James M. Woods, Ph.D. - US grants

DESCRIPTION (provided by applicant): Dysregulated monocyte sequestration and angiogenesis clearly play a deleterious role in the joints of rheumatoid arthritis (RA) patients. The CX3C chemokine, fractalkine (fkn), is structurally unique and functionally versatile. In conjunction with its receptor, CX3CR1, fkn can act directly as both an adhesion molecule and a chemoattractant for monocytes, thus suggesting it may contribute to the sustained inflammatory response occurring in the RA synovium. Data presented here suggest that )1 the soluble form of fkn (sfkn) is sufficient to induce endothelial cell (EC) chemotaxis, EC tube formation on Matrigel in vitro and angiogenesis in vivo consistent with a pro-angiogenic role for sfkn; 2) CX3CR1 s expressed by endothelial cells; 3) the fkn present in RA, synovial tissue is capable of inducing angiogenesis; and 4) the sfkn in RA synovial fluid is capable of inducing monocyte migration and angiogenesis. Therefore, we propose to examine the role of the seven transmembrane CX3CR1 receptor as well as the G proteins with which it associates. Our hypothesis is: (1) fkn- and sfkn-induced adhesion, chemoattraction, and angiogenesis in RA each occur via proteins in monocytes or ECs will inhibit fkn-associated functions induced by RA synovial fluid and synovial tissue. Through the use of overexpressed dominant-negative peptides that can inhibit the association of G proteins with their cognate receptors, we will accomplish three of our specific aims, which are to delineate which G proteins are responsible for mediating sfkn- and RA synovial fluid-induced monocyte chemotaxis, monocyte/ endothelial cell adhesion, and angiogenesis. As a first step towards identifying novel inhibitors of CX3CR1 and potential new therapies, our fourth specific aim will then utilize the above information to identify short peptide sequences that can specifically inhibit CX3CR1 from associating with G proteins. Finally, we will use these new inhibitors to determine the impact of uncoupling CX3CR1 from its associated G proteins in chemotaxis, adhesion, and angiogenesis assays induced by sfkn and RA SF. These experiments should provide extensive new insights about the mechanism of fkn signaling through CX3CR1 and its relationship to cellular function.

DESCRIPTION (provided by applicant): Chronic inflammation is a hallmark of late stage rheumatoid arthritis (RA), driven by a multitude of factors including cytokines and chemokines, ultimately resulting in joint destruction. Targeting of individual cytokines in this process has shown efficacy in RA, however, these treatments are not effective for all patients suggesting that new therapies are required. Manzamines are a unique class of natural marine products with significant biological activity isolated from sponges collected from the ocean floor. Over 50% of novel compounds patented from all marine organisms describe sponge-derived molecules from this same chemical class, demonstrating tremendous potential for this group of molecules. Recent cell culture studies strongly suggest that one manzamine in particular, manzamine A (MZA), has potent anti-inflammatory properties on activated human macrophages, in the absence of toxicity. Studies using MZA to treat malaria in rats demonstrate exciting changes that are consistent with desired effects for RA patients. For example, two mediators which promote inflammation were significantly lowered, including tumor necrosis factor (TNF)-1 and interferon-3. TNF-1 is often over-produced in the RA joint late in disease, contributing to inflammation. On the other hand, levels of a protein which can naturally act to reduce inflammation in the joint, known as interleukin-10, were increased by MZA. Our preliminary data suggests that these three beneficial changes are only the "tip of the iceberg", and that MZA holds many additional, yet unrecognized, anti-inflammatory properties specifically relevant to RA. While literally thousands of marine products are being explored for the treatment of cancer, there are very limited comparable studies for the treatment of arthritis. No studies testing the anti-inflammatory potential of any manzamine have been conducted in RA. Oral and intravenous studies in rats given MZA suggest it is an excellent compound for pharmaceutical development. It has a very long half-life, good stability, fine absorption, adequate oral bioavailability, and low toxicity. The outlook for large-scale production of MZA is also very good, with many groups working to synthesize it from scratch or to isolate it from new sources. We developed a unique collaboration with an oceanographic institute and obtained more than enough purified compound to study the effects of MZA in arthritis. Here, we propose to examine the potential application of MZA to RA with regards to its: 1) toxicity;2) ability to alter inflammatory mediators in a manner that benefits RA patients;3) ability to reduce the influx of immune system cells to the joint;and 4) mechanism. Based on our preliminary data and the background we review, we are very optimistic about the future potential of MZA as a novel treatment for RA. PUBLIC HEALTH RELEVANCE. While many new therapies have become available in the rheumatologists'arsenal in the last decade to treat rheumatoid arthritis (RA), significant RA-associated morbidity still exists. Therefore, the search for additional and improved anti-inflammatory agents with low side effects is warranted. This application has high relevance to RA in that it will specifically focus on the anti-inflammatory potential of a unique compound, manzamine A, on RA synovial tissue and other relevant cell types.