Kevin Lynch - US grants

We propose to characterize the different forms of angiotensinogen mRNA in the rat, measure the relative accumulation of these mRNAs in various tissues and determine whether the various angiotensinogens encoded by these mRNAs are capable of acting as renin substrates. Angiotensinogen is a 55,000-60,000 dalton serum glycoprotein, whose only known function is to act as a precursor to the decapeptide, angiotensin I, which is in turn rapidly converted to the octapeptide hormone, angiotensin II. Ang II is a potent vasopressor and regulates mineral balance, primarily through regulation of mineral corticoid synthesis. We have recently discovered multiple forms of angiotensinogen mRNA in the rat and will characterize these mRNAs by determining the nucleotide sequence of the corresponding cDNAs. This sequence information will be used to develop hybridization probes specific for each form of angiotensinogen mRNA. To determine whether the angiotensinogens encoded by the mRNAs have the potential to be renin substrates we will synthesize each angiotensinogen mRNA in vitro, translate these mRNAs in vitro or, if necessary, in ovo, and test the secreted angiotensinogen for susceptibility to proteolysis by renin. This research has the potential of describing new roles for angiotensinogen as a precursor to other peptide hormones.

The ability of the brain to synthesize angiotensinogen, angiotensin I and angiotensin II is now well established and it is widely accepted that angiotensin I and II produced by the brain itself can affect neuronal activity, autonomic function and various behaviors, most prominently thirst and sodium appetite. The brain circuitry involved in cardiovascular homeostasis is clearly affected by both circulating angiotensin II (via its action on circumventricular organs) and by the activity of the brain's own angiotensin system. Some evidence also suggests that an increase in the activity of the central angiotensin system is one of the factors involved in the resetting of the homeostatic regulation of arterial pressure (and blood volume) to higher levels in various forms of experimental hypertension. Yet on a cellular level, the reality of the central angiotensin system(s) remains obscure. Unlike classical peptide euromodulators, the prohormone angiotensinogen is reportedly found at 100-fold molar excess over the active hormone, angiotensin II (Ang II), is present extracellularly and may be synthesized by glia or the microvasulature as well as by neurons. Morever, the enzymatic processing of angiotensinogen in the brain is quite controversial with regards to the nature of the protease that releases brain angiotensin. It is also not known whether brain angiotensinogen gene expression varies in response to physiologic status. The purpose of the proposed research is therefore to use a molecular biologic approach to resolve a specific set of issues concerning the cellular site of synthesis of brain angiotensinogen, angiotensinogen gene expression in the brain during hypertension or changes in blood osmolarity and the nature of the brain angiotensinogen processing enzyme. It is proposed to use our angiotensinogen cDNA to determine what brain cells (neurons, glia) synthesize central angiotensinogen. In addition, sites of brain angiotensinogen accumulation will also be determined after development of anti-angiotensinogen sera. Brain isorenin will be described by isolating and characterizing its cDNA. The role of the angiotensin system in hypertension will be explored by quantitating brain angiotensinogen mRNA accumulation in individual nuclei in the spontaneously hypertensive rat and its normotensive control rat. Changes in brain angiotensinogen gene expression will also be examined as a function of changes in blood osmolarity. The results of these studies will significantly advance the state of knowledge in this field.

Alpha2-Adrenergic receptors mediate a large portion of known inhibitory effects of catecholamines on central and peripheral neurons. The inhibitory properties of alpha2-adrenergic receptor agonists make several of these drugs useful as antihypertensives, in potentiating volatile anesthetics and in blunting the autonomic and affective symptoms of opiate withdrawal. Classic pharmacologic approaches (ligand binding, transmitter overflow) have recently contributed towards the partial characterization of several subtypes of alpha2-adrenergic receptors. However, the specific pharmacologic profile of these receptors remains imprecise and their anatomical Ioaction in neural tissue needs to be explored in detail. The recent molecular cloning of three alpha2-adrenergic receptor DNAs makes possible new and powerful approaches to understanding the specific biologic role of these important proteins. We propose to use these novel reagents to identify the precise pharmacologic profile and the neuroanatomical locations of alpha2-adrenergic receptor subtypes. Each subtype will be expressed individually in the same cellular environment and their pharmacologies defined in terms of a wide variety of potentially active compounds. Subtype-specific antisera, raised against recombinant fragments of each receptor, will be generated and rigorously tested. These antisera will be used to map brain receptor subtypes by examining immunohistochemically stained brain sections by both light and electron microscopy. Correlates of the immunohistochemical mapping will include electrophysiologic analyses in conjunction with subtype-specific compounds and hybridization histochemistry. Although the entire CNS and will be examined, we will concentrate our efforts on several areas with high adrenergic activity including the locus coeruleus, rostral ventral lateral medulla, hippocampus and intermediolateral cell column of the spinal cord. In addition to defining the pharmacology and anatomical location of the alpha2-adrenergic receptors, our results might aid in the rational design of pharmaceuticals to be used as antihypertensives, in conjunction with inhalational anesthetics and to alleviate the noxious symptoms of opiate withdrawal. Finally, our results might help address an important issue in receptor biology, i.e. why are there multiple, apparently closely related, receptor subtypes for each of the cationic amines?

[unreadable] DESCRIPTION (provided by applicant): Four years ago we proposed an integrated program of synthetic chemistry and molecular pharmacology with the goals of gaining a better understanding of the structure activity relationships (SAR) in lysophosphatidic acid (LPA) and discovering LPA receptor selective agonists and antagonists. We have achieved this goal, as well as the goal of defining these compounds at recombinant LPA receptors and lipid phosphate phosphohydrolases (LPP). Our collaborators have shown that our lead LPA receptor antagonist is efficacious in several animal models. Further, we exceeded the goals by our discovery of synthetic compounds active at individual sphingosine 1-phosphate (S1P) receptors. [unreadable] [unreadable] Our present Aims can be stated succinctly as: (1) Developing further the SAR of LPA mediators whereby we will define the pharmacophore systems responsible for the agonist and antagonist activities at individual LPA receptors; (2) Profiling our synthetic compounds for inhibition of LPA metabolic enzymes and (3) Using our compounds to discover new lysophospholipid receptors. [unreadable] [unreadable] The strength of our program remains the combination of synthetic chemistry and molecular pharmacology - an interaction strengthened by the molecular definition of target proteins including the LPA receptors, lysophospholipase D and lipid phosphohydrolases. Minimally, our efforts will extend significantly the SAR for receptors, phosphatases and synthetic enzymes and optimize the existing lead antagonist and enzyme inhibitor structures. Optimally, we will discover new classes of LPA receptor antagonists and agonists, identify lysophospholipase D inhibitors and describe additional LPA receptors. Our work will enable a determination of the effects of blockage or mimicry of LPA signaling and thus provide crucial information as to what LPA signaling pathways might be valid targets for therapeutic intervention. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goals of the predoctoral Training Program in Pharmacological Sciences at the University of Virginia are to provide a broad education in modern pharmacology that includes fundamental aspects of human physiology, pharmacokinetics, biostatistics and whole animal pharmacology; to provide rigorous training in scientific inquiry; and to imbue each trainee with a professional, scholarly attitude. Our program emphasizes critical thinking and technical skills as well as oral and written presentations of experimental findings and ideas. A training grant Steering Committee administers the training program and oversees student progress through performance in course work, qualifying examinations, presentations in the Pharmacology journal club and written reports from semi-annual committee meetings. There are currently 20 trainees; we recruit 5-6 new trainees each year. Students (40-45 each year, nearly all TGE) enter graduate school through an umbrella program. During the first year, students enroll in a core knowledge course and perform three research rotations. They choose their dissertation mentor and PhD program mid-way in their second semester. On joining the laboratories of the 43 Pharmacological Sciences Training Grant preceptors, they can be nominated for appointment to the training grant for their second and third years in graduate school. Although the majority of trainees appointed are Pharmacology PhD students, in the past 10 years the TG has supported students in the Biochemistry & Molecular Genetics, Biomedical Engineering, Chemistry, Pathology and Physiology PhD programs. Training program-specific activities include courses in human physiology, general pharmacology, analysis of drug targets, biostatics and the responsible conduct of research as well as participation in the weekly Pharmacology journal club. Non-Pharmacology PhD students are required to matriculate in one pharmacology course, biostatics and the Pharmacology journal club while appointed to the training grant and are strongly encouraged to continue this participation for the duration of graduate school. All faculty preceptors are tenure- track faculty with robust research programs. A plurality of preceptors have primary appointments in Pharmacology; the other preceptors represent nine additional academic units; seven preceptors are active clinicians. Forty trainees have been awarded the PhD in past 10 years and 17 more remain in training. Twenty trainees were awarded individual predoctoral fellowships and our trainees have published, on average, three original research papers (one as first author) while in training. The time-to-degree of our trainees is 5.4 years. With this application for renewal of the training grant, we request continued support for five years (-39 to -44) for nine trainees yearly.

Description (Adapted from the Applicant's Abstract): Dr. Lynch and colleagues have used classical medicinal chemistry to generate a library of LPA structural analogs. In testing these novel chemical entities as cloned receptors and as substrates for the LPA ecto-phosphatases, the investigators identified compounds that are stable, receptor subtype-selective agonists and inhibitors of the phosphohydrolases. These tools allow, for the first time, delineation of the function of LPA signaling through endogenous receptors. The investigators now intend to use their existing compounds and others in development to learn whether LPA promotes prostate cancer progression. They will assign signaling events in LNCaP and PC-3 cells to specific LPA receptors thus both prioritizing LPA receptors for antagonist development and identifying compounds for testing on prostate cancer cell growth in vivo. Finally they will identify and quantify the species of LPA produced by prostate cancer cells to determine it suitability as a biomarker.

[unreadable] DESCRIPTION (provided by applicant): Sphingosine 1-phosphate (SIP) is a pleiotropic lipid mediator that is most commonly associated with cell migration, cell survival and vasculogenesis. The recent discovery that the novel sphingosine-like drug, FTY720, is a pro-drug that after phosphorylation targets S1P receptors provides a fascinating insight into S1P biology. FTY720 treatment sequesters lymphocytes in secondary lymphoid tissue and away from inflamed peripheral tissues and graft sites. The drug is protective in both allogenic transplant and autoimmune disease models and was found to be safe and effective in a human renal transplantation trial. Importantly, FTY720-treated animals are resistant to systemic viral infection; if confirmed in humans, this represents a striking advantage over existing immunosuppressive therapeutic regimens. However, the precise molecular targets - the activating kinase, the S1P receptor types and the inactivating phosphatase - remain undefined. Further, FTY720 is not without problems, i.e. about 30% of patients experience a transient, asymptomatic bradycardia with onset of therapy. Thus our present Aims can be stated succinctly as: (1) synthesize sphingosine-like and S1P-like compounds that mimic the FTY720- evoked lymphopenia due to lymphocyte sequestration, (2) characterize these new chemical entities regarding activity at individual S1P receptors and metabolic enzymes and (3) discover the kinase that activates FTY720 and like compounds by phosphorylation. The strength of our program is the combination of synthetic chemistry and molecular pharmacology - an interaction strengthened by the molecular definition of target proteins including the S1P receptors and S1P phosphohydrolases. Minimally, our efforts will extend significantly knowledge of the structure activity relationships (SAR) for S1P receptors, phosphatases and lipid kinases. Optimally, we will discover new FTY720-1ike entities with enhanced selectivity and lessened toxicity and we will discover additional lipid kinases. Our work will also lead to the discovery of S1P receptor selective antagonists and agonists - indeed we have already found several such compounds. These agents will enable a determination of the effects of blockage or mimicry of S1P signaling and thus provide crucial information as to what additional S1P signaling pathways might be valid targets for therapeutic intervention. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): AGK, along with the sphingosine (SPHK1, 2) and ceramide (CERK) kinases and the ceramide kinase like protein (CERKL), comprise the sphingolipid kinase family. AGK is unique among members of this family in that it is a mitochondrial protein. We discovered that mice lacking a functional AGK allele die early in embryogenesis due to failure to implant, which is also unlike SPHKs, CERK or CERKL where null mice are viable and fertile. However, the lipid substrate of phosphoryl transfer reaction catalyzed by AGK is uncertain. The research program we propose will discover that substrate/product and in doing so will define a lipid metabolic pathway that is most likely crucial to mitochondrial survival. Specifically, we will: (Aim 1) Generate matched pairs of cell cultures and tissues wherein AGK expression is markedly different and use mass spectrometry to characterize the lipidome of those cells and tissues so as to ultimately identify the reaction catalyzed by AGK and (Aim 2) characterize AGK function in mitochondria by determining its sub- organelle location, studying mitochondrial physiology in cells deficient in AGK and using conditional deletion of AGK alleles to determine the fate of cell lineages in the mouse. Our extensive experience studying lysophospholipid chemical biology including sphingosine kinases coupled with expertise in mitochondrial physiology will enable us to solve this problem. Minimally, the experiments proposed will reveal a new branch of sphingolipid metabolism. Maximally, we will define a new pathway that is integral to mitochondrial function. PUBLIC HEALTH RELEVANCE: A number of serious pathologies are characterized by defects in the body's energy factories, which are cell organelles named mitochondria. Depending on the organ system affected in mitochondrial diseases, symptoms might include poor growth, muscle weakness, diabetes, neurological problems such as seizures, respiratory disorders and dementia. In this project, we study a protein named AGK, which is thought to be involved in lipid metabolism. AGK is a mitochondrial protein and we discovered that mice lacking this protein die early in embryo formation, which indicates that AGK is essential for development in mammals including humans. We are researching AGK to learn the nature of the lipid pathway that is affected by AGK and why this pathway is so important to mitochondrial survival. Our intention is to use this knowledge to provide better understanding, diagnosis and, eventually, treatment of mitochondrial disease.

Sphingosine kinases (SphK1, SphK2) catalyze the formation of an important extracellular mediator, sphingosine 1-phosphate (S1P). A fundamental aspect of S1P biology is the large difference in S1P abundance between blood or lymph (high) and tissue (low), which is termed the S1P vascular gradient. This gradient maintains vascular endothelial barrier function and facilitates lymphocyte mobilization from lymphoid tissues. Indeed, S1P1 receptor agonist drugs (e.g. fingolimod) are therapeutically beneficial because S1P signaling is highly sensitive to changes in S1P gradient. We used our SphK2 inhibitors to demonstrate that interdicting S1P signaling at the level of synthesis steepens the S1P vascular gradient by slowing S1P clearance from the blood. This result suggests that SphK2 inhibitors will be extremely useful in treating conditions where the endothelial barrier is compromised, e.g. acute kidney injury and sepsis. Although our recently discovered SphK2 inhibitors are active in vivo, improvements in potency, oral availability and chemical diversity are needed to advance them to the clinic. We will accomplish these goals by generating additional inhibitors on our current chemical scaffold and by developing a novel second scaffold. The current scaffold has also yielded a few SphK1 inhibitors but these lack potency at mouse SphK1, which precludes their testing for efficacy in some key disease models. In contrast to SphK2, inhibition of Sphk1 decreases the S1P vascular gradient and to probe the resulting physiological consequences, multiple inhibitors are needed. We will use iterative rounds of synthesis and testing to generate a library of SphK1 inhibitors with emphases on increasing their potency at mouse SphK1 and discovering inhibitors that have suitable pharmacokinetic properties in rodents. To understand the molecular mechanism of SphK inhibition as well as to inform the synthetic chemistry strategies, we will solve the structures of both isozymes with bound inhibitors using X-ray crystallography. Finally, we will discover a blocker of the S1P exporter, SPNS2, which provides the S1P to lymph and thereby maintains the S1P vascular gradient that is required for lymphocyte egress from lymphoid organs to lymph. Currently, due to the unavailability of SPNS2 inhibitors, this particular approach to the manipulation of S1P gradient and subsequent immunomodulation remains completely unexplored. The strength of our program is the synergism in the combination of chemistry (Santos) and pharmacology (Lynch) to which we now add structural biology (Faham). Our central theme of is to understand the therapeutic potential of manipulating the S1P gradients either at the level of synthesis (SphK inhibition) or transport (SPNS2 blockade). We have a track record of productivity that enabled a fundamental insight into S1P biology, e.g. our discovery that SphK2 inhibition modulates S1P signaling to protect endothelial function, a new therapeutic strategy. Now, we propose the development and detailed characterization of greatly improved SphK inhibitors and to make the chemical tools necessary to interrogate SPNS2 as a potential drug target.

Acute kidney injury (AKI) after cardiac surgery requiring cardiopulmonary bypass (CS-AKI) occurs in up to one- third of patients. Currently, there are no pharmacologic or biologic treatments to either prevent or treat AKI. The pathogenesis of AKI involves ischemia-reperfusion injury (IRI), endothelial cell dysfunction and activation of immune cells in the cardiopulmonary bypass circuit. In experimental AKI models, two complementary, intrinsic systems have been identified that A) promote native resistance to kidney injury and B) can be targeted pharmacologically to inhibit the development of AKI. The sphingosine 1-phosphate (S1P) vascular gradient is an important determinant of susceptibility to AKI in the mouse kidney IRI model. Similarly, a deficit of Tregs predisposes mice to kidney IRI. For both protective mediators (S1P and Tregs): therapies that increase their intra-vascular abundance are efficacious at preventing renal injury and dysfunction in the mouse AKI model. S1P acts directly on the endothelium to strengthen the endothelial barrier, while Tregs act on the innate immune cells that infiltrate the injured kidney and exacerbate the damage. We propose to measure Treg number and whole blood and plasma S1P levels in 200 adult cardiac surgery patients that undergo cardiopulmonary bypass; blood will be obtained both just before and 24 hr after surgery. Our main research question is whether relatively low levels of either mediator prior to surgery correlates with increased incidence of post-surgical AKI. If either variable correlates negatively with AKI, we be motivated to investigate the potential therapeutic use/targeting of Tregs and/or S1P-modulating agents for prevention of AKI in this clinical setting. These could be breakthrough therapies for protecting future patients from CS-AKI. We have established a multidisciplinary collaboration to investigate these kidney-protective mediators in patients undergoing cardiac surgery that requires cardiopulmonary bypass. As Co-PIs, Drs. Rosner, Lynch and Kinsey bring complementary expertise in clinical AKI, S1P and Treg biology, respectively. Dr. Kern (Division Chief of Cardiothoracic Surgery) will provide input from the surgeon's perspective and ensure access to potential study subjects and Sandra Burks, RN, CCRC (Associate Director of the Surgical Therapeutic Advancement Center) will manage the study, obtain informed consent from the patients and collect the specimens. Dr. Ma (Professor of Biostatistics) has extensive experience with clinical studies in multiple types of kidney disease and will offer support in study design and perform statistical analyses. Our overall hypotheses are 1) that high circulating Tregs and S1P correlates with reduced AKI, 2) measuring their levels prior to surgery can help predict those patients at higher risk for AKI and 3) therapeutic strategies based on Tregs and/or S1P might be useful to reduce the incidence of CS-AKI.

Acute; Acute Renal Failure with Renal Papillary Necrosis; Adverse effects; Adverse event; Affect; Agonist; Animal Model; Autoimmune Diseases; base; Biological Assay; Biological Availability; Biological Testing; Biology; Blood; Blood Vessels; Bradycardia; Cardiac; cell motility; Cells; chemical synthesis; Chemicals; Chronic Kidney Failure; Clinic; Clinical; Clinical Trials; Complex; Coupled; Cytoplasm; Data; desensitization; Disease model; Dose; Drug Kinetics; Drug Targeting; edg-1 Protein; Endothelial Cells; Endothelium; Environment; Enzymes; Erythrocytes; Experimental Autoimmune Encephalomyelitis; extracellular; Generations; Genetic study; Goals; Immune; Immune response; Immune system; Immune System Diseases; immune system function; immunomodulatory drugs; Immunooncology; immunoregulation; Immunosuppressive Agents; in vivo; inhibitor/antagonist; Inorganic Phosphate Transporter; kinase inhibitor; Lead; Leukocytes; Lipids; Lymph; lymph nodes; Lymphocyte; Lymphocyte Count; Lymphoid Tissue; Lymphopenia; Masks; Medicine; meetings; melanoma; Melanoma Cell; Metastatic Neoplasm to the Lung; Methods; migration; Modeling; Modification; Multiple Sclerosis; multiple sclerosis treatment; Mus; Mutant Strains Mice; Oral; ototoxicity; Pathway interactions; Peripheral Blood Lymphocyte; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phosphorylation; Physiological; Plasma; Positioning Attribute; pre-clinical; Process; programs; Property; Proteins; renal ischemia; Reperfusion Injury; Role; Saccharomyces cerevisiae; Saccharomycetales; screening; Second Messenger Systems; Series; Signal Transduction; Signaling Molecule; small molecule libraries; Sphingosine; sphingosine 1-phosphate; sphingosine kinase; Sphingosine-1-Phosphate Receptor; SPHK1 enzyme; success; targeted agent; targeted biomarker; Testing; Therapeutic; Therapeutic Agents; therapeutic target; Thymus Gland; Tissues; tool; Toxic effect; trafficking; transport inhibitor; Treatment Efficacy; Validation;

Sphingosine kinases (SphK1, SphK2) catalyze the formation of an important extracellular mediator, sphingosine 1-phosphate (S1P). A fundamental aspect of S1P biology is the large difference in S1P abundance between blood or lymph (high) and tissue (low), which is termed the S1P vascular gradient. This gradient maintains vascular endothelial barrier function and facilitates lymphocyte mobilization from lymphoid tissues. Indeed, S1P1 receptor agonist drugs (e.g. fingolimod) are therapeutically beneficial because S1P signaling is highly sensitive to changes in S1P gradient. We used our SphK2 inhibitors to demonstrate that interdicting S1P signaling at the level of synthesis steepens the S1P vascular gradient by slowing S1P clearance from the blood. This result suggests that SphK2 inhibitors will be extremely useful in treating conditions where the endothelial barrier is compromised, e.g. acute kidney injury and sepsis. Although our recently discovered SphK2 inhibitors are active in vivo, improvements in potency, oral availability and chemical diversity are needed to advance them to the clinic. We will accomplish these goals by generating additional inhibitors on our current chemical scaffold and by developing a novel second scaffold. The current scaffold has also yielded a few SphK1 inhibitors but these lack potency at mouse SphK1, which precludes their testing for efficacy in some key disease models. In contrast to SphK2, inhibition of Sphk1 decreases the S1P vascular gradient and to probe the resulting physiological consequences, multiple inhibitors are needed. We will use iterative rounds of synthesis and testing to generate a library of SphK1 inhibitors with emphases on increasing their potency at mouse SphK1 and discovering inhibitors that have suitable pharmacokinetic properties in rodents. To understand the molecular mechanism of SphK inhibition as well as to inform the synthetic chemistry strategies, we will solve the structures of both isozymes with bound inhibitors using X-ray crystallography. Finally, we will discover a blocker of the S1P exporter, SPNS2, which provides the S1P to lymph and thereby maintains the S1P vascular gradient that is required for lymphocyte egress from lymphoid organs to lymph. Currently, due to the unavailability of SPNS2 inhibitors, this particular approach to the manipulation of S1P gradient and subsequent immunomodulation remains completely unexplored. The strength of our program is the synergism in the combination of chemistry (Santos) and pharmacology (Lynch) to which we now add structural biology (Faham). Our central theme of is to understand the therapeutic potential of manipulating the S1P gradients either at the level of synthesis (SphK inhibition) or transport (SPNS2 blockade). We have a track record of productivity that enabled a fundamental insight into S1P biology, e.g. our discovery that SphK2 inhibition modulates S1P signaling to protect endothelial function, a new therapeutic strategy. Now, we propose the development and detailed characterization of greatly improved SphK inhibitors and to make the chemical tools necessary to interrogate SPNS2 as a potential drug target.