An Enabling Technology to Compare Kinase Compound Residency Time with Potency Within Living Cells in a HTS Format Gary Allenby1, Craig Malcolm2 and Matthew Robers2 1 Aurelia Bioscience Ltd, Biocity, Pennyfoot street, Nottingham, U.K. 2 Promega Corporation, Headquarters: Madison Wisconsin, U.S.A.
Introduction Kinase Tracer Affinity Determination As a Pharmacologist screening compounds for your kinase target what parameters are important? Binding affinity and a) b) Kinase Tracer Concentration (uM) selectivity are used for Structure Activity Relationships 3 1 0.33 0.11 0.037 0.012 0.0041 Table. 1. IC50 values (nM) of (SAR) progression but what about the kinetics of unlabelled compound ABL 22 10 8 8 4 measured at multiple fixed FGR 49 33 20 31 compound binding? The association (Kon) and dissociation concentrations of tracer (uM) EPHA8 14 6 3 2 2 (Koff) rates of compounds are important consideration. Is DDR-1 8 5 4 3 2 1nM binding and 60 minute occupancy ‘better’ than 100nM binding and 8 hour occupancy in living cells? How A pan-agonist compound (K4) was fluorescently labelled and used as the competing tracer for would occupancy time influence off-target effects and evaluation of binding on four kinases; Abl, FGR, EPAH8 and DDR-1. Initially the affinity of the could these be removed by studying binding kinetics? tracer for each kinase was determined (left panel), subsequently the tracer was used at fixed When choosing which compounds to progress this may concentrations (right panel) to examine competition of the unlabelled compound to determine the IC for the compound on each kinase (Table. 1.) influence your SAR decisions. Using Promega Target 50 Engagement reagents we have developed assays designed to study the interaction (K and K ) of compounds on E ffe c t o f in h ib ito rs o n A B L k in a s e on off 4 0 kinase targets in living cells at physiological ATP 3 0
o D a s a tin ib - IC 5 0 1 1 n M
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dissociation of compounds for kinase in living cells. F o re tin ib - IC 5 0 E s t 2 u M 1 0 P o n a tin ib - IC 5 0 4 8 0 n M
0 ABL EPHA8 FGR DDR-1 -1 1 -1 0 -9 -8 -7 -6 -5 [c o m p o u n d ] (M ) Dasatinib 11 <1 45 4 How the technology works Nilotinib 340 164 IA 3 Foretinib ~2,000 107 IA 12
Ponatinib 480 27 ~600 67 a) b) Table. 2. IC50 values (nM) for the binding of Dasatinib, Nilotinib, Foretinib and Ponatinib to ABL, FGR, EPHA8 and DDR-1
Left Panel: Tracer affinity was measured by treating transfected cells with Right Panel: Binding activity of each compound was determined in living cells. Cells were increasing concentrations of tracer in the presence or absence of molar excess of transfected with each of four kinase; ABL, FGR, EPHA8 and DDR-1. Cells were treated unlabelled compound. Right Panel: Affinity of unlabelled compound was measured with exemplar kinase compounds including dasatinib, nilotinib, foretinib and ponatinib The technology uses Bioluminescence Energy Transfer (BRET) at multiple fixed concentrations of tracer, where the IC50 (shown in the table) at as a dose response for each compound competed against a fixed concentration of to measure compound engagement with the kinase target. the recommended tracer concentration (also in table) is depicted in orange. fluorescent tracer K4 at the concentration depicted in orange (left panel). The kinase cDNA plus the Nanoluc enzyme cDNA are transiently transfected (24hrs) on a single plasmid into cells. When expressed, the kinase protein is connected to the Screening – HTS on one Kinase and Selectivity Screening against many Kinases luminescence enzyme (NanoLuc). In the presence of a cell
DasatinibT ra n s bindingfo rm o f toD a DDRs a tin-1ib in o nnon D D-Kcryo1 tr apreservedc e r 4 cells
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M ( and the photons excite the fluorescent tag resulting in one was cryo-preserved while R 0 other cells was cultured for an -1 2 -1 0 -8 -6 -4 fluorescence emission at 600+nm and generating BRET. C o n c e n tr a tio n (M ) Compound engagement is measured in a competitive format additional 24 hrs then evaluated using dasatinib and Dasatinib binding to DDR-1 in cryo preserved cells of tracer versus compound. Binding of the test compound tracer K4. Frozen cells were T ra n s fo rm o f D a s a tin ib o n D D K 2 0 0 D A S R e p 1 defrosted, cultured for 24 hrs D A S E C 5 0 = 1 .5 n M results in a loss of NanoBRET™ signal between the target D A S R e p 2
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a D A S R e p 3 R protein and the tracer inside living intact cells. For analysis of and tracer K4. The IC for
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target engagement by a test compound, cells are treated with dasatinib between fresh and R
B BRET Ratio BRET a fixed concentration of NanoBRET™ tracer that is near the A subset of 80 random compounds were arrayed onto a 96 well plate and discrete wells frozen cells was very similar. 5 0 were spiked with an active compound. All were tested at 10uM on each of the four 0 EC value of the NanoBRET™ tracer dose response curve. To -1 4 -1 2 -1 0 -8 -6 -4 50 kinase in the target engagement assay. Most compounds as expected showed a C o n c e n tr a tio n (M ) determine test compound affinity, cells are titrated with distribution centred around zero percent inhibition (no binding) and all spiked active varying concentrations of the test compound in the presence compounds were detected in each plate tested. The Z factor for each plate was above Data suggests it is possible to transfect and cryo-preserve cells allowing these to be 0.7. Data suggests that all four kinase assays could be used as a high throughput screen rapidly defrosted and used, improving the efficiency of selectivity testing of a single of a fixed concentration (EC50–EC80) of tracer. to detect compounds that binding to the kinase target in living cells compounds against many kinase targets
Kinetics of Binding – On rates and Off rates of compounds inside cells Summary and Conclusion
HEK-293 cells transiently expressing ABL, FGR, EPHA8 or DDR-1 were HEK-293 cells transiently expressing ABL, FGR, EPHA8 or DDR-1 were • We have developed high throughput screening o incubated for 2hrs at 37 C with a nominal tracer K4 concentration of incubated for 2hrs with a 10X IC50 concentrations of dasatinib (red), assays in living cells looking at the competition of 0.33uM, 1uM, 0.012uM and 0.062uM respectively (see figure above in nilotinib (green), foretinib (blue), ponatinib (purple) or DMSO (orange), compounds to displace the fluorescent tracer orange) to allow the tracer to bind to the kinase. Simultaneously both washed to remove the compounds from media surrounding the cells with good assay quality and reproducibility (Z Nanoluc substrate and 10X IC50 concentrations of dasatinib (red), then treated with a fixed concentration of tracer K4 in the presence of nilotinib (green), foretinib (blue), ponatinib (purple) or DMSO (orange) the Nanoluc substrate and the plate incubated in the reader with factor = 0.7 and above) were added to the media surrounding the cells and the plate incubated readings taken every 5 mins. Data suggest that tracer K4 competes for • By using cryo-preserved cells we can simply and in the reader with readings taken every 5 mins. Compounds compete binding to the kinase targets and therefore the rate of dissociation (Koff) for binding to the kinase by displacing the tracer, as such the rate of of each compound can be determined. efficiently perform selectivity screening taking a
association (Kon) can be determined. single compound and testing for binding against many kinase targets stored in the cryostore
A s s o c ia tio n R a te A B L K in a s e A s s o c ia tio n R a te D D R 1 K in a s e • We can determine the binding (as IC50’s) of
3 0 8 0 A B L F o rtin ib D D R 1 F o rtin ib exemplar compounds such as dasatinib,
A B L D a s a tin ib D D R 1 D a s a tin ib
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B B 2 0 • We can measure both the association constant (K ) and dissociation (K ) for compounds within 0 0 on off 0 5 0 1 0 0 1 5 0 2 0 0 0 5 0 1 0 0 1 5 0 2 0 0 T im e (m in s ) T im e (m in s ) living cells at physiological ATP concentrations A s s o c ia tio n R a te E P H A 8 K in a s e A s s o c ia tio n R a te F G R K in a s e • These assays can be used to examine binding 8 0 8 0 E P H A 8 F o rtin ib F G R F o rtin ib and kinetics for individual kinase targets and off- E P H A 8 D a s a tin ib F G R D a s a tin ib
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t target kinase toxicity testing
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E P H A 8 D M S O F G R D M S O The cDNA construct can be designed to work
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B B 2 0 2 0 within a CRISPR Cas9 system to replace the
0 0 endogenous kinase in different cell backgrounds 0 5 0 1 0 0 1 5 0 2 0 0 0 5 0 1 0 0 1 5 0 2 0 0 T im e (m in s ) T im e (m in s ) • This technology represents a significant step forward for SAR screening as compound kinetics By studying both the association and dissociation rate of compounds from each of the target kinase it is of binding can, in addition to potency, be used to possible to consider introducing kinetic binding parameter evaluation during compound SAR development. rank compounds in projects