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Irreversible Inhibition Kinetics!!!EGFR inhibition by covalent drugs""-Full automation: Five passes through raw data..0Full automation: Sharing of intermediate results11+Full automation: Implementation - Scripting6," :Quality control of raw data: Piecewise linear fit - Method;;;Quality control of raw data: Piecewise linear fit - Results<<;Quality control of raw data: Piecewise linear fit - Summary<<7Local algebraic fit to determine initial rates - Method888Local algebraic fit to determine initial rates - Results99'Algebraic fit of initial rates - Method(((Algebraic fit of initial rates - Results))(Global fit of reaction progress - Method)))Global fit of reaction progress - Results** Irreversible Inhibition Kinetics!!Possible cellular mechanism/Possible cellular mechanism in DynaFit software00lDynaFit simulation output: Afatinib  strong inhibitor77%Simulate multiple injections - Method&&/Multiple injections: Implementation - Scripting60& Multiple injections: ResultsvMultiple injections: Results  Increase injection frequency<<vMultiple injections: Results  Decrease injection frequency<<7Simulating multiple injections: Summary and conclusions88/pC    0` ̙33` ` ff3333f` 333MMM` f` f` 3>?" dd@,|?" dd@   " @ ` n?" dd@   @@``PR    @ ` ` p>> <4(    6ܒ P  T Click to edit Master title style! !  0ߒ   RClick to edit Master text styles Second level Third level Fourth level Fifth level!     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S  4 T$ y y r   p* a00aa 4 T ) y y r3   r* a00aaH 4 0ηo~ ? ̙3380___PPT10.pU(X p\x(  \ \ N ]e]e      p* AAA \ N ]e]e %     r* AAA  \ T ]e]e     p* AAA \ T ]e]e %    r* AAAH \ 0ηo~ ? ̙3380___PPT10.CP)  0 #0(  X  0A?`m  6~  aAutomation and Simulation,33XB  0D$  <8~@W t >Petr Kuzmi , Ph.D. BioKin, Ltd.^      XB  0D0  <~0 D<4___PPT9 .`Automate the determination of biochemical parameters PK/PD simulations with multiple injectionsza- 6) @`r  S H~`  ~ H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0 !@  (   X  0A?`m  6,P  aAutomation and Simulation,XB  0D$  <e@W t >Petr Kuzmi , Ph.D. BioKin, Ltd.^      XB  0D0  <q0 D<4___PPT9 &`Automate the determination of biochemical parameters PK/PD simulations with multiple injectionsra, 6) @`x  c $`   H  0޽h ? ̙33y___PPT10Y+D='  = @B ++  0L0  } P   (   x  c $~`   XB  0D`X  0A?`mJ  0؏"` Schwartz, P.; Kuzmic, P. et al. (2014)  Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance Proc. Natl. Acad. Sci. USA. 111, 173-178. Issue 1, January 7    *    <     ,2)  Z >?"6@ NNN?Nv  ,$ 0 bInitial estimates Suitable initial estimates of rate constants were discovered by trial and error.Fc@ @`l     ,$D 0   Z >?"6@ NNN?N6  .Outlier rejection Certain  defective progress curves were manually excluded from analysis.F\) @`   Z >?"6@ NNN?N GPRACTICAL CHALLENGES:  Z >?"6@ NNN?N0 j@ ,$  0 This  manual method is not ideally suited for routine production environment.OOH  0޽h ? ̙336.___PPT10+!9D' = @B D=' = @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<* D{' =%(D#' =%(D' =A@BBBB0B%(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<* D{' =%(D#' =%(D' =A@BBBB0B%(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<* +p+0+ 0 ++0+ 0 +A  0 h``  (  x  c $8̆0   XB  0D`X   0A?`m |  `DΆ >?"6@ NNN?N^E[ vPiecewise linear fit: Eliminate  defective progress curves*<' }  `pԆ >?"6@ NNN?NH&K  Local algebraic fit of reaction progress: Determine offsets and initial rates*P+%B  ZD>?"0@NNN?Nb R   `ن >?"6@ NNN?N(  RAlgebraic fit of initial rates: Determine Ki(app) for initial non-covalent complexnS   ! *#B  ZD>?"0@NNN?N L   ` >?"6@ NNN?N p  ~|Global numerical fit of reaction progress: Pass #1 Determine kinact, Ki, and kinact/Ki under rapid-equilibrium approximation}2     & >= 'B  ZD>?"0@NNN?N"       ` >?"6@ NNN?N k  6zGlobal numerical fit of reaction progress: Pass #2 Estimate lower limits for kon and koff under steady-state approximationl{2  ! ,M"B  ZD>?"0@NNN?N   H  0޽h ? ̙33y___PPT10Y+D='  = @B +=  0 .. 8G pS.(   ~  s *t^0   XB  0D`X  0A?`m%   `a >?"6@ NNN?N  WPiecewise linear fit &f   `g >?"6@ NNN?Nx V Local algebraic fit of reaction progress &,*B  ZD>?"0@NNN?N p=   `pl >?"6@ NNN?N0  oAlgebraic fit of initial rates 4  B  ZD>?"0@NNN?N <   `4q >?"6@ NNN?N+ Ts  nGlobal numerical fit: Pass #1 4 B  ZD>?"0@NNN?N0 <   `Lw >?"6@ NNN?Nh ]  nGlobal numerical fit: Pass #2 4 B  ZD>?"0@NNN?Np ` ol 'h  'h,$D 0   Z}|?"6@`NNN?N'h Mmark-up of raw data filesB  B  fD|?"0@NNN?Nl @  " @ ,$D 0B   ZD|?"0@NNN?N``B   # lD|?"0@NNN?N`B   TD|?"0@NNN?NB    fD|?"0@NNN?NB   TD|?"0@NNN?N B    fD|?"0@NNN?N  B   TD|?"0@NNN?N@@ B    fD|?"0@NNN?N@  cl   5  ,$D 0Y@         `4|?"6@`NNN?N A initial ratesB    fD|?"0@NNN?N22   ZЉ|?"6@`NNN?N  Dbaseline offsetsB   ND|?"0@NNN?N00B ! B  `D|?"0@NNN?N0l ``  9 `` ,$D 0B #  ND|?"0@NNN?N B $ B  `D|?"0@NNN?N`  B %  ND|?"0@NNN?N`` B & B  `D|?"0@NNN?N` ` 'l @C  6 @C ,$D 0G (   `H|?"6@`NNN?N@  qKi(app)4  B )   fD|?"0@NNN?NB /  ND|?"0@NNN?N# C B 0 B  `D|?"0@NNN?N`C C l `   8  ` ,$D 0. 2  ZX|?"6@`NNN?N   ^Ki& =@ `   7 `   -   `|?"6@`NNN?Na   kinact, kinact/KiZ   $ B .   fD|?"0@NNN?N`@ P@ B 3  ND|?"0@NNN?N { B 4  `D|?"0@NNN?N`  - >  `|?"6@`NNN?Np 9 D _kon& B ?  fD|?"0@NNN?N p B N(|?"6@`NNN?Np? D `koff&  C Nԯ|?"6@`NNN?Np pD bkinact&  E N >?"6@ NNN?N@` `B F @ TD>?"0@NNN?N`  G Z >?"6@ NNN?N@ Hlower limit estimateH  0޽h ? ̙33___PPT10+1Dn'  = @B D)' = @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<*" %(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*5 %(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*6 %(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 %(+  0 0  T (   ~  s *Lĕ0   XB  0D`X  0A?`m4 9  `Xƕ >?"6@ NNN?N fMaster script (Perl)64 :  `̕ >?"6@ NNN?N:  fPerl script: QA/QC8B ;  `D>?"0@NNN?NP0  <  `hҕ >?"6@ NNN?NP ADynaFitB =  `D>?"0@NNN?N< >  `@֕ >?"6@ NNN?N nPerl script: initial rates8 ?  `ܕ >?"6@ NNN?NP ADynaFitB @  `D>?"0@NNN?NPPx A  `\ڕ >?"6@ NNN?N   Perl script: Ki(app)X    B  `, >?"6@ NNN?NP  ADynaFitB C  `D>?"0@NNN?N0 0  D  `, >?"6@ NNN?N ! ^  Perl script: kinact, Kif  ,  E  ` >?"6@ NNN?N P^  ADynaFitB F  `D>?"0@NNN?N   G  `  >?"6@ NNN?NR N 0  Perl script: kon, kofff  ,  H  `  >?"6@ NNN?NR _0  ADynaFitB I  `D>?"0@NNN?N  B J @  fD>?"0@NNN?NB K @  fD>?"0@NNN?NB L @  fD>?"0@NNN?N  B M @  fD>?"0@NNN?Nb R B N  `D>?"0@NNN?NPPPB O  `D>?"0@NNN?N0 P 0 B P TD>?"0@NNN?NPP B Q  `D>?"0@NNN?N P B R  `D>?"0@NNN?N P0 B S ZD>?"0@NNN?NRPRB T TD>?"0@NNN?NPPPH  0޽h ? ̙33y___PPT10Y+D='  = @B +S  0 zr  (   x  c $% `p    XB  0D`X  0A?`m  Z,(  >?"6@ NNN?Nd D<4___PPT9 ~Fit progress curves to three linear segments. Examine the linear slopes in each segment. If the slope in either the second or the third segment is negative reject the entire progress curve. Reject also corresponding curves from remaining replicates.Pk&` @`H  0޽h ? ̙33y___PPT10Y+D='  = @B +a  0   (   x  c $< `p    XB  0D`X  0A?`m  ZA >?"6@ NNN?Np   ZA >?"6@ NNN?N    Z\?  >?"6@ NNN?N i  <Accept  ZC  >?"6@ NNN?N f  <RejectH  0޽h ? ̙33y___PPT10Y+D='  = @B +   0 2*  (   x  c $TK `p    XB  0D`X  0A?`m  ZA >?"6@ NNN?Nt   ZB  >?"6@ NNN?Nt H,$  0 FNOTE: Each assay will require its own of set of heuristic QA/QC rules!$GCH  0޽h ? ̙33___PPT10+DL)D' Q = @B DD' = @BA?%,( < +O%,( < +D{' =%(D#' =%(D' =A@BBBB0B%(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 +  0  =(   x  c $,Z `p    XB  0D`X  0A?`m  <$\ WP_ 6Fit fluorescence vs. time to an exponential equation 27   kF  ]    #` x   S A ??#" `?      <c  0 ]  !Mt ... time vi ... initial reaction rate kobs ... first-order rate constantN          * p   S A ??#" `?@h    <n ] c F ... fluorescence signal at time t F0 ... instrument baseline rP ... concentration-to-signal scaling parameter [P] ... product concentration at time t"        W   @Yl 0t   0t ,$D 0>   Z |  >?"6@ NNN?N0 it  n8Reused in subsequent steps of the fully automated system99   N >?"6@`NNN?N pu   N >?"6@`NNN?N UpB   TD>?"0@NNN?NpB   TD>?"0@NNN?N B   ND>?"0@NNN?Np00B   ND>?"0@NNN?N00 B  B TD>?"0@NNN?N`  B  B TD>?"0@NNN?N`0 0 H  0޽h ? ̙33~___PPT10^+Y DB'  = @B D' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(+  0    (   x  c $܇ `p    XB  0D`X  0A?`m  ZA >?"6@ NNN?N`  N 8c?"6@`NNN?NN,$@ 08  Z\  >?"6@ NNN?N Gt,$ 0 <reused  T o?"6@`NNN?N,$D 09  Z  >?"6@ NNN?NI,$ 0 =ignoredH  0޽h ? ̙33d \ ___PPT10< +I\PD'  = @B Dk' = @BA?%,( < +O%,( < +D' =%(Du' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =%(Du' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(+p+0+ 0 ++0+ 0 +  0    ) . (  ( x ( c $ `p    XB ( 0D`X ( 0A?`m ( Bx    ( BP hh   ) B( x x     ) B hh   ) Bر x x    ) ZA >?"6@ NNN?N n ) Z|  >?"6@ NNN?N@@ p Morrison equation for tight-binding enzyme inhibition:99 ) Z\  >?"6@ NNN?N 6p,$  0 A little twist: Optimize [E]0 but only within a narrow range (up to [E]nominal). See Kuzmic P., et al. (2000) Anal. Biochem. 286, 45-50. *  ,F &H ( 0޽h ? ̙33___PPT10+uD'  = @B DD' = @BA?%,( < +O%,( < +D{' =%(D#' =%(D' =A@BBBB0B%(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 +   0 B :    (   x  c $\ `p    XB  0D`X  0A?`m  Bd     B< hh    B hh    ZA >?"6@ NNN?N0pf  Z  >?"6@ NNN?N . Ki(app) = (6.3 0.8) nM>  $2  T>?"6@`NNN?N@  Z  >?"6@ NNN?NP  LUsed to make the initial estimate of k(off) in global fit of progress curvesHM%"%#  ZD  >?"6@ NNN?N nT  00k(off) = Ki(app) k(on)6H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0    "(   x  c $ `p    XB  0D`X  0A?`m  B     B hh    B x x     B  hh    B x x     Z$  >?"6@ NNN?N@  Generalized mechanism (no longer simplified  Hit-and-Run model):DD  ZA >?"6@ NNN?N   Z >?"6@`NNN?N_ ,$D  0 [mechanism] ;  T = ATP,  D = ADP E + T <==> E.T : kaT kdT S + E.T <==> S.E.T : kaS kdS S.E.T ---> P + E + D : kcat E + I <==> E.I : kaI kdI E.I ---> E-I : kinact S + E.I <==> S.E.I : kaS kdS S.E.I ---> S.E-I : kinact S.E-I <==> S + E-I : kdS kaS Jz V3   B  Z,  >?"6@ NNN?N ? ,$  0 FDynaFit notationH  0޽h ? ̙33  ___PPT10 +(D\ ' 0 = @B D ' = @BA?%,( < +O%,( < +DN' =%(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' =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<* +p+0+ 0 ++0+ 0 +l  0    @ (   x  c $@= `p    XB  0D`X  0A?`m  B\A     B4E hh    B I x x     BL x x     ZA >?"6@ NNN?N`B  TD|?"0@NNN?Nz z ,$@ 0  ZO  >?"6@ NNN?Ny W ,$ 0 k(off) little or no correlationL    Z8W  >?"6@ NNN?N ?Correlation of biochemical rate constants with cellular potency^@  B  TD|?"0@NNN?NP0` ,$D 0  Z _  >?"6@ NNN?N `,@ ,$ 0 k(on) strong correlationH H  0޽h ? ̙33d \ ___PPT10< +H-D'  = @B Dk' = @BA?%,( < +O%,( < +D' =%(Du' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =%(Du' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<* %(+p+0+ 0 ++0+ 0 +  0 !`  (   X  0A?`m  6p   aAutomation and Simulation,XB  0D$  <Pt @W t >Petr Kuzmi , Ph.D. BioKin, Ltd.^      XB  0D0  <d| 0 D<4___PPT9 &`Automate the determination of biochemical parameters PK/PD simulations with multiple injectionsra. 6) @`x  c $ `    H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0   U(   x  c $ `p    XB  0D`X  0A?`mX  0A?"9G   < Pp$,$ 0 Jprotein re-synthesis  <  P` ,$ 0 Iprotein degradation  <L  p ` ,$ 0 Fdrug elimination  6>"`P 0p ,$D 0  <ܠ  9` ,$ 0 Iprotein degradation  <ԃ  OREALISTIC PK/PD MODEL MUST ACCOUNT FOR METABOLISM OF PROTEIN AND DRUG MOLECULESPP 3 DH  0޽h ? ̙33  ___PPT10a +2!pODe '  = @B D ' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =-o6Bdissolve*<3<* DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(++0+ 0 ++0+ 0 ++0+ 0 ++0+ 0 +!  0 H@  (   x  c $ `p    XB  0D`X  0A?`m0  <Ĺ  DYNAFIT USES  SYMBOLIC REPRESENTATION OF ARBITRARY MOLECULAR MECHANISMHH 3  T  >?"6@`NNN?Np N[task] task = simulate data = progress [mechanism] E + I <==> E.I : kon koff E.I ---> E~I : kinact I ---> X : kout ---> E : ksyn E ---> X : kdeg E~I ---> X : kdeg ...\' J  Z  >?"6@ NNN?N d HExample DynaFit input:H  0޽h ? ̙33y___PPT10Y+D='  = @B +   0 , $    (   x  c $ `p    XB  0D`X  0A?`m  C `A*data-model-t001-g001Data and model0 B   fD>?"0@NNN?N0    `l  >?"6@ NNN?NpK  Otarget concentration, %B   `D>?"0@NNN?NpP p0  Zh  >?"6@ NNN?N t h time, seconds (total = 72 hours),! B   `D>?"0@NNN?Npp 0   Z >?"6@`NNN?Np   Nincreasing [inhibitor]  T  >?"6@ NNN?Nd@0 *kon = 18 koff = 0.044 kinact = 0.0024j+   6   #  Z  >?"6@ NNN?NLG%  [ Afatinib: H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0   C(   x  c $\ `p    XB  0D`X  0A?`m  Z(  >?"6@ NNN?Nv D<4___PPT9 q'Set initial concentrations of [Enzyme] and [Inhibitor] Run a DynaFit simulation for one injection Record concentrations at the end of the run Increase [Inhibitor] concentration by next injection amount Set initial concentrations to the final values (after adjusting [I]) Go to step #2 above(( @`B   fD>?"0@NNN?NppB  TD>?"0@NNN?NpP B  TD>?"0@NNN?NP P H  0޽h ? ̙33y___PPT10Y+D='  = @B +"  0 . & @  &  (   ~  s *l 0    XB  0D`X  0A?`m4   `  >?"6@ NNN?NK  fMaster script (Perl)6   `  >?"6@ NNN?N  ADynaFitB   `D>?"0@NNN?N.B   `D>?"0@NNN?N~K B " TD>?"0@NNN?N..B # TD>?"0@NNN?N.B % Z  >?"6@ NNN?N,$ 0 FMaster script input: & ZT   >?"6@ NNN?N&,$ 0 kon = 198.954 ; binding koff = 0.0472361 ; dissociation kinact = 0.0016792 ; covalent inactivation kelim = 0.0000641803 ; 3 h drug half-life kpsyn = 0.000000001605 ; 0.0001 uM per 12 h * ln(2) kpdeg = 0.00001605 ; 12 h protein half-life E = 0.0001 EI = 0 EJ = 0 I = 0.01 ReinjectI = 0.01 Mesh = linear from 0 to 43200 step 600 ; 12 hours total Injections = 10 ...#'., = ^H  0޽h ? ̙33___PPT10t+ WD'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*& %(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*% %(+p+0+% 0 ++0+& 0 +  0 =5P   (   ~  s *L9 0    XB  0D`X  0A?`mP  C (Aenzyme 0` P  C (A enzyme ` 8  Z<  >?"6@ NNN?N : pCompound 2: strong inhibitor8 6  ZB  >?"6@ NNN?N M6 nCompound 4: weak inhibitor8 !  ZG  >?"6@ NNN?N4  Y'simulate 10 injections @ 12 hours each:((H  0޽h ? ̙33y___PPT10Y+D='  = @B +   0 / ' p   (   ~  s *O 0    XB  0D`X  0A?`m>  ZR  >?"6@ NNN?Np v"Compound 5: intermediate inhibitor8#    C A enzymefile:///J:/fil/diary_dv10/2014/1406/140613/Celgene/001-dynafit-PKPD/_scripts/output/Cpd-5/enzyme.gif     C A enzymefile:///J:/fil/diary_dv10/2014/1406/140613/Celgene/001-dynafit-PKPD/_scripts/output/Cpd-5_8h/enzyme.gif  ` !  ZY  >?"6@ NNN?N   Yinject every 12 hours(     Z]  >?"6@ NNN?N g Xinject every 8 hours( B  ND|?"0@NNN?N 0 B  ND|?"0@NNN?N  H  0޽h ? ̙33y___PPT10Y+D='  = @B +   0 2 *   (   ~  s *`g 0    XB  0D`X  0A?`m>  Zli  >?"6@ NNN?Np v"Compound 5: intermediate inhibitor8#   C A enzymefile:///J:/fil/diary_dv10/2014/1406/140613/Celgene/001-dynafit-PKPD/_scripts/output/Cpd-5/enzyme.gif  !  ZPp  >?"6@ NNN?N   Yinject every 12 hours(  !   Zhk  >?"6@ NNN?N  Yinject every 24 hours(  B   ND|?"0@NNN?N 0     C A enzymefile:///J:/fil/diary_dv10/2014/1406/140613/Celgene/001-dynafit-PKPD/_scripts/output/Cpd-5_24h/enzyme.gif @  B  ND|?"0@NNN?N @ H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0     f (   ~  s *t~0  ~ XB  0D`X  0A?`m   Z~ >?"6@ NNN?N b\ Q DynaFit does not have to be enhanced or modified to do PK/PD simulations PK/PD module can be implemented as a simple Perl script Perl scripts are simple text files: can be modified by any programmer6x G,8~P   Z\~ >?"6@ NNN?N   ,$ 0 TRESULTS (not shown):$    Z̒~ >?"6@ NNN?NZ CIMPLEMENTATION:|  Z~ >?"6@ NNN?N( 00 ,$ 0 * Association ( on ) rate constants are very important for PK/PD outcome Dissociation ( off ,  residence time ) rate constants appear less important:Z  Zp~ >?"6@ NNN?N ,$  0 CAVEAT: Highly reliable values for  on /  off rate constants are needed!,KEH  0޽h ? ̙33W O ___PPT10/ +-Dk ' ~= @B D& ' = @BA?%,( < +O%,( < +D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*  %(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<* %(D{' =%(D#' =%(D' =A@BBBB0B%(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<* ++0+  0 ++0+ 0 ++0+ 0 + 0 @ <( @Y@  X  C 4     S (:49 f   >@  H  0ηo~ ? ̙33, 0 ` |(   d  c $4      3 r$ ss49 f     H  0ηo~ ? ̙33 0 P  T(   d  c $4      s *D 49 f    >@  H  0ηo~ ? ̙33 0 p  T(   d  c $4      s * 49 f    >@  H  0ηo~ ? ̙33 xU;OPUWqh4! h$#quq_`tpSG]] h|䴜s~|.LA`cʊ eaж:a/ yn)h(y㗥ڥ@e p[S͊8u柦w:ֱU1AReO^_(9z'W_jzATI۠YͮȰhJ^=|tc7#I_0 'fCfV1o;~N\$V y3!hL}h-=1Vas!Ivjȧ!:ZF.R"mc&xvl.h1~4NO0&\,N$2!_ݭ'aY*YH 0)'F߁=ޞspQV? xp^RЀ3ÿ lHbP  LPÄ!1 4axĐ% y@( XyT0cH!-ػf#WyFf V30d'2b`bDD`;PtGC y'bOzC? rB x}V({ LLhc(#00sAzEi% %y z <@)`73|THLJf4d-jv1ɒ(f: ڴE0hS4P4)X?PWBRD`LgK<`AyeD #>W"1 Om0bRr:C5J>OCy] h`Vjih/:SlpCP,<O1Oh+'0T hp  Algebraic Model..480Microsoft PowerPoint@0@E1(@s}*@0"̉GSg  )'    """)))UUUMMMBBB999|PP3f333f3333f3ffffff3f̙3ff333f333333333f33333333f33f3ff3f3f3f3333f33̙33333f333333f3333f3ffffff3f33ff3f3f3f3fff3ffffffffff3ffff̙fff3fffff3fff333f3f3ff3ff33f̙̙3̙ff̙̙̙3f̙3f333f3333f3ffffff3f̙3f3f3f333f3333f3ffffff3f̙3f3ffffffffff!___www4'A x(xKʦ """)))UUUMMMBBB999|PP3f3333f333ff3fffff3f3f̙f3333f3333333333f3333333f3f33ff3f3f3f3333f3333333f3̙33333f333ff3ffffff3f33f3ff3f3f3ffff3fffffffff3fffffff3f̙ffff3ff333f3ff33fff33f3ff̙3f3f3333f333ff3fffff̙̙3̙f̙̙̙3f̙3f3f3333f333ff3fffff3f3f̙3ffffffffff!___www¼rrIrrrrrnnrr%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% ՜.+,0    iOn-screen Show.$  Times New RomanVerdanaArialSymbol Courier NewDefault DesignMicrosoft Equation 3.0!Irreversible Inhibition Kinetics!Irreversible Inhibition Kinetics"EGFR inhibition by covalent drugs.Full automation: Five passes through raw data1Full automation: Sharing of intermediate results,Full automation: Implementation - Scripting;Quality control of raw data: Piecewise linear fit - Method<Quality control of raw data: Piecewise linear fit - Results<Quality control of raw data: Piecewise linear fit - Summary8Local algebraic fit to determine initial rates - Method9Local algebraic fit to determine initial rates - Results(Algebraic fit of initial rates - Method)Algebraic fit of initial rates - Results)Global fit of reaction progress - Method*Global fit of reaction progress - Results!Irreversible Inhibition KineticsPossible cellular mechanism0Possible cellular mechanism in DynaFit software7DynaFit simulation output: Afatinib strong inhibitor&Simulate multiple injections - Method0Multiple injections: Implementation - ScriptingMultiple injections: Results<Multiple injections: Results Increase injection frequency<Multiple injections: Results Decrease injection frequency8Simulating multiple injections: Summary and conclusions  Fonts UsedDesign TemplateEmbedded OLE Servers Slide Titles_e0..  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmopqrstuwxyz{|}Root EntrydO)PicturesCurrent UservSummaryInformation(CTPowerPoint Document(JDocumentSummaryInformation8n