A novel method for detecting and monitoring the reactive nitrogen species peroxynitrite in normal physiology and human pathology. The invention offers significant advantages over traditional methods for detecting and monitoring the cellular production of peroxynitrite by overcoming limitations of other sensors. This is important because current methods are less sensitive and less robust, especially for studies of living cells such as its involvement in immunology. This invention may yield better information about the effects of peroxynitrite in a variety of diseases including infectious disease, cancer, neurodegeneration, and others.
This invention is a technique to monitor and detect cellular peroxynitrite. Peroxynitrite is an endogenous reactive nitogen species in the body that is produced by some immune cells to kill foreign pathogens but that can also damage normal cells when generated aberrantly during disease. The inventors have designed a novel fluorescent sensor that may increase understanding of peroxynitrite in cancer and infectious disease and enable more complex testing and monitoring of cellular processes.
The invention supports the potential to serve as an effective tool to study peroxynitrite. Peroxynitrite is closely associated with cancer and immunology associated with cancer and infectious disease. Peroxynitrite is difficult to study, observe, and assess, as it is highly unstable and reactive, and acts at very low concentrations while in the body. As a result, the demand for the development of techniques to monitor and detect peroxynitrite is of great interest.
How it works:
The method involves a novel, small molecule fluorophore that is used to target a specific interaction with peroxynitrite, rather than testing all peroxynitrite available in a given environment. The current invention is an optimized sensor, which inventors have determined is highly selective for peroxynitrite compared to other reactive species.
Although a wide range of sensors of peroxynitrite have been described, detection of low levels of endogenous peroxynitrite, such as those produced during phagocytosis of pathogens by macrophages, remains a challenge. Previously reported fluorescent sensors capable of detecting endogenous peroxynitrite during phagocytosis, such as fluorescein boronate, require additional stimulation of cells by cytokines such as interferon-gamma (IFN-γ) and lipopolysaccharide (LPS). However, immune cells are likely to be able to kill pathogens in vivo in the absence of these types of additional stimulants. To better understand the biology of peroxynitrite in vivo, more sensitive fluorescent sensors are needed. This invention is a small molecule fluorophore that selectively reacts with peroxynitrite and can detect peroxynitrite in living cells without additional stimulants.
Why it is better:
This invention represents a promising method in the fight against cancer. While testing for peroxynitrite is fairly new and still evolving, it is not novel on the market. There are several kits that enable peroxynitrite monitoring in live cells. Two popular kits that are used for peroxynitrite testing include Amplite and Cell Meter, each of which use Pon green (DAX-J2 PON) in a kit that measures peroxynitrite to enable real time, quantification of peroxynitrite in living cells. These options selectively react with peroxynitrite, but generally throughout the cell. The proposed invention selectively targets peroxynitrite associated with the endoplasmic reticulum in cells. As a consequence, the proposed invention can detect peroxynitrite under conditions where it is undetectable with existing sensors. The inventors theorize, and early testing has shown, that the current invention has a higher level of specificity for peroxynitrite and may yield better information about cancer and other diseases during diagnosis and marketing.