MECC2009 Oct 10, 2009

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

MECC2009 Oct 10, 2009

Abstract

Tyrosine kinases are involved in many signaling pathways useful for chemotherapeutic intervention of chronic diseases. Because the family of tyrosine kinases have conserved active sites, the ability to identify selective inhibitors can be challenging. One tyrosine kinase that is important for selectivity is Epidermal Growth Factor Receptor (EGFR). EGFR possesses a conserved cysteine residue near its active site which can react irreversibly with compounds containing strategically-placed electrophillic components. This conserved cysteine is shared with only 10 other tyrosine kinases, making such an irreversible inhibition strategy attractive. We have developed a method to calculate kinact/Ki for irreversible compounds using a kinetic competition binding assay that generates a TR-FRET (time-resolved fluorescence resonance energy transfer) signal. In some cases it is possible to additionally determine K i and kinact separately. This combination of inactivation rate (kinact) along with affinity (Ki) can then be used to help identify lead chemical series for further investigation in vivo and clinical trials.

MECC2009 Oct 10, 2009

IntroductionThe binding of an irreversible inhibitor to its target enzyme is typically time-dependent. Since the amount of active enzyme changes with respect to inhibitor concentration over time, potency cannot be expressed simply in terms of a K i value. Instead, potency is expressed by taking the inactivation rate with respect to inhibitor concentration (k inact / Ki).

Determination of kinact / Ki for several tyrosine kinases have been developed in-house to be used as part of a selectivity panel. While working with Epidermal Growth Factor Receptor (EGFR) we observed that the progress curves for known irreversible inhibitors were not fit well with a first-order exponential decay function when complete inhibition was assumed. Instead, the binding kinetics resembled those expected that of a slow-binding reversible compound. Several experiments were designed to ascertain potential causes for this effect and are discussed in this poster. These experiments look at

– order of addition– extended time points to monitor binding

– probe concentration with respect to its Kd

– activity of enzyme in the presence of probe

– correlation of IC50 determined by either the binding or Caliper mobility shift assay.

MECC2009 Oct 10, 2009

Caliper Off Chip Mobility Shift Assay

Reference: Caliper, LabChip Assay: Off Chip Incubation, Mobility Shift Protein Kinase A Assay, Apllication Note 207.

Figure 1. Caliper Mobility Shift Assay. A fluorescently labeled peptide in assayed in the presence of a kinase, metal and ATP. The phosphorylated state (P or product) is separated from the non-phosphorylated form (S or substrate) using a capillary sipper CHIP and electrophoresis. The Caliper instrument then applies a laser which quantifies the amount of each over time and plots the results (left panel). Using the HTS Analyzer software, percent of product formed is calculated. The right panel is an example from Caliper of a staurosporine titration against PKA. This assay format can be run in either end-point or kinetic modes.

MECC2009 Oct 10, 2009

LanthaScreenTM Eu Binding Assay

Reference: Invitrogen, LanthaScreen™ Eu Kinase Binding Assay

Figure 2. Invitrogen’s Binding Assay Kits. The concept involves a time-resolved fluorescence-energy resonance transfer (TR-FRET) between an Eu-label antibody that binds to a tagged-kinase and competition between a test compound and an AlexaFluro-labeled probe. In the top example (High TR-FRET) where the probe is bound to the enzyme and not challenged by an inhibitor, the energy emitted from the Eu excited at 340nm is transferred to the probe providing a signal output at 665nm. In the bottom example, (Low TR-FRET) an irreversible compound competes for binding and blocks the energy transfer. In this later case, only a signal at 615nm can be measured, not 665nm The ratio (655/615) is calculated and multiplied by 10K to provide whole numbers from which to generate plots. This assay format can be run in either end-point or kinetic modes.

MECC2009 Oct 10, 2009

Time-dependent irreversible inhibition requires Ki and kinact to describe potency

Figure 3. Ki and kinact. While it is often difficult to determine the absolute value of K i (see above), it is always possible to determine kinact/Ki, the second order rate constant for inhibition. The TR-FRET binding assay had been successfully used to measure kinact/Ki 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

MECC2009 Oct 10, 2009

Kd determination of 2 potential tracers

Figure 4. Kd determination. In standard assay buffer (20mM HEPES, pH 7.4, 10mM MgCl2, 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 Kd values of 35nM and 153nM for KT-178 and KT-236 respectively.

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Capacity 345 .9556 6 .9992

Kd value 34 .8535 2 .1273

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Capacity 358 .2287 26 .8352

Kd value 152 .6089 22 .1477

MECC2009 Oct 10, 2009

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.

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MECC2009 Oct 10, 2009

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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.

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MECC2009 Oct 10, 2009

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.

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MECC2009 Oct 10, 2009

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 Ki(app) and kinact/Ki(app) is about what would be expected.

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MECC2009 Oct 10, 2009

IC50 Correlation Between Assay Formats

Figure 9. IC50 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 IC50 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.

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MECC2009 Oct 10, 2009

DiscussionTo be able to calculate kinact/Ki of an irreversible compound with the competitive binding assay it is important to observe progress curves that extrapolate to complete inhibition at extended time. If the curves level out and run parallel with each other, this would be suggestive of a reversible inhibitor, or a reversible inhibitor that binds slowly. This has held true for a number of tyrosine kinases tested in our labs. However, with EGFR, the binding kinetics for irreversible inhibitors appeared different. Although showing time-dependence, the TR-FRET progress curves did not appear to be extrapolating to complete inhibition of signal.

Order of addition experiments showed that the same final inhibitor concentrations that gave incomplete loss of signal would result in complete loss of signal if the inhibitor was pre-incubated with the enzyme prior to probe addition. By extending the length of the incubation time of the binding assay it was shown that additional time did not change this observation. This suggested that there was not an additional, reversible site on EGFR that might be generating the residual TR-FRET signal.

Another possibility was that the probe, itself, was interfering in some highly unorthodox way with the inhibition of EGFR. In support of this, the loss of signal in the binding assay was not affected by increased concentrations of probe in a manner consistent with simple competition. For this reason the effect of probe on the inhibition progress curves in the Caliper enzymatic assay was investigated. In contrast to the binding assay, the rate of inactivation was affected in a predictable manner, consistent with simple competition.

The TR-FRET binding assay and Caliper assay gave IC50 values that were somewhat in agreement. However, the perplexing observations of effect order of addition and probe competition on the binding assay suggest that significant additional investigation is needed before its reliability as an assay can be assessed. In the meantime, the Caliper assay can be used to determine inactivation rate, with the understanding that the throughput will be much lower / slower.