Evaluation of assay technologies for the identification of protein-Peptide interaction antagonists.
An increasing number of assay detection technologies are routinely used in small molecule drug discovery and lead optimization. These assays range from solid-phase heterogeneous assays such as enzyme-linked immunosorbent assay and dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Boston, MA) to solution phase, bead-based assays such as electrochemiluminescence assay (BioVeris [Gaithersburg, MD] technology) and amplified luminescent proximity homogeneous assay (AlphaScreen, PerkinElmer Life and Analytical Sciences) to completely solution-based homogeneous assays such as time-resolved fluorescence resonance energy transfer and fluorescence polarization. The aim of this study is to compare these assay technologies and assess the advantages and disadvantages of each in the context of our efforts to develop small molecule antagonists to the melanoma inhibitor of apoptosis protein. In this study, seven peptides have been evaluated for their potencies in each assay format. Our results indicate that these assay technologies produce similar relative potencies; however, some methods may be more susceptible to interference than others. Consequently, the choice of the method used frequently depends on a number of factors in addition to assay reproducibility and performance, such as throughput of the assay, cost, compound interference, and ease of use.
Kadkhodayan
Saloumeh
S
Department of Bioanalytical Research and Development, Genentech, Inc., South San Francisco, CA 94080, USA. sallyk@gene.com
Elliott
Linda O
LO
Mausisa
Grace
G
Wallweber
Heidi Ackerly
HA
Deshayes
Kurt
K
Feng
Bainian
B
Fairbrother
Wayne J
WJ
D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization; D010455:Peptides / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000737:chemistry*; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*
D005452:Fluoresceins; D010455:Peptides; D011506:Proteins; C024098:6-carboxyfluorescein; D001710:Biotin