High-throughput screening for small molecule modulators of motor protein Kinesin.
The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.
1 Department of Neuroscience, The Scripps Research Institute , Jupiter, Florida.
Beena M
BM
Kadakkuzha
Timothy P
TP
Spicer
Peter
P
Chase
Jeffery B
JB
Richman
Peter
P
Hodder
Sathyanarayanan V
SV
Puthanveettil
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D016547:Kinesins / Q000187:drug effects*; D049449:Luminescence; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
D007202:Indicators and Reagents; D054852:Small Molecule Libraries; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins