A poster presented at the MECC 2009 conference in Chicago, IL on October 10, 2009.

1. Determination of kinact / Ki for EGFR Irreversible Inhibitors Using Competition Binding Studies SUSAN FOLTIN , Ann Wrightstone, Jeffrey Hirsch, and Arthur Wittwer Pfizer Global Research and Development Pfizer, Inc. 700 W Chesterfield Parkway Chesterfield, MO 63017

6. Time-dependent irreversible inhibition requires Ki and kinact to describe potency Figure 3. K i and k inact . While it is often difficult to determine the absolute value of K i (see above), it is always possible to determine k inact /K i , the second order rate constant for inhibition. The TR-FRET binding assay had been successfully used to measure k inact /K i for a number of JAK3 irreversible inhibitors (see neighboring poster by Ann Wrightstone ). These inhibitors all have strategically-placed electrophillic components capable of reacting with a non-catalytic cysteine residue conserved in the active sites of JAK3, EGFR, and several other tyrosine kinases. 3 nM JAK3 + irreversible inhibitor* *Compound 4 – Pan, Z. et al. “Discovery of Selective Irreversible Inhibitors for Bruton's Tyrosine Kinase.” ChemMedChem. 2007 Jan 15;2(1):58-61

7. K d determination of 2 potential tracers Figure 4. K d determination. In standard assay buffer (20mM HEPES, pH 7.4, 10mM MgCl 2 , 0.01% BSA, 1mM DTT and 0.005% Tween-20) GST-tagged EGFR from Invitrogen at 5nM was combined with 2nM Eu-labeled anti-GST antibody, 2% DMSO and either 10uM staurosporine or serially diluted probe in a 384-well PerkinElmer proxi-plate, sealed and incubated for 3 hours at room temperature. The wells are read on an LJL Analyst and analyzed by subtracting the background ratio (10 uM staurosporine) from the probe ratio resulting in the corrected ratio. The above diagram plots the corrected ratio against probe concentration for K d values of 35nM and 153nM for KT-178 and KT-236 respectively. nM probe 0 20 40 60 80 100 120 140 160 180 200 220 240 260 corrected ratio 0 100 200 300 KT-178 KT-236 KT-178 KT-236 Kd determination for probes against EGFR Parameter Value Std. Error Capacity 345 .9556 6 .9992 Kd value 34 .8535 2 .1273 Parameter Value Std. Error Capacity 358 .2287 26 .8352 Kd value 152 .6089 22 .1477

8. Order of addition—different results Figure 5. Irreversible compound did not show complete inhibition of TR-FRET signal in the assay if added after the probe, but does show complete inhibition if pre-incubated prior to probe addition. In standard assay buffer (see figure 4) 5 nM GST-tagged EGFR from Invitrogen was pre-incubated at room temperature for 2 hours in the presence of 2 nM Eu-labeled anti-GST antibody, 2% DMSO and 40 nM KT-178 probe (final concentrations). At t=0 this mixture was added to inhibitor at the final indicated concentrations in a 384-well PerkinElmer Proxi-plate (20 µL final volume) and mixed. The wells were then read for 3 hours. In contrast to the results seen with this compound and JAK3 (Figure 3) the drop in TR-FRET signal does not appear to extrapolate to background for the intermediate inhibitor concentrations, but rather starts to level off around 70 minutes (left panel). A possible explanation is that a second site for probe binding is available that would be reversibly inhibited by the compound. To answer this question, the enzyme was allowed to be pre-incubated with the compound first, then read after addition of probe (right panel). All concentrations tested under these latter conditions were at background, indicating no secondary binding of probe. Compound added last Probe added last 0 uM 0.046 uM 0.015 uM 1.250 uM 0.42 uM 0.14 uM 0.074 uM 0.025 uM 0 uM 2.00 uM 0.68 uM 0.22 uM

9. Extending time does not result in complete inhibition of TR-FRET signal Figure 6. Extended incubation by several more hours does not result in complete inhibition of TR-FRET assay signal. The left panel are data collected during the first 3 hours of reading using the same assay setup as described in figure 5. At the end of 3 hours, the plate was re-inserted into the reader and allowed to continue to read an additional 3 hours. The data points continue to display a leveling off of signal. Initial 3 hour read Extended 3 hour read 0.556 uM 0.021 uM 0.007 uM 0 uM 0.185 uM 0.062 uM

10. Varying concentration of probe Figure 7. Inhibition of EGFR in the presence of varying probe levels. Interaction of inhibitor with enzyme could be affected significantly if the Kd of KT-178 was in error. To verify the effect of probe concentration on inhibition rate, probe was included at final concentrations of 100, 80, 60, 40 and 20 nM. While the TR-FRET signal increased with probe concentration, as expected, the rate at which signal was lost was relatively unaffected. This was true for all probe concentrations tested (middle doses not shown). Curiously, signal appeared to be lost somewhat MORE rapidly at 100 nM (left) compared to 20 nM probe (right) (see graph at far right). This the opposite of what would be expected from a simple competition of probe and inhibitor. 100nM KT178 20nM KT178 0.556 uM 0.021 uM 0.007 uM 0 uM 0.185 uM 0.062 uM 0.556 uM 0.021 uM 0.007 uM 0 uM 0.185 uM 0.062 uM

11. Inhibition of EGFR in enzymatic assay in presence and absence of binding assay probe Figure 8. Progress curves showing inhibition of EGFR in the Caliper assay. Curves are fit well with an exponential function leading to full inhibition both in the presence (left) and absence (right) of KT-178 probe. The effect of probe on K i(app) and k inact /K i(app) is about what would be expected. No probe 40 nM probe k inact = 0.0221 min -1 K i(app) = 0.0293 µ M k inact /K i(app) = 0.0754 k inact = 0.0158 min -1 K i(app) = 0.0609 µ M k inact /K i(app) = 0.0259

12. IC 50 Correlation Between Assay Formats Figure 9. IC 50 correlation between Caliper Off Chip Mobility and LanthaScreen Eu Binding Assay. 12 compounds (a mixture of reversible and irreversible) were first evaluated in the Caliper 40-minute assay with ( W PI ) or without ( no PI ) a 60-minute pre-incubation with enzyme. These same compounds were later tested with the Eu-labeled binding assay and had their IC 50 values determined using the same time points. These data are plotted in the above correlation chart with circles and arrows used to identify reversible and irreversible compounds, respectively. Due to lack of competition (inhibition) in the binding assay, one (1) of the 12 compounds did not have a complete data set for plotting.