Jerod S. Denton, Ph.D. - US grants

SUMMARY Swelling caused by edematous fluid retention is a common, life-threatening symptom of heart failure (HF) and chronic kidney disease (CKD). Loop diuretics are often prescribed as a first-line therapy to quickly reduce the extracellular fluid volume burden in HF and CKD patients. This class of diuretic works by inhibiting NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop, and increases the delivery of NaCl and fluid to the distal nephron comprised of the distal convoluted tubule (DCT) and collecting duct (CD). In response to the increased NaCl and fluid load, the DCT and CD increase their NaCl reabsorbing capacity by upregulating the expression of specific ion transporters and channels. This compensatory mechanism diminishes the effectiveness of loop diuretics and gives rise to loop diuretic resistance. A growing consensus among nephrologists is that distally acting diuretics that inhibit sodium (Na+) reabsorption in the DCT (i.e. thiazide diuretics) or CD (potassium-sparing diuretics) downstream of the TAL should be administered in an effort to overcome loop diuretic resistance. However, both diuretic classes have critical limitations that highlight the need to discover more effective, safer, and novel-mechanism distal diuretics for circumventing loop diuretic resistance. In this application, we propose to discover the first potent and selective inhibitors of heteromeric Kir4.1/5.1 potassium channels, which have emerged recently as key regulators of NaCl reabsorption and kinase signaling in the distal nephron. In Aim 1, we will employ a fully validated, fluorescence-based thallium-flux assay to screen approximately 110,000 structurally diverse compounds from the Vanderbilt Institute of Chemical Biology library for novel inhibitors of Kir4.1/5.1 channels heterologously expressed in HEK-293 cells. A series of secondary thallium-flux assays, as well as high-throughput automated patch clamp electrophysiology, will then be used to evaluate the potency and selectivity of confirmed inhibitors for Kir4.1/5.1 over an extensive panel of related inward rectifier potassium (Kir) channels. In Aim 2, we will select the most promising Kir4.1/5.1 inhibitors based on their potency, selectivity, chemical structure, and in vitro metabolic stability properties to develop analog libraries using state-of-the-art medicinal chemistry techniques with the goal of optimizing the pharmacological properties of inhibitors for in vivo administration. In Aim 3, we will use single channel analysis to test the activity lead inhibitors against native rat and human Kir4.1/5.1 channels in freshly isolated kidney tubules. In addition, we will test the hypothesis that inhibition of Kir4.1/5.1 induces renal excretion of Na+, K+, and water in rats. Completion of these aims will provide critically needed tool compounds for evaluating the therapeutic potential of Kir4.1/5.1 channels as a diuretic target in the setting of loop diuretic resistance in HF and CKD patients.

PROJECT SUMMARY The ductus arteriosus (DA) is an essential fetal artery connecting the aorta and pulmonary artery, which shunts blood away from the developing lungs in utero . Circulatory adaptation at birth requires rapid constriction of the DA to facilitate proper perfusion of the newly inflated lungs. Persistent patency of the neonatal DA (PDA) is a significant clinical problem that is inefficiently managed with currently available therapies. Pharmacology-based PDA therapeutics non-specifically target the prostaglandin pathway, have worrisome off target effects on other vascular beds, and are ineffective in approximately 30% of patients. While surgical ligation and catheter-based closure are effective alternatives, these mechanical approaches come with their own risks and limitations. Consequently, there is a significant need to identify and rigorously validate novel drug targets for manipulating DA tone. An emerging body of physiological and genetic data from our group and others has implicated vascular ATP-regulated potassium (KATP) channels as novel drug targets for regulating DA tone. Specifically, we show here for the first time that KATP channels comprised of pore-forming Kir6.1 and regulatory SUR2B subunits are highly enriched in smooth muscle cells of the PDA and regulate DA tone in response to pharmacological modulation. Unfortunately, the lack of specific Kir6.1/SUR2B inhibitors (and activators) has precluded a rigorous assessment of the therapeutic potential of DA KATP channels for treating PDA. In this multi-PI collaboration, which will benefit from complementary expertise in potassium channel drug discovery (Drs. Denton/Lindsley) and DA physiology and pharmacology (Dr. Shelton), we propose to employ high- throughput screening (HTS) and medicinal chemistry to develop an extensive ?tool kit? of vascular-specific KATP channel modulators for validating Kir6.1/SUR2B channels for regulating DA tone in vitro and in vivo. In Aim 1, we will employ a fully validated HTS assay to interrogate ~110,000 small molecules for potent and selective Kir6.1/SUR2B modulators. In Aim 2, we will use medicinal chemistry to optimize lead compounds for selectivity and potency and determine compound metabolism and pharmacokinetic properties. In Aim 3, we will evaluate the efficacy of lead compounds to regulate mouse and human DA tone using isolated vessel myography assays and in vivo mouse models of PDA. The successful completion of these aims will generate critically needed tool compounds for modulating DA tone and validate Kir6.1/SUR2B channels as novel therapeutic targets for treating PDA.

? DESCRIPTION (provided by applicant): Ion channels and transporter proteins are ubiquitous molecules that serve a variety of important physiological functions, provide targets for many types of pharmacological agents, and are encoded by genes that can be the basis for inherited diseases affecting the nervous system and other tissues. This proposal describes the continuation of a Training Program in Ion Channel and Transporter Biology that will provide multidisciplinary research training for postdoctoral scientists. This highly focused training program involves 28 NIH-funded preceptors (aggregate funding >$44,000,000 direct costs/year) affiliated with 13 different academic departments and 10 research centers at Vanderbilt University with strong records of accomplishments in the ion channel and transporter field, and with a deep commitment to training postdoctoral fellows. This interdepartmental training program capitalizes on a over 20-year history of institutional and multidisciplinary strength in ths research field. The program began initially in 2001 with only 19 faculty and has successfully filled all funded positions since that time. Although the training program originally included predoctoral training, since 2010 the program has focused solely on postdoctoral training. Postdoctoral trainees are selected from the pool of applicants that apply to preceptor laboratories as well as to participating centers and departments at Vanderbilt University. A multi-faceted recruitment strategy will continue to attract highly qualified individuals from underrepresented groups (URM). During the current funding cycle, 3/12 (25%) of trainees were URMs. In addition to intensive research experiences, trainees will have didactic course requirements that include a focused course on grant writing, an innovative program group meetings devoted to individualized training in grant writing, mentoring and career guidance. The high caliber of faculty mentors, the interdisciplinary nature of training opportunities, the strong institutional strengths combine to foster a unique environment suited to the goal of the training program, which is to develop scientists with strong commitments to biomedical research in the area of ion channel and transporter biology.

PROJECT SUMMARY The volume-regulated anion channel (VRAC) is expressed ubiquitously in vertebrate cells where it mediates the efflux of Cl- and organic solutes required for cell volume regulation, an essential physiological process. VRACs are activated and inactivated by cell swelling and shrinkage, respectively. They also detect changes in intracellular ionic strength, which modifies their sensitivity to cell volume changes. VRACs and the genes that encode them are implicated in multiple diseases including diabetes, obesity, cancer and immunity. Whole genome RNA interference screening led to the demonstration in 2014 that VRACs are encoded by five members of the Lrrc8 gene family, Lrrc8a?e. VRAC/LRRC8 channels are hexaheteromers and require co-assembly of the essential subunit LRRC8A with one or more other LRRC8 proteins. Subunit assembly order and stoichiometry are unknown. Cryo-electron microscopy (EM) structures of homomeric LRRC8A and LRRC8D channels were recently determined. However, LRRC8A and LRRC8D homomers do not exist in Nature. Furthermore, LRRC8A homomeric channels have non-native functional properties and LRRC8D channel properties are undefinable because they are not trafficked to the plasma membrane. Existing cryo-EM structures thus have limitations for understanding VRAC/LRRC8 structure-function relationships. Directly translating LRRC8A and LRRC8D cryo-EM structural information into functional understanding is further constrained by the unknown and likely variable stoichiometry and assembly of hexaheteromeric VRAC/LRRC8 channels. Our laboratory, funded by DK51610, has studied VRAC extensively and was the first to demonstrate many of the channel's unique functional properties. Most recently, we described novel LRRC8 chimeric channel constructs that allow detailed molecular study of homomeric channels with physiologically relevant functional properties and defined stoichiometry and assembly. Our chimera studies uniquely demonstrated that 1) the LRRC8A intracellular loop, IL1, has unique structural features, 2) it is required for cell volume sensing, 3) the LRRC8 C-terminus is required for sensing changes in intracellular ionic strength and 4) both the LRRC8A IL1 and C-terminus are required for correct cellular processing of VRAC/LRRC8 channels. The overarching goal of this R01 renewal application is to utilize these novel LRRC8 chimeras to better elucidate VRAC/LRRC8 channel structure-function relationships. We will characterize the roles of the LRRC8A C-terminus in VRAC/LRRC8 channel regulation and will test the hypothesis that the LRRC8 IL1 determines VRAC/LRRC8 channel pore properties and regulates channel gating. We will also determine the cryo-EM structure of a unique LRRC8 chimera in multiple physiologically relevant conformations. Our studies will provide novel insights into the regulation and function of VRAC/LRRC8 channels and will provide a higher confidence foundation for detailed mutagenesis-based structure-function analyses.