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S  0  ``   X*  0h  `    l*(    0  `    `* H  0޽h ? ̙33 Default Design 0 4 (  4 4 Nȃ y y .    p* a00aa 4 N@ y y 3 .   r* a00aad 4 c $ ?   4 4 N y y 9 f   RClick to edit Master text styles Second level Third level Fourth level Fifth level!     S  4 T y y r    p* a00aa 4 Tܧ y y r3    r* a00aaH 4 0ηo~ ? ̙3380___PPT10. !8X p\x(  \ \ N ]e]e      p* AAA \ ND ]e]e %     r* AAA  \ Tp ]e]e     p* AAA \ T ]e]e %    r* AAAH \ 0ηo~ ? ̙3380___PPT10. `D~  0 . & 0 $(  X  0A?`m   6   MOptimal Experiment Design for Dose-Response Screening of Enzyme Inhibitors:N 1S  <D   APetr Kuzmic, Ph.D. BioKin, Ltd. WATERTOWN, MASSACHUSETTS, U.S.A.hB    7XB  0D0  T|  ?< 3  ? Most assays in a typical screening program are not informative&@0XB  0D04 ! T  ?L F   n Abandon "batch design" of dose-response experiments Use "sequential design" based on D-Optimal Design Theory&oW " T   ?P : Save 50% of screening time, labor, and material resources@;1 # T(  ?p AhD  ?PROBLEM $ T  ?x @L  @SOLUTION H  0޽h ? ̙33  0L0   I L (   r  S  `   XB  0D`X  0A?`m  < 3 3BATCH VS. SEQUENTIAL DESIGN OF ANY RESEARCH PROJECT`4 3 3  3 3 3l v0 B 0 ,$D 0I <  HL  ? D<4___PPT9 }decide beforehand on the design of a complete series of experiments perform all experiments in the series without analyzing interim results analyze entire batch of accumulated data issue final report%() @` ?  T+  ?v0 kBATCH DESIGN OF EXPERIMENTS4 l v C  ,$D 0@ @  H'  ? D<4___PPT9 tdecide on the design of only one (or a small number of) experiment(s) perform one experiment analyze interim results; did we accumulate enough experiments? if not, go back to step 1, otherwise ... issue final reportf e$ @` A  T>  ?v  p SEQUENTIAL DESIGN OF EXPERIMENTS4!  l P   H P  ,$D 0xB D B H D8c?  xB F H D8c?P  ~B G  N D8c?P P ! I BxJ G\HWQ?@P,$D 0 {+design = choice of screening concentrations4,   H  0޽h ?I  ̙33QI___PPT10)+0ӧD' , = @B D' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*B %(D' =+4 8?dCB0-#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*B D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*B Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*C %(D' =+4 8?dCB0-#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*C D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*C D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<*H %(D' =-o6Bdissolve*<3<*H D' =%(D' =%(D@' =A@BB BB0B%(D' =1:Bvisible*o3>+B#style.visibility<*I %(D' =-o6Bdissolve*<3<*I +8+0+I 0 +U  0L0    @ (  x  c $] `   XB  0D`X  0A?`m  <_  MADAPTIVE CLINICAL TRIALS (ACT): ADJUST THE EXPERIMENT DESIGN AS TIME GOES ON >N 3 30 3P  Txf  ?} HBorfitz, D.: "Adaptive Designs in the Real World" BioIT World, June 2008<I2   $+  Tl  ?PR B assortment of statistical approaches including  early stopping and  dose-finding interim data analysis reducing development timelines and costs by utilizing actionable information soonernJ33B'3YrP  T4x  ?0 \ ,$D  0 " experts: Donald Berry, chairman of the Department of Biostatistics University of Texas MD Anderson Cancer Center software vendors: Cytel, Tessela industry pioneers: Wyeth 1997  Learn and Confirm model of drug development  nSb5R3/   TL ?@,$D 0 w5 "slow but sure restyling of the research enterprise"&65H  0޽h ? ̙335 - ___PPT10 +=[WD'  = @B D<' = @BA?%,( < +O%,( < +D{' =%(D#' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*D' =%(D' =%(D@' =A@BB BB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =-o6Bdissolve*<3<* +p+0+0 ++0+ 0 +  0L0   q (  r  S | `   XB  0D`X  0A?`m  <  NTHE "RESPONSE" IS INDEPENDENT OF "DOSE": NOTHING LEARNED FROM MOST DATA POINTSOO 3p  H A ?@< vB  N D>? P   TT  ?P @ $ ilog10 [Inhibitor]< vB   N D>?\pp    Tԯ  ?  Presidual enzyme activity|   T  >?HtjB   B D?PP@   Tس  ?  g%"control" data point: [Inhibitor] = 0&&  3 z  p   ,$D 0  T  |?  fxB  H D>? 6,6  T`  ? p :this point alone would suffice to conclude: "no activity"<; +z  p;   | ; ,$D 0  T  >? pp rB  B D?@ @   T\  ? p;  /to make sure, let's use two points not just one<0   rB  B D?@  H  0޽h ? ̙33___PPT10+}fD~' D = @B D9' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(+  0L0 t l   (  p  H A ?@P x  c $ `   XB  0D`X  0A?`m  < :  b,THE SAME STORY: MOST DATA POINTS ARE USELESS-- 3vB  N D>? P   T  ?P @ $ ilog10 [Inhibitor]< vB   N D>?\pp    T|  ?  Presidual enzyme activity|   T  >?HtjB   B D?PP@   T|  ?  g%"control" data point: [Inhibitor] = 0&&  3 l 0 P  0  ,$D 0  Z  |?0  ~B  N D>?   Z  ?P Pthese points would sufficexB  H D>?  zl @ V   @ V ,$D 0  Z  8c?@ @   T  ? V  `these points are useless,33xB  H DjJ?@  H  0޽h ? ̙33___PPT10+D~' D = @B D9' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(+!  0L0 # (  x  c $ `   XB  0D`X  0A?`m  < W  Z$IN CASE THE REASONS ARE NOT OBVIOUS:%% 3  Tt  ? = Academia:   T  ?; H time money fame  T  ?`   = Industry:    T,  ?   L time money securityXz  l    1 ,$D  0   T  ?  W!Beat "the competition" to market.""    T  ? 0d _)Spend less on chemicals, hire a post-doc.**    T!  ? ll d.Invent a drug, avoid closure of Corporate R&D.// _  + # ,$D  0  T&  ? + c-Publish your paper on time for grant renewal...   T+  ? 0d _)Spend less on chemicals, hire a post-doc.**   T/  ? l Y#Invent a drug, get the Nobel Prize.$$   H A ?JZ ,$D  0  T A u? ? `8 u$D  0H  0޽h ? ̙33"___PPT10+ED' = @B D' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<* D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<* Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?\CB#ppt_xBCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?dCB0-#ppt_h/2BCB#ppt_yB*Y3>B ppt_y<*+#   0L0  (  x  c $C `   XB  0D`X  0A?`m  <E B RTHERE ARE VERY GOOD REASONS TO GET SCREENING PROJECTS DONE AS QUICKLY AS POSSIBLE <S  3  3@ 3p  H A# f?   H A% f?@ t,$D 0p  H A& ? @Q  TL  ?p P  OLeishmania major Photo: E. Drberov Academy of Sciences of the Czech Republic6P * $  +H  0޽h ? ̙33___PPT10+d<D|' = @B D7' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?dCB0-#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*+'  0L0 ph!Q(  x  c $_ `   XB  0D`X  0A?`m  <`  <DO NOT USE IC50. THE INHIBITION CONSTANT IS MORE INFORMATIVEP=  3 3 3. 3 ' Tb  ? } (`Kuzmi  et al. (2003) Anal. Biochem. 319, 272 2791       $l    P  ,$D  0l 2 < A ?   7 Tq  ? Xm I"Morrison equation"cl  0 Q 0,$D  0l 3 < A ? 0   8 Thv  ?   V Four-parameter logistic equation!!x 9 H ?   ; T  8c?0 > xB < H DjJ?   = T{  ? 0 q)"Hill slope" no clear physical meaning !,*  p > H ?  l   O ,$D 0l 5 < A  ? xB ? H DjJ?0  A Tl  ?m =slope 1 xB B H DjJ?  D T  ?` .=  =slope 2  E T  ?P$ 7E + IpB F H D?@pB G@ H D?@ H T  ?P$ REI0z I T  ?D  Ki = [E]eq[I]eq /[E.I]eqr    H J T  ?D Ki ... equilibrium constant0 Jl 0 @D  N@  ,$D 0xB K H DԔ?0 P  xB L H DԔ?0   M T  ?`@D BP$<<H  0޽h ? ̙33___PPT10+QzD' = @B D_' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*O%(D' =+4 8?dCB0-#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*OD' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*ODn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*P%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*PD' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*PDn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*Q%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*QD' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*QD4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*N%(+Z&  0L0 &(  x  c $ `   XB  0D`X  0A?`m.  <  HAN APPROXIMATE VALUE OF THE INHIBITION CONSTANT FROM A SINGLE DATA POINT^I 3  3) 3 3  3  T8  ?3 } $\Kuzmi  et al. (2000) Anal. Biochem. 281, 62 67/       $   H A x? ? xpB @ H D?`   Tx  ?@D JV0,pB  @ H D? `    TL  ? T  IV,   T$  ?0H- ? "control"     T  ?D6,$D 0 Relative rate Vr = V/V0  pB @ H D?    TH  ?0   K[I],Ml ! p $! p,$D 0~B  N D?pK  T  ?`!   Ki = 12 nM t      Ll  P  # P ,$D 0~B  N D?P J  T  ?P   Ki = 9 nM t      Ml @   %@  ,$D 0~B  N D?@`  K  T  ? `   Ki = 11 nM t      Ll  *  & * ,$D 0~B  N D? P ` J   T  ? * P   Ki = 8 nM t      l p  "p ,$D 0  s ,A ??@  8 $D 0 ! T  ?pD  GSingle-point formula:H  0޽h ? ̙33___PPT10+$RD{' D = @B D6' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*"%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*#%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*$%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*&%(+8+0+ 0 +  0L0    &b (  x  c $( `   XB  0D`X  0A?`m  <*   j4NOT ALL POSSIBLE EXPERIMENTS ARE EQUALLY INFORMATIVE55 3  T/  ?" :BOOKS: " T3  ? h Fedorov (1972) "Theory of Optimal Experiments" Atkinson & Donev (1992) "Optimum Experimental Designs"Pi!38,4&l }   % } ,$D 0   T<  ?} j A EDITED BOOKS: # ZDA  ?p   Endrnyi (Ed.) (1981) "Kinetic Data Analysis: Design and Analysis of Enzyme and Pharmacokinetic Experiments" Atkinson et al. (Eds.) (2000) "Optimum Design 2000" 33Z3  >: Dl  U & U,$D 0 ! TQ  ?   EJOURNAL ARTICLES: $ TV  ?M U q Thousands of articles in many journals. Several articles deal with experiments in enzymology / pharmacology. fr K  H  0޽h ? ̙33___PPT10+OD~' D = @B D9' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*&%(+C   0L0 )(  x  c $i `   XB  0D`X  0A?`m  <l  l6SIMPLE LIGAND BINDING AND HYPERBOLIC SATURATION CURVES77 3]   Tq  ? } 3Endrnyi & Chang (1981) J. Theor. Biol. 90, 241-263^4     $  TPx  ?Z0 . Y% Protein (P) binding with ligand (L) &&  Tdt  ?fe0: 7P + LpB  H D?upB @ H D?u  T  ?l c@ RPL0  T|  ? ^Kd& pB  H D?0   Ť  ?* J Kdissociation constant   T(  ?7p$,$D 0 O Vary total ligand concentration [L] Observe bound ligand concentration [LB]P  l 0*  (0* ,$D 0  T؜  ?0{ j,$D 0 S Fit data to nonlinear model:   d  S A  ??*    T  ?@= <SUMMARY:     S A  ?? Pc8 $D  0*l  `  ) ` ,$D 0[  T  ?  u  ETWO OPTIMAL LIGAND CONCENTRATIONS (we need at least two data points):nF   fB # 6DjJ ` d $ S A  ??P B  xB & H D?P` @`  ' H  ?@ p  qmaximum feasible [Ligand]<    H  0޽h ? ̙33  ___PPT10 +p D= '  = @B D ' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*(%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*)%(Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =+4 8?\CB#ppt_xBCB#ppt_xB*Y3>B ppt_x<* D' =+4 8?dCB1+#ppt_h/2BCB#ppt_yB*Y3>B ppt_y<* +8+0+0 +!  0L0 + (  x  c $ `   XB  0D`X  0A?`m  < f ITHIS TREATMENT APPLIES BOTH TO "TIGHT BINDING" AND "CLASSICAL" INHIBITORSJJ 3f  T  ? } NKuzmi  (2008) manuscript in preparation^(     "  T0  ?Z0 . [' Enzyme (E) binding with inhibitor (I) ((  T  ?fe#: 7E + IpB   H D?upB  @ H D?u   T  ?l V@ REI0   T  ? ^Ki& pB   H D?0   T  ?* J Kdissociation constant x  T  ?7C$,$D 0 d Vary total inhibitor concentration [I] Observe residual enzyme activity, proportional to [E]free@e%:   T  ?@= <SUMMARY:  ?l  `  ) ` ,$D 0d  Z  ?  A  HTWO OPTIMAL INHIBITOR CONCENTRATIONS (we need at least two data points):nI!   lB  <DjJ ` ~B  N D?P` @`   N'  ?@ P  k#control experiment (zero inhibitor),$  d % S A  ??    ' 2A ?8c?P +8 $D  0bl 0E  +0E ,$D 0 t 0  $0 ,$D 0  Zl'  ?0{ T,$D 0 S Fit data to nonlinear model:   d " S A ??p   * T '  ?E}  I"Morrison Equation" H  0޽h ? ̙33  ___PPT10 +LD= '  ' = @B D ' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*+%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*)%(Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*'%(D' =+4 8?\CB#ppt_xBCB#ppt_xB*Y3>B ppt_x<*'D' =+4 8?dCB1+#ppt_h/2BCB#ppt_yB*Y3>B ppt_y<*'+8+0+0 +w"  0L0 A95 (  x  c $L' `  '  XB  0D`X  0A?`m  <X' q  wA CLASSIC CHICKEN & EGG PROBLEM<  3  3  3]  T%'  ? } 3Endrnyi & Chang (1981) J. Theor. Biol. 90, 241-263^4     $  S A  ??p j8 $ 0b  c &A  ?? Z `l  %,$D 02  T  >?H82  T  >?2  T  >?2  T  >?@ 0 2   T  >?@ 0 2 ! T  >?2 " T  >?2 # T  >?2 $ T  >? & TX0'  ?` t JPROTEIN/LIGAND BINDINGV ' T5'  ? }  NKuzmi  (2008) manuscript in preparationN(    " ( T;'  ? t  EENZYME INHIBITION ) 2A" ?8c? ? 8 $ 0b / c &A  ??  y  2 0 T  >?  ,$D 0& 1 N@' ? T,$D 0 tBWe must guess the answer before we begin designing the experiment.CCl    5  ,$D  0~B 2B N D>?   3 NF'  ?   ba model parameter ("final answer") we are trying to determine by the experiment ... being planned!cc xB 4 H D>?   H  0޽h ? ̙33___PPT10+*d DJ' P' = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<*%%(D' =-o6Bdissolve*<3<*%D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<*0%(D' =-o6Bdissolve*<3<*0Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*5%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*5D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*5D' =%(D' =%(D@' =A@BB BB0B%(D' =1:Bvisible*o3>+B#style.visibility<*1%(D' =-o6Bdissolve*<3<*1+8+0+10 +   0L0   6 (  x  c $Z' `  '  XB  0D`X  0A?`m   <\'  7PUT TOGETHER OPTIMAL DESIGN AND THE SINGLE-POINT METHODN8  3 3  3 38   0P p2  T8d'  ?  qchoose next concentration   >A" ?8c? _ 8 $ 02  Th'  ?  }!collect single data point at [I]@" 2 ! Zo'   ? {choose first concentration [I]@  p $ H ? `v & N ? P 8 ` 1 p2 # T`v'  ?` estimate Ki @    ( >A ??M8 $D 0p * H ?p0p0v 2 NK ? P  3 T}'  ? C  Esingle point method  4 T '  ? Goptimal design theory  6 Th'  ?  8repeatH  0޽h ? ̙33S  0L0 H{(  x  c $' `  '  XB  0D`X  0A?`m  <'  |FPUTTING IT ALL TOGETHER: "SINGLE-POINT METHOD" + OPTIMAL DESIGN THEORYGG 3&  T'  ?  :perform "preliminary" assays (n=3, sequentially optimized)L;  pB ! H D? p " TР'  ?p0 0 Hdetectable activity? * T'  ?I t  4NOB + T@'  ? 0 Hreport "no activity"pB , H D?  - T'  ?  5YES . T'  ?0 0`  Gmoderate activity ? / Td'  ?` 4  5YESpB 0 H D?` P  1 T'  ?P   X add control point ([I] = 0) assemble accumulated dose-response perform nonlinear fit&YX B 4 T'  ?  report best-fit value of Ki4 # 5 T'  ?`  )perform "follow-up" assays (n = 2, batch)Z*   jB 6 B D?00@0pB 7 H D?0@@ pB >@ H D?0  @ T'  ?00i 4NO A T'  ?*P  Ki L       B T<'  ?   Ki [E]L        D T'  ?r DFOR EACH COMPOUND:  E T'  ? NEXTREMELY TIGHT BINDING! jB F B D?` PP@ pB G H D?@ P@ pB H H D? H  0޽h ? ̙33  0L0 c[ >(  x  c $' `  '  XB  0D`X  0A?`m  <'  6ASSAY EVERY COMPOUND AT THREE DIFFERENT CONCENTRATIONS<7 3 3 3R * T-  ?0p  e#choose a starting concentration [I]&$# F + T-  ?P  *measure enzyme activity at [I]: Vr=V[I]/V0Z+!     " , T -  ?P  estimate Ki: @  , \ - S A ??B  pB . H D? PPpB / H D?PP 0 T<-  ?P @  hchoose next concentration 2pB 1 H D?` PPP \ 2 S A ?? * p pB 3 H D? PP  4 T-  ?  `  ecompleted three cycles?2  jB 5 B D?0 jB 6 B D?   7 T"-  ? `  5YESjB 8@ B D?  pB 9 H D?0P0 : T&-  ?` P4  4NOpB ; H D?   < T*-  ? 0 0 Hdetectable activity?pB = H D?pB > H D?P 0P H  0޽h ? ̙33  0L0 SK03(  x  c $8- `  -  XB  0D`X  0A?`mz  0:-  DWE DO THIS ONLY FOR EXTREMELY TIGHT BINDING COMPOUNDS (Ki << [E]tot)E 3 3  3 3 3 3 3 37 F $ T,E-  ?|< ` *measure enzyme activity at [I]: Vr=V[I]/V0Z+!     pB % H D?`||PpB & H D?|| ' T`O-  ?|<  Rchoose [I] = [E]&  ( HT-  ?  6optimal [I] at Ki approaching zero: [I]opt = [E] + Ki7            ,"jB ) B D?}@ }F * T^-  ?0 |<  *measure enzyme activity at [I]: Vr=V[I]/V0Z+!     pB + H D? || pB , H D?@||0  - T,i-  ?P|< @ Tchoose [I] = [E]/2&  . H4-  ? - A"rule of thumb" jB / B D?<  0 T(q-  ?cs BEXTRA POINT #1  1 Tu-  ? c  BEXTRA POINT #2 h 2 T,w-  ? `@  combine with three "preliminary" data points add control point ([I] = 0) assemble accumulated dose-response curve perform nonlinear fit ("Morrison equation")& pB 3 H D?@H  0޽h ? ̙33  0L0 ph@x(  xx x c $- `  -  XB x 0D`X x 0A?`m x Ť-  ?`0h   We need safeguards against concluding too much from marginal data: - greater than 95% inhibition, or - less than 5% inhibition. We need safeguards against falling outside the feasible concentration range. We use other safeguards and rules of thumb. The overall algorithm is a hybrid creation: - rigorous theory, and - practical rules, learned over many years of consulting work.L5  x Z-  ? M g1 The actual "designer" algorithm is more complex:22H x 0޽h ? ̙335   0L0   P] (  x  c $- `  -  XB  0D`X  0A?`m  0-  #EA REAL-WORLD SCREENING PROGRAM AT AXYS PHARMA (LATER CELERA GENOMICS)F" 3 3 3 3 33 3 3 3 3 3q  H-  ? DATA COURTESY CRAIG HILL & JAMES JANC, CELERA GENOMICS PRESENTED IN PART (BY P.K.) AT 10TH ANNUAL SOCIETY FOR BIOMOLECULAR SCREENING, ORLANDO, 2004JX    "    H A ? ?P    T|-  ?0 v  z 10,008 dose response curves Maximum concentration 0.5 50 M Serial dilution ratio 1:4 Eight data points per curve 3% Random error of initial rates Enzyme concentration 0.6 10 nM h |  T  8c?.jB  B D?pPp  T-  ?"2  X"completely inactive compounds (8%)## |  T  1? jB  B D?@p@  T-  ?p  Upositive control on every plate  H  0޽h ? ̙33   0L0 H @ ` (  x  c $- `  -  XB  0D`X  0A?`m  0-  #EA REAL-WORLD SCREENING PROGRAM AT AXYS PHARMA (LATER CELERA GENOMICS)F" 3 3 3 3 33 3 3 3 3 3p  H A ? p Xp  H A ?` p  H A ? r" p  H A ? RH  T-  ?   pKi = 10 Ki = 0.1 nMV        6  T-  ?m ? no activity    T-  ?  @ weak binding  6  T2  ? g  pKi = 4.5 Ki = 30 MV        $  T<2  ? ;  Dmoderate binding 3  T|-  ?   pKi = 6 Ki = 1 MV        $  T2  ?   A tight binding H  0޽h ? ̙33;  0L0 pc(  x  c $82 `  2  XB  0D`X  0A?`m  0 2  JSIMULATE A POPULATION OF INHIBITORS THAT MATCHES THE AXYS/CELERA CAMPAIGN KK 3  T%2  ?P D<4___PPT9  Simulate 10,000 pKi values that match Celera's "two-Gaussian" distribution Simulate enzyme activities assuming 3% random experimental error Virtually "screen" the 10,000 compounds using the sequential optimal method Compare resulting 10,000 pKi values with the "true" (assumed) values Repeat the virtual "screen" using the classic serial dilution method Compare accuracy and efficiency of sequential and serial-dilution methods $ YT> @`  T=2  ?@P!  }1PLAN OF A HEURISTIC MONTE-CARLO SIMULATION STUDY:02   XB  0D0`0H  0޽h ? ̙33$  0L0 bZ#9(  x  c $(O2 `  2  XB  0D`X  0A?`m  0O2  9A TYPICAL MODERATELY POTENT (SIMULATED) ENZYME INHIBITOR <:  3 3 36   ThV2  ?`PP "true" Ki = 181 nMP ,   Zd'2   ?PP@ l [I] = 1.0 MD     Zc2   ?@P0 ` [E] = 1 nM  v   N  ?`0   TTi2  ?  z Ki = 0.18 M8        H o2  ?  p Morrison Equation + Random Error4!  3   T$u2  ?` p #"Experimental" rate #1 V/V0 = 0.127P$    Tw2  ? ESingle Point Method N  T2  ?`@p Estimated Ki = 146 nMP  > p / H ?Pl P @& 9P @&,$D 0x2 # H ?P 0  ' T2  ?   UOptimal Design Theory$   ' ( Z|2   ? P !Next concentration [I] = 183 nM >"   ) T2  ?` P  z Ki = 0.18 M8       . T2  ? ` @p %"Experimental" rate #3 V/V0 = 0.511 P&   2 Hأ2  ? P & p Morrison Equation + Random Error4!  3  3 Tx2  ?  ESingle Point Method V 4 T2  ?` @p Estimated Ki = 191 nMP  > x 5 H ? YP # l   8} ,$D 0  Z42  ?c UOptimal Design Theory$   -   `L2   ?@  !Next concentration [I] = 147 nM >"    Z2  ? P  $"Experimental" rate #2 V/V0 = 0.554D%   "  Z<2  ? P  Ki = 0.18 MD      ~" ! N ?` \ % Z2  ?P  Estimated Ki = 182 nMP  >  0 NP2  ? P  p Morrison Equation + Random Error4!  3  1 Z2  ?`  ESingle Point Method ~2 6 N ?H  0޽h ? ̙33___PPT10+{eD~' D = @B D9' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*9%(+  0L0 :(  x  c $2 `  2  XB  0D`X  0A?`m  02  EA TYPICAL MODERATELY POTENT (SIMULATED) ENZYME INHIBITOR - CONTINUED ^F  3 3  3  3 36  T2  ? "true" Ki = 181 nMP ,  Z 6   ?p ` [E] = 1 nM     ZD6   ?p` \V0 = 1008  jB ' B D?X X , Nt 6  ? p = # 1 2 3 4 2  - N86  ? =p Q], M 0.0 1.0 0.147 0.183 2 . N 6  ?@d MRate 100 12.7 55.4 51.1 2 / N6  ? `p Ynote negative control arbitrary initial [I] optimally designed [I] optimally designed [I]Z 2Z 0 N\6  ? = X$ASSEMBLE AND FIT DOSE-RESPONSE CURVE% 2% pB 1 H D?  2 NH"6  ?PF  Jfrom preliminary phase 2 v 3 N  ? jB 4 B D?p 6 H A ?  0pB 8 H D? `   9 N'6  ?}  Mfrom nonlinear regression 2 ! : T ,6  ? 0  Ki = (178 9) nMD  $ H  0޽h ? ̙33*  0L0  R(   x   c $L=6 `  6  XB   0D`X   0A?`m   0X?6  4A TYPICAL TIGHT-BINDING (SIMULATED) ENZYME INHIBITOR<5  3  3 38   TE6  ? "true" Ki = 0.021 nMP ,    Z N6   ?p ` [E] = 1 nM     Z4R6   ?p` \V0 = 1008  jB   B D?X X   NW6  ?   A # 1 2 3 4 5 6 2   N[6  ? =  ^([I], M 0.0 1.0 0.04 0.0016 0.001 0.0005) 2)   N ]6  ?@  URate 100 -3.3 1.6 3.1 13.1 49.5 2    NDd6  ?   Hnote negative control arbitrary initial [I] maximum jump 25 maximum jump 25 optimally designed rule of thumbPo 2;#   Ntk6  ? = X$ASSEMBLE AND FIT DOSE-RESPONSE CURVE% 2% pB   H D?    N@p6  ?PF  Jfrom preliminary phase 2 v   N  ? P jB   B D?P pB   H D? `     N0u6  ?}  Mfrom nonlinear regression 2 '   Tz6  ?   Ki = (0.033 0.011) nMD $p   H A ?  0H   0޽h ? ̙33  0L0 Z R .( (  (p %( H A ?k/rx ( c $6 `  6  XB ( 0D`X ( 0A?`m4 ( 06  HDISTRIBUTION OF "TRUE" pKi VALUES IS SIMILAR TO THE AXYS/CELERA CAMPAIGNNI 3 3 3/ 3.l  u -( @,$D  0@  u *( u &( Tt6  ? u RmM 3 '( T6  ?p s u 8M 3 (( Tȝ6  ? "u RnM 3 )( T6  ? u RpM 3rB +( B D?@@ ,( T6  ? u fKi.33 .( T86  ?0E |0SEQUENTIAL OPTIMAL DESIGN n = 3(or 5) + control01  H ( 0޽h ? ̙33___PPT10+z;*D|' = @B D7' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*-(%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*-(D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*-(+  0L0   0G (  0p 0 H A ?k)lx 0 c $6 `  6  XB 0 0D`X 0 0A?`m4 0 0t6  HDISTRIBUTION OF "TRUE" pKi VALUES IS SIMILAR TO THE AXYS/CELERA CAMPAIGNNI 3 3 3/ 3.z  u 0  @,$D  0N  u 0  u  0 TT6  ? u RmM 3  0 T6  ?p s u 8M 3  0 T6  ? "u RnM 3  0 T6  ? u RpM 3rB  0 B D?@@ 0 T6  ? u fKi.33B 0 T6  ?PP^(  V [I]max = 50 M Dilution 4 Eight wellsL,  0 T6  ?0E s'SERIAL DILUTION DESIGN n = 8 + control0(  H 0 0޽h ? ̙33___PPT10+`D|' = @B D7' = @BA?%,( < +O%,( < +Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*0%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*0D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*0+7   0L0   8_ (  8x 8 c $6 `  6  XB 8 0D`X 8 0A?`m 8 06  a+HOW MANY WELLS / PLATES DO WE END UP USING?,, 3Z 8 H$;  ?   total 96-well plates compounds per plate control wells per plate wells with inhibitors control wells ([I] = 0) total wells wells per compound2l(E 8 T;  ? 9SCREEN 10,000 COMPOUNDS (DOSE-RESPONSE) TO DETERMINE Ki'sJ: 5 8 T\;  ?   YSERIAL DILUTION.  8 T ;  ?,` p [SEQUENTIAL DESIGN. jB 8 B D?@jB 8 B D?P@ 8 T;  ?g p  X$909 11 8 79992 7272 87264 8.73%% 8 T@;  ?GP  X$343 88 8 30042 2744 32786 3.28%% 8 T`";  ?  8,62.3 % 62.4 % 62.3 % 62.4 % 62.4 %-3 3 3 33 33 33 33,!jB 8 B D?P@ 8 T/;  ?p= =SAVINGSH 8 0޽h ? ̙33  0L0 #P#@(  @x @ c $<; `  ;  XB @ 0D`X @ 0A?`m @ 0>;  1PROPOSAL FOR FULLY AUTOMATED OPTIMIZED SCREENING <2 3  3  3 @ ZD;  8c?pp0  UROBOT liquid handling$  @ TdI;  ?  XOPTIMAL DESIGN ALGORITHM$  @ ThG;  ? @  n.DATABASE store/retrieve results between plates$/ & vB @ N D8c? @ vB @@ N D8c?` @ `  @ ZL;   ?p   0ANALYSIS SOFTWARE fit dose-response determine Ki$1 . @ ZPY;  8c?p L PLATE READER$   | @ T  8c?  #vB @ N D8c? |  @ T ;  ?   Rreprogram robot for next platevB @@ N D8c?`0 ` @ Tdb;  ?3 ; odispense optimal concentrations4 3vB  @ N D8c?& v  !@ Hh;  ?l  = export data  "@ Te;  ? m71. Accumulate minimal (optimized) dose-response curves 88 #@ T,k;  ?Si v6COMPUTER SUBSYSTEM: INSTRUMENT-CONTROL & DATA-ANALYSIS$7 # H @ 0޽h ? ̙33B   0L0   hj (  hx h c $; `  ;  XB h 0D`X h 0A?`m h 0;  JHOW MANY WELLS / PLATES DO WE END UP USING WITH FEWER COMPOUNDS TO SCREEN?KK 3Z h H;  ?   total 96-well plates compounds per plate control wells per plate wells with inhibitors control wells ([I] = 0) total wells wells per compound2l(A h T;  ? 5SCREEN 88 COMPOUNDS (DOSE-RESPONSE) TO DETERMINE Ki'sJ6  1 h T;  ?   YSERIAL DILUTION.   h T|;  ?,` p [SEQUENTIAL DESIGN. jB  h B D?@jB  h B D?P@  h Tȝ;  ? p  P8 11 8 704 64 768 8.73  h Th;  ?P  P3 88 8 264 24 288 3.27 h TĢ;  ?  8,62.5 % 62.5 % 62.5 % 62.5 % 62.5 %-3 3 3 33 33 33 33,!jB h B D?P@ h T;  ?p= =SAVINGSH h 0޽h ? ̙332  0L0 <4'p (  px p c $( `  ;  XB p 0D`X p 0A?`m p 0;  JHOW MANY WELLS / PLATES DO WE END UP USING WITH FEWER COMPOUNDS TO SCREEN?KK 3 p T;  ?$vv ESERIAL DILUTION p T,;  ? i ..., 8 plates@ p T;  ?H] KInhibitors #1 through #11  p T;  ?@ ] LInhibitors #12 through #22  p TL;  ?] CEtc., through #88  "p Bx; ? 6CT 2  #p Tl;  ? ; = control  l 0 p9 'p0 p9,$D 0 p T;  ?0   GSEQUENTIAL DESIGN p T A ? ?0P 4    p T A ? ?|   !p T A ? ?p  J $p T;  ?  p9 dAll 88 inhibitors on every plate. 3 consecutive plates with progressively optimized concentrations.>e#  -  %p T A! ? ?    &p T A$ ? ?   H p 0޽h ? ̙33~___PPT10^+kOpDB' D = @B D' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*'p%(+   0L0 q i H (  Hx H c $; `  ;  XB H 0D`X H 0A?`m H 0@  3PROPOSAL FOR A FULLY AUTOMATED OPTIMIZED SCREENING <4 3  3  3 H T@  ?  XOPTIMAL DESIGN ALGORITHM$  H T @  ? @  n.DATABASE store/retrieve results between plates$/ & vB  H@ N D8c?    H T@  ?p   0ANALYSIS SOFTWARE fit dose-response determine Ki$1 .|  H T  8c?  # H T@  ?Si v6COMPUTER SUBSYSTEM: INSTRUMENT-CONTROL & DATA-ANALYSIS$7 #  H T,@  ?k  Q2. Analyze accumulated datavB H N D8c? @ vB H@ N D1?` @ `  H Z<@  8c?pp0  UROBOT liquid handling$  H Z%@  8c?p L PLATE READER$   H H 0޽h ? ̙33   0L0 w o 'P (  Px P c $43@ `  @  XB P 0D`X P 0A?`m P 0@5@  NTHE WAY WE SCREEN TODAY: 3vB P N D8c?p  P T|:@  ?p p0  BHUMAN OPERATOR P T8#@  ?p   0ANALYSIS SOFTWARE fit dose-response determine Ki$1 . P ZtC@  8c?pp0  UROBOT liquid handling$   P Z=@  8c?p L PLATE READER$   vB !P@ N D8c?`0 ` "P TM@  ?3 ; m!dispense arbitrary concentrations0"   #P TS@  ?   A program robotvB %P N D8c?PPp pB &P H D?0 p pB 'P H D? 0  H P 0޽h ? ̙33  0L0   X: (  Xx X c $a@ `  @  XB X 0D`X X 0A?`m X 0c@  OSEARCH KEYWORDS: "OPTIMAL DESIGN", "OPTIMUM DESIGN", "OPTIM* EXPERIMENT DESIGN"PP 3p X H A ?npB X H DjJ? pB X H DjJ?o X Tj@  ? } -Franco et al. (1986) Biochem. J. 238, 855-862.       el @s X@s,$D 0~ X N A jJ?0@sxB X H D?p` p X T,t@  ? Z  Wuncertainty of model parameters  l ` `   X` `  ,$D 02 X T  jJ?` p P xB X H D?  X Ny@ ? `   {3fewer experiments = better results ("less is more"),43/3H X 0޽h ? ̙33___PPT10+7m D~' D = @B D9' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*X%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*X%(+  0L0 0 `(  `x ` c $Ċ@ `  @  XB ` 0D`X ` 0A?`m ` 0Ќ@  4OPTIMAL DESIGN IS IMPORTANT FOR MECHANISTIC ANALYSIS<5  3 3 3o  ` T@  ? } -Franco et al. (1986) Biochem. J. 238, 855-862.       p  ` H A ?  |  ` T  8c?0 pB ` H D?P0 F ` T@  ? gC Xtry to decide on molecular mechanism (e.g., competitive vs. non-competitive inhibition)NY3333 3 ` Tԣ@  ?0 `z  optimal design03 3H ` 0޽h ? ̙33_  0L0     (  x  c $@ `  @  XB  0D`X  0A?`m  0Ĵ@  MTHE BATCHKI SOFTWARE IS WELL SUITED FOR PROCESSING "SMALL", OPTIMAL DATA SETS<N 3 3C 3.   T@  ?`n  Automatic initial estimates of model parameters Kuzmi  et al. (2000) Anal. Biochem. 281, 62-67 Automatic active-site titration (for ultra-tight binding compounds) Kuzmi  et al. (2000) Anal. Biochem. 286, 45-50 Automatic detection of chemical impurities in samples Kuzmi  et al. (2003) Anal. Biochem. 319, 272-279 Automatic handling of outlier data points ("Robust Regression") Kuzmi  et al. (2004) Meth. Enzymol. 281, 62-67 Handles enzyme inhibition and cell-based assays Fifteen years of experience Approximately 100,000 compounds analyzed by this consultant alone] -        $         3                 u7YKX v   N  ?`@   TH@  ? j"ALGORITHMS theoretical foundation,#  3 3XB  0D ` H  0޽h ? ̙33  0L0 q i @ (  x  c $@ `  @  XB  0D`X  0A?`m  0D  MSEQUENTIAL OPTIMAL DESIGN FOR INHIBITOR SCREENING HAS BEEN TESTED "IN SILICO"NN 3   TD  ?2  reduce material expenditures by more than 50% reduce screening time by more than 50% increase accuracy & precision of the final answer (Ki)@   TD  ? d l(Advantages of sequential optimal design:() 3 xl vL  vL ,$D 0  HD  ? LN  v works best for large number of compounds (n > 100) has not been tested in practice to avoid programming liquid handler manually, needs "closing the loop": robot reader computerv+|33   TLD  ?v h  l(Disadvantages, limitations, and caveats:()  xB  H D?p D p rB  B D?p  rB  B D?D  l p    p ,$D 0  T  jJ?p    T($D  ?   LCollaboration, anyone?   H A ?,$D  0H  0޽h ? ̙33  ___PPT10 +xiD ' = @B D^ ' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bdissolve*<3<*Dn' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =+4 8?dCB1+#ppt_w/2BCB#ppt_xB*Y3>B ppt_x<*D' =+4 8?\CB#ppt_yBCB#ppt_yB*Y3>B ppt_y<*+  0L0 jbP(  x  c $5D `  D  XB  0D`X  0A?`m  T7D  ?  \Craig Hill & James Janc Theravance Inc. South San Francisco, CA formerly Celera Genomics  South San Francisco formerly Axys Pharmaceuticals formerly Arris Pharmaceuticals.*m P/ H  0޽h ? ̙33[  0L0 ` (  x  c $LD `  D  XB  0D`X  0A?`m  TND  ?@>  5 Questions ? More info: www.biokin.com Contact:  ( ` $ ,    HPD  ? 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