Why IC 50 s Are Bad for You And Other Surprises Petr Kuzmi , Ph.D. - PDF document

Why IC 50 ’s Are Bad for You And Other Surprises Petr Kuzmi č , Ph.D. BioKin, Ltd. What is enzyme inhibition on the molecular level COMBINATION OF TWO MOLECULES TO FORM AN ENZYME-INHIBITOR COMPLEX “Drugs produce their inhibitory action by combining with the enzyme [molecules].” “One molecule of drug will inhibit the activity of one [molecule] of enzyme.” Easson, L. H. & Stedman, E. (1936) Proc. Roy. Soc. B 121 , 142-151. + I E E I inhibitor molecular enzyme molecule complex molecule IC50's are bad for you 2 1

What is the inhibition constant (K i ) DISSOCIATION EQUILIBRIUM CONSTANT OF THE ENZYME-INHIBITOR COMPLEX [ E ] [ I ] E + I E·I = eq eq K i ⋅ [ E I ] eq • low K i (“dissociation”) means high binding activity • dimension = concentration (moles/liter, M ) • “good” inhibitors have K i ’s around 10 -9 moles/liter or better ( nano molar) 10 -3 mili- m M 10 -6 micro- μ M “better” inhibitor 10 -9 nano- n M 10 -12 pico- p M 10 -15 femto- f M 10 -18 atto- IC50's are bad for you 3 A thought experiment: Effect of K i on “fraction bound” Assume equal amounts: [E] 0 = [I] 0 = 1 nM E + I E·I 1.0 0.8 [ E.I] / [E] 0 0.6 0.4 0.2 at nano molar [E] 0 = [I] 0 0.0 and femto molar K i , 1e-15 1e-14 1e-13 1e-12 1e-11 1e-10 1e-9 1e-8 1e-7 1e-6 10 -15 10 -12 10 -9 10 -6 there is no free enzyme left K i , M IC50's are bad for you 4 2

A thought experiment: Titrate 1 nM enzyme, K i = 0.000001 nM remaining enzyme v activity V 0 recall: one molecule of inhibitor inhibits one molecule of enzyme 0.5 V 0 recall: at [I] 0 = [E] 0 and K i << [E] 0 , there is no free enzyme left 0.0 0.5 1.0 1.5 2.0 2.5 [E] 0 /2 [E] 0 [I] 0 , nM IC50's are bad for you 5 Thought experiment, continued: What is the IC 50 ? CONCENTRATION OF INHIBITOR THAT PRODUCES HALF-MAXIMUM INHIBITORY EFFECT remaining [E] 0 = 1 nM enzyme v K i = 1 fM = 0.000001 nM activity V 0 IC 50 in this case: IC 50 ~ 0.5 nM IC 50 ~ [E] 0 / 2 IC 50 in every case: IC 50 > [E] 0 / 2 0.5 V 0 0.0 0.5 1.0 1.5 2.0 2.5 [E] 0 /2 [E] 0 [I] 0 , nM IC50's are bad for you 6 3

What is the difference between K i and IC 50 ? IC 50 DEPENDS ON ENZYME CONCENTRATION AND IS ALWAYS HIGHER THAN THE K i [ E ] = + ( app ) IC 0 K 50 i 2 ( app ) = + competitive K K ( 1 [ S ] / K ) i i M ( app ) = + uncompetitive K K ( 1 K /[ S ]) i i M ( app ) = noncompetitive K K i i + [ S ] K ( app ) = K M mixed-type i α + [ S ] / K K / K i M i Cha, S. (1975) “Tight binding inhibitors. I. Kinetic behavior” Biochem. Pharmacol. 24 , 2177-2185. IC50's are bad for you 7 Implications for drug discovery: “Hitting the IC 50 wall” NO MATTER HOW TIGHTLY THE INHIBITOR BINDS, THE IC 50 CAN NEVER GET LOWER THAN [E] 0 /2 (app) = K i (1 + [S]/K M ) Assume: K i • competitive • competitive • [E] = 5 nM • [E] = 60 nM • [S] 0 = K M • [S] 0 = K M K i , nM IC 50 , nM K i , nM IC 50 , nM 1,000 2,002.5 1,000 2,030 100 100 202.5 230 10 22.5 10 50 The IC 50 wall. 1 1 4.5 32 2.6 30 .2 0.1 0.1 0.01 2.52 0.01 30 .02 2.502 30 .002 0.001 0.001 IC50's are bad for you 8 4

What is “tight binding” THERE IS NO SUCH THING AS A “TIGHT BINDING INHIBITOR” EXPERIMENTAL CONDITIONS “Tight binding” kinetics applies when the enzyme concentration in any given assay is greater than the inhibition constant. [E] / K i ~ 1 tight binding is beginning to show up [E] / K i ~ 10 tight binding is definitely present [E] / K i ~ 100 highly tight binding [E] / K i ~ 1000 extremely tight binding IC50's are bad for you 9 How prevalent is “tight binding” in a screening campaign? EXAMPLE: A PROTEASE CAMPAIGN A typical data set: ~ 10,000 compounds Completely inactive: ~ 1,100 ... NOT SHOWN Tight binding: ~ 400 2000 about 4% of compounds 1500 N 1000 500 0 -12 -9 -6 -3 0 Data courtesy of log K i * Celera Genomics 2003-2005 IC50's are bad for you 10 5

A word about the Cheng-Prusoff Equation IT DOES NOT TAKE INTO ACCOUNT “TIGHT-BINDING”! Cheng, Y.-Ch. and Prusoff, W. H. (1973) “Relationship between the inhibition constant (K i ) and the concentration of inhibitor which causes 50 per cent inhibition (IC 50 ) of an enzymatic reaction” Biochem. Pharmacol. 22 , 3099-3108. competitive inhibition: IC 50 = K i (1 + [S]/K M ) Cha, S. (1975) “Tight binding inhibitor. I. Kinetic behavior” Biochem. Pharmacol. 24 , 2177-2185. competitive inhibition: IC 50 = K i (1 + [S]/K M ) + [E] 0 / 2 Many J. Med. Chem. papers still use the Cheng-Prusoff equation. If the conditions are tight binding ([E] > K i ) this produces wrong K i ’s. IC50's are bad for you 11 Misuse of the “fold increase” plot: A case study unnamed kinase WT FGFR2 inhibition mode by IC 50 comparison 0.04 0.035 compound “X” 14741087 known ATP competitor 0.03 Cedirinib 0.025 IC50 (µM) 0.02 0.015 0.01 0.005 0 0 1000 2000 3000 4000 5000 6000 ATP concentration (µM) CONCLUSION: Compound “X” is an ATP insensitive inhibitor not IC50's are bad for you 12 6

A word about the “fold increase in IC 50 ” plot IT CAN BE VERY MISLEADING – IF “TIGHT BINDING” DOES OCCUR SIMULATED KINASE EXAMPLE: • competitive • K M (ATP) = 100 µM • K i = 0.5 nM = 66 nM • [E] 0 Very shallow slope, “unexpected”. [E] 0 >> Ki. IC50's are bad for you 13 Another word about the “fold increase in IC 50 ” plot you don’t know if there is “tight binding” until you get the K i (app) SOMETIMES IT DOES WORK , BUT ... SIMULATED KINASE EXAMPLE: • competitive • K M (ATP) = 100 µM • K i = 0.5 nM = 0.25 nM • [E] 0 Steep slope, as “expected” [E] 0 < Ki. IC50's are bad for you 14 7

Compound “X” is an ATP-competitive inhibitor after all 7 6 10-fold increase in IC 50 5 IC 50 , nM 4 3 2 1 0 0 200 400 600 800 1000 1200 [ATP], µM IC50's are bad for you 15 Rules of thumb do not always work What is the “IC 50 Rule of Thumb”: IC 50 for a competitive inhibitor should increase about 10 × going from [ATP] = K m to physiological [ATP]. What is “Tight Binding”: Experimental conditions where the enzyme concentration is comparable with the inhibition constant. An important fact: The “Rule of Thumb” does not apply under the conditions of “Tight Binding”. How do we know this: Many journal articles on “Tight Binding” : Morrison (1969), Cha (1974), ..., Kuzmic (2000, 2003, 2011) IC50's are bad for you 16 8

Final word about the “fold increase in IC 50 ” plot: Do not use it. SOMETIMES IT WORKS AND SOMETIMES IT DOESN’T. NO WAY OF TELLING IN ADVANCE IF IT WILL. [E] 0 = 0.25 nM [E] 0 = 66 nM SAME INHIBITOR – DIFFERENT ASSAY CONDITIONS IC50's are bad for you 17 Other people still use the “fold increase in IC 50 ” plot A RECENT PAPER FROM NOVARTIS What is the main difference between Novartis and ArQule ? Chene, P. et al. (2009) BMC Chem. Biol. 9 :1, doi:10.1186/1472-6769-9-1 IC50's are bad for you 18 9

Review: Measures of inhibitory potency THE INHIBITION CONSTANT IS AN INTRINSIC MEASURE OF POTENCY: Δ G = -RT log K i Depends on Example: DEPENDENCE ON EXPERIMENTAL CONDITIONS Competitive inhibitor [S] [E] 1. Inhibition constant NO NO K i * = K i (1 + [S]/K M ) K i 2. Apparent K i YES NO YES YES 3. IC 50 IC 50 = K i (1 + [S]/K M ) + [E]/2 [E] « K i : IC 50 ≈ K i * "CLASSICAL" INHIBITORS: [E] ≈ K i : IC 50 ≠ K i * "TIGHT BINDING" INHIBITORS: IC50's are bad for you 19 Summary: IC 50 ’s are bad for your drug discovery efforts 1. You can hit the “IC 50 Wall” You could be looking at picomolar K i inhibitors and yet they would look “ nanomolar ” to you, if you are screening at nanomolar [E]. A thousand-fold difference in true vs. “apparent” potency. 2. You could get very confused about the “mode” of inhibition A competitive inhibitor can give you almost no “fold increase” in IC 50 if the experiment is done under tight binding conditions. 3. Muddled communication channels within your enterprise The question “What is the biochemical IC 50 for compound X?” does not make sense for a competitive inhibitor, without specifying [ATP] level . Don’t use IC 50 as measure of potency in biochemical assays. IC 50 ’s are perfectly good for cell-based assays. IC50's are bad for you 20 10

What else is there, other than the IC 50 ? Use intrinsic molecular measures of potency ( K i , ...) IC50's are bad for you 21 Example: Correlation between k inact /K i and cellular IC 50 “Molecular Underpinnings of Covalent EGFR Inhibitor Potency [...]” SUBMITTED inhibition of EGFR-L858R/T790M autophosphorylation in H1975 tumor cells cell-based potency k inact K i IC50's are bad for you 22 11