IC50 - Wikipedia
The relationship between IC50 and deltaG is not linear so the assumption that the zero molecular size limit for IC50 and Kd is 1 M. Are The way I look at ligand efficiency is nicely summarized by a famous quote by George. The expected Kd is ~ nM as determined by a radioligand binding assay (Lax, I . et al. ). Based on the data, the calculated IC50 is nM. View protocol. IC50 is an operational parameter defined as the concentration of inhibitor .. the difference between IC50 and Ki,app, and the drawbacks surrounding IC
What diseases are associated with mutant kinases that my compound inhibits? Mutated kinases have been implicated as causative agents in a diverse range of diseases, including many forms of cancers.
These same mechanisms can also confer resistance to therapeutic agents thereby rendering them ineffective or lead to relapse after initial positive response. The availability of disease relevant mutant kinases affords investigators an important tool to elucidate the mechanisms of action for their compounds, reveal novel therapeutic opportunities, or as starting points for next generation drugs. For a list of mutant kinases available in our panel and associated disease-relevance, please visit our Mutant Kinase Reference page.
Does the affinity of the ligand for a respective kinase used in the assays affect affinity measurements of my compounds? Binding assays are performed under conditions in which binding constants measured for the interaction between kinases and test compounds are independent of the affinity of the immobilized ligand for the kinase. Test compound binding affinity did not change with the immobilized ligand used and confirms that results are independent of the affinity of the immobilized ligand for the kinase.
Additional details regarding compound binding constant relative to affinity of bait can be found on p. Although off-target liabilities can be program and target specific, there are kinases which have been described in literature as being associated with varying degrees of toxicity. For example, Olaharski, A. What is the standard immobilized ligand used in your competition binding assays? We utilize over 30 different small molecule active-site directed ligands, each extensively tested and validated to deliver high quality, consistent data.
A few of the inhibitors we use include Staurosporine and Purvalanol B. What is the naming convention used your assays? This naming convention is frequently used to denote the presence of an amino acid mutation or deletion in the sequence and its location. But in solution, the fragments rapidly recombine to form active enzyme that is capable of hydrolyzing substrate and producing a chemiluminescent or fluorescent signal. Because Arrestin recruitment is independent of G-protein signaling, these assays offer a powerful and universal screening and profiling platform that can be used for virtually any Gi- Gs, or Gq-coupled receptor.
How do PathHunter Internalization cell-based assays work?
Sigmoidal Fitting/Dose Response Curves
These non-imaging, non-antibody-based chemiluminescent assays, provide a direct and quantitative measurement of internalized GPCR localized to intracellular endosomes and allow the fate of unlabelled, activated GPCRs to be monitored in live cells without the need for expensive microscopy.
Stimulation of the receptor results in Arrestin binding to the activated GPCR, internalization of the receptor and trafficking to cellular endosomes.
GPCR activation results in internalization of the receptor and trafficking to endosomes. The enzyme is split into two complementary portions. The active enzyme can convert a chemiluminescent substrate to generate an output signal detectable on a standard microplate reader. In this assay, a calcium sensitive dye is loaded into cells to allow for the real time detection of intracellular calcium mobilization. Activation of a GPCR by injection of a compound results in the release of calcium from intracellular stores and an increase in dye fluorescence that is measured in real time.
Fit the data only, ignoring the Blank control values. Average the Blank control values, and set the parameter Top to be a constant value equal to the mean of the blanks. Enter the blank values as if they were part of the dose-response curve.
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Simply enter a low dose, perhaps or You can't enter zero, because zero is not defined on a log scale. The results will be very similar with any of these methods, because the data form a complete dose-response curve with a clear top plateau that is indistinguishable from the blank. I prefer the third method, as it analyzes all the data, but that is not a strong preference.
Similarly, there are three ways to deal with the bottom plateau: That is the ideal situation. There is no ambiguity about what IC50 means. A situation where IC50 can be defined in two ways This figure shows an unusual situation where the inhibition curve plateaus well above the control values NS defined by a high concentration of a standard drug.
This leads to alternative definitions of IC Clearly, a single value cannot summarize such a curve. You'd need at least two values, one to quantify the middle of the curve the drug's potency and one to quantify how low it gets the drug's maximum effect.
Dose Response Relationships/Sigmoidal Curve Fitting/Enzyme Kinetics
The graph above shows two definitions of the IC The relative IC50 is by far the most common definition, and the adjective relative is usually omitted. The NS values are totally ignored with this definition of IC This definition is the one upon which classical pharmacological analysis of agonist and antagonist interactions is based.
With appropriate consideration of the biological system and concentrations of interacting ligands, estimated Kd values can often be derived from the IC50 value defined this way not so for the "so-called absolute IC50" mentioned below.
This term is not entirely standard. The concept but not the term "absolute IC50" is used to quantify drugs that slow cell growth. The abbreviation GI50 is used for what we call here the absolute IC They don't use the terms relative and absolute.
Incomplete dose-response curves Any attempt to determine an IC50 by fitting a curve to the data in the graph above will be useless. A curve fitting program might, or might not, be able to fit a dose-response curve to the data. But if the curve fits, the value of the IC50 is likely to be meaningless and have a very wide confidence interval.
The data simply don't form a top plateau which would define or a bottom plateau which would define 0. If data haven't defined or 0, then 50 is undefined too, as is the IC If you also have control values that define and 0, then the curve can be easily fit.
The curve below was created by fitting a dose response curve, but constraining the Top plateau to be a constant value equal to the mean of the Blanks values, and the Bottom plateau equal to the mean of the NS values.