FLOURESCENCE-BASED DETECTION ASSAY METHODS FOR DRUG DISCOVERY PROCESSES
Introduction:
Cancer, neural disorder, inflammatory diseases and metabolic problems are some of the Pathological conditions in which protein kinases are involved in. Therefore this makes protein kinases such as IRAK-4 one of the most promising groups of drug targets. Irak-4 stands for (interleukin-1 receptor-associated kinase 4). IRAK-4 is involved in the signaling innate responses from Toll-like receptors. IRAK4 is part of the phosphoryl transferases family that transfer the γ -phosphate of ATP to the serine, threonine, or tyrosine conserved residues on specific substrate proteins. The quantification of this phosphoryl transfer has been detected and measured using traditional biochemical based method wchi involves the use if radioisotope labeled-ATP. But due to drawbacks associated with the use of radioisotope materials scientists have been trying alternative ways to facilitate HTS formats. Some of these alternative methods which have been used instead instead of radioactivity involve the use of fluorescence emission. As well, methods that use fluorescence-based detection methods have become widely used more because they are automation friendly, easy to use, relatively low cost, and widely available. Examples of these technologies which will be discussed review include T fluorescence intensity (FI), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF), time resolved fluorescence resonance energy transfer (TR-FRET). I try to discuss the advantages and disadvantages of these methods and discussing the challenges of discovering kinase inhibitors.
KEY WORDS: fluorescence intensity (FI), fl...
... middle of paper ...
...reproducibility, signal/background ration as well as assay sensitivity and came to a relevant conclusion that there is no such thing as a perfect assay. Each fluorescent immunoassay can be used towards the pursuit of drug discovery and each one of these assays work in their own rapid and efficient way which is different in comparison to each other. Every assay has its advantages and disadvantages which must be evaluated when one is making a choice for which technology to select.
When selecting the assay formats for HTS not just the final goals of the screens should be taken into account but as well as the limitation of resources. There is no single technology that will totally satisfy all kinase drug discovery needs. But fortunately, there is always a possibility to find a suitable assay format for specific needs and targets in the drug development process.
The transducer in the assay was the Shimadzu UVmini-1240 UV-Vis Spectrophotometer. It is used to measure the absorbance of ferricyanide in solution. Ferricyanide is a yellow species that be measured and compared to the glucose concentration of the sample. Electrochemical glucometers look like the most common type of transducer for commercial use. It utilizes electrodes and flowing current measured by a voltmeter.2
Fluorescence measurement provides very important information about the photochemistry of a particular molecule. The first part of this experiment was dealing with the fluorescence behavior of a Leucophor PAF. Information from both spectrophotometry and fluorimetry was used to measure the quantum yield as well as to explain why Leucophor PAF was use as commercial optical brightener. The second part of this experiment dealing with fluorescence quenching of quinine bisulphate solution (QBS) is the presence of sodium chloride.
Irradiation in the red/near-infrared spectrum (R/NIR, 630 – 1000 nm) has been recently used as a potential therapeutic strategy to treat different diseases and injuries such as Mitochondrial Disease, Degenerative Eye Diseases, Neurodegenerative Diseases, Cardiovascular Disease and Stroke, Metabolic Diseases (Eells et al., 2003), wound healing, central nervous system injury, and for restless leg syndrome (Fitzgerald et al., 2013).
Case 1: In this case. As a certified public accountant, Erickson oversaw and initiated an arbitrary adjustment to increase cash and decrease accounts receivable. Also, Erickson signed Form 10-K with full knowledge that the financial statements include therein incorporated entries misstating revenues. As we can see from this case, Erickson’s behavior not only violate the Business and Professions Code, Division 3, Chapter 1, § 5100(g) and (i), but also against the ethical theories.
During this time, it could only be used in a lab with semi-intense supervision. Now, fast forward a few decades and there are D.I.Y. at home kits. The process of Electrophoresis starts with an electric current being run through a gel containing the molecules of interest. The molecules will then travel through the gel in different directions and speeds, based on their size and charge, allowing them to be separated from each other. Dyes, fluorescent tags, and radioactive labels can all enable the molecules on the gel to be seen after they have been separated. Because of these identification markers, they appear as a band across the top of the gel. Electrophoresis can be used for many different things. It is used to identify and study DNA or DNA fragments, and helps us to better understand the molecular components of both living and deceased organisms. Electrophoresis can also be used to test for genes related to specific diseases and life altering diagnoses such as Multiple Sclerosis, Down’s Syndrome, kidney disease, and some types of cancer. Electrophoresis also plays a major role in the testing of antibiotics. It can be used to determine the purity and concentration of one specific type of antibiotic or several general antibiotics at a time. Electrophoresis is also extremely useful in the creation and testing of
Isoniazid (also referred to as isonicotinic acid hydrazide or INH), one of the top two first-line drugs, is a powerful anti-TB drug that works by inhibiting the action of InhA, a crucial enzyme in the process of manufacturing the cell wall. In recent years however, there have emerged strains of TB that are increasingly resistant to Isoniazid. Instead of searching for new targets, there is a goal to expand on this already terrific target, and by doing so, find drugs that inhibit InhA, but do it by a different mechanism, thereby, showing efficacy against MDR-TB strains.
3. Corey, E. J., Barbara Czako, and Laszlo Kurti. Molecules and medicine. New Jersey: John
The objective of this experiment will be to combine various substances, liquids and metals, and to observe their behavior when they are combined. The types of reactions observed shall determine the nature of these reactions: physical or chemical.
Alternative methods are sometimes more reliable, more accurate, cost-effective, practical, and expedient Alternative testing can be used for in preclinical studies .These methods are vitro methods (human cells and tissues), silico models (advanced computer-modeling techniques), studies with human volunteers (microdosing, advanced brain imaging and recording techniques), stem cell, genetic testing methods, computerized patient-drug databases ,virtual drug trials and human-patient simulators can be used for the assessment of the safety of drugs, chemicals, cosmetics, medical devices, consumer and investigational products.
N, Ranganathan, and I. J. Kuppast. "A Review on Alternatives to Animal Testing Methods in Drug Development." Ebscohost. N.p., 16 Oct. 2012. Web. 13 Nov. 2013.
Today, there are more advanced lab tests to help doctors classify ALL so they no longer have to rely on just the cell’s characteristics. These new lab tests aid in the grouping of ALL based on the type of lymphocyte the leukemia stems from (B cell or T cell) and how mature the cancer cells are (American Cancer Society, 2013)54.... ... middle of paper ... ... Diseases & Conditions - Medscape Reference.
Westgard, J. O. (2013). Perspectives on Quality Control, Risk Management, and Analytical Quality Management. Clinics in Laboratory Medicine, 33(1), 1-14.
An alternative approach of affinity chromatography with extremely significant results is dye-ligand affinity chromatography. In this type of affinity chromatography, dyes compose the group of ligands than are employed in the technique (Hage et al., 2012).
Its history is long and successful. Additionally, its sensitivity and simplicity, spatial and temporal resolution have all played a part in its importance that has led to its persistence as the gold standard in disease detection (Kiernan, 1999; Boekelheide, K. & Schuppe-Koistinen, I. 2012)
Moreover, It is quite obvious that animal testing has done a lot for medical research in the past, and the use of an animal was needed. The main purpose of animal testing is to “Gain basic knowledge; for fundamental medical research; for the discovery and development of drugs and vaccines and medical advances” (Estimates for Worldwide Laboratory Animal Use in 2005 1). However, while that may have been true in the 1900’s, it is certainly not the case in the 21st century. With technology constantly advancing and expanding, researchers have found other alternatives that do not need a live animal body. Such alternatives, like computer models and in vitro testing, give the same amount of medical research without the nereed of harming an animal. Computer programs use specialized models to help design new products. These generated simulations are used to “predict the various possible biological and toxic effects of a chemical or potential drug candidate” (Alternatives to Animal testing: a review 225). It is unreasonable to assume that in the 21st century, animal testing is still the best option for medical research. With technology currently being used for many medical advances, future discoveries of medical research can and should be made without animal