Anjaparavanda P. Naren - US grants

DESCRIPTION (provided by applicant): The broad goal of this proposal is to identify specific amino acid residues critical for syntaxin 1A-CFTR interactions and to use this information to augment C1- current activity in vivo and ex vivo by maneuvers that disrupt their interactions with syntaxin 1A in whole animals (WT, G551D and R117H CFTR mice). Our preliminaiy results indicate that syntaxin 1A binds to G55 1 D CFTR and inhibits its activity in Xenopus oocytes. In addition, syntaxin 1A is present in native tissues affected in CF (in particular, airway epithelium), and we will show that an 18 a.a peptide of CFTR (p46-63) that can block the syntaxin 1A-CFTR interaction can augment CFTR activity in polarized colonic epithelial cells (HT29-CL1 9A). We have preliminary data to indicate the feasibility of these experiments in whole animals as well. A few organic molecules have been screened and we have identified one positive hit (SRI 1725) that can disrupt syntaxin 1A-CFTR interaction. Functional data suggesting an augmentation of CFTR activity upon pre-treating the cells by this molecule will be presented. These preliminary data form the basis for this proposal. The main aims of this project are (1). To identify residues that are essential for syntaxin 1A-CFTR interaction and to disrupt this interaction in order to augment the activity of WT and partial loss of function CFTR mutants in whole animals. (2). To identify small organic molecules that disrupt CFTR-syntaxin 1A interaction. (3). To determine the specificity of these organic molecules on other syntaxin 1A interacting proteins (SNAP-23, VAMP-2, Munc-18, Calcium channels and ENaC) and to monitor the effects of these organic molecules (that specifically disrupt only syntaxin 1A-CFTR interaction) in whole animals. These studies should help us identify critical residues involved in CFTR and syntaxin 1A interaction. Also, these studies may help us understand how syntaxin 1A down regulates the C1- channel function in whole animals. These experiments will also provide a test of the concept that mutant CFTR function can be potentiated by maneuvers that block this protein-protein interaction. Finally, these studies should help us identify a few novel cell permeant organic molecules that can augment CFTR activity of partial loss of function CFTR mutants that will eventually be useful in CF therapy.

[unreadable] DESCRIPTION (provided by applicant): The hypothesis to be tested is that lysophosphatidic acid (LPA) inhibits secretory diarrhea through CFTR-dependent protein interactions. The long-term objectives of this laboratory as related to this grant are (i) to gain a better understanding of the dynamic protein-protein interactions that regulate LPA-dependent inhibition of CFTR and (ii) to understand the relevance of these interactions in secretory diarrhea. The specific aims of the grant are (AIM 1) to test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice and (AIM 2) to test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. To advance the research mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the proposed research will yield important basic science information essential to understanding, treating, and preventing digestive diseases such as secretory diarrhea. In Aim 1, we will test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice. In subaim 1a, we will test whether LPA inhibits CFTR function in cultured gut epithelial cells and in excised mouse intestinal tissue. In subaim 1 b, we will test whether LPA inhibits cholera toxin-induced CFTR-dependent secretory diarrhea. In subaim 1c, we will test whether LPA does not inhibit CFTR function in LPA2 receptor knockout mice. In Aim 2, we will test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. In subaim 2a, we will determine if LPA2, CFTR, and NHERF2 are assembled in a macromolecular complex in vitro. In subaim 2b, we will cross-link the components of the preexisting macromolecular complex (LPA2, CFTR, and NHERF2) in cultured epithelia and in mouse intestinal epithelial cells. In subaim 2c, we will test whether LPA inhibits the CFTR Cl-transporter due to a physical interaction between LPA2, and CFTR (mediated by NHERF2). At present, the molecular mechanisms responsible for LPA-mediated inhibition of secretory diarrhea are unclear. This project is a critical step in understanding the molecular mechanisms underlying the beneficial effects of LPA, thereby making possible improved treatments in the prevention of secretory diarrhea. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cholera is a serious diarrheal disease and is caused by Vibrio cholerae a gram negative bacterium and has the potential to be used as a bioterror weapon by contaminating water and food supplies. The National Institute of Allergy and Infectious Diseases (NIAID) has categorized V. cholerae as category "B" pathogen that posses a potential bioterrorism threat. Lysophosphatidic acid (LPA) is a lipid mediator present in blood and foods. LPA has been shown to be an LPA-receptor agonist and is beneficial in reducing weight loss in inflammatory bowel disease and diarrhea. We will provide preliminary data to show that intestinal fluid secretion induced by cholerae toxin (CTX) in mice is significantly reduced by LPA administration. Based on this observation, the proposed research will test the hypothesis that foods rich in LPA and stabilized synthetic LPA-receptor agonists can inhibit CTX-induced secretory diarrhea in mice. Therefore the use of lipid mediators such as LPA and its anologs in addition to oral rehydration therapy would have value in treating V. cholerae pandemic. [unreadable] [unreadable] The specific aims of this proposal are: [unreadable] Aim 1: To test if foods rich in LPA will inhibit CTX-induced diarrhea in mice. [unreadable] Aim 2: To test if metabolically stabilized synthetic LPA-receptor agonists exhibit potent anti- diarrheal properties (compared to LPA). [unreadable] [unreadable] These studies will demonstrate that cholera toxin-induced diarrhea can be controlled by consuming foods rich in LPA. It will also help us test and develop drugs that may have therapeutic potential. It will help move us toward the long-term goal of understanding the lipid mediators which (e.g., LPA) can regulate the CFTR Cl- channel function. The results of these studies will provide us with possible alternative methods of treating diseases of the gastrointestinal tract such as secretory diarrhea, that can be caused due to an act of bioterror such as contaminating food and water supplies with V. cholerae or due to natural causes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The hypothesis of this proposal is that the functional activities of the cAMP transporter (MRP4) and CFTR Cl- channel are physically and functionally coupled within the gut epithelial cells. The long-term objectives of this laboratory are (I), To define the mechanism of how protein-protein interactions regulate CFTR Cl- channel function and (II), To dedicate our efforts toward making a long-term contribution in understanding gastrointestinal disorders related to diarrheal diseases. The specific aims of this proposal are: Specific Aim 1. To test the hypothesis that MRP4 is an apical cAMP transporter in the gut and inhibition of this transporter potentiates cholera toxin (CTX) induced diarrhea. Four subaims will be tested, they are (1a). To test whether MRP4 is an apical cAMP transporter in gut epithelial cells and to characterize the cyclic nucleotide transport using HPLC. (1b). To test whether MRP4 inhibition and MRP4 silencing (using Si-RNA) augments CFTR-dependent short circuit currents in the apical membrane of polarized gut epithelial cells and in excised mouse intestine. (1c). To test whether MRP4 inhibition can potentiate cholera toxin (CTX)-induced and CFTR-dependent secretory diarrhea in mice and to test if CFTR knock out mice fail to respond to CTX and MRP4 inhibition. (1d). To test whether MRP4 knock out mice are more susceptible to CTX-induced secretory diarrhea and to test if MRP5 inhibitors fail to induce secretion. Specific Aim 2. To test the hypothesis that there is a physical and functional coupling of cAMP transporter (MRP4) and CFTR Cl- channel at or near the apical plasma membrane of gut epithelial cells. Three subaims will be tested, they are (2a). To test whether the cAMP transporter (MRP4) is in a macromolecular complex with PDZK1 and CFTR and to define the stoichiometries of CFTR:PDZK1 and MRP4:PDZK1 complex in the plasma membrane of gut epithelial cells. (2b). To test if the disruption of the cAMP transporter containing macromolecular complex inhibits CFTR function and to test if the lateral mobility of CFTR and MRP4 increases (high diffusion rates) at the plasma membrane. (2c). To test whether cAMP accumulates at or near the plasma membrane (using a membrane associated cAMP sensor) upon inhibition of the cAMP transporter. These studies will demonstrate that the two ABC transporters (CFTR and MRP4) can be functionally and physically coupled and that MRP4 inhibition can augment CFTR transporter function. The results of these studies will provide us with possible alternative methods and targets for treating certain diseases of the gastrointestinal tract such as secretory diarrhea and IBD. These studies will, therefore, have clinical relevance in individuals suffering from certain forms of diarrhea. PUBLIC HEALTH RELEVANCE. The proposed research will test the hypothesis that the two ABC transporters MRP4 (functions as cAMP transporter) and CFTR Cl- channel are physically and functionally coupled within the gut epithelial cell. This proposal will also test if inhibition of the MRP4 transporter function augments CFTR Cl- channel function thus potentiating cholera toxin (CTX) induced diarrhea in mice.

ABSTRACT Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations. Clinically, chronic lung disease is the main cause of morbidity and mortality for CF patients. Among the 2000+ disease-causing mutations, ?F508 is the most common mutation and associates with a severe form of CF disease. The ideal therapy for CF associated with ?F508 requires increasing the quantity of ?F508-CFTR protein at the plasma membrane, potentiating the impaired channel gating properties, and improving its stability. This notion was supported by the approval of two CFTR modulating drugs, Orkambi® (VX-809 + VX-770) and Symdeko® (VX- 661 + VX-770), to treat CF patients homozygous for ?F508, and by trials with triple combinations (VX-445 + VX- 661 + VX-770; VX-659 + VX-661 + VX-770). It is to be noted that the clinical benefits of approved drugs are modest and the mechanisms of action of these CFTR correctors are poorly understood. In this grant, we plan to study the mechanisms of how CFTR correctors promote the maturation of ?F508-CFTR and stabilize the mutant protein at the plasma membrane. We will focus on VX- CFTR correctors (VX-661, VX-809, VX-445, and VX-659) and study the subject from the perspective of CFTR-containing macromolecular complexes. The hypotheses to be tested are: (i) CFTR correctors (e.g., VX- CFTR correctors) bind directly to ?F508-CFTR to exert their rescue effects. A high-affinity binding will produce a better rescue outcome. (ii) The instability of ?F508-CFTR (with a short half-life) at the plasma membrane is, at least in part, due to its reduced ability to interact with binding partners and consequently cannot form a stable macromolecular complex, which leads to its rapid internalization and targeted for degradation. (iii) CFTR correctors not only help fold ?F508-CFTR in the ER to promote its maturation, but also stabilize the mutant protein at the plasma membrane by enhancing its interaction with binding partners and facilitating the formation of a stable macromolecular complex. And (iv) by isolating the corrector-associated- and ?F508-CFTR-containing complexes under different conditions and using proteomics, we can identify effectors and pathways important in the rescuing process. Click chemistry and photo-affinity labeling will be used to investigate the interactions and macromolecular complexes formation in various CF model systems. This study will help us (i) better understand the mechanisms of action of CFTR correctors, (ii) identify novel targets in ?F508-CFTR-containing complexes, (iii) develop more potent drugs to combat CF, and (iv) understand the molecular basis of other human diseases resulting from insufficiently folded and processed proteins (e.g., P-glycoprotein) and find ways to treat these diseases.