Small Molecules

Using protein microarrays to examine small molecule inhibitory specificity

Investigator: Fernanda Festa, Ph.D.

Collaborators: Nathaniel Gray (HMS, DFCI)

The human kinome contains more than 500 proteins well known for their importance in normal cell physiology and for their role in many diseases. Using kinase NAPPA arrays we demonstrated that the kinases on the array display autophosphorylation activity. To demonstrate this, anti-pTyr antibodies detect phosphotyrosines on the kinases on the arrays, which disappear after treatment with phosphatase and then returns after incubation in kinase buffer and ATP.

To determine if the kinase activity could be affected by inhibitors, the proteins' autophosphorylation activity was also evaluated when the reactions were performed in the presence of global kinase inhibitors such as ADP and Staurosporine. In this case, the observed reduction of the kinase activity suggested that the proteins on the array can be inhibited by general kinase inhibitors, as expected. Ongoing experiments are using kinase NAPPA arrays to study the effect of specific kinase inhibitors (small molecules) on the kinase activity and determine the small molecule selectivity and efficacy among many tested kinases, all performed in a single experiment. Determined IC50s agree well with published results for solution based assays. This method may provide a high throughput rapid approach for evaluating drug selectivity.

Tyrosine kinases printed on NAPPA arrays demonstrate autophosphorylation activity as demonstrated with anti-pTyr antibodies.