A real-time fluorescent assay of guanylyl cyclase activity

Guanylyl cyclase is an enzyme that controls many physiological processes. Malfunction in guanylyl cyclase activity is implicated in a range of diseases, including hypertension, toxic shock, and neurodegenerative diseases such as stroke and Alzheimer's. It is therefore important to understand how guanylyl cyclase works, both in order to understand how the enzyme functions in healthy and pathological states and to develop drugs that can enhance or inhibit its activity. Current methods for measuring guanylyl cyclase activity are laborious and time-consuming. We have developed a new chemical indicator which gives a continuous read-out of enzyme activity without the need for further processing or additional reagents. In principle, this offers a very useful tool for probing the mechanism of activation of guanylyl cyclase at a molecular level of detail, as well as a means for rapidly and efficiently testing drugs that influence the enzyme. However, the current indicator has limitations that make it difficult to use in some experimental conditions, and we therefore plan to develop and refine the chemistry of the compound to circumvent these problems. If successful, the proposed research will result in a new tool that will accelerate the rate at which biological, pharmacological and pharmaceutical research into guanylyl cyclase can be carried out. We hope that this tool will increase the speed at which drugs are developed to treat the numerous disease states in which the enzyme is implicated.

Guanylyl cyclases catalyse the formation of cyclic guanosine 3'-5' monophosphate (cGMP) from guanosine triphosphate (GTP) and are classified into two families, particulate and soluble, which are activated by natriuretic peptides and nitric oxide, respectively. Once formed, cGMP acts on a range of downstream targets: cGMP-dependent protein kinase, several isoforms of phosphodiesterases, and cyclic nucleotide gated (CNG) and modulated (HCN) ion channels. Through these signalling pathways, guanylyl cyclases coordinate a diverse range of physiological processes, and dysfunction in cGMP dynamics is implicated in numerous diseases. The aim of the proposed work is to develop a promising lead compound that offers a real-time assay of guanylyl cyclase activity. The principle is that labelling the GC substrate, GTP, with an environment sensitive fluorophore (MANT), yields a change in fluorescence intensity as a result of the bonding rearrangements during cyclization. Although preliminary results are promising, as a fluorophore MANT has some limitations: the spectral change is strongest in the far UV (280 nm), and the maximal increase in emission intensity is modest (2-fold). We therefore propose screening a range of fluorescently tagged GTP analogues, to determine the chemical basis of the observed fluorescent change, and to synthesize alternative indicators with more useful optic properties. If successful, we will test the indicators in cell based and cell free preparations, with the overall ambition being to find a GTP analogue that allows the continuous measurement of GC activity in space and time.