David L. Roman, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Regulators of G-protein signaling (RGS) proteins are important modulators of signals initiated through G- protein coupled receptors (GPCRs). The action of RGS proteins is the acceleration of the deactivation of G- protein signals through modulation of G? subunit GTPase activity, which causes a termination of signal. While GPCRs have been classical drug targets, many cellular processes can be modulated by altering the action of RGS proteins. I propose to measure the interaction affinity of a variety of RGS proteins for their effector proteins (i.e. G?) using a novel high throughput flow cytometric method. With these parameters established, a library of diverse chemical compounds (~35,000) will be screened to identify modulators of RGS activity using the high throughput flow cytometric method. Once modulators are identified, complete quantitative structure-activity relationships will be evaluated and additional similar compounds will be characterized in both biochemical and cell-based assays. The use of these pharmacological modulators would provide utility for studying various disease in which RGS proteins could play a role, including Parkinson's disease (RGS9), Schizophrenia (RGS4), as well as cell proliferation and metastasis (LARG). [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is in response to PA-10-213 titled Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic discovery. Our overall goal is develop novel biochemical and cell-based assays that will be used to identify inhibitors of Regulator of G protein signaling 17 (RGS17). RGS17 has been identified as being over-expressed in lung and prostate tumors, and facilitates tumor cell growth. Reports indicate that knockdown of RGS17 in lung and prostate cancer cell lines reduces their rate of growth and results in shrinkage of xenograft tumors in nude mice. Unfortunately, there are no reports of small molecule RGS17 inhibitors, and only a handful of reports of small molecules that target any RGS protein. Therefore, RGS17 presents an unexplored target for pre-therapeutic development and represents a novel path to develop lead molecules in the fight against cancer. Thus, the discovery of RGS17 inhibitors would provide a new avenue for both understanding the role of RGS17 in cancer progression as well as provide new small molecule leads for anticancer drug development. In order to facilitate the discovery and optimization of RGS17 inhibitors, we propose to develop assays for an HTS campaign using novel biochemical and cell-based assays. Specifically, we aim to develop the following 1) a high-density (384-1536 well) biochemical assay for the RGS17:G protein alpha subunit protein: protein interaction, and 2) two novel cell based assays for detection of RGS effects on cAMP levels and GIRK channel kinetics in real-time. Achievement of these aims will provide both novel methodology for use in interrogating small molecule libraries for RGS inhibitors in HTS campaigns as well as provide new chemical probes for RGS17 protein function in cancer cell models. After development and validation, our goal is to perform a modest in-house HTS screen to identify and optimize lead molecules as RGS17 inhibitors. The outcome of this project will be both novel screening methodology for RGS protein ligands and new probe molecules that will serve as tools for studying RGS17 protein function and provide initial molecules for pre-therapeutic optimization. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it aims to probe a novel target, RGS17 using two novel assay methodologies to interrogate small molecule libraries in order to identify probes and pre-therapeutic molecules for studying and treating prostate and lung cancers. RGS17 plays an important role in tumor cell proliferation and has not been targeted in previous campaigns to identify new anticancer leads. Identifying new lead molecules targeting RGS17 is relevant to NIH

ABSTRACT Chronic pain is a major concern in public health with financial costs projected to surmount $600 billon in the next year. Patients afflicted with chronic pain endure extreme emotional, physical, and social burdens, resulting in severely diminished quality of life. Unfortunately, drugs currently used for chronic pain management, such as NSAIDs, opioids, neuronal stabilizers, and antidepressants, do not typically provide sufficient relief to restore full quality of life, and in many instances these treatments themselves limit patients, such as opioid treatment preventing a patient from legally driving. Recent preclinical studies have identified neuronal adenylyl cyclase type 1 (AC1) as a novel target for treating chronic pain. AC1 is highly expressed in neuronal tissues associated with pain processing and neuronal plasticity, and studies using AC1 knockout mice provide direct evidence linking AC1 to chronic inflammatory pain conditions. Furthermore, AC1 inhibitors would lack the side effects associated with other agents (e.g. opioids) used to treat chronic inflammatory pain. The development of AC1 inhibitors represents a unique challenge, as demonstrated by a prior preclinical AC1 inhibitor, NB001. NB001 has significant shortcomings, including modest selectivity over other adenylyl cyclase isoforms, likely due to its adenine-like structure. Compounds of this type are called P-site inhibitors and act by binding to the active site of AC that is conserved among all isoforms. Additional concerns for adenine-containing molecules like NB001 include effects on other cellular processes such as DNA synthesis. We hypothesize that developing a small molecule inhibitor of AC1 will allow us to mimic the AC1 knockout phenotype and provide a new avenue for the treatment of chronic inflammatory pain. We designed our studies to target NOT the conserved P-site or forskolin-binding site, but rather a novel approach, targeting the unique protein-protein interaction of AC1 and calmodulin (CaM). AC1 and AC8 are both activated by CaM, however, the CaM binding domains are unique in structure and location providing an unprecedented opportunity to achieve AC1 selectivity. Thus, the goals of this proposal are to: 1) develop a novel AC1/CaM biochemical screening assay, 2) implement this novel assay in a high throughput screen to interrogate a library of 100,000 compounds for inhibitors of the AC1/CaM protein- protein interaction, and 3) validate and chemically optimize lead molecules using cellular assays focused on selectivity and potency to guide medicinal chemistry efforts. To date, we have completed initial studies to develop the novel screening assay, established a subset of the necessary assays, and cemented the collaboration between the University of Iowa and Purdue University for the successful completion of our aims. We anticipate the identification of selective AC1 inhibitors that ultimately be improved and applied in models of chronic inflammatory pain.

NON-TECHNICAL SUMMARY

To effectively treat disease, drug molecules must accumulate in sufficient amounts at a specific site in the body. To achieve this, tiny vehicles called carriers are often needed, but unwanted interactions within the body often limit their effectiveness. This project uses knowledge of the molecular interactions between bacterial/viral pathogens and human cells during human infection to design bioinspired polymer coatings for drug carriers. The project will provide deeper insight into chemical structures that simultaneously limit biofouling on the carrier surface and enhance attachment of carriers to human cells. Longer term, these results will enable the development of nanocarriers that can provide better treatment of disease by decreasing the amount of drug required for treatment and limiting side effects. This project will have a direct impact on the educational experience of students at various levels. Two graduate students will perform research for their Ph.D. theses and several undergraduate students from underrepresented groups will participate in the research. Further, a K-12 outreach program in engineering and science will utilize concepts from the research during camps throughout the state of Iowa and provide graduate and undergraduate students with opportunities for teacher training. Research results and discoveries will be disseminated through publications in peer-reviewed journals and presentations at local, national, and international conferences.

TECHNICAL SUMMARY

Current drug delivery strategies to target specific cells within the body are plagued by low levels of drug accumulation in the areas they are most needed. When drug carriers enter the body and interact with biological fluids, biofouling of the carrier surface occurs and new surface properties develop that control the carrier?s subsequent bio-interactions and fate. Our lack of knowledge of how to design drug carriers with appropriate properties to trigger and mediate their interactions within complex biological environments has significantly hindered progress towards efficient drug delivery. The goal of this proposal is to design a surface coating for nanocarriers to simultaneously address issues of biofouling and cell uptake in complex biological environments. Bioinspired polymeric ligands containing molecular structures that mimic the surfaces of respiratory pathogens known to efficiently penetrate human fluids and the lung epithelium will be developed. The ability of ligand-coated nanoparticle to target lung epithelial cells submerged in natural secretions will be experimentally observed and quantified. Two independent techniques will be pursued to evaluate the molecular recognition of the ligands. First, a high-throughput, optical biosensor technique will quantify the real-time cellular responses to receptor-ligand interactions in living cells. Second, a 3-D homology modeling approach will elucidate the ability of the ligands to dock into the receptor binding pocket. Results from these studies will provide insight into chemical structures that mediate the binding and activation of receptor-mediated processes. Through biomolecular mimicry, the proposed studies will lead to more efficient design of ligands that will enhance nanocarrier delivery.

This project is jointly funded by the Biomaterials Program in the Division of Materials Research, the Established Program to Stimulate Competitive Research (EPSCoR), and the Polymers Program in the Division of Materials Research.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.