1) Structural Biochemistry of soluble guanylate cyclase
The overall goal of this research project is to characterize the structural biochemistry underlying the catalytic activity, regulation, and assembly of soluble guanylate cyclase (sGC). Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) enzymes and plays key roles in neurotransmission, blood pressure regulation, and the immune response. NO-induced vasodilation depends primarily on the activity of sGC, via the NO-sGC-cGMP pathway. sGC converts GTP (guanosine-5′-triphosphate) to cGMP (cyclic guanosine-3′,5′-monophosphate), which acts as a second messenger to modulate the activity of kinases, cGMP-gated ion channels, and cGMP-regulated phosphodiesterases. Basal sGC activity is increased ~200-fold by binding of nanomolar concentrations of NO to the heme cofactor. As both reduced NO bioavailability and response to endogenous NO both contribute to cardiovascular disease, compounds that activate cGMP production by sGC have a high therapeutical potential.
Understanding the structural basis for the mechanisms leading to sGC assembly and regulation should facilitate the development of these therapeutic agents. We use interdisciplinary analyses combining mutagenesis, x-ray crystallography, spectroscopy, and small-angle x-ray scattering.
Our results will advance fundamental understanding of sGC activity and regulation, and provide novel tools to develop next-generation small molecule sGC activity modulators that will be used to treat cardiovascular diseases, including hypertension, impotence, heart failure, and atherosclerosis.
Project funded by the American Heart Association, Scientist Development Grant.
2) Reporter compounds for quantitative imaging of biomolecular interactions using coherent x-ray scattering.A novel experimental methodology for the characterization of molecular interactions within cells and, better yet, within living organisms will represent a scientific breakthrough with fundamental applications for the molecular understanding of biological processes, for the design of high-specificity pharmacological approaches, and for the immediate assessment of response to therapy. The proposed research will explore the feasibility of BICXS (Biomolecular Interactions using Coherent X-rayScattering), a novel technology based on small-angle coherent x-ray scattering (SAXS) signatures of reporter agents that are indicative of the interaction between two targeted proteins or small molecules. This novel technology complements other methods under development for in vivo biomolecular interaction imaging: positron emission tomography and bioluminescence resonance energy transfer. In comparison, BICXS has the potential to provide increased spatial resolution and higher specificity while allowing for deep tissue imaging. The project goal is to provide a proof-of-concept for this novel biomolecular interaction imaging technology.
To explore the feasibility of the BICXS technology, we will seek to demonstrate the fundamental assumption behind this concept, namely, that the coherent x-ray scattering signatures of biomolecular systems of interest tagged with reporter agents can be indicative of molecular interactions. This method seeks to use coherent x-ray scattering signatures of reporters not to gain additional information for estimating the object anatomical and conformational properties, but as a new, non-invasive contrast imaging technology to determine the presence of interaction. The experimental approach will be divided into two steps. First, SAXS data will be collected for monomeric, dimeric, and oligomeric forms of reporter agents. Next, we will measure the scattering signature of a biomolecular interacting model system tagged with the reporter agent. Since we expect signal contamination from other scatterers, we will develop analysis techniques to extract the useful information related to the interaction levels from the noisy signals. Our studies will be complemented with a computational and experimental optimization study to investigate the design of a prototype imaging system with variables including x-ray energy, spread of input spectrum, image acquisition, imaging geometry, detector specifications, and data analysis methodologies with a focus on in vivo applications of this methodology.
This project is a multidisciplinary collaboration among Dr Daniel-Onuta (UMBC-Chem; reporter agents, nanotechnology), Dr Elsa Garcin (UMBC-Chem; biomolecular model system and SAXS studies), Dr Maricel Kann (UMBC-Biol; bioinformatics, systems biology) and researchers at the FDA.
Project funded by NSF/FDA SIR.