Developing Small Molecule Inhibitors to Study the Actin Cytoskeleton

Luke PeyralansNolen Lab (IMB)

August 2012

Courtesy UCSF

Actin

G-actinF-actin

Branched actin

The Actin Cytoskeleton Plays Critical Roles in Diverse Cellular

ProcessesCrucial for movement, cell division, intracellular

transport

Immune system, wound healing

C. elegans embryo undergoing mitosisActin in red, coutesy of Bowerman lab (IMB)

Small Molecules can Inhibit Actin Branching

Arp2/3 complex enables formation of branched

filaments

CK666 AI-015

Small Molecules can Inhibit Actin Branching

Arp2/3 w/ bound inhibitor

Efficacy of inhibitors should be evaluated in vivo

• Cell permeability?

• Off-target effects?

Need in vivo assay for inhibitors

Summary

1. Branched actin is required for biologically important processes

2. Arp2/3 is the only known actin branch nucleator

3. Small molecules (AI015, CK666, etc) can inhibit Arp2/3 and serve as a tool

What is the efficacy of these inhibitors in vivo?

Experiment OutlineSpeckle microscopy used

to observe actin dynamics in live Drosophila m. S2 cells

Compare dynamics before & after inhibitor addition

Velocity of speckles used as a measure of Arp2/3 inhibition

Time

Space

Slope = velocity (dependent on activity of Arp2/3)

Effect of CK666 on Activity of Arp2/3 Complex, in vivo

Conclusions & Future Directions

CK666 shows comparable activity in vivo and in vitro

How does this compare to in vivo activity of AI015?

AcknowledgementsNational Institute of Child Health & Human

Development NIH-1R25HD070817 Training Grant

Dr. Peter O’Day

Dr. Brad Nolen (IMB)

Dr. Shih-Yuan Liu (Organic Chemistry)

Su-Ling Liu, Byron Hetrick, Luke Hegelson, Julianna Han