IC50 determination

The lack of information conveyed by total brain concentration is indicated by studies on KA-672 [6], a lipophilic benzopyranone acetylcholinestrase inhibitor. The compound achieved total brain concentrations of 0.39 iM at a dose of 1 mg kg" equivalent to the IC50 determined in vitro (0.36 juM). Doses up to 10 mg kg were without pharmacological effect. Analysis of CSF indicated concentrations of the compound were below 0.01 juM readily explaining the lack of activity. These low concentrations are presumably due to high (unbound) free drug clearance and resultant low concentrations of free drug in the plasma (and CSF). 

An activity threshold of 30% inhibition at 10 M for follow-up IC50 determination was selected based on the following considerations ... 

The rate of confirmation between the inhibitory effect at 10 M in the screening and the percent inhibition obtained at the same concentration in the subsequent IC50 determination defines a success rate for each assay. This success rate is deemed satisfactory when the inhibitory effect in the primary screening step is above 50% inhibition reasonable between 30% and 50% inhibition but weak below 30% inhibition. 

A solubility assay with a detection range from 10 to 2 x 10 is performed as a part of the BioPrint profile and shows that about one third of the BioPrint compounds are insoluble at 10 " M. Thus, aqueous solubility limits the high-end concentration for IC50 determination to 10 " M. The final DMSO concentration tolerated in the assay reaction medium must not exceed 1%. This also limits the high-end concentration to 10 M. 

The retained approach for compound testing is therefore a first screening at 10 M, in duplicate. Every compound that displays more than 30% inhibition is further tested at 8 concentrations ranging from 10 " M to 10 M, in duplicate, for the IC50 determination. 

Preliminary IC50 determinations may guide the design of A) determinations and the inhibitor concentration should bracket the IC50 value. 

IC50 determinations were performed as described in the text. Values are rounded to two significant figures, and are the average of three or more determinations. Values in parentheses represent the relative standard deviation. 

Manages results for bioassays. Response curve fitting and EC50/IC50 determination. Table pivoting for multiple compounds and targets. Provides links to compounds, notebooks. 

Based on the result from the IC50 determination, determination of additional kinetic parameters such as Ki and the inhibition mode are useful (variation of the substrate concentration e.g. Km/4 1 Km with time). Transformation of the Michaelis-Menten equation are used both for calculation the Ki value as well as for graphical depiction of the type of inhibition (e.g. direct plot ([rate]/[substrate], Dixon plot [l/rate]/[inhibitor], Linewaver-Burk plot [l/rate]/[l/substrate] or Eadie-Hofstee plot [rate]/[rate/substrate]). 

The starting point of all successful assay development processes is a protein preparation that ideally contains only the enzyme of interest. The amount of enzyme needed for the whole assay development process depends mainly on the catalytic efficiency of the enzyme with the substrate. Only 10 pg of a protease can be sufficient for proper determination of the kcalIKu value, the determination of the endpoint linearity by an enzyme titration, and the assay validation by IC50 determination for... 

Turner, R.J. and Charlton, S.J. 2005. Assessing the minimum number of data points required for accurate IC50 determination. Assay Drug Dev. Technol. 3, 525-531. 

Fluorescent AMC product formation was monitored 1 hour at ambient temperature by measuring the fluorescence emission at 460 nm using an excitation wavelength of 360 nm and the IC50 determined where Cbz-ValAlaAsp-H was used as the reference. Testing results are summarized in Table 2. 

Selected experimental agents were incubated with 0.25 p,g human 20S proteasome in the assay buffer (25 mM Hepes, 500 jjlM EDTA, 3% SDS, pH 7.6) for 10 minutes in a final volume of 200 jjlI. The rate of AMC released was measured by monitoring the increase of fluorescence having an excitation of 390 nm and an emission of 460 nm. For IC50 determination, a selected experimental agent was added so that a final concentration between 5 and 0.005 jjlM was achieved. Testing results are provided in Table 1. 

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