated a mechanism by which multifunctional CaM kinase II may be switched to a Ca2+ -independent species in a stimulus frequency- dependent manner. In collaboration with Dr. Richard Tsien we will correlate activation of the kinase at various frequencies that elicit LTD or LTP in hippocampal cultures. Immunocytochemistry with phosphoselective Ab and biochemical analysis will compare antagonism between CaM kinase II and calcineurin, a Ca2+-dependent phosphatase. We will examine how Ca2+ can change the sign of the synaptic strength by favoring activation of CaM kinase II to elicit a potentiation or favoring calcineurin to elicit a depression. We will examine Ca2+ -based signaling pathways from the glutamate receptors on synaptic spines to the phosphorylation of the transcription factor CREB in the nucleus with Dr. Tsien. Transgenic animals with reporter genes driven by promoters for regulatory elements for CREB and other transcription factors will be generated to examine transcription at different stimulus frequencies. Single cell PCR will be used to correlate synaptic plasticity and induciton of genes in single cells examined morphologically under the microscopy. Finally, we have developed a method termed indexing which will be optimized to a single cell level and will allow us to compare cDNA from control, LTD or LTP neurons and thereby clone novel plasticity genes.

DESCRIPTION (Investigator's abstract): CaM kinase II is a major mediator of Ca2+ -linked signal transduction systems; it phosphorylates diverse substrates located in cytosolic, nuclear, cytoskeletal, and membrane compartments. How is response specificity achieved if any rise in Ca2+ activates all Ca2+ -dependen processes throughout the cell and if CaM kinase II responds by phosphorylating all its substrates? They have found temporal and spatial elements of signal transduction which may enable response specificity by CaM kinase II. Temporal regulation: Many cell stimuli subject the kinase to oscillations in the concentration of intracellular Ca2+. Although information is thought to be encoded in the stimulus frequency, no frequency decoder has previously been identified. They have now demonstrated that CaM kinase II is sensitive to the frequency of Ca2+ oscillations in vitro with the use of a pulse flow device that exposes immobilized kinase to Ca2+ pulses under precise kinetic control and which they can now exploit. They will examine the molecular basis for this biochemical behavior, defining the relative contribution of various steps in kinase activation and deactivation to the frequency response. They will use kinase constructs as probes of effective Ca2+/CaM concentration to test of whether cellular CaM is limiting and assess its contribution to the frequency response. They will then test whether CaM kinase is sensitive to the frequency of Ca2+ oscillations in situ using several cell lines with defined Ca2+ signaling pathways and intracellular targets of the kinase. Spatial regulation: They have found that CaM kinase isoforms are targeted to distinct cellular localization and that cell signals can have preferential regulation of the kinase based on its localization. They will explore our recent finding that targeting of a nuclear isoform of the kinase is regulated by Ca2+-dependent autophosphorylation as well as phosphorylation by other kinases. They will test the ability of various cell stimuli to spatially direc their signals by engineering cells with CaM kinase II targeted to distinct cellular localization as a spatial probe to cell signaling. Signal transductio pathways may both control activation of the kinase as well as its intracellula targeting and thereby regulate the specificity of cellular responses to stimulation.

DESCRIPTION (provided by applicant): Biomarkers are urgently needed for the early and accurate diagnosis of Alzheimer's disease (AD). SurroMed is using a high throughput, multiplexed proteomics approach to detect, identify and quantify a large number of putative biomarkers in animal models and human cerebrospinal fluid (CSF) specimens. Aim I proposes to develop methods to fractionate CSF and identify peptides and proteins in CSF using micro-scale liquid chromatography and mass spectrometry (MS). We will use proprietary bioinformatics to identify and quantify metabolites, peptides, proteins in mass spectra. Aim II proposes to identify specific candidate biomarkers related to underlying pathophysiological processes with known or hypothesized involvement in AD, specifically the ubiquitin proteasome system (UPS). We will develop conditions to simulate AD using proteasome inhibition in rodent primary neuronal cell cultures since proteasome inhibition is suspected to occur as an early event in AD pathology. We will use a variety of affinity techniques to capture peptides or proteins that accumulate due to UPS inhibition. Captured proteins will be identified by MS, and added to a list of putative biomarkers. Taken together, these studies will demonstrate the feasibility of our broad phenotypic analysis approach to identifying and quantifying biomarkers and provide us with validated methods and tools for studies of human CSF samples. PROPOSED COMMERCIAL APPLICATION: We propose to develop a global solution for proteomic analysis using an innovative technology platform for probing the molecular signatures of Alzheimer's disease (AD). The analysis will be coupled with proprietary bioinformatics to identify and validate putative biomarkers for AD. This research program will lead to commercialization of diagnostic kits, therapeutic targets, instrumentation, and bioinformatics.

DESCRIPTION (provided by applicant): There is need to develop an agent that can be used to prevent or treat arterial or venous thrombosis without increasing hemorrhage. The human placental anticoagulant protein annexin V inhibits coagulation by binding to phosphatidylserine on the outer surface of activated platelets in which phospholipid asymmetry is lost. Recombinant human annexin V decreases arterial and venous thrombosis in experimental animal models without increasing hemorrhage. However, annexin V (32 kDa) rapidly passes from the circulation into the urine limiting its usefulness as an anti-thromobic agent. The investigators propose to increase the molecular weight of annexin V by conjugation with polyethylene glycol. It is predicted that this modification will prolong the half-life of annexin V in the circulation and its anticoagulant activity, thereby producing a clinically useful anti-thrombotic agent. Pegylated annexin V may also attenuate reperfusion injury. The investigators' milestone for Phase I is development of a pegylated annexin with significantly prolonged serum half-life and which retains its intrinsic anticoagulant activity; clinical efficacy in thrombosis will be tested in a subsequent SBIR-Phase II proposal.

DESCRIPTION (provided by applicant): Biomarkers are urgently needed for the early and accurate diagnosis of Alzheimer's disease (AD). SurroMed, Inc. is using high throughput, metabolomic and proteomic approaches to differentially quantify hundreds to thousands of molecules in human cerebrospinal fluid (CSF), serum, and urine to identify biomarkers of AD. The low molecular peptide and metabolite fraction (the metabolome) contains a rich diversity of possible biomarkers of AD, yet it has never been systematically examined. We propose to develop methods to fractionate the metabolome of CSF, serum, and urine and identify peptides and metabolites using micro-scale liquid chromatography and mass spectrometry. This discovery-based approach will be complemented with a hypothesis-based approach in which we examine molecules implicated in AD pathophysiology using immuno-affinity capture and/or MS. Phase I will therefore establish a metabolomic platform for detection and identification of AD biomarkers at an unprecedented scale and with high throughput. This technology will be applied in a subsequent Phase II application aimed at discovering biomarkers for early stages of AD in a clinical study with mild cognitively impaired and AD patients.

DESCRIPTION (provided by applicant): A biosignature based on long-term plasma protein changes specifically associated with chronic drug exposure can be used to identify patients predisposed to repeated drug use and thereby facilitate early intervention. We will test the feasibility of finding such a signature using chronic exposure of rats to addictive drugs and differential proteomic expression. Rats will receive treatment with cocaine or a non-addictive dopamine transporter inhibitor, or morphine, or saline according to a 14 day escalating dose schedule (chronic exposure) and plasma collected up to 75 days following last drug treatment. Proteomic analysis will involve stepwise depletion of abundant plasma proteins followed by liquid chromatography coupled online with mass spectrometry to analyze both the depleted proteome consisting of very low abundance proteins and the bound proteins of intermediate abundance. Proteins whose altered expression is maintained for several weeks, e.g. 30 days, and eventually return to baseline would be candidate biomarkers that could be used to detect recent exposure at a time when the addictive drug can no longer be readily measured. Top candidate will be validated by multiple reaction monitoring in a quantitative multiplexed assay that does not require antibodies to the proteins. Proteomic changes will be examined for temporal correlation with behavioral and neurochemical sequelae of chronic drug treatment that will be measured in parallel. Statistical analysis and data mining will be used to identify a biosignature with the desired properties. A successful feasibility study would stimulate and inform similar studies on human subject samples. PUBLIC HEALTH RELEVANCE: There is an unmet need for analytical biomarkers, a biosignature, of chronic drug exposure that would help to identify patients predisposed to repeated drug. An effective test could become part of routine medical care that would identify patients in need of treatment for substance use disorders and mitigate the personal and societal burdens of drug addiction.

DESCRIPTION (provided by applicant): Biomarkers for diseases of the nervous system can benefit from advanced proteomic analysis of cerebral spinal fluid (CSF) but some basic biological and pre-analytical parameters of CSF collection and handling, and reproducibility and accuracy of proteomic bioanalysis must first be systematically evaluated. Label-free differential expression profiling involves separation of tryptic digests of the CSF proteome depleted of the most abundant proteins by cation exchange chromatography followed by liquid chromatography coupled online with mass spectrometry. The effect of time from lumbar puncture to freezing at different temperatures with variable blood contamination, freeze thaw cycles, circadian time of collection and other variables will be investigated. The project will generate best practices for future collections and develop a CSF Integrity test for stored samples. Best practices will be applied to biomarker discovery in three pilot case-control studies-frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and schizophrenia, to find new biomarkers and inform power calculations and platforms for future studies. A resource for the neuroscience community will be established with an annotated in-depth catalog of the CSF proteome and research tools to access information about CSF proteins. Finally, the catalog will include information on tryptic peptide for each proteins that will enable academic and pharma investigators to construct a panel of peptides for multiplexed measurements of CSF proteins of choice without the need for antibody by multiple reaction monitoring (MRM), and thereby promote and facilitate biomarker discovery in many diseases of the nervous system. PUBLIC HEALTH RELEVANCE: Biomarkers for diseases of the nervous system can benefit from advanced proteomic analysis of cerebral spinal fluid (CSF) but some basic biological and analytical parameters of CSF collection, handling, and analysis must first be systematically evaluated. The project will generate best practices for future collections and develop a CSF Integrity test for stored samples. Elaboration of an annotated CSF proteome and biomarker discovery in frontotemporal lobar degeneration (FTLD), Alzheimer's disease, and schizophrenia will accelerate biomarker discovery to speed therapeutic development for these major diseases.

DESCRIPTION (provided by applicant): Heart disease converts to the clinical syndrome of heart failure when the cardiac output is inadequate to meet metabolic requirements. Evidence now supports that pharmacological targeting of Ca2???? dependent protein kinase II (CaMKII), a sensor of dysregulated calcium homeostasis, will inhibit conversion of early stages of cellular pathophysiology to symptomatic heart failure and sudden death. Inhibition of the kinase with research inhibitors, or by genetic knock-out of the major cardiac isoform, blocks this chain of events in animal models. We propose a strategy to modify a small molecule inhibitor of CaMKII to increase its potency and selectivity, test it biochemically to ensure it has the desired mechanism of action, then test for inhibition of characterized markers of hypertrophy and for apoptosis in neonatal mouse cardiomyocytes. We start with an allosteric CaMKII inhibitor used to demonstrate its cardiovascular functions but has never been pharmacologically optimized and thus have low potency. Guided by our analysis of new crystal structures and structural insights we have developed from docking inhibitors to its active site we have designed a set of compounds that target a unique feature of the allosteric pocket in the active site of CaMKII. Our overall goals for Phase I are to retain and improve the selectivity of the inhibitor while markedly increasing its potency, and to test the lead inhibitor compounds for efficacy then test for inhibition of characterized markers of hypertrophy and for apoptosis it in neonatal mouse cardiomyocytes PUBLIC HEALTH RELEVANCE: Heart failure is a global burden, with the lifetime risk in the developed world above 20% and a consuming focus for patients, clinicians, scientists, and policymakers. Heart disease converts to the clinical syndrome of heart failure when the cardiac output is inadequate to meet metabolic requirements. Evidence now supports that pharmacological targeting of intracellular signaling, in particular of Ca2???? dependent protein kinase II, a sensor of dysregulated calcium homeostasis will inhibit conversion to symptomatic heart failure and sudden death. We propose a strategy to modify a small molecule inhibitor of this protein kinase in ways that increase its potency, analyze it biochemically to ensure it has the desired mechanism of action, then test for inhibition of characterized markers of hypertrophy and for apoptosis it in neonatal mouse cardiomyocytes.

DESCRIPTION (provided by applicant): Asthma is a major public health problem affecting 25 million American adults and children in the US and over 300 million people globally. It is characterized by reversible airflow obstruction due to bronchial smooth muscle contraction and a diseased epithelial cell phenotype of airway hyper-reactivity (AHR) to environmental stimuli, increase in goblet cell, and increased luminal mucous secretion. The allergic response is associated with an increase in reactive oxygen species (ROS) in bronchial epithelium that precedes and promotes epithelial cell dysfunction. There is an unmet need for disease modifying therapy that reduces reliance on glucocorticoids, potentially by targeting pathways of oxidative damage. Our recent studies position Ca2???? (CaM)-dependent protein kinase II (CaMKII) as a key sensor, amplifier, and mediator of pathological ROS responses in asthma. We aim to optimize a small molecule inhibitor of CaMKII based on new insights from CaMKII crystal structures and thereby improve its drug-like properties. We will increase its potency and then test our lead compound in a validated mouse model of allergic asthma. Our overall goals for Phase I are to perform sufficient lead optimization to conduct a pre-clinical proof-of-concept that CaMKII inhibition suppresses AHR and other asthma phenotypes. We have designed inhibitors based on new crystal structure of the enzyme that together with prior SAR suggests how it binds the kinase and identifies nearby residues that new compounds can interact with to produce more potent and more selective inhibitors. Optimized lead inhibitors will then be tested for efficacy in the ovalbumin mouse model of allergic asthma, monitoring its efficacy in AHR, goblet cell metaplasia, and mucous secretion. This previously unrecognized disease pathway offers a novel and innovative therapeutic opportunity for asthma. Our overall Phase I aim is to test an improved lead compound in the ovalbumin model, that if successful would justify a Phase II proposal to optimize drug-like properties of the lead compound and perform the ADME/Tox and other studies necessary to reach IND. PUBLIC HEALTH RELEVANCE: Asthma is a major public health problem affecting 25 million American adults and children in the US and over 300 million people globally. The reduced lung function and disease phenotype of airway hyper-reactivity to environmental stimuli and increased luminal mucous secretion is preceded by oxidation and inflammation of bronchial epithelium. Our recent studies implicate Ca2????dependent protein kinase II (CaMKII) as a key sensor, amplifier, and mediator of pathological oxidation and inflammation in asthma. We aim to optimize a small molecule inhibitor of CaMKII based on new insights of its structure to fill an unmet need for disease modifying therapy that reduces reliance on glucocorticoids, potentially by targeting pathways of oxidative damage.

DESCRIPTION (provided by applicant): Gating the activation and tuning the Ca[2+] frequency response of CaM kinase II Ca[2+] functions as a second messenger for many signaling molecules, including neurotransmitters, hormones and growth factors. One of the central mediators of Ca[2+]/CaM action is the multifunctional CaM kinase II (CaMKII), a ubiquitous Ser/Thr protein kinase that phosphorylates dozens of key cellular proteins and enzymes in the cytosol, plasma membrane, and nucleus. The kinase has been the focus of considerable attention because i) it has a unique architecture with 12 kinase subunits that determine its Ca[2+]/CaM sensing, intracellular targeting, and substrate specificity; ii) it displays a form of molecular memory in which Ca[2+]-dependent autophosphorylation at a Thr residue and/or oxidation at a nearby Met residue switches it to a Ca[2+]-independent (autonomous) state that participates in neuronal memory and other functions; iii) it can respond to the frequency of Ca[2+]-linked stimulation, such as heart rate, and modifies cell function accordingly. Understanding the mechanism and structural basis by which CaMKII decodes the frequency of Ca[2+] spikes is therefore critical to understanding both its physiological and pathological functions. Based on a recent crystal structure and functional analysis of the kinase we hypothesize that the kinase undergoes an equilibrium between a compact structure where its catalytic domains are tightly packed into a central hub composed of its association domain and a more extended structure that is more readily activated by CaM. We will test whether the length of linker sequences between the catalytic and association domains tune the kinase to different frequencies of Ca[2+] spikes and how this is affected by oxidation. We will further examine the effects of gating of the autoinhibitory domain by a pharmacological inhibitor and by a SNP that is associated with increased risk of sudden cardiac arrest. We propose to test its remarkable properties by determining whether the kinase decodes the frequency of Ca[2+] stimuli delivered to cardiomyocytes to increase its autophosphorylation and phosphorylation of its substrates. Finally, we will use our structural and regulatory insights to develop an activator of CaMKII that can be used to evaluate and discover and delineate new CaMKII functions in diverse cell types.

DESCRIPTION (provided by applicant): Ventricular arrhythmias have diverse and complex etiologies that include excessive stimulation of multiple neurohumoral pathological pathways, suggesting that no single upstream blocker will be sufficiently efficacious. Ca[2+]/calmodulin-dependent protein kinase II (CaMKII) is a common downstream mediator of these neurohumoral pathways and its hyperactivity contributes to the mechanisms of ventricular arrhythmia. We propose that the kinase is a novel therapeutic target, an idea supported by the demonstration that pharmacological and genetic reduction of CaMKII activity decreases arrhythmia in animal models and in human cardiomyocytes from patients with arrhythmia and elevated CaMKII. We have developed small molecule inhibitors of CaMKII and propose to increase potency and selectivity of the lead compound then test the best compounds for inhibition of arrhythmia in a robust mouse model. Guided by insights we developed from docking inhibitors to its active site in our new crystal structures we propose a set of compounds designed for lead optimization. Our goal is to improve potency 10-fold and reach IC50s below 15 nM. The preclinical proof-of concept study will test the in vivo efficacy of the top inhibitors using a calcineurin over expressing mouse model that is relevant to larger animal models, and likely to diseased human myocardium as well. The mice have severe heart failure and high levels of ambient arrhythmias resulting from increased CaMKII expression and our milestone is to show efficacy with at least one CaMKII inhibitor. If successful we will be positioned to extend the preclinical development by optimizing the inhibitors for ADME/Tox and other drug-like properties, testing their efficacy in other animal models, and perform IND enabling studies for an IND application aimed at arrhythmia in heart failure. PUBLIC HEALTH RELEVANCE: Sudden cardiac death is a major public health problem, which is estimated to kill 500,000 Americans each year. Most sudden cardiac death is due to rapid ventricular arrhythmias and patients with heart failure are at highest risk. Evidence now supports that pharmacological targeting of intracellular signaling, in particular of Ca2+/calmodulin-dependent protein kinase II, a sensor of dysregulated calcium homeostasis, will inhibit arrhythmia in heart failure, and it is thus a novel target in arrhythmia. We propose to modify a small molecule inhibitor we developed for this protein kinase in ways that increase its potency, analyze the new inhibitors biochemically to ensure they have the desired properties, and then test for inhibition of arrhythmia in a mouse model as a proof-of-concept.

DESCRIPTION (provided by applicant): Osteosarcoma is the most common primary bone cancer in children and adolescents. A key feature of osteosarcoma is their inherent high growth rates and the increased vasculature to enable rapid growth. Recent data implicate Ca2+/CaM-dependent protein kinase II (CaMKII), a major mediator of Ca2+ signaling, in both the unregulated proliferation of osteosarcoma and in the angiogenesis that supports its growth. Hyperactivity of CaMKII is associated with cell proliferation and resistance to apoptosis, while inhibition of CaMKII suppresses growth of osteosarcoma in animals. We aim to focus medicinal chemistry and preclinical development to generate improved CaMKII inhibitors that incorporate an allosteric site interaction. Our strategy is to start with a potent lead ATP site inhibitor of the kinase and extend it to interact with the helical inhibitory domain of the kinase. Biochemical analysis of inhibitors will measure their interaction with the inactive and active conformations. Medicinal chemistry will be used to develop inhibitors that span the catalytic site with preferential binding to the inactive conformation that is characteristic of allosteric interactions. While our current lead compounds can be used to test efficacy in osteosarcoma, the allosteric inhibitors will be more broadly useful because of greater selectivity. The best inhibitor will be tested for efficacy for its cellular action followed by efficacy on human osteosarcoma xenografted in mice. This will provide a clear path for a Phase II proposal to further improve its potency and other drug-like properties up to IND filing. CaMKII inhibitors may present a new paradigm in osteosarcoma-targeted agents that effectively treat the tumor by the dual mechanism of slowing its rapid growth and blocking its access to nutrients. PUBLIC HEALTH RELEVANCE: Osteosarcoma is the most common primary bone cancer in children and adolescents. A key feature of osteosarcomas is their inherent high growth rates and the increased vasculature to enable rapid growth. Recent data implicate Ca2+/CaM-dependent protein kinase II (CaMKII), a major mediator of Ca2+ signaling, in both the unregulated proliferation of osteosarcoma and in the increased blood vessel formation that upports its growth. We aim to modify an existing potent small molecule inhibitor of CaMKII to increase its selectivity, test it biochemically to ensure it has the desired mechanism of action, then test it on xenografted human osteosarcoma.

DESCRIPTION (provided by applicant): Stroke is a major public health problem, with a US mortality rate that ranks third behind other diseases involving the cardiovascular system and cancer. We propose to pursue neuroprotection in ischemic stroke by targeting a key regulator of Ca2+ homeostasis, Ca2+/CaM-dependent protein kinase II (CaMKII). Inhibition of CaMKII represents a novel paradigm in ischemic stroke-neuroprotection based on blocking the effects of CaMKII on Ca2+ overload as well as its more direct effect on mediators of neurotoxicity. Two developments provide us with the rationale and opportunity to test potent small molecule CaMKII inhibitors in ischemic stroke. First, hyperactivity of CaMKII has been shown to promote cell death in glutamate excitotoxicity while inhibitors of the kinase are neuroprotective in situ and in the middle cerebral artery occlusion (MCAO) model. Second we have taken the opportunity to restart a CaMKII inhibitor program initially advanced at a pharm and which we are now accelerating based on insights from the first crystal structures of the human enzyme. Our proposal is relatively straightforward, to increase CNS penetration of the potent inhibitors we developed and test the optimized lead compound in a permanent occlusion MCAO model. We have designed a number of modifications that increase lipophilicity of our compounds with the aim of increasing their CNS penetration. The compounds will be analyzed for kinase inhibition and selectivity in vitro, and for inhibition and protection from glutamate excitotoxicity in neuronal cultures. The inhibitor with the greatest potential for CNS penetration will be selected based on bidirectional permeability through MDR----MDCK monolayers and its brain/plasma distribution in vivo will be quantified. The lead CNS penetrating inhibitor will be tested in the rat permanent occlusion MCAO model. We will monitor the degree of CaMKII inhibition achieved in vivo using biomarkers of kinase activity and quantify the effect of the inhibitor on infarct size. A successfully proof----of----concept in ischemic stroke will position us for a Phase II SBIR proposal to optimize the drug----like properties and conduct IND enabling studies of the lead compound. PUBLIC HEALTH RELEVANCE: Stroke is a major public health problem with a US mortality rate that ranks third behind other diseases involving the cardiovascular system and cancer. Recent data implicate hyperactivity of Ca2+/CaM- dependent protein kinase II (CaMKII), a major mediator of Ca2+ signaling, in ischemic damage. We aim to increase the brain penetration of our potent CaMKII inhibitors and test the optimized lead compound for efficacy in an animal model of human ischemic stroke.ischemic stroke.

? DESCRIPTION (provided by applicant): Atrial fibrillation (AF) is the most common cardiac arrhythmia, a condition that predisposes individuals to heart failure and stroke and is a major contributor to cardiovascular mortality. There is an unmet need to treat AF and the underlying electrical and structural changes in heart. There is a long unsuccessful history of ion channel blockers in AF; invariably they are proarrhythmic. We have identified a new target for atrial fibrillation, Ca2+/CaM-dependent protein kinase II (CaMKII), whose hyperactivity is pro-arrhythmic via multiple pathways and systems implicated in the genesis of AF and ventricular arrhythmia (VA). Targeting CaMKII is an innovative new paradigm - treating AF while also being anti-arrhythmic in ventricle. In humans and mice CaMKII made hyperactivity by autophosphorylation and oxidation elicits a diastolic Ca2+ 'leak' from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) hyperphosphorylation, leading to AF. Atrial tissue from patients with AF is marked by elevated CaMKII activity, while CaMKII inhibition prevents aberrant RyR2 Ca2+ release. Furthermore, inhibition of the kinase with our lead compound or genetic ablation of CaMKII or its phosphorylation site on RyR2 blocks this chain of events and reduces the frequency of AF in mice. We aim to modify our potent and selective lead compound to i) reduce its rapid liver microsomal metabolism in rodents, which will facilitate preclinical development toward IND, and ii) limit any central nervous system (CNS) penetration to mitigate concerns regarding CaMKII inhibition in brain. Following lead optimization we will test efficacy in an established in vivo mouse model of induced AF and in an isolated rabbit heart model. The Ryr2R176Q/+ AF mouse model exhibits a Ca2+ leak and susceptibility to ectopic activity, reentry, and AF triggered by atrial pacing. Importantly, these models share mechanisms with post-op AF, a significant inpatient indication and an achievable entry point for Allosteros Therapeutics in cardiovascular therapeutics. The study will also include assay of biomarkers to assess in vivo inhibition of CaMKII and reduction in site-specific phosphorylation of its targets, RyR2 and phospholamban. The Phase I work will position us for lead selection and IND-enabling studies and enable us to reach metabolic and drug disposition milestones sought by investors and pharma who now recognize that CaMKII is a consensus target in AF and VA.

Type 2 diabetes mellitus has become a worldwide epidemic with significant human and economic burdens. Diabetic patients are particularly at risk of mortality from myocardial infarction and it is therefore important that glucose lowering is achieved by drugs that do not further compromise the heart. Evidence now supports Ca2+/calmodulin-dependent protein kinase II (CaMKII), a mediator Ca2+ signaling in liver and heart, as a novel target for inhibitors with the unique dual action of lowering glucose while providing cardioprotection. Genetic approaches have rigorously demonstrated that a CaMKII-p38a-MK2/3 pathway mediates the action of glucagon on glucose production in liver while a CaMKII-Ryanodine receptor pathway underlies dysregulated Ca2+ signaling in heart. Genetic suppression of CaMKII lowers blood glucose, and decreases progression from hypertrophy to heart failure and arrhythmia, while its hyperactivity increases blood glucose and promotes heart disease. We propose to advance our highly selective allosteric small molecule CaMKII inhibitors via lead optimization and test the feasibility that they lower hepatic glucose production. Activated CaMKII is prone to pathological hyperactive states in diabetes, due to oxidation and other post-translational modifications, all of which are blocked by our allosteric inhibitors. We propose a structure-based medicinal chemistry strategy of lead optimization to increase inhibitor potency and limit any CNS penetration while retaining their high kinase selectivity. We will evaluate their biochemical and cellular properties to ensure they have the desired characteristics prior to a mechanistic POC in lowering hepatic glucose output in ob/ob mice. Successful completion of our Phase I milestones will set a clear path for a Phase II proposal to further advance its pharmacokinetics and other drug-like properties and extend the proof-of-concept for cardioprotection and long-term glucose control. Pharma and venture capitalists have shown significant interest in our allosteric CaMKII inhibitor program?their funding is dependent on achieving a more advanced lead compound that does not affect brain CaMKII while showing preclinical efficacy in glucose control. Our proposed studies and an SBIR award will position the program for funding or a partnership to advance through IND enabling studies and an IND.