Summary And Conclusion

With more than 500 members, the proteases constitute one of the largest families of enzymes in the human genome and are an important class of targets for drug discovery. Biochemical assays based on fluorescence readouts are the most frequently used technologies to elaborate the enzymatic mechanisms of proteases and to test the inhibitory potencies of drug-like chemical compounds. These assay formats exist for all classes of proteases and substrate specificities. The development of protease...

Buffers and Solvents

The concentrations of detergents, buffers, carrier proteins, reducing agents, and divalent cations can affect the specific signals and apparent potencies of compounds in concentration response curves (Schroter et al., 2000). In general, it is good to stay as close to physiological conditions as possible, but there are many exceptions. For instance, sometimes carrier proteins are required for enzyme stability and to reduce unspecific binding to reaction vessels. A very good summary of solvent...

Assay Readout Technologies

The keys to protein kinase and protein phosphatase assay development lie in the ability to choose an appropriate readout technology to have ample quantities of enzymes, cell lines, antibodies, and reference compounds and to optimize the assay for buffer conditions, reagent concentrations, timing, stopping, order of addition, plate type, and assay volume. We will discuss these aspects in separate sections below. The readout technologies summarized in Table 1.1 present many options for assay...

Fluorescence Resonance Energy Transfer FRET

FRET-based readouts are the most prominent methods used for endopeptidase and carboxypepti-dase activity assays. In general, a huge number of biological assays have been developed based on the FRET principle (Van der Meer et al., 1994 Andrews and Demidov, 1999). FRET substrates extend on both sides of the scissile bond. This is important for proteases, where the S' binding site significantly contributes to the binding affinity of the substrate. For example, the activity of human FIGURE 2.4 FRET...

Equilibration Period after Dispensing Cells

An equilibration period is often used after dispensing cells to allow uniform attachment before addition of test compound. The length of equilibration can be easily determined and optimized to reach an acceptable window between positive and negative control responses. In some cases, the equilibration step is eliminated when compounds are dispensed immediately after seeding cells and in other cases the test compounds may have already been dispensed into the assay plates prior to dispensing...

Time Resolved Tr Fret

TR-FRET measurements are interesting alternative readouts to the above described FRET quench. The technology is very popular due to its sensitivity, ease of use, and ease of assay development. The key aspect of this readout technology is the use of donor fluorophores with large Stokes shifts and extremely long fluorescence emission half-lives. The fluorescence emission measured after energy transfer to the acceptor fluorophore has a typical half-life (t1 2) in the microsecond to millisecond...

BaF3 System

The BaF3 cell-based assay is very useful for HTS targeting the receptor-associated protein tyrosine kinases. The BaF3 cells are a mouse interleukin 3 (IL3)-dependent pro-B cell line. In the normal case, the BaF3 cells require interleukin 3 for proliferation, but when an oncogenic tyrosine kinase is overexpressed, the normal IL3 pathway is subverted by the oncogenic kinase and growth is driven by the recombinant protein. Alternatively, receptor tyrosine kinases can be expressed in these cells...

Enzyme Assays

Kinase and phosphatase assay technologies can be divided roughly into those employing whole cells and those employing purified enzymes. Protein kinase enzyme assays require ATP, magnesium (and sometimes manganese) cofactors and a peptide or protein substrate. The requirements for phosphatase enzyme assays are simpler and well described by Montalibet et al. (2005). One must have a method to detect the conversion of substrate by determining either the formation of HTS Assay Development for...

Tetrazolium Reduction Assays

Among the first cell viability assays developed for HTS was the MTT bromide tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymi-dine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced...

Fluorophores Frequently Used for Protease Assays Based on FI Readout

Fluorophore Excitation Wavelength (nm) Emission Wavelength (nm) Reference Okun et al., 2006 Harris et al., 2000 Gurtu et al., 1997 Grant et al., 2002 FIGURE 2.3 Dose-response curves for protease inhibitor with autofluorescence characteristics. (a) Result of standard fluorescence intensity-based assay employing an AMC-labeled substrate at a concentration of 2 M. Profiling data could not be obtained due to the interference of the compound's fluorescence with the readout. (b) Result obtained from...

Substrate Finding Approaches

In vitro protease activity assays are based on monitoring the cleavage of the substrate, either a short peptide or a whole protein. Peptide substrates are sufficient if the peptide sequence in its denatured form carries the accurate specificity information of the protease's active site. Proteases prefer whole proteins as substrates if native components of structural elements for recognition and or activation of the protease are required. In these cases, substrate design must be...

Disadvantages of Metabolic Reduction Assays

Dual Reporter Assay Hours Iso

Some of the major disadvantages of both the tetrazolium and resazurin reduction assays are related to the requirement to incubate a substrate with viable cells for a sufficient time to generate a measurable signal. This is a very important feature to consider when designing assays for HTS. In addition to the extra plate handling steps needed to return the cells to a 37 C incubator, the extended incubation of the detection reagents with viable cells increases the possibility of undesirable...

Introduction

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes serine threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threo-nine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent...

Fluorescence Lifetime

Fluorescence lifetime (FLT) is a recent addition to the portfolio of readouts for biochemical protease assays. Lifetime is an intrinsic property that corresponds to the average time fluorophore electrons remain in the excited state before relaxing to the ground state. The FLT assay principle is described in Figure 2.7. In the case of a single emitting species, the probability of observing a photon at a certain time point after the fluorophore is excited follows an exponential decay (Moger et...

Determination of Kinetic Parameters Km and kcat under Initial Velocity Conditions

The Michaelis constant, KM, can be obtained from measurements conducted at a constant enzyme concentration and different substrate concentrations. Under conditions in which the substrate concentration S is significantly higher than the enzyme concentration and the substrate consumption is below 20 , the initial enzymatic velocity v0 can be approximated by the Henri-Michaelis-Menten equation where vmax is the maximal velocity at saturating substrate concentrations. The initial enzyme velocity is...

Fluorescence Polarization FP

FP is an alternative readout principle for endopeptidase activity assays. FP or anisotropy measurements allow the detection of changes in the rotational correlation time of particles. These differences in the rotational correlation (or relaxation) time are related to different masses of particles. The experimental determination of steady-state fluorescence anisotropy requires the linear polarization of the light used for the excitation of the probe as well as linear polarization of the emitted...

Radioactive Assay Technologies

The earliest assays were based on the use of 32P as a label either on the ATP cofactor for kinases or on a peptide substrate for phosphatases. With kinases, the transfer of the 32P from the g position of ATP to a peptide or protein substrate resulted in a 32P-labeled peptide or protein that would be separated away from the ATP by capture on a filter and subsequent washing. The quantity of phos-phoprotein could be quantified by scintillation counting. The availability of a new version of...

Lb

4> & & A< 3 p W V cf FIGURE 4.5 Development of automated electrophysiology assay for KV1.3. (A) Description of IonWorks process for KV1.3 assay (PP PatchPlate). (B) Distribution of pre-DMSO and post-DMSO K+ current amplitudes in typical PatchPlate PPC. (C) Distribution of pre- and post-compound seal resistances in typical PatchPlate PPC. (D) Family of K+ currents recorded from a representative single well of a PatchPlate PPC. Currents were evoked by a series of 300-ms test pulses,...

Flow Chart For Assay Development

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 of a protease can be sufficient for proper determination of the kcatIKM value, the determination of the endpoint linearity by an enzyme titration, and the assay validation by IC50 determination for a...

Contents

About the Chapter 1 Assay Development for Protein Kinases and Chapter 2 Fluorescence-Based Biochemical Protease Assay Chapter 3 Assay Development for Nuclear Hormone Chapter 4 Assay Development for G Protein-Coupled Receptors and Ion Channels 59 Joseph G. McGivern, Yi-xin Qian, and Paul H. Lee Chapter 5 Assay Development for Heat Shock Chapter 6 Assay Development for Cell Viability and Apoptosis for High-Throughput Terry L. Riss, Richard A. Moravec, and Andrew L. Niles Chapter 7 Assay...

References

Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits. Biol. Pharm. Bull. 19, 1026-1031. Andrews, D.L. and Demidov, A.A. 1999. Resonance Energy Transfer. Chichester John Wiley amp Sons. Angliker, H. et al. 1995. Internally quenched fluorogenic substrate for furin. Anal. Biochem. 224, 409-412. Attucci, S. et al. 2002. Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates. Biochem. J....

Development of Scintillation Proximity Assay for GPR23

GPR23 P2Y9 is a novel receptor that binds lysophosphatidic acid LPA , although its physiological importance is not fully understood Noguchi, Ishii, and Shimizu 2003 . In order to develop a GPR23 binding assay, we prepared membranes from a Chinese hamster ovary CHO cell line that expressed human GPR23 under the control of a tetracycline-inducible expression system. Under normal conditions, these cells expressed GPR23 at a very low level but could be induced by tetracy-cline or a derivative to...

Contributors

National Human Genome Research Institute National Institutes of Health Bethesda, Maryland and Therapeutics St. Jude Children's Research Hospital Memphis, Tennessee Enabling Technologies Axxam SpA Milan, Italy Digital Fox Consulting, LLC Chapel Hill, North Carolina Novartis Institutes for BioMedical Research Cambridge, Massachusetts Novartis Institutes for BioMedical Research Emeryville, California Novartis Institutes for BioMedical Research Emeryville, California The Rockefeller University New...

Cellular Enzyme Linked Immunoadsorbent Assay ELISA

The technique of cellular ELISA is to treat the cells with a test compound, and by modulating kinase or phosphatase activity, cause a change in a particular signaling pathway. The change in phosphorylation state of the signaling components can be detected by lysing the cells and measuring the phosphorylation state of the analytes by capturing them between an anti-analyte antibody and an anti-phosphopeptide or anti-phosphotyrosine antibody Minor, 2003 . The ELISA plates are coated with the...