The role of MicroScale Thermophoresis (MST) in Drug Discovery of protein kinase inhibitors

UNIVERSITA’ DEGLI STUDI DI PARMA DOTTORATO DI RICERCA IN “SCIENZE DEL FARMACO” CICLO XXXV THE ROLE OF MICROSCALE THERMOPHORESIS (MST) IN DRUG DISCOVERY OF PROTEIN KINASE INHIBITORS Coo dina o e: Chia .mo P o . Ma co Mo Tu o i: Chia .mo P o . Ma co Mo Do .ssa Ba ba a Pioselli Do o ando: Elena Picchi Anni Accademici 2019/2020 – 2021/2022 2 3 TABLE OF CONTENTS ABSTRACT .............................................................................. 5 1. INTRODUCTION ..................................................................... 7 1.1 PROTEIN INTERACTIONS ....................................................................... 7 1.1.1 PROTEIN INTERACTIONS KEY ROLE AND CHARACTERIZATION ................................................. 7 1.1.2 BINDING THEORY: THE LAW OF MASS ACTION ........................................................................ 8 1.1.3 CORRELATION BETWEEN KD, Ki and IC50: THE CHENG-PRUSOFF EQUATION .......................... 10 1.1.4 FITTING MODELS .................................................................................................................... 11 1.1.5 KEY CRITERIA TO MEASURE BINDING REACTIONS .................................................................. 14 1.1.6 MODES OF INHIBITOR INTERACTIONS .................................................................................... 15 TIGHT BINDING ....................................................................................................................... 16 SLOW BINDING ....................................................................................................................... 19 COVALENT BINDING ............................................................................................................... 20 1.2 THE MICROSCALE THERMOPHORESIS (MST) ....................................... 21 1.2.1 GENERAL DESCRIPTION .......................................................................................................... 21 1.2.2 MST APPROACHES .................................................................................................................. 25 1.3 PROTEIN KINASES AS DRUG TARGETS................................................. 27 1.3.1 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR).................................................................. 29 1.3.2 RHO-ASSOCIATED PROTEIN KINASE (ROCK) ........................................................................... 31 2. AIM OF THE WORK .............................................................. 33 3. MATERIALS AND METHODS ................................................. 35 3.1 EGFR .................................................................................................. 35 3.1.1 MATERIALS ............................................................................................................................. 35 3.1.2 MATERIAL HANDLING AND STORAGE .................................................................................... 36 3.1.3 GENERAL PROTOCOL FOR EGFR INTERACTION ASSAYS ......................................................... 36 3.1.4 STOICHIOMETRY ASSAYS ........................................................................................................ 38 3.1.5 REVERSIBLE LIGAND RETENTION ASSAY ................................................................................. 40 3.2 ROCK .................................................................................................. 42 3.2.1 MATERIALS ............................................................................................................................. 42 3.2.2 MATERIAL HANDLING AND STORAGE .................................................................................... 42 3.2.3 GENERAL PROTOCOL FOR ROCK COMPETITION ASSAYS ........................................................ 43 3.2.4 LABELING PROTOCOL ............................................................................................................. 45 3.2.5 GENERAL PROTOCOL FOR ROCK BINDING ASSAYS ................................................................. 45 3.3 MST-ON TIME CHOICE ........................................................................ 47 4 3.4 DATA ELABORATION AND FITTING MODELS ....................................... 57 4. RESULTS AND DISCUSSION .................................................. 59 4.1 EGFR (Epide mal G ow h Fac o Recep o ) ......................................... 59 4.1.1 INTERACTION WITH KINASE TRACER 199 ............................................................................... 60 4.1.2 COMPETITION ASSAYS ............................................................................................................ 72 GEFITINIB - TIGHT BINDING CONDITIONS .............................................................................. 72 LAPATINIB - SLOW BINDING TYPE II INHIBITOR ...................................................................... 80 OSIMERITINIB - IRREVERSIBLE BINDING ................................................................................. 87 4.2 ROCK (Rho-associa ed p o ein kinase) ................................................ 95 4.2.1 COMPETITION ASSAYS ............................................................................................................ 96 INTERACTION WITH KINASE TRACER 236 ............................................................................... 96 OPTIMIZATION OF THE INTERACTION WITH COMPOUND A .................................................. 97 COMPETITION WITH ALLOSTERIC COMPOUNDS .................................................................... 99 4.2.2 DIRECT BINDING ASSAYS ...................................................................................................... 102 ROCK-1 COVALENT LABELING AND BINDING COMPETENCE TEST ....................................... 102 INTERACTION WITH ALLOSTERIC COMPOUNDS ................................................................... 107 5. CONCLUSIONS ................................................................... 111 6. BIBLIOGRAPHY .................................................................. 113 5 ABSTRACT Mic oScale The mopho esis (MST) is one o he biophysical echniques commonly used o he cha ac e iza ion o ligand/p o ein in e ac ions. The phenomenon o " he mopho esis" is he di ec ed mo emen o molecules along a empe a u e g adien gene a ed by an IR lase and MST can de ec changes in cha ge, size, and hyd a ion shell o con o ma ion o a biomolecula complex caused by he in e ac ion be ween a a ge p o ein and he cogna e ligand. This phenomenon can be quan i ied by i a ing he ligand o ob ain a binding cu e om which he dissocia ion cons an (KD) can be de i ed. E en hough he main applica ion o MST is o de e mine binding pa ame e s, he echnique can also be employed o gain insigh s in o o he aspec s o p o ein in e ac ions such as s oichiome y, con o ma ional s a es, ime dependency, selec i i y o e mu a ions, and he modynamics. In his p ojec di e en ypes o in e ac ions we e in es iga ed by using wo model p o ein kinases o pha maceu ical in e es , EGFR (Epide mal G ow h Fac o Recep o ) and ROCK (Rho-associa ed p o ein kinase) in he p esence o hei well-known inhibi o s. The main pu pose was o in es iga e MST abili y o cha ac e ize a ious binding modes wi h a pa icula emphasis on po en inhibi o s, slow binde s, co alen binde s, and allos e ic binde s. Fi s , EGFR sys em enabled he cha ac e iza ion o po en inhibi o s while acing hei in insic limi a ion o esul ing in igh binding expe imen al condi ions. Second, a ime dependence analysis e ealed he MST po en ial o desc ibing con o ma ional changes in p o ein kinases, whe eas a a ge ed s oichiome y expe imen s acili a ed he iden i ica ion o a ious p o ein s a es as well as a di e ence in he p opensi y o Type I and Type II inhibi o s o bind hem. Finally, an al e na i e me hod o Jump Dilu ion o di e en ia ing e e sible and i e e sible inhibi o s was de eloped using MST. ROCK sys em, on he o he hand, has been used o di e en ia e be ween o hos e ic and allos e ic binde s by combining o hogonal app oaches o compe i ion and di ec binding assays. Fu he mo e, an analysis o he bes labeling condi ions e ealed di e ences be ween he wo examined allos e ic compounds, indica ing a possible dis inc in e ac ion mechanism due o hei di e en sensi i i y o he dye posi ion on he p o ein. These esul s enabled he de elopmen o a wide ange o knowledge in he ield o p o ein kinase in e ac ions, p o iding a sui able backg ound o u u e and unknown in e ac ion sys ems. 6 7 1. INTRODUCTION 1.1 PROTEIN INTERACTIONS 1.1.1 PROTEIN INTERACTIONS KEY ROLE AND CHARACTERIZATION P o eins a e mac omolecules pe o ming essen ial oles in he cell, including biochemical (enzymes), s uc u al (cy oskele on), mechanical (muscle), and cell signaling (ho mones) unc ions[1]. P o ein in e ac ions a e he basis o e e y biological unc ion, om he simples biochemical signal o he mo e complex cellula ea angemen . They a e usually e y speci ic and can ei he in ol e small molecules and co ac o o o he p o eins and mac omolecules[2]. The in ica e sys em o pa hways and speci ic in e ac ions ypically con ibu e o he main enance o he co ec homeos asis o a whole o ganism bu in ce ain cases can be he cause o undesi ed al e ed s a es, leading o disease mechanisms[3]. Indeed, almos e e y d ug wo ks by binding p o eins (speci ic a ge ) and in e e e wi h hei biological unc ion. The e o e, a deep unde s anding o p o ein in e ac ions mechanism and i s cha ac e iza ion play a c ucial ole in biochemis y s udies and D ug Disco e y pipeline. To do his, o hogonal biophysical measu es could be applied, allowing o a comp ehensi e desc ip ion o he in e ac ions in e ms o a ini y, kine ics, and he modynamics. A comple e cha ac e iza ion also includes da a on con o ma ional changes, s oichiome y, and he iden i ica ion o speci ic binding egions[4]. The mos app op ia e biophysical echnique o a speci ic goal mus ake in o accoun a a ie y o ac o s, including he a ailabili y o p o ein and ligand in e ms o quan i y, pu i y, and concen a ion, all p io knowledge abou he in e ac ion sys em, and, mos impo an ly, which ou pu would bes answe a speci ic ques ion[2]. Once hese aspec s a e cla i ied, i is possible o op imize an adequa e expe imen al design and p oceed wi h he assays. A leading app oach is o en chosen as he p ima y in es iga i e me hod, while a ew o he echniques a e applied o c oss- alida ion and o gain complemen a y in o ma ion[5]. 8 1.1.2 BINDING THEORY: THE LAW OF MASS ACTION Mos o he binding in e ac ions occu be ween p o eins and smalle molecules gene ically called ligands. These in e ac ions a e ypically e e sible and can be quan i ied using he law o mass ac ion, a simple physical-ma hema ical model in which a molecule A binds a molecule B o o m he complex AB[6]: A + B  AB The binding p ocess is desc ibed by he binding kine ics and depends on he a e o associa ion and dissocia ion o he wo binding pa ne s. When a p o ein molecule (P) and i s speci ic ligand molecule (L) ind each o he in solu ion he in e ac ion sys em can be desc ibed as ollow: whe e PL is he p o ein-ligand complex, kon is he associa ion a e cons an and ko is he dissocia ion a e cons an . The uni s o measu emen o hese wo pa ame e s a e espec i ely M-1s-1 and s-1. When he eac ion eaches he equilib ium s a e he associa ion a e is equal o he dissocia ion a e: He e he squa e b acke s indica e he concen a ion o he molecule in solu ion. The a e o associa ion kon depends on he concen a ion o bo h ee p o ein and ee ligand in solu ion (o he “ac i e masses” o he eac an s), because he highe he numbe o molecules, he highe he p obabili y o hei collision. Mo eo e , because la ge p o eins ha e a la ge su ace a ea o in e ac ion, he associa ion a e will be highe . Al hough no e e y encoun e esul in a bene icial in e ac ion, once he wo binding pa ne s a e in p oximi y, elec os a ic o ces can d aw hem owa ds one o he and di ec he cha ged ligand on o he binding si e. Fu he mo e, he o ganiza ion o he su ace chemical g oups on he p o ein can o ce a ligand ha collides anywhe e on he p o ein's su ace owa ds he binding si e. On he con a y, i only a ac ion o he p o ein and ligand a e in a binding-compe en s a e, he a e o associa ion may be educed[2]. The a e o dissocia ion is a s ochas ic e en and desc ibes he p obabili y o a complex o dissocia e he nex second and i is only p opo ional o he concen a ion o he complex. 9 The e o e, a equilib ium i is possible o ea ange he equa ion: 𝐾𝐷=𝑘𝑜𝑓𝑓 𝑘𝑜𝑛 𝐾𝐷=[𝑃][𝐿] [𝑃𝐿] whe e KD is he dissocia ion cons an , a pa ame e di ec ly ela ed o he a ini y o he ligand o he p o ein. When a ligand concen a ion is equal o he KD, he amoun o he ligand p esen in he solu ion would be s a is ically enough o bind hal o he o al numbe o he binding si es. Knowing ha he o al p o ein concen a ion is he sum o he ee p o ein in solu ion and he p o ein in complex wi h he ligand as desc ibed below: [𝑃]=[𝑃]𝑡𝑜𝑡−[𝑃𝐿] he equa ion can ea ange as ollow: [𝑃𝐿]=[𝐿][𝑃]𝑡𝑜𝑡 [𝐿]+𝐾𝐷 Conside ing [PL] as a unc ion o [L], his equa ion is ha o a hype bola called "Binding Iso he m", becoming a sigmoidal cu e using a semi-loga i hmic scale, he mos ly used model o desc ibe biophysical, biochemical and pha macological in e ac ion da a (Figu e 1). 0 2×10-6 4×10-6 6×10-6 8×10-6 -50 0 50 100 150 5×10-5 1×10-41.5×10-4 [ligand] M binding obse able -10 -8 -6 -4 -2 -50 0 50 100 150 Log[ligand] binding obse able Figu e 1 Binding iso he m ep esen ed as a ec angula hype bola (on he le ) and as a sigmoidal cu e (on he igh ). I is impo an o emphasize ha he law o mass ac ion is based on some p e equisi es: 1. The eac ion mus be e e sible, so an equilib ium s a e can be eached. 2. E e y p o ein molecule needs o be equally accessible o he ligand. 3. The p o ein and he ligand mus exis only in he bound o in he unbound s a e. 4. The in e ac ion mus no al e he p o ein o he ligand. 16 TIGHT BINDING[14][16][17][18][19] The igh binding expe imen al condi ions occu when he analyzed inhibi o has an inhibi ion cons an , Ki, lowe han he speci ic p o ein concen a ion selec ed o he assay. As a esul , igh binding is no an absolu e cha ac e is ic inhe en o he po en compound, bu a he a ela i e condi ion s ic ly dependen o he expe imen al chosen concen a ions. When igh binding occu s, a inhibi o concen a ions lowe han he p o ein concen a ion e e y ligand molecule added o he sys em is seques e ed by he p o ein i sel . The e o e, he p o ein- inhibi o in e ac ion will be go e ned by he amoun o p o ein in he sys em a he han he ac ual a ini y o he small molecule. When an in e ac ion expe imen is pe o med, he de i ed phenomenological IC50 alue becomes s ongly dependen on he p o ein concen a ion and, since he IC50 is he inhibi o concen a ion equi ed o achie e 50% binding, i will ne e be less han hal he p o ein concen a ion. In classical binding condi ions, when Ki > [P], i is possible o assume ha IC50 ~ Ki. Ins ead in igh binding condi ions he p o ein concen a ion becomes a ele an pa ame e . I Ki/[P] is be ween 0.01 and 10, he IC50 depends on bo h he p o ein concen a ion, [P], and he Ki, acco ding o he ollowing equa ion: 𝐼𝐶50=[𝑃] 2+ 𝐾𝑖 When Ki/[P] is lowe han 0.01 he IC50 becomes independen om he Ki alue and he equa ion is ea anged: 𝐼𝐶50=1 2 [𝑃] In hese condi ions he sys em has en e ed he i a ion egime, he “IC50 wall” has been hi and he IC50 will no all below he hal he p o ein concen a ion (i i does, he la e has been p obably o e es ima ed in e ms o ac i e ac ion). 17 Figu e 3 Di e en aspec o in e ac ion cu es: in he “A” sec ion is epo ed he classical binding cu e (when KD > [P]), while in he “B” sec ion is indica ed he i a ion egime when KD << [P]. The s eepness o he cu e becomes highe and he bes i ing model becomes he quad a ic. Adap ed om: Ja moskai e, I. e al. (2020) ‘How o measu e and e alua e binding a ini ies’, eLi e, 9, pp. 1–34. doi: 10.7554/ELIFE.57264. When he sys em is in igh binding condi ions, he da a can be elabo a ed using he quad a ic Mo ison equa ion, which includes he p o ein concen a ion as a pa ame e in o he i ing model. 18 One issue o deba e is whe he o lea e he [P] loa and so conside i as a a iable pa ame e , o o keep i cons an when applying he Mo ison model. Kuzmic (2000) and Mu phy (2004) epo ed dis inc simula ion s udies in which hey concluded ha as he a io o Ki o [P] d ops, he inaccu acy in calcula ing Ki inc eases by ixing he p o ein concen a ion. The p oposed solu ion in ol es wo king in wo s eps: i s , ixing he p o ein concen a ion o i s nominal alue and obse ing he esul ing inhibi ion cons an : i he Ki alue is g ea e han he ixed [P] alue, he inal esul can be accep ed. Al e na i ely, he i ing analysis mus be epea ed while allowing he model o i he p o ein concen a ion as a a iable pa ame e . O he wise, acco ding o Copeland (2005 and 2013), allowing he p o ein concen a ion o luc ua e may esul in physically incohe en es ima ions o [P] as well as e o s in es ima ing Ki. Howe e , bo h poin s o iew ag ee on he impo ance o accu a ely es ima ing he p o ein ac i e ac ion. The p o ein popula ion is assumed o exis in a leas wo s a es, one o which is binding compe en and he o he un olded o dena u ed. The compe en s a e may be in u n popula ed by di e en con o ma ional s a es. The ac i e ac ion can be de e mined by exploi ing he igh binding si ua ion i sel . One o he wo binding pa ne s has o be ixed a a concen a ion g ea e han Ki ( he op imum would be a ac o 200) o achie e igh binding condi ions. The concen a ion o he o he binding pa ne mus be i a ed o e a na ow ange, which includes he i s one. As a esul , he e will be wo dis ibu ions o poin s ha can be i by wo di e en linea eg essions. The dis ibu ion a high i an concen a ions indica es he o ally bound condi ion, whe eas he dis ibu ion a lowe i an concen a ions indica es he i a ing egime. Assuming a s oichiome y o 1:1 he amoun o he ac i e ac ion is de e mined by he b eaking poin be ween he wo dis ibu ions. By inc easing he igh binding condi ion, he b eaking poin becomes sha pe , and he ac i e ac ion is mo e p ecisely de ined (Figu e 4). 19 Figu e 4 The igu e is e e ed o a case s udy epo ed by Ja moskai e (2020) in which RNA is used as biological a ge and es ed a he ixed concen a ion o 100 nM (on he le ) and 10 nM (on he igh ). The measu e o he p o ein ac i e ac ion was conduc ed by i a ing he p o ein Pu 4 while in igh binding condi ions. By using a highe RNA concen a ion (on he le ), he wo dis ibu ions o poin s a e be e desc ibed, and he b eaking poin is sha pe . Ja moskai e, I. e al. (2020) ‘How o measu e and e alua e binding a ini ies’, eLi e, 9, pp. 1–34. doi: 10.7554/ELIFE.57264. Once he amoun o ac i e p o ein has been iden i ied, he binding da a can be i ed by using he Mo ison equa ion, by including he ac ual amoun o p o ein capable o binding in o he model. SLOW BINDING[17] Slow binde s a e compounds ha ei he associa e o dissocia e slowly om he p o ein. The e o e, due o hei ime dependence, a ini y and po ence need o be es ablished only a e he eaching o equilib ium. Slow binding can occu wi h di e en mechanisms: - Simple e e sible slow binding: mechanism in which he associa ion, he dissocia ion o bo h a e slow; - Induced- i : wo-s ep eac ion wi h a i s apid in e ac ion and a ollowing slow in e con e sion o he p o ein o a o m ha be e accommoda es he ligand; - Con o ma ion selec ion: wo-s ep eac ion wi h a i s slow con o ma ional in e con e sion o he p o ein and hen a apid in e ac ion wi h he inhibi o . Usually a comple e cha ac e iza ion o slow binde s yields he inhibi ion mechanisms, he ue a ini y based on enzyma ic assays and he indica ion o he esidence ime, he pe iod o ime ha he ligand spends bound o i s a ge . 20 COVALENT BINDING[17][20][21][22] Co alen inhibi o s a e molecule wi h a eac i e wa head aimed o o m co alen bond wi h speci ic esidues o a ge p o eins. They o en in ol e a wo-s ep eac ion, he i s o which is a e e sible in e ac ion be ween he inhibi o and i s binding pocke while he co alen bond i sel is o med as a esul o he second ollowing eac ion. The possible di icul y in selec i i y is balanced by a longe du a ion o ac ion, imp o ed ligand e iciency, and he capaci y o a oid d ug esis ance. The e o e, he co alen in e ac ion consis s o wo componen s: he binding a ini y be ween he inhibi o and he p o ein (Ki) and he chemical eac i i y desc ibed by he a e o enzyme inac i a ion (kinac ), he equi ed ime o co alen ly modi y hal o he p o ein[23]. Usually co alen inhibi ion is desc ibed wi h he co alen e iciency cons an (kinac /Ki) a p e e ed pa ame e desc ibing he inhibi o y po ency wi hou he ime dependence in e e ence. Howe e , in some cases he inhibi o y concen a ion a a speci ic ime, IC50( ), can be ca e ully used as su oga e o he pa ame e kinac /Ki, o can mo e easily co ela e da a wi h o he ac i i y assays[24]. Finally, he e i ica ion ha he i e e sible eac ion has occu ed is a c i ical elemen o ake in o accoun . The me hods usually employed o his pu pose a e mass spec ome y analyses o iden i y he co alen adduc and he enzyma ic es o ac i i y eco e y a e a massi e dilu ion (Jump Dilu ion). 21 1.2 THE MICROSCALE THERMOPHORESIS (MST)[25][26][27][28] Among he di e en biophysical app oaches aimed a he cha ac e iza ion o p o ein-ligand in e ac ions (e.g. ITC, SPR, luo escence spec oscopy, e c.), he Mic oScale The mopho esis eme ges as a e sa ile echnique o he de ec ion o binding be ween a la ge a ie y o molecula species. The app oach main ad an ages a e i s minimal sample consump ion, quickness o analysis, abili y o u ilize almos any ype o bu e , and abili y o disco e binding o any na u e (o hos e ic, allos e ic, e c.) as long as a su icien ligand-dependen change in he mopho e ic mobili y occu s. 1.2.1 GENERAL DESCRIPTION The Mic oScale The mopho esis (NanoTempe ) is an immobiliza ion- ee biophysical echnique o he cha ac e iza ion o bimolecula in e ac ions. The me hod is based on a physical phenomenon called he mopho esis, acco ding o which e e y molecule in a luid and subjec ed o a empe a u e g adien will s a o mig a e along he g adien , wi h a a e di ec ly ela ed o i s physical cha ac e is ics; in pa icula i has been demons a ed he h ee aspec s ha mainly impac on he he mopho e ic mig a ion a e: - Size - Cha ge - Hyd a ion shell These cha ac e is ics a e s ongly in luenced by ex e nal pe u ba ion o he sys em; indeed, by conside ing he single molecule o a p o ein, i s size, cha ge and con o ma ion may signi ican ly change in he p esence o a speci ic in e ac o like an inhibi o . The e o e, i is by i a ing he p o ein wi h a ligand molecule ha is possible o moni o he change in he mopho esis mig a ion o he esul ing complex and o use ha in o ma ion as obse able o ob ain a binding cu e and speci ic in e ac ion pa ame e s. In he Mic oScale The mopho esis (MST), he molecule mo emen s a wi h he ac i a ion o an in a ed lase (wa eleng h 1480 nm) and i s ene gy abso p ion by he wa e molecule in solu ion. This apidly esul s in he gene a ion o a empe a u e g adien along he capilla y ube inside o which he sample is loaded; he molecules s a o mig a e om he ho e o he colde zone as a consequence o he so-called So e ’s e ec . The mo emen con inues un il i would be con as ed by he back-di usion o he molecules. 22 The So e ’s e ec is desc ibed by he ollowing equa ion: ST=A kBT(−∆shyd(T)+βσe 2 4εε0T λDH) whe e ST is he So e coe icien , A is he molecule su ace, λDH is he Debye leng h, kB is he Bol zmann cons an , ∆shyd is he pa icle-a ea-speci ic hyd a ion en opy, σe is he e ec i e su ace cha ge densi y, ε is he dielec ic cons an , β is he coe icien ha desc ibe he co ela ion be ween he dielec ic cons an and he Debye leng h, T is he empe a u e (exp essed in Kel in)[29]. Once he IR ay has ac i a ed and gene a ed he empe a u e g adien , he mo emen o he molecules is de ec ed by hei in insic luo escence o h ough he use o luo opho es co alen ly a ached o he p o ein. Indeed, a luo escence de ec o is placed in he same spo o he IR ac i a o and collec s he in o ma ion o dec ease in he obse ed luo escence in he cen e o he empe a u e g adien . The IR lase in ensi y is de e mined by he MST powe , which may be egula ed in h ee modes: low, medium, and high. The i adia ion sample olume is app oxima ely 2 nL, wi h a hea ed zone diame e o 100 µm, and he empe a u e g adien ampli ude inc eases wi h in ensi y. In Figu e 5 is epo ed a ep esen a ion o e e y s ep o an MST expe imen . Figu e 5 Rep esen a ion o an MST expe imen and i s phases. Adap ed om M. Je abek-Willemsen e al., “Mic oScale The mopho esis: In e ac ion analysis and beyond,” J. Mol. S uc ., ol. 1077, pp. 101–113, Dec. 2014, doi: 10.1016/j.mols uc.2014.03.009. 23 The ime along which he IR lase emains ac i e is named MST-ON ime and usually he expe imen e can a bi a ily choose among six di e en MST-ON ime, (1.5, 2.5, 5, 10, 15 and 20 seconds). Immedia ely a e he IR ac i a ion he sys em de ec s a consis en d op in ela i e luo escence called T-Jump. The ini ial change in luo escence is mos likely caused by he g ea e luo opho e collisional quenching a ele a ed empe a u es (TRIC, Tempe a u e Dependen In ensi y Change) a he han he ac ual he mopho esis o he molecules[28]. The la e mig a ion imes, ins ead, a e mos likely o be go e ned by he global p ope ies o he molecule o complex in e ms o size, cha ge and hyd a ion shell. The luo escence dec ease a e e y MST-ON ime (Fho ) is compa ed o he ini ial luo escence (Fcold) and plo ed as no malized luo escence (FNo m), on a ac ional o pe mille scale: 𝐹𝑁𝑜𝑟𝑚= 𝐹ℎ𝑜𝑡 𝐹𝑐𝑜𝑙𝑑 ‰ As al eady men ioned, he he mopho e ic mobili y o a p o ein should change in he p esence o a ligand ha modi ies he mig a ion cha ac e is ics o he p o ein alone. The e o e, by i a ing he in e ac o om an app op ia e s a ing concen a ion, a ound 50-100- old he es ima ed KD, i is possible o ob ain a symme ical dis ibu ion a ound he ligand concen a ion co esponding o he KD alue. Then, by choosing an a bi a y MST-ON ime, he ela i e expe imen al poin s se can be i ed o ob ain an in e ac ion cu e and he ela i e pa ame e s (Figu e 6). Figu e 6 Rep esen a ion o how a binding cu e is de i ed om he mopho esis aces. 24 The di e ence be ween he MST signal o he unbound and bound s a es is called Response Ampli ude and depends on bo h he MST-ON ime (la e imes a e always cha ac e ized by a highe Response Ampli ude) and on he MST powe . An indica ion o he quali y o he binding cu e is he signal- o-noise a io (S/N) de ined as ollow: 𝑆 𝑁= 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝐴𝑚𝑝𝑙𝑖𝑡𝑢𝑑𝑒 𝑁𝑜𝑖𝑠𝑒 whe e he Noise is he s anda d de ia ion o he measu emen and i is de ined as he MST signal a ia ion do no caused by he in e ac o : 𝑁𝑜𝑖𝑠𝑒= √∑(𝑟𝑖− 𝑟)2 𝑖𝑛−1 wi h i indica ing he esidual o he i , 𝑟 he a e age o all esiduals and n he numbe o da a poin s[30]. Ano he me hod o indica e he Noise is he s anda d de ia ion o he esiduals (Sy.x), epo ed by so wa e like G aphPad P ism: 𝑆𝑦.𝑥= √∑(𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙2) 𝑖𝑛−𝐾 whe e he esidual is he e ical dis ance (Y uni s) o he expe imen al poin s om he cu e and n-K is he numbe o deg ees o eedom o he eg ession. 25 1.2.2 MST APPROACHES The Mic oScale The mopho esis can be used o wo di e en app oaches: he compe i ion assay and he di ec binding assay. The compe i ion assay elies on he use o a luo escen molecule o bind he p o ein a ge ; hen, a second non- luo escen molecule is i a ed o displace he i s one, and an indi ec indica ion o he a ini y o he i a ing molecule is collec ed. The di ec binding assay elies on he use o a luo escen molecule as one o he binding pa ne s o ob ain a ini y in o ma ion on he in e ac ion sys em wi h a non- luo escen molecule which is i a ed. To employ he di ec binding s a egy, one o he in e ac o s mus be luo escen , ei he in insically o ex insically, and all wi hin he emission-exci a ion ange in o de o be de ec ed co ec ly h oughou he expe imen s. In Figu e 7 a e epo ed di e en ypes o luo escen dyes and hei exci a ion and emission anges. Figu e 7 Di e en ypes o dyes and hei exci a ion and emission anges. The ins umen used o he analysis o his wo k and he po en ially applicable luo opho es o measu emen s on he de ice a e indica ed in he ed box. Adap ed om NanoPedia - Monoli h NT.115, NanoTempe Technologies GmbH. The binding app oach equen ly equi es co alen labeling o he p o ein, which can be a di icul s ep due o he likelihood o dye in e e ence wi h he s uc u e and unc ionali y o he p o ein, and hus wi h i s binding compe ence[31]. Fu he mo e, he deg ee o labeling mus be op imized because a low amoun o dye co alen ly a ached o he p o ein may esul in a low luo escence 32 33 2. AIM OF THE WORK This wo k aims o alida e he Mic oScale The mopho esis (MST) as a biophysical echnique o ob ain aluable in o ma ion on di e en binding modes in he ield o p o ein kinase inhibi o s. The ele ance and he he apeu ic po en ial ha kinase inhibi o s assumed in pha maceu ical esea ch a e due o he essen ial ole hese p o eins play in li e p ocesses and in se e al pa hologic condi ions. The e o e, in he las decades medicinal chemis y di ec ed i s e o s o he cha ac e iza ion o kinase-inhibi o in e ac ion in o de o op imize d ug design. Mos o he in o ma ion add essing SARs (S uc u e-Ac i i y Rela ionship) choices esul om in i o s udies[3]. P o ein kinase inhibi o s ha e a wide ange o inhibi ion mechanisms, hus ha ing a biophysical pla o m om which ob aining eliable in o ma ion on hese mode o ac ion a ie ies is impo an o bo h a deepe unde s anding o al eady known mechanisms and a mo e us able in es iga ion o new po en ial d ugs. Mic oScale The mopho esis is in mos cases only applied o gain he a ini y in o ma ion o KD o IC50, bu his pe spec i e migh be limi ing conside ing he po en ial o he echnique and i s low sample consump ion and apidi y o analysis. In his s udy, he MST has been employed o cha ac e ize, om di e en pe spec i es, he in e ac ion modali ies o wo model p o ein kinases o pha maceu ical in e es (EGFR and ROCK) and hei well-known inhibi o s o c ea e a wide ange knowledge o be used in u u e and unknown in e ac ion sys ems. 34 35 3. MATERIALS AND METHODS The ollowing me hods a e e e ed o he in e ac ion expe imen s pe o med wi h he ins umen Monoli h NT.115 (NanoTempe ). 3.1 EGFR 3.1.1 MATERIALS The EGFR (Epide mal G ow h Fac o Recep o ) in e ac ion sys em has been in es iga ed h ough he compe i ion assay and he iso o ms conside ed we e he ca aly ic domains o EGFR wild ype and he mu a e o m EGFR L858R. The es ed inhibi o s we e he h ee o hos e ic compounds Ge i inib, Lapa inib and Osime inib. Fo he compe i ion assay he selec ed luo escen species was he Kinase T ace 199. In he able below a e epo ed he cha ac e is ic o he s a ing ma e ial pu chased o he expe imen s. I em Supplie P oduc Numbe Lo Tag Sequence (s a -end) MW Da Concen a ion Pu i y S o age bu e EGFR (E bB1) Wild Type The mo Fishe PR7295B 2281133H GST 668-1210 90500 4.86 µM (0.44 g/L) 70 % 50 mM T is (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.02% T i on® X-100, 2 mM DTT and 50% Glyce ol EGFR (E bB1) L858R The mo Fishe PR7447A 2468986C GST 668-1210 90500 3.09 µM (0.28 g/L) 70 % 50 mM T is (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.02% T i on® X-100, 2 mM DTT and 50% Glyce ol Kinase T ace 199 The mo Fishe PV5830 - - - - 25 µM - DMSO Ge i inib (I essa®) UniPR - - - - 446.90 - - Powde Lapa inib (GW572016) Med Chem Exp ess HY-50898 - - - 581.06 10 mM - DMSO Osime inib (AZD9291) Med Chem Exp ess HY-15772 - - - 499.61 10 mM - DMSO The employed p o eins a e ecombinan and used o glu a hione S- ans e ase (GST- ag), a commonly used me hod o imp o e pu i ied p o ein solubili y[51]. The cons uc s a e he same employed in wo ci ed wo ks[52][53] and his allowed a di ec compa ison be ween he epo ed esul s and he di e en in e ac ion sys ems o u he alida e MST as a aluable echnique o hogonal o o he biophysical app oaches. 36 3.1.2 MATERIAL HANDLING AND STORAGE Bo h p o eins and small molecules in DMSO a e s o ed a -80°C upon hei a i al. The Kinase T ace 199 is s o ed a -20°C. P o eins Fo he i s use (and o e e y hawing cycles) he p o eins we e hawed in ice, gen ly pipe ed, and hen cen i uged a 5000 pm, 0°C, 5 minu es. Then, he p o eins we e aliquo ed in PCR ubes 10 µL each and used only wi h a single u he eeze- haw cycle, besides he i s one. Small molecules A 10 mM s ock solu ion in DMSO was p epa ed also o he Ge i inib, p o ided in powde . The s ock solu ions o he h ee inhibi o s we e s o ed a -80°C in 100 µL aliquo s and hawed and o exed p io he use o e e y expe imen al session. The s ock solu ion 10 mM in DMSO is he s a ing solu ion o he assays. Kinase T ace 199 An in e media e solu ion o T ace 199 is p epa ed a 250 nM by dilu ing 2 µL o s ock solu ion (25 µM) in 198 µL o bu e 50 mM HEPES (pH 7.5), 150 mM NaCl. The solu ion is hen s o ed in ial a - 20°C and hawed and o exed p io he use o e e y expe imen al session. This in e media e solu ion is he s a ing solu ion o he assays. 3.1.3 GENERAL PROTOCOL FOR EGFR INTERACTION ASSAYS The in e ac ion assays o he EGFR sys em a e pe o med as ollow: 1) Thawing o he p o ein, he Kinase T ace 199 and he inhibi o o in e es ; cen i uga ion o he p o ein a 5000 pm, 0°C, 5 minu es and o ex o he T ace and he small molecules. 2) P epa a ion o he in e ac ion bu e : 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, by adding esh TCEP and Tween e e y expe imen al session o he base bu e 50 mM HEPES (pH 7.5), 150 mM NaCl (s o ed a +4°C). 3) P epa a ion o h ee solu ions o he i a ion: (T) o (PT) → Fluo escen molecule: T ace alone o in complex wi h he p o ein. (P) o (L) → Ti a ing molecule: he p o ein alone o he inhibi o o in e es . 37 (C) → Con ol solu ion a he same composi ion o he i a ing molecule solu ion. NOTE: E e y solu ion has o be p epa ed a wice he inal desi ed concen a ion. • BINDING ASSAY BETWEEN EGFR AND THE KINASE TRACER 199 (25 nM o 5 nM) SOLUTION FUNCTION COMPOSITION INITIAL CONCENTRATION (2X) FINAL CONCENTRATION (IN THE ASSAY) (T) FLUORESCENT TRACER 199 50 nM o 10 nM 25 nM o 5 nM (P) TITRANT EGFR 3.76 µM 1.88 µM – 0.45 nM (C) CONTROL GLYCEROL 50 % glyce ol 25 % glyce ol • COMPETITION ASSAY WITH THE ORTHOSTERIC INHIBITORS SOLUTION FUNCTION COMPOSITION INITIAL CONCENTRATION (2X) FINAL CONCENTRATION (IN THE ASSAY) (PT) FLUORESCENT EGFR + TRACER 199 40 nM + 10 nM 20 nM + 5 nM (L) TITRANT GEFITINIB o LAPATINIB o OSIMERTINIB 200 nM 100 nM – 0.02 nM (C) CONTROL DMSO 0.002 % 0.001 % 4) P epa a ion o he i a ion on 8 poin s, wi h a dilu ion ac o o 4; he las poin is lacking in i a ing molecule and is aken as con ol o he luo escen molecule/complex he mopho e ic mobili y. - Numbe 8 PCR ubes om 1 o 8. - Add an app op ia e olume (a leas 10 o 15 µL) o (C) o he 2 - 8 ubes and he same olume o (L) o (P) o he ube numbe 1. - Ti a e (L) o (P) om he ube 2 o he ube 7, by using a olume 3- imes lowe han he (C) olume. Then disca d he las olume wi hou adding i o 8. - Add (T) o (PT) o e e y ube, om 1 o 8, in he same olume used o (C). - Cen i uge he ubes a 15000 g, 22°C, 5 minu es. 5) Loading o he solu ions in o he capilla ies ( wo capilla ies o e e y concen a ion poin ) and se 10 minu es incuba ion inside he ins umen a he desi ed empe a u e. 6) The assay is pe o med applying he ollowing ins umen se up. 38 Capilla ies ype P emium coa ed Exci a ion Powe 100 %* MST-Powe Medium Tempe a u e 25°C *as men ioned in he In oduc ion pa ag aph 1.2.2, he choice o using 100 % o he Exci a ion Powe was ponde ed o e e y expe imen o his wo k, in o de o maximize he yield a low luo opho e concen a ion, once he main enance o a good signal- o-noise a io had been e i ied. 3.1.4 STOICHIOMETRY ASSAYS The s oichiome y assays a e pe o med in a na ow ange o i an concen a ion and a high non- i an concen a ion, in o de o exploi ligand deple ion and o ob ain s oichiome y in o ma ion. This ype o assay is pe o med as ollow: 1) Thawing o he p o ein, he Kinase T ace 199 and he inhibi o o in e es ; cen i uga ion o he p o ein a 5000 pm, 0°C, 5 minu es and o ex o he T ace and he small molecules. 2) P epa a ion o he in e ac ion bu e : 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, by adding esh TCEP and Tween e e y expe imen al session o he base bu e 50 mM HEPES (pH 7.5), 150 mM NaCl (s o ed a +4°C). 3) P epa a ion o h ee solu ions o he i a ion: (T) o (PT) → Fluo escen molecule: T ace alone o in complex wi h he p o ein. (P) o (L) → Ti a ing molecule: he p o ein alone o he inhibi o o in e es . (C) → Con ol solu ion a he same composi ion o he i a ing molecule solu ion. NOTE: E e y solu ion has o be p epa ed a wice he inal desi ed concen a ion. • STOICHIOMETRY TITRATING THE PROTEIN OVER THE TRACER 199 SOLUTION FUNCTION COMPOSITION INITIAL CONCENTRATION (2X) FINAL CONCENTRATION (IN THE ASSAY) (T) FLUORESCENT TRACER 199 100 nM 50 nM (P) TITRANT EGFR 2 µM 1 µM – 35.18 nM (C) CONTROL GLYCEROL 50 % glyce ol 25 % glyce ol • STOICHIOMETRY TITRATING THE INHIBITOR OVER THE TRACER 199/EGFR COMPLEX SOLUTION FUNCTION COMPOSITION INITIAL CONCENTRATION (2X) FINAL CONCENTRATION (IN THE ASSAY) (PT) FLUORESCENT EGFR + TRACER 199 280 nM + 10 nM 140 nM + 5 nM (L) TITRANT GEFITINIB o LAPATINIB 200 nM 100 nM – 0.02 nM (C) CONTROL DMSO 0.002 % 0.001 % 39 4) P epa a ion o he i a ion on 16 poin s, wi h a dilu ion ac o o 1.25: - Numbe 16 PCR ubes om 1 o 16. - Add an app op ia e olume (a leas 5 µL) o (C) o he 2 - 16 ubes and he same olume o (L) o (P) o he ube numbe 1. - Ti a e (L) o (P) om he ube 2 o he ube 16, by using a olume 4- imes highe han he (C) olume. Then disca d he las olume. - Add (T) o (PT) o e e y ube, om 1 o 16, in he same olume used o (C). - Cen i uge he ubes a 15000 g, 22°C, 5 minu es. 5) Loading o he solu ions in o he capilla ies (one capilla y o e e y concen a ion poin ) and se 5 minu es incuba ion inside he ins umen a he desi ed empe a u e. 6) The assay is pe o med applying he ollowing ins umen se up. Capilla ies ype P emium coa ed Exci a ion Powe 100 % MST-Powe Medium Tempe a u e 25°C NOTE: In hese expe imen s he ime dependence (10 minu es and 3 hou s) has been explo ed in wo ways: 7) P epa a ion o he solu ions (bulle poin s om 1 o 4) and se ing o wo ime s, 10 minu es and 3 hou s. Once pas he 10 minu es loading o he i s se o capilla ies and incuba ion o u he 5 minu es inside he ins umen be o e he analysis. A e 3 hou s loading o he second se o capilla ies om he same solu ions, incuba ion o u he 5 minu es inside he ins umen be o e he analysis. 8) P epa a ion o he (PT) s a ing solu ion and se ing o wo ime s, 10 minu es and 3 hou s. P oceed wi h he bulle poin s om 4 o 6 wo imes, a e 10 minu es and hen a e 3 hou s. 40 3.1.5 REVERSIBLE LIGAND RETENTION ASSAY This assay has been de eloped as an al e na i e o he Jump Dilu ion o he di e en ia ion o e e sible and i e e sible ligands. The app oach is based on he size exclusion e en ion o small e e sible molecules, while he la ge molecules and e e y small molecule co alen ly a ached o i will no be e ained and will be mechanically sepa a ed om he o he s wi hou s ong change in hei concen a ion. The expe imen s ha e been pe o med as ollow: 1) Thawing o he p o ein, he Kinase T ace 199 and he inhibi o o in e es ; cen i uga ion o he p o ein a 5000 pm, 0°C, 5 minu es and o ex o he T ace and he small molecules. 2) P epa a ion o he in e ac ion bu e : 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, by adding esh TCEP and Tween e e y expe imen al session o he base bu e 50 mM HEPES (pH 7.5), 150 mM NaCl (s o ed a +4°C). 3) P epa a ion o h ee solu ions (a leas 320 µL each): (P) → P o ein alone wi h he same concen a ion o DMSO han he o he wo solu ions. (PG) → P o ein in complex wi h he Ge i inib. (PO) → P o ein in complex wi h he Osime inib. SOLUTION FUNCTION COMPOSITION CONCENTRATION (P) CONTROL EGFR 20 nM (PG) REVERSIBLE LIGAND EGFR + GEFITINIB 20 nM + 10 nM (PO) IRREVERSIBLE LIGAND EGFR + OSIMERTINIB 20 nM + 10 nM 4) Incuba ion o he solu ions o 60 minu es and 150 minu es. 5) P elimina y Binding Check a e e e y ime poin and be o e he Spin Column passages: addi ion o 0.6 µL T ace 199 (s ock solu ion 250 nM) o 29.4 µL o e e y solu ion in o de o ob ain 30 µL a T ace concen a ion o 5 nM. Cen i uga ion a 15000 g, 22°C, 5 minu es. E e y solu ion is used o load h ee capilla ies and he capilla ies a e hen incuba ed o 5 minu es inside he ins umen be o e he analysis. 6) Size exclusion: o each ime poin 9 size exclusion Zeba™ Spin Desal ing Columns, 7K MWCO, 0.5 mL, The mo a e used. E e y Zeba™ Spin Desal ing Column needs h ee s eps o condi ioning wi h he dilu ion bu e (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20). A e he condi ioning, 130 µL o each solu ion (P), (PG), and (PO), 41 endu es a passage in a Zeba Column each and hen he elu ed solu ions unde go wo u he sequen ial passages in as many Zeba Columns ( h ee Zeba column a e used o e e y solu ion). 7) Final Binding Check in he same condi ions o poin numbe 5 a e e e y incuba ion ime and passage s eps h ough he Zeba Spin Columns. 8) The assay is pe o med applying he ollowing ins umen se up. Capilla ies ype P emium coa ed Exci a ion Powe 100 % MST-Powe Medium Tempe a u e 25°C 48 P ism so wa e o he i ed pa ame e s pIC50, Hill Slope, Top and Bo om, o he han he S/N (ob ained by di iding he Response Ampli ude o he Sy.x) and he R2. The MST-ON ime we e hen anked on he basis o he na owes in e al con idence and goodness o i o choose he mos ep oducible ime poin o ha sys em. The same expe imen al se has been epea ed o e e y di e en sys em o de ec ion such as he co alen labeling o he p o ein. EGFR COMPETITION Fo he EGFR sys em (bo h wild ype and L858R) h ee p o ein concen a ions (70 nM, 20 nM, 5 nM) ha e been es ed in o de o consolida e he obus ness o he me hod. The chosen e e ence is he Ge i inib, i a ed s a ing om 100 nM. [EGFR] 70 nM, 20 nM, 5 nM [INHIBITOR] 100 nM – 0.02 nM + CONTROL POINTS [TRACER 199] 5 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005 % Tween 20 TEMPERATURE 25°C The binding cu es we e i s analyzed as sepa a ed eplica es in o de o e alua e he mac oscopic di e ences in i ing and pa ame e s. EGFR WILD TYPE - EGFR WT, 70 nM + GEFITINIB 49 F om he da a collec ed a 70 nM i can be no iced ha , excep o he 5 s (coinciding wi h he c oss- o e o he MST aces), all he MST-ON ime yield a compa able pIC50 and Hill Slope in o ma ion. The la e MST-ON imes howe e gi e a mo e obus i ing wi h a highe R2 alue. A his high p o ein concen a ion, he ac ion o he ace ha is bound o he p o ein is nea ly equal o he o al amoun o he ace leading o a high signal/noise. In hese si ua ions, e e y MST-on ime gi es a solid i . The h ee eplica es o each MST-ON ime da a se a e hen indi idually no malized om 0 o 100 and he ob ained alues a e ea ed in G aphPad P ism as eplica es alues in side-by-side sub columns o ge eliable con idence in e als (CI, 95%). E e y MST-ON ime is now anked om 1 o 6, gi ing 1 o he ime wi h he na owes CI 95% o e e y i ing pa ame e (pIC50, Hill Slope, Top, Bo om) and he highes S/N and R2 alue, and so on up o he wo s MST-ON ime in e ms o con idence in e als and i ing a which is assigned he six h alue. The lowes sum o he anked posi ions o e e y conside ed pa ame e gi es he MST-ON ime ha yields he mos ep oducible in o ma ion on h ee iden ical eplica es. 1.5s 2.5s 5s 10s 15s 20s 8.0 8.5 9.0 pIC50 pIC50 1.5s 5s 10s 15s 20s 2.5s 1.5s 2.5s 5s 10s 15s 20s 0 1 2 3 Hill Slope Hill Slope 1.5s 2.5s 5s 10s 15s 20s 1.5s 2.5s 5s 10s 15s 20s 0.7 0.8 0.9 0.97 0.98 0.99 1.00 R2 R2 1.5s 2.5s 5s 10s 15s 20s 50 In his case he bes MST-ON ime in e ms o ep oducibili y is he 20 s. - EGFR WT, 20 nM + GEFITINIB 51 Also by using 20 nM o p o ein he bes MST-ON ime esul s being he 20 s. The loss o i ing a 5 s is due o he c oss-o e o he he mopho e ic aces. - EGFR WT, 5 nM + GEFITINIB 1.5s 2.5s 5s 10s 15s 20s 9.0 9.5 10.0 pIC50 pIC50 1.5s 5s 10s 15s 20s 2.5s 1.5s 2.5s 5s 10s 15s 20s 0.0 0.5 1.0 1.5 2.0 10 20 30 Hill Slope Hill Slope 1.5s 5s 10s 15s 2.5s 20s 1.5s 2.5s 5s 10s 15s 20s 0.5 0.6 0.7 0.8 0.96 0.98 1.00 R2 R2 1.5s 2.5s 5s 10s 15s 20s 52 By using a low p o ein concen a ion i is mo e e iden ha choosing he T-Jump egion ins ead o he The mopho esis egion may lead o di e en conclusions in e ms o po ency and Hill Slope, especially when he numbe o eplica es o a single condi ions a e no high. In his case a signi ican amoun o T ace is ee in solu ion, he e o e he noise is high and iden i ying he mo e obus MST- ON ime become ele an o ely he in e ac ion in e p e a ion on us wo hy i ing pa ame e s. By applying he anking me hod, he 20 s esul ed once again as he bes MST-ON ime in e ms o ep oducibili y. The e o e, i seems ha he p o ein concen a ion had a li le impac on which MST-ON ime yield he mos obus i ing pa ame e s, e en i a high concen a ions, and consequen ly high signal- o- noise a io, e e y ime poin seems o be able o yielding compa able in o ma ion. The choice has 1.5s 2.5s 5s 10s 15s 20s 8 9 10 11 pIC50 pIC50 1.5s 5s 10s 15s 20s 2.5s 1.5s 2.5s 5s 10s 15s 20s 0.0 0.5 1.0 1.5 2.0 20 40 60 80 100 Hill Slope Hill Slope 1.5s 5s 10s 15s 20s 2.5s 1.5s 2.5s 5s 10s 15s 20s 0.0 0.1 0.2 0.3 0.75 0.80 0.85 0.90 0.95 1.00 R2 R2 1.5s 5s 10s 15s 20s 2.5s 53 mo e impac a low p o ein concen a ions, condi ion ha is he one mo e ele an o he in es iga ion o po en inhibi o s. - EGFR WT, 20 nM + LAPATINIB To con i m ha he MST-ON ime ep oducibili y was no dependen on he inhibi o , a iplica e wi h Lapa inib and EGFR WT 20 nM has been pe o med as well. Only he anking able is epo ed below. The 20 s is con i med o be also in his case he mos ep oducible MST-ON ime o he EGFR wild ype in e ac ion sys em in compe i ion wi h he T ace 199. 54 EGFR L858R The mu an o m o EGFR was also es ed in iplica es wi h Ge i inib o iden i y he bes MST-ON ime in e ms o ep oducibili y. Only he anking ables a e epo ed below. - EGFR L8583 70 nM + GEFITINIB - EGFR L8583 20 nM + GEFITINIB - EGFR L8583 5 nM + GEFITINIB F om he collec ed da a i is possible o conclude ha also o he mu a ed o m o EGFR he bes MST-ON ime o he compe i ion assays wi h T ace 199 is he 20 s. 55 ROCK-1 COMPETITION Fo he ROCK-1 compe i ion sys em he iplica e has been pe o med wi h he ollowing expe imen al condi ions and he inhibi o used o his pu pose is he o hos e ic Compound A. [ROCK-1] 50 nM [INHIBITOR] 1 µM – 0.24 nM + CONTROL POINTS [TRACER 236] 15 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01 % Tween 20 TEMPERATURE 22°C The anking able is epo ed below. On he basis o he epo ed esul s, he 10 s was he selec ed MST-ON ime o he ROCK-1 compe i ion expe imen s. ROCK-1 LABELED The ROCK-1 co alen ly labeled sys em has been in es iga ed wi h he anking me hod o bo h labeling eac ion in he p esence o he ac i e si e p o ec o : #1 and #3. - ROCK-1 LB #1 (w/ ac i e si e p o ec ion) 56 - ROCK-1 LB #3 (w/ ac i e si e p o ec ion) I is in e es ing o no e ha wo di e en labeling eac ions, as a as esul ing in wo equally compe en labeled p o eins ha yield compa able po ency in o ma ion, s ill could ely hei mos obus i ing pa ame e s on dis inc MST-ON imes. Indeed, in he case o ROCK-1 LB #1 he in e ac ion wi h he Compound A is be e desc ip ed by he 5 s iming, while o he ROCK-1 LB #3 he 2.5 s esul ed he bes MST-ON ime o choose. The e o e, o g aphically compa e wo binding cu es o igina ing in he wo di e en labeled sys ems, i has been employed a no maliza ion om 0 o 100 and he y-axis became indica ed as “F ac ion Bound”. The anking es was no pe o med o he p o ein labeled in he absence o an ac i e si e p o ec o (ROCK-1 LB #2) since he in o ma ion acqui ed om he expe imen wi h ha labeled p o ein was used as quali a i e indica ion o he in luence o he p o ec o du ing he labeling phase. As a esul , he MST-ON ime o 5 s was a bi a ily chosen o he analysis o he binding cu es in hose condi ions. 57 3.4 DATA ELABORATION AND FITTING MODELS The main da a elabo a ion has been pe o med by using G aphPad P ism 8.1.0 and he binding o compe i ion da a se ha e been i ed by using he Nonlinea eg ession (cu e i ), log(inhibi o ) s esponse - Va iable slope ( ou pa ame e s) model. Quad a ic i ing was pe o med h ough Excel Sol e Add-In o wi h he MST analysis so wa e (MO.A ini y Analysis). The Akaike's In o ma ion C i e ion (AIC)[15], a me hod de eloped o compa ing models and assis ing in de e mining which model is mo e likely o be co ec , has been used o help decide whe he o ea he p o ein concen a ion in he quad a ic equa ion as a cons an o as an adjus able pa ame e . The me hod elies on h ee pa ame e s: - N: he numbe o da a poin s - K: he numbe o pa ame e s i by nonlinea eg ession plus 1 (cons ained pa ame e s a e no coun ed) - SS: sum-o -squa e o he nonlinea eg ession The h ee alues a e hen combined in o he AIC equa ion: AIC=Nln(SS N)+2K+ 2K(K+1) N−K−1 Once he alue has been calcula ed o all he model o compa e, he AIC de i ing om he di e en models is obse ed: he model ha ing he lowe AIC sco e is mo e likely o be co ec . By conside ing he di e ence be ween wo AIC sco es (ΔAIC), i is possible o calcula e he E idence Ra io, a pa ame e indica ing how many imes one model is likely o be co ec in compa ison wi h he o he : E idence Ra io= 1 e−0.5 ΔAIC I is impo an o no e ha his me hod is no a s a is ical app oach, i is an indica ion o which model is mo e likely o be co ec and how much mo e likely. The e o e, in his wo k i has been conside ed as side suppo o be e in es iga e he dependence o po ency om he ixed pa ame e o p o ein concen a ion. 64 The expe imen al condi ions o his assay a e epo ed in he ollowing able. [EGFR] 1 µM – 35.18 nM [TRACER 199] 50 nM TITRATION 4:1, 16 POINTS DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20 TEMPERATURE 25°C Figu e 18 S oichiome y assay in which EGFR WT is i a ed om 1 µM wi h a dilu ion ac o o 1.25 o e a ixed concen a ion o T ace 199 (50 nM). The b eaking poin indica ed by he a ow is he concen a ion o EGFR WT si es capable o binding 50 nM o T ace . The sys em clea ly shows wo di e en ends o poin s and he in e sec ion o he wo linea eg ession is he b eaking poin (Figu e 18). The pa ame e s o he linea eg essions a e indica ed in he able below. LINEAR REGR. #1 LINEAR REGR. #2 B eaking Poin (nM) Slope -0.087 0.0087 390.77 Y-in e cep 762 724.4 R2 0.96 0.64 The nominal concen a ion o ace is ixed a 50 nM, he e o e i he b eaking poin appea s a 390.77 nM he pe cen age o he p o ein ha is able o bind he T ace is he 12.8% o he o al amoun o he p o ein. 0 500 1000 720 730 740 750 760 770 STOICHIOMETRY - EGFR WT EGFR WT conc (nM) FNo m 20s 390.77 nM 65 % ac i e si es (EGFR WT)= 50 nM 390.77 nM100=12.8 % The same expe imen al se has been applied o he EGFR L858R: Figu e 19 S oichiome y assay in which EGFR L858R is i a ed om 1 µM wi h a dilu ion ac o o 1.25 o e a ixed concen a ion o T ace 199 (50 nM). The b eaking poin indica ed by he a ow is he concen a ion o EGFR WT si es capable o binding 50 nM o T ace . In his case wo dis inc b eaking poin s a e egis e ed: one a 135.18 nM and he second a 642.44 nM (Figu e 19). I is in e es ing o no ice ha he non-changing T ace mobili y alues a e in be ween he wo b eaking poin s; his sugges s ha he change in mobili y a highe p o ein concen a ions may be due o a massi e a ia ion in some o he p o ein physicochemical ea u es. A possibili y is ha in hese condi ions MST egis e ed p o ein oligome iza ion. Indeed, he T ace mobili y a p o ein concen a ions abo e 642.44 nM could be a combina ion o he T ace comple ely bound o he monome and he T ace bound o he dime , a o m ha inc eases wi h p o ein concen a ion. As a esul , he second b eaking poin migh be he concen a ion a which he sys em begins o be popula ed by ha new ype o in e ac ion o m (Figu e 20). 0 500 1000 740 760 780 800 820 840 STOICHIOMETRY - EGFR L858R EGFR L858R conc nM FNo m 20s LINEAR REGR. #1 LINEAR REGR. #2 LINEAR REGR. #3 B eaking Poin #1 (nM) B eaking Poin #2 (nM) Slope -0.21 0.018 0.19 135.18 642.44 Y-in e cep 777.8 746.5 633.2 R2 0.95 0.67 0.99 135.18 nM 642.44 nM 66 This hypo hesis is consis en wi h he consolida ed knowledge abou he highe endency o L858R o dime iza ion[41] and wi h he obse a ion made du ing he T ace a ini y de e mina ion expe imen o EGFR L858R, in which he highes concen a ion poin s exhibi ed anomalous beha io in compa ison o he sigmoidal end o he lowe concen a ion poin s, indica ing ha abo e 500 nM he he mopho e ic mobili y begins o be go e ned by e en s beyond he simple T ace -p o ein in e ac ion. Figu e 20 In e p e a ion o he s oichiome y assay wi h EGFR L858R: he i s linea eg ession (o ange)is ela ed o he combina ion o T ace mobili y alone and bound o he monome ; he second linea eg ession (blue) is ela ed o he mobili y o he T ace comple ely bound o he monome ; he hi d linea eg ession (pink) could be ela ed o he coexis ence o T ace mobili y bo h bound o EGFR monome and dime . Rega ding he ac i e ac ion o he p o ein, also in his case he nominal concen a ion o he T ace is ixed a 50 nM, he e o e he b eaking poin ha appea s a 135.18 nM should be he pe cen age o he p o ein in monome ic o m ha is able o bind he T ace . The alue is he 37 % o he o al amoun o he p o ein. % ac i e si es (EGFR L858R)= 50 nM 135.18 nM100=37 % These esul s indica e ha he wo iso o ms o EGFR, wild ype and L858R, exhibi wo di e en pe cen age o ac i e si es and wo di e en endency o dime iza ion. 0 500 1000 740 760 780 800 820 840 STOICHIOMETRY - EGFR L858R EGFR L858R conc nM FNo m 20s T ace bound o he monome + T ace bound o he dime T ace bound o he monome T ace bound o he monome + T ace ee 67 % ac i e si e (able o bind he T ace 199) EGFR WT 12.8 % EGFR L858R 37.0 % As a esul , he analysis o he di ec binding da a be ween he wo iso o ms o he EGFR and he T ace 199 needs an adjus men in e ms o p o ein concen a ion (x-axis). EGFR WT (nM nominal conc) 12.8 % ac i e si es (nM) Log10 (M) 1880 240.64 -6.6 470 60.16 -7.2 117.5 15.04 -7.8 29.4 3.8 -8.4 7.3 0.9 -9.0 1.8 0.2 -9.6 0.5 0.06 -10.2 0.001* 0.0001* -12 * ic ional alue assigned o he ze o concen a ion o ob ain a eal Log10 alue EGFR WT ACTIVE FRACTION ADJUSTMENT (12.8%) T199 25 nM T199 5 nM LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING pKD 7.97 8.01 7.99 8.12 KD (nM) 10.7 9.9 10.2 7.6 R2 0.99 0.99 0.96 0.96 HILL COEFF 0.85 / 1.34 / RESPONSE AMPLITUDE 58.04 54.89 53.45 55.75 MST-on TIME 20 20 20 20 i ing pe o med by le ing “ loa ” he T ace concen a ion as adjus able pa ame e . By conside ing he eal ac ion o he p o ein compe en o he T ace binding he pKD o EGFR WT inc eases by an o de o magni ude. E en in his case bo h he logis ic and he quad a ic i ing yields qui e he same in o ma ion: a 25 nM he sys em is p obably in igh binding condi ion bu he ac ha he [T ace ]/2 alue is 12.5 nM, hus close o he eal a ini y o he T ace , migh mask he a ini y dependence o he T ace concen a ion. EGFR L858R (nM nominal conc) 37 % ac i e si es (nM) Log10 (M) 2160 799.2 -6.1 540 199.8 -6.7 135 50.0 -7.3 33.8 12.5 -7.9 8.4 3.1 -8.5 2.1 0.8 -9.1 0.5 0.2 -9.7 0.001* 0.0004* -12 * ic ional alue assigned o he ze o concen a ion o ob ain a eal Log10 alue 68 EGFR L858R ACTIVE FRACTION ADJUSTMENT (37%) T199 25 nM T199 5 nM LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING pKD 7.72 8.20 8.55 8.65 KD (nM) 18.9 6.3 2.8 2.2 R2 0.87 0.85 0.94 0.94 HILL COEFF 1.37 / 0.76 / RESPONSE AMPLITUDE 33.15 32.67 21.51 19.09 MST-on TIME 20 20 20 20 i ing pe o med by le ing “ loa ” he T ace concen a ion as adjus able pa ame e . In he case o EGFR L858R he a ini y gain is less e iden because he ac i e ac ion o he p o ein is la ge and hus al eady close o i s nominal concen a ion. Obse ing he esul s a he lowe T ace concen a ion, 5 nM, bo h he logis ic and quad a ic i ing gi e simila in o ma ion, mos likely o he same eason as he wild- ype expe imen a 25 nM: he eal a ini y would be mos p obably close he [T ace ]/2 alue, so, despi e he igh binding condi ions, a eliable i ing wi h he logis ic i ing is s ill possible. Rega ding he [T ace ] = 25 nM expe imen s, he quad a ic i ing highligh s he igh binding condi ions ha he logis ic i ing pa ially masked (wi h he excep ion o he high Hill coe icien ). In Figu e 21 and 22 i is possible o obse e he pKD alues and hei changes by conside ing he nominal p o ein concen a ion o i s ac i e ac ion. NOMINAL PROTEIN CONCENTRATION [TRACER] EGFR WT (nominal conc.) EGFR L858R (nominal conc.) 25 nM 5 nM Figu e 21 Compa ison be ween he pKD (ob ained conside ing he nominal concen a ion o he p o ein) a 25 nM and 5 nM o he T ace . In ligh yellow he alue ob ained by he logis ic i ing and in ligh pu ple he quad a ic. LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR WT pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR L858R pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR WT pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR L858R pKd 69 ACTIVE FRACTION ADJUSTMENT [TRACER] EGFR WT (12.8%) EGFR L858R (37%) 25 nM 5 nM Figu e 22 Compa ison be ween he pKD ob ained (conside ing he adjus ed p o ein concen a ion on he basis o he s oichiome y assay) a 25 nM and 5 nM o he T ace . In ligh yellow he alue ob ained by he logis ic i ing and in ligh pu ple he quad a ic. In summa y, om his MST cha ac e iza ion eme ged ha EGFR wild ype has less ac i e si es ha can bind T ace 199 han EGFR L858R; e en when his di e ence is aken in o conside a ion in he da a in e p e a ion o he di ec binding be ween he T ace and he p o eins, he EGFR L858R seems o ha e a highe a ini y o he ace han he EGFR WT, a condi ion ha is mo e e iden a lowe T ace concen a ions. Howe e , i was obse ed om hese da a ha , in some cases, quad a ic i ing yielded meaningless and inde e mina e pa ame e s, so he da a esul ing om hose i ing (and epo ed in he ables as blue i alic on ) was ob ained by ea ing he T ace concen a ion as a loa ing pa ame e . The eason o his inde e mina ion could be a non-su icien ly p ecise de e mina ion o he ac i e ac ion o he ac ha he sys em has eached a le el o complexi y whe e a quad a ic i ing is insu icien o eliably desc ibe he in e ac ion. A mo e complex i ing model, such as a cubic equa ion, may be equi ed in hese cases. The ollowing compe i ion assays wi h po en inhibi o s will in oduce a u he elemen o complexi y and a possible double igh binding condi ion will occu leading o an e en mo e di icul pa ame e de e mina ion. The e o e, o he ollowing pa ag aph (4.1.2 COMPETITION ASSAYS), he Akaike's In o ma ion C i e ion (AIC) has been applied o help decide whe he o ea he p o ein concen a ion in he quad a ic equa ion as a cons an o as an adjus able pa ame e and he alues LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR WT pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR L858R pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR WT pKd LOGISTIC QUADRATIC 7.0 7.5 8.0 8.5 9.0 EGFR L858R pKd 70 epo ed in he esul s ables will ake in o accoun his in o ma ion. Fu he conside a ions on he in e ac ion sys em in e p e a ion will ollow a he end o he a o emen ioned pa ag aph. EGFR AND TRACER 199 TIME DEPENDENCE Ano he aspec e alua ed was he change in T ace he mopho e ic mobili y o e ime. The assay was pe o med by p e-incuba ing o h ee hou s an app op ia e olume o h ee solu ions: - T ace 199 alone 5 nM - T ace 199 5 nM + EGFR WT 20 nM - T ace 199 5 nM + EGFR L858R 20 nM Du ing his ime ou capilla ies o e e y condi ion we e loaded and analyzed a e e y selec ed ime poin (10, 40, 60, 90, 120, 150 and 180 minu es). As usual, he he mopho e ic mobili y on he y- axis is e e ed o he T ace mobili y. Figu e 23 Time dependence o he complex mobili y p o ein-T ace . In o ange is epo ed he he mopho e ic mobili y o T ace 199 alone du ing ime as con ol. In g een is epo ed he mobili y o e ime o he complex EGFR WT + T ace 199 and in pink he mobili y o e ime o he complex EGFR L858R + T ace 199. In Figu e 23 i is e iden ha , also in his case, EGFR WT and EGFR L858R show di e en beha io s. Since he T ace mobili y in he bound s a e ge s close o he T ace alone mobili y, i is possible o assume he wild ype unde goes some con o ma ional change ha ei he could cause: - A loss in T ace binding. - A change in he complex mobili y ha casually app oaches he T ace alone. 0 760 780 800 820 PRE-INCUBATION: PROTEIN + T199 min FNo m 20s T199 T199 + WT T199 + L858R 10 40 60 90 120 150 180 71 In bo h cases he he mopho e ic mobili y eaches a s eady s a e a e wo hou s o p e-incuba ion. EGFR L858R also unde goes a con o ma ional change esul ing in he mopho e ic mobili y ha is simila o T ace alone, bu he p ocess is much slowe . To de e mine i he con o ma ional change was caused solely by he p esence o he T ace , he same expe imen was epea ed wi h he p e-incuba ion o he p o ein alone and he T ace addi ion igh be o e loading he capilla ies a each ime poin . Figu e 24 Time dependence o he complex mobili y p o ein-T ace p io incuba ion o he p o ein alone. In o ange is epo ed he he mopho e ic mobili y o T ace 199 alone du ing ime as con ol. In g een is epo ed he mobili y o e ime o he complex EGFR WT + T ace 199 and in pink he mobili y o e ime o he complex EGFR L858R + T ace 199. In Figu e 24 i is e iden ha , e en in he absence o T ace 199 du ing he p e-incuba ion phase, EGFR WT unde goes anyway a con o ma ional modi ica ion ha b ings T ace -p o ein complex mobili y e en close o mobili y o T ace alone. The EGFR L858R seems o unde go he same p ocess, albei a a slowe and less p onounced a e. The e o e, T ace 199 p esence in he sys em is no he di ec eason o he con o ma ional change. Howe e , in bo h cases, ime plays an impo an ole in he con o ma ional changes o he wo EGFR iso o ms and mus be conside ed du ing compe i ion assays wi h o hos e ic inhibi o s o o he expe imen s aimed a desc ibing he ime dependence o a speci ic inhibi o . Fu he mo e, i is impo an o emphasize ha he Response Ampli ude o a compe i ion assay eco ded a longe ime poin s (> 120 minu es) will end o dec ease; indeed, he unbound poin s co espond o T ace -p o ein mobili y, whe eas he high inhibi o concen a ion poin s co espond o T ace alone mobili y. This could be p oblema ic in any assay ha su e s om low Response Ampli ude om he beginning. 0 760 780 800 820 PRE-INCUBATION: PROTEIN ALONE min FNo m 20s T199 T199 + WT 10 40 60 90 120 150 180 T199 + L858R The absence o T ace 199 du ing he incuba ion led o a mo e e iden change in he complex mobili y leading i close o he T ace alone mobili y 72 4.1.2 COMPETITION ASSAYS The T ace 199 – EGFR in e ac ion sys em is now desc ibed and i is possible o bene i o his in o ma ion o app oach he cha ac e iza ion o h ee EGFR inhibi o s ha ing di e en binding modes: a. Ge i inib: ype I, e e sible po en binde b. Lapa inib: ype II, e e sible slow binde c. Osime inib, i e e sible binde As p e iously men ioned in pa ag aph 4.1, he common ea u e o hese inhibi o s is o be o hos e ic and o compe e wi h he ATP, hence wi h he T ace 199, o he binding o he ATP binding pocke . In a compe i ion assay he p o ein and T ace a e ixed a a con enien concen a ion ha enables o minimize luo escence noise caused by ee T ace in solu ion. The o hos e ic inhibi o is hen i a ed om a sa u a ing concen a ion and compe es wi h he T ace o p o ein binding. The egis e ed he mopho e ic mobili y will a y om he T ace signal when ully associa ed o he p o ein and he signal ela ed o he ee T ace in solu ion when he inhibi o concen a ion allows i s comple e displacemen . GEFITINIB - TIGHT BINDING CONDITIONS Ge i inib is a e e sible Type I inhibi o ha is EGFR L858R selec i e[40][42][43]; he e o e, he aim o his sec ion was o in es iga e he Ge i inib-EGFR sys em wi h bo h he wild ype and he mu a ed o m o be e unde s and how MST can desc ibe highly po en in e ac ions and i a good expe imen al design could highligh a di e ence in Ge i inib L858R selec i i y o e he wild ype. A c i ical aspec o ake in o conside a ion du ing he design o a compe i ion assay wi h a po en inhibi o is he co esponding a ini ies o T ace and he inhibi o i sel . To educe he noise p oduced by ee T ace in solu ion, he p o ein and T ace concen a ions may be ixed excessi ely high, esul ing in a igh binding condi ion as soon he po en inhibi o is added o he sys em. On he o he side, i is possible ha by dec easing oo much he p o ein/T ace concen a ions he luo escen di e ence be ween he bound and he unbound s a e will be ha dly no iceable, leading o an unce ain si ua ion. 73 A aluable s a egy could be o pe o m a p elimina y compe i ion expe imen by using high p o ein/T ace concen a ion which ce ainly will esul s in a good signal- o-noise a io. Once he binding o he inhibi o is con i med is possible o dec ease he p o ein/T ace concen a ion and o obse e he ela i e pIC50: i he sys em is in igh binding condi ions he pIC50 will a y wi h he p o ein concen a ion. I is hen possible o dec ease he p o ein concen a ion un il he s abiliza ion o he pIC50 is eached o ei he when he noise becomes oo high o ob ain eliable in o ma ion. In bo h cases he quad a ic i ing could help o be e in e p e he compe i ion expe imen s and o highligh an a ini y alue nea o he eal one. The Figu e 25 epo s a decision scheme ha is possible o apply when s udying a po en compound. Figu e 25 Decision scheme o help in app oaching a po en compound o a oid igh binding condi ions. 80 LAPATINIB - SLOW BINDING TYPE II INHIBITOR Lapa inib has been chosen o he in es iga ion, h ough MST, o ime dependen Type II inhibi ion in EGFR wild ype sys em. Since Lapa inib is known o ha e a ime-dependence mechanism o he in e ac ion wi h EGFR[42][44], i s in e ac ion du ing ime has been moni o ed and he selec ed ime poin s o he analysis we e 10, 40, 75, 120 and 180 minu es. Mo eo e , a u he 2.5-hou p e-incuba ion o he complex p o ein-T ace alone was done be o e adding he inhibi o , based on p e iously egis e ed p o ein-T ace changes in he mopho e ic mobili y o e ime. The in e ac ion was hen examined o wo hou s a e he inhibi o was added. In summa y, he wo expe imen s we e pe o med as ollow: 1. No p eincuba ion: p epa a ion o he i a ion (EGFR WT, T ace 199, inhibi o ) and capilla y loading o he same solu ion a he de ined ime poin s o 10, 40, 75, 120 and 180 minu es. 2. P eincuba ion (2.5h): p epa a ion o he solu ion EGFR WT-T ace 199 and p eincuba ion o 2.5h. Then addi ion o he i a ed inhibi o and capilla y loading o he same solu ion a he de ined ime poin s o 10, 40, 75, and 120 minu es. The goal o he wo analyses was o obse e he in e ac ion change o e ime while he p o ein- T ace equilib ium was changing, and hen o compa e he in e ac ion ime dependency a e he p o ein-T ace equilib ium was eached. Ge i inib was also es ed in he same condi ions as e e ence. [EGFR WT] 20 nM [INHIBITOR] 100 nM – 0.02 nM + CONTROL POINTS [TRACER 199] 5 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20 TEMPERATURE 25°C INTERACTION TIME (no p eincuba ion) 10’, 40’, 75’, 120’, 180’ INTERACTION TIME (a e 2.5h p eincuba ion) 10’, 40’, 75’, 120’ 81 NO PREINCUBATION GEFITINIB LAPATINIB pIC50 IC50 (nM) Hill slope R2 pIC50 IC50 (nM) Hill slope R2 10’ 9.47 0.3 1.18 0.99 8.62 2.4 2.15 0.99 40’ 9.46 0.4 1.36 0.98 8.57 2.7 2.15 0.99 75’ 9.48 0.3 1.25 0.98 8.45 3.5 2.64 0.98 120’ 9.60 0.3 1.52 0.97 8.54 2.9 1.80 0.96 180’ 9.61 0.2 1.03 0.96 8.48 3.3 2.33 0.96 2.5H PREINCUBATION (PROTEIN-TRACER) GEFITINIB LAPATINIB pIC50 IC50 (nM) Hill slope R2 pIC50 IC50 (nM) Hill slope R2 10’ 9.46 0.3 1.59 0.99 8.99 1.0 1.29 0.99 40’ 9.47 0.3 1.13 0.97 8.98 1.0 1.90 0.98 75’ 9.43 0.4 1.10 0.96 9.05 0.9 2.10 0.98 120’ 9.40 0.4 1.08 0.95 8.95 1.1 2.13 0.97 -14 -12 -10 -8 -6 780 790 800 810 820 830 10' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib -14 -12 -10 -8 -6 780 790 800 810 820 830 40' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 780 790 800 810 820 830 75' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 790 795 800 805 810 815 820 120' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 795 800 805 810 815 820 180' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 800 810 820 830 10' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 800 810 820 830 840 40' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 800 810 820 830 75' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib -14 -12 -10 -8 -6 805 810 815 820 825 830 120' Log[inhibi o ] FNo m 20s Ge i inib Lapa inib Osime inib 10’ 40’ 75’ 120’ 180’ 10’ 40’ 75’ 120’ 82 By conside ing he "no p eincuba ion" analysis, he pIC50 alue o Ge i inib (ligh blue) and Lapa inib (yellow) does no change signi ican ly o e ime. In e es ingly, allowing he sys em p o ein-T ace 199 o equilib a e o 2.5 hou s p io o he addi ion o he inhibi o s e ealed a signi ican di e ence be ween he wo compounds: whe eas Ge i inib kep i s pIC50 alue cons an , Lapa inib showed a 0.5 loga i hm inc ease in po ency. This new pIC50, howe e , is main ained in he ime dependency analysis ha ollows. By obse ing he in e media e concen a ion o 1.5 nM o e ime ( ed ci cle in Figu e 29 and Figu e 30), his di e ence becomes mo e e iden : Figu e 29 Compa ison be ween compe i ion cu es ob ained wi h Ge i inib (blue) and Lapa inib (yellow). Up he condi ion wi hou p eincuba ion while down he condi ion wi h 2.5h o p eincuba ion be ween p o ein and T ace . In he ed ci cles is indica ed he 1.5 nM concen a ion. Figu e 30 Focus on he 1.5 nM concen a ion and i s con ibu ion o he change in he mopho e ic mobili y o he T ace o e ime. 83 As a esul , he inc eased po ency o e ime appea s o be a ibu ed o he slow con o ma ional change o he p o ein alone o e ime, a he han he addi ion o he inhibi o i sel , which appea s o s abilize he sys em in wha e e s a e i would be in when added. This obse a ion is consis en wi h he li e a u e e idence indica ing he slow mechanism o Lapa inib as a esul o he ime equi ed o EGFR con o ma ional change o he inac i e s a e[34][36][41]. The second explo ed aspec was he di e ence in binding mode o Ge i inib and Lapa inib. Ge i inib is known o bind EGFR in i s ac i e s a e (Type I inhibi o ), while Lapa inib is a Type II inhibi o , binding he inac i e o m o he p o ein[42][57]. Thus, he goal o he ollowing expe imen al sec ion was o unde s and i Mic oScale The mopho esis could di e en ia e he binding o Type I and Type II inhibi o s. To do so, s oichiome y expe imen s we e pe o med o highligh di e ences o Ge i inib and Lapa inib in T ace 199 displacemen . Di e en ly om he s oichiome y expe imen used o iden i y he ac i e ac ion o he p o ein, in which EGFR was i a ed o e a ixed T ace concen a ion, he i a ing agen in his case is he inhibi o (Ge i inib o Lapa inib), and he p o ein- T ace complex is ixed a a concen a ion ha gua an ees igh binding condi ions. Wi h his expe imen al se up he b eaking poin s will highligh he inhibi o concen a ion capable o displace T ace 199. [EGFR WT] 140 nM [TRACER 199] 5 nM [INHIBITOR] 100 nM – 3.5 nM TITRATION 4:1, 16 POINTS DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20 TEMPERATURE 25°C 84 Figu e 31 S oichiome y assay in which Ge i inib (blue) o Lapa inib (yellow) a e i a ed o e a ixed concen a ion EGFR WT and T ace 199. GEFITINIB LINEAR REGR. #1 LINEAR REGR. #2 B eaking Poin (nM) Slope 9.12 0.095 12.75 Y-in e cep 693.4 808.4 R2 0.96 0.71 LAPATINIB LINEAR REGR. #1 LINEAR REGR. #2 LINEAR REGR. #3 B eaking Poin #1 (nM) B eaking Poin #2 (nM) Slope 0.57 4.47 0.29 14.18 35.07 Y-in e cep 700.4 645.1 791.7 R2 0.47 0.96 0.87 In e es ingly he da a show a di e en beha io o Ge i inib and Lapa inib (Figu e 31). By conside ing he high inhibi o concen a ions, bo h d ugs demons a e he abili y o displace T ace 199, indeed he T ace mobili y does no change, se ling i s alue a ound 800 uni s o no malized luo escence. Then, by dec easing he inhibi o concen a ion, he wo compounds s a o di e en ia e: Ge i inib allows he binding o he i s small amoun o T ace 199 a 12.75 nM, while Lapa inib a 35.07 nM. Lapa inib hen eaches he concen a ion o 14.18 nM a which T ace 199 is 0 10 20 30 40 50 60 70 80 90 100 650 700 750 800 850 STOICHIOMETRY GEFITINIB s LAPATINIB Inhibi o concen a ion (nM) T ace Mobili y FNo m 20s Ge i inib Lapa inib 12.75 nM 35.07 nM 14.18 nM 85 no displaced anymo e, leading o a second pla eau. This can be explained by conside ing he p esence o di e en p o ein s a es: - By i a ing Ge i inib, he b eaking poin a 12.75 nM co esponds o he inhibi o concen a ion necessa y o bind all he si es ha Ge i inib sha es wi h T ace 199. Unde ha concen a ion a su icien amoun o si es a e a ailable o he T ace binding. This ac ion amoun ed o 9 %. % Ge i inib(like) si es= 12.75 nM 140 nM 100=9 % - In he Lapa inib expe imen , he lowes concen a ions o inhibi o up o 14.18 nM esul ed non capable o T ace displacing, hen Lapa inib s a s o bind he same si es o T ace 199 displacing i . Thus, since he sys em is in igh binding condi ions and Lapa inib mus be seques e ed and deple ed e en a low concen a ions, unde 14.18 nM he Lapa inib molecules mus be bonded a a di e en si e popula ion han T ace 199. As a esul , a concen a ions below 14.18 nM T ace 199 is comple ely bound o i s si es (pla eau a 700 FNo m), whe eas Lapa inib is bound o a no isible p o ein ac ion ha T ace 199 does no ha e he abili y o bind: he 10 % o he o al p o ein amoun . % Lapa inib(like)si es= 14.18 nM 140 nM 100=10 % The i s b eaking poin co esponds o he concen a ion o Lapa inib ha sa u a es he i s popula ion (Lapa inib-like si es); abo e ha , Lapa inib begins o bind he T ace -sha ed si es (p obably he ac i e con o ma ion), o which he inhibi o has a lesse endency o bind, p e e ing o sa u a e he o he s i s . Then, he second b eaking poin a 35 nM is he Lapa inib concen a ion ha does no allow T ace binding anymo e: hus, i co esponds o he o al amoun o Lapa inib si es ( he sum o Lapa inib-like and T ace -like). This ac ion is a ound 25 % o he o al amoun . % Global Lapa inib si es= 35.07 nM 140 nM 100=25 % Finally, he esidual amoun , i.e. 75 %, is e e ed o he p o ein ac ion no capable o binding T ace 199 and no highligh ed by Lapa inib binding. In his ac ion can be lis ed he p o ein amoun dena u ed and all he p o ein molecule in a con o ma ional s a e no able o bind T ace 199 and hus no isible. 86 The e o e, some assump ion can be made: - Lapa inib binds wo di e en p o ein s a es (i is possible o specula e he ac i e and he inac i e s a e) one o which is sha ed wi h T ace 199 (o he wise displacemen could no be possible); howe e Lapa inib p e e s o bind he inac i e s a e a low concen a ions and s a s o occupy ac i e si es only a e he sa u a ion o he inac i e o m. - T ace 199 binds only one o he wo s a es highligh ed by Lapa inib (p esumably he ac i e s a e), o he wise he pla eau s a e a low Lapa inib concen a ion would no be isible. - Ge i inib binds i s all he T ace si es bu i is no possible o know by hese expe imen s i i occupies also o he si es wi h a lowe a ini y once he T ace si es a e comple ely sa u a ed. - The highe p o ein ac ion is howe e cons i u ed by p o ein non-ac i e in e ms o T ace 199 binding. These conside a ions a e summa ized in Figu e 32. Figu e 32 Rep esen a ion o he di e en EGFR popula ion highligh ed om Ge i inib and Lapa inib s oichiome y assays. Fo Lapa inib g aph (on he igh ) he shadowed sec ion is e e ed o he ac ion ob ained o di e ence om he Global and he Lapa inib-like si es. I is easonable o assume ha his ac ion o e laps wi h he ac ion iden i ied by Ge i inib s oichiome y assay (on he le ). In conclusions, MST allowed he di e en ia ion o Type I and Type II inhibi o s, by highligh ing he p esence o mul iple con o ma ional s a es and he inhibi o s di e en p opensi y o bind hem. % GEFITINIB-LIKE SITES % LAPATINIB-LIKE SITES 9 % GEFITINIB-LIKE 91 % OTHER 75 % OTHER 25 % GLOBAL 10 % LAPATINIB-LIKE 87 OSIMERITINIB - IRREVERSIBLE BINDING As discussed in he pa ag aph 1.1.6 - MODES OF INHIBITOR INTERACTIONS - COVALENT BINDING, he i e e sible mechanism is usually desc ibed om a kine ic poin o iew, wi h he kinac /Ki cons an , a po ency indica ion ha akes in o conside a ion he inhibi o a ini y (Ki) and he i e e sible inac i a ion a e (kinac ). By pe o ming a classical binding o compe i ion assay, is s ill possible o ob ain a sigmoidal cu e and an appa en IC50 alue ha , in his case, is dependen on he incuba ion ime. Indeed, a p ope po ency measu emen elies on speci ic kine ic assays, bu ha does no di e much om a e e sible inhibi o binding cu e in e ms o shape and i ing. As i can be obse ed in Figu e 33 (in which he same sample p epa a ions ha e been loaded and es ed in MST a 10’ and a e h ee hou s o incuba ion a oom empe a u e), he di e ence in binding mechanisms o Ge i inib ( e e sible) and Osime inib (i e e sible) eme ges only by obse ing hei po ency o e ime, no in any pa icula changes in hei sigmoidal shape. [EGFR] 20 nM [INHIBITOR] 100 nM – 0.02 nM + CONTROL POINTS [TRACER 199] 5 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20 TEMPERATURE 25°C Figu e 33 Compe i ion cu es wi h Ge i inib (le ) and Osime inib ( igh ) moni o ed o e ime a 10’ and 180’. GEFITINIB OSIMERTINIB pIC50 IC50 (nM) Hill slope R2 pIC50 IC50 (nM) Hill slope R2 10’ 9.5 0.3 1.33 1 8.7 2.2 0.80 1 180’ 9.7 0.2 1.33 0.98 9.2 0.7 0.78 0.97 The e o e, an impo an aspec o e alua e when dealing wi h unknown po en ially co alen inhibi o s, is o ind a way o es he e e sibili y o he binding. This ea u e is usually in es iga ed -14 -12 -10 -8 -6 0 50 100 GEFITINIB Log[inhibi o ] M F ac ion Bound 10' 180' -14 -12 -10 -8 -6 0 50 100 OSIMERTINIB Log[inhibi o ] M F ac ion Bound 10' 180' 88 wi h enzyma ic assays which indica e he ac i i y eco e y o a a ge a e he incuba ion wi h he inhibi o o in e es and he subsequen ly apid and massi e dilu ion o he p o ein-inhibi o complex (Jump-Dilu ion)[17]. Ne e heless, his ype o app oach is challenging o pe o m in a compe i ion se ing like he one ha has been applied so a . The main issues in ol e he p o ein and inhibi o concen a ions and he high po ency o he inhibi o . Indeed, he ypical p o ocol would equi e a p e-incuba ion o he p o ein a a concen a ion 100- old highe han he concen a ion used in he ollowing compe i ion assay and he inhibi o a a concen a ion 10 imes highe han i s IC50. By conside ing he EGFR WT sys em and he Ge i inib as he e e ence compound, he ini ial condi ions should be: Ini ial conc. Final conc. [EGFR WT] 2 µM 100x he inal conc. 20 nM [Ge i inib] 3 nM 10x i s IC50 (0.3 nM) 0.03 nM [T ace 199] / 5 nM Good inal concen a ions bu no ideal ini ial concen a ions due o he massi e p o ein/inhibi o a io. I is clea ha in hese expe imen al condi ions (p o ein:inhibi o > 600- old) he bound ac ion o he p o ein can be neglec ed also in he ini ial s a e and wi h e e y ype o inhibi o making he measu emen incapable o dis inguishing be ween e e sible and i e e sible in e ac ion: T ace 199 would be able o bind i s small amoun o p o ein e en in he p esence o Ge i inib. This p oblem would be o e come wi h a less po en inhibi o o which he 10- old IC50 ini ial concen a ion migh be nea o he p o ein concen a ion. On he o he hand, by dec easing he p o ein concen a ion a nanomola alues o allow Ge i inib binding a a signi ican numbe o si es (e.g. 40 nM, a which he ac i e si es will be 5 nM, based on he 12.8% ac i e ac ion), he inal concen a ion a e a 100- old jump dilu ion would be oo small (0.4 nM), and he addi ion o T ace 199 as luo escen ma ke a i s consolida ed concen a ion o 5 nM would comple ely co e he complex signal. Mo eo e , dec easing T ace 199 concen a ion below 5 nM would esul in a poo luo escence signal. Ini ial conc. Final conc. [EGFR WT] 40 nM 100x he inal conc. 0.4 nM [Ge i inib] 3 nM 10x i s IC50 (0.3 nM) 0.03 nM [T ace 199] / 5 nM Good ini ial concen a ions bu no ideal inal concen a ions due o he low [EGFR WT] ela ed o he [T ace 199]. 89 Fo hese easons in he con ex o he p esen wo k an al e na i e me hod has been de eloped o he di e en ia ion o e e sible and i e e sible inhibi o s in he EGFR wild ype sys em. This me hod, summa ized in Figu e 34, in ol es h ee phases: 1. P e-incuba ion a wo di e en imes, 60 and 150 minu es, o he p o ein and he inhibi o ( e e sible o i e e sible) a a low concen a ion ha s ill allows he sa u a ion o all he p o ein ac i e si es. The sys em is hen p elimina y es ed in a Binding Check expe imen by adding 5 nM o he T ace 199 o con i m he comple e sa u a ion o he p o ein. 2. Pu i ica ion o he complex h ough a size exclusion spin column (Zeba™ Spin Desal ing Columns, 7K MWCO, 0.5 mL, The mo), which should e ain e e y molecule smalle han 7 kDa no co alen ly bound o he p o ein. 3. A second Binding Check expe imen wi h he addi ion o he T ace 199 5 nM a e he pu i ica ion, in o de o iden i y which inhibi o s ha e been e e sibly emo ed om he sys em and now allow he binding o he T ace o he p o ein. Figu e 34 Scheme o he de eloped me hod o he di e en ia ion o e e sible and i e e sible ligands. Fo he se up o he expe imen wo inhibi o concen a ions ha e been es ed, 100 nM and 10 nM o con i m in a single concen a ion expe imen ha bo h we e sa u a ing concen a ions. A e he p o ein and inhibi o p e-incuba ion o 10 minu es, 5 nM o T ace 199 we e added. In he g aph e e y do co esponds o a single capilla y scanned in o he ins umen and he y-axis is always e e ed o as he T ace mobili y ha in e e y condi ion is added a a concen a ion o 5 nM. As i can be seen in Figu e 35, when he Ge i inib is added o he sys em a bo h 100 nM (pink do s) and 10 nM (ligh blue do s) he esponse is he same, he T ace canno bind he p o ein; so [EGFR WT] 20 nM [Ge i inib] 100 nM o 10 nM 10’ p e-incuba ion o p o ein-inhibi o [T ace 199] 5 nM 96 ALLOSTERIC INTERACTION Figu e 41 Wi h allos e ic compounds a disc epancy be ween Compe i ion and Di ec Binding assays could occu , due o he missing ATP luo escen displacemen du ing he Compe i ion Assay. 4.2.1 COMPETITION ASSAYS INTERACTION WITH KINASE TRACER 236 The in e ac ion be ween ROCK-1 and i s luo escen ATP analog (Kinase T ace 236, The mo) has also been in es iga ed in his case. In analogy wi h he EGFR sys em, wo T ace concen a ions ha e been es ed, 25 nM and 5 nM, and he esul s a e epo ed in Figu e 42. [ROCK-1] 15 µM – 3.6 nM + CONTROL POINTS [TRACER 236] 25 nM / 5 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01% Tween 20 TEMPERATURE 22°C Figu e 42 Binding assay be ween ROCK-1 and T236 a 25 nM (on he le ) and 5 nM (on he igh ). -14 -12 -10 -8 -6 -4 800 810 820 830 T ace 236 25 nM Log[ROCK-1] (M) FNo m 10s T236 25nM T236 5nM -14 -12 -10 -8 -6 -4 815 820 825 830 835 T ace 236 5 nM Log[ROCK-1] (M) FNo m 10s T236 25nM T236 5nM 97 ROCK-1 T236 25 nM T236 5 nM LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING pKD 6.44 6.45 6.06 6.22 KD (nM) 359.4 352.0 878.11 598.27 R2 0.99 0.99 0.97 0.97 HILL COEFF 1.05 / 0.73 / RESPONSE AMPLITUDE 22.31 22.52 13.68 11.85 MST-on TIME 10 s 10 s 10 s 10 s In his case, he quad a ic and he logis ic i ing a 25 nM yield he same a ini y alues, well abo e he nominal T ace concen a ion, sugges ing ha he sys em should no be in igh binding condi ions. By dec easing he T ace concen a ion o 5 nM, he a ini y alue does no inc ease, con i ming ha he sys em is no in igh binding condi ions and he logis ic i ing is su icien o desc ibe he in e ac ion. OPTIMIZATION OF THE INTERACTION WITH COMPOUND A Th ee p o ein doses we e es ed o be e cha ac e ize he in e ac ion be ween ROCK-1 and he o hos e ic Compound A: 200 nM, 50 nM, and 20 nM (Figu e 43). The expe imen al condi ions a e de ailed in he able below. The T ace concen a ion o 15 nM was chosen since 5 nM ied in he p elimina y es ing p oduced an insu icien luo escence signal. [ROCK-1] 200 nM, 50 nM, 20 nM [INHIBITOR] 1 µM – 0.24 nM + CONTROL POINTS [TRACER 236] 15 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01% Tween 20 TEMPERATURE 22°C 98 Figu e 43 Compe i ion assays wi h ROCK-1 es ed a h ee concen a ions (200 nM, 50 nM and 20 nM), T ace 236 and Compound A. ROCK-1 (nominal conc.) LOGISTIC FITTING ( ou pa ame e s) QUADRATIC FITTING pIC50 IC50 (nM) Hill slope R2 pIC50 IC50 (nM) R2 200 nM 7.11 77.3 1.81 0.97 7.02 94.5 0.96 50 nM 8.22 6.0 0.75 0.95 8.64 2.3 0.93 20 nM 7.92 12.0 0.37 0.77 7.79 16.3 0.69 When he p o ein concen a ion is educed om 200 nM o 20 nM, he noise inc eases due o he inc eased concen a ion o ee T ace 236 in solu ion. E en wi h he bes R2 alue, he 200 nM condi ion has a Hill slope g ea e han one, indica ing ha he expe imen al condi ions a e no ideal o desc ibing he sys em: he high p o ein concen a ion likely causes inhibi o deple ion, which is e lec ed in he isible amoun compe ing wi h he T ace , howe e small i may be due o he ace low a ini y o he p o ein. As e idence, dec easing he p o ein concen a ion changes he pIC50 alue o Compound A. In his case, 50 nM is p obably he bes comp omise be ween a good i and eliable in o ma ion on po ency, bu due o he low a ini y o he T ace 236 o he p o ein, i is impo an o conside ha e e y po en inhibi o migh be unde es ima ed in e ms o pIC50. The e o e, as p e iously men ioned, in hese condi ions, he compe i ion assay could be solely applied o ob ain quali a i e in o ma ion abou in e ac ion, elying on he di ec binding assay o ob ain quan i a i e in o ma ion. -14 -12 -10 -8 -6 -4 805 810 815 820 825 ROCK1 200 nM / T236 15 nM Log[Compound A] (M) FNo m 10s -14 -12 -10 -8 -6 -4 810 812 814 816 818 820 ROCK1 50 nM / T236 15nM Log[Compound A] (M) FNo m 10s -14 -12 -10 -8 -6 -4 805 806 807 808 809 810 ROCK1 20 nM / T236 15nM Log[Compound A] (M) FNo m 10s 99 COMPETITION WITH ALLOSTERIC COMPOUNDS Once he expe imen al condi ions o he compe i ion assay a e chosen, he allos e ic inhibi o s (Compound B and Compound C) ha e been es ed (Figu e 44). [ROCK-1] 50 nM [INHIBITOR] 1 µM – 0.24 nM + CONTROL POINTS [TRACER 236] 15 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01% Tween 20 TEMPERATURE 22°C Figu e 44 Compe i ion assay wi h ROCK-1, T ace 236 and Compounds A, B and C. pIC50 IC50 (nM) Hill slope Response Ampli ude R2 COMPOUND A 7.11 77.3 1.81 5.8 0.97 COMPOUND B ~ 9.6 ~ 0.2 1.50 1.5 0.34 COMPOUND C - - - - - As can be seen in Figu e 44, he only compound gi ing a obus compe i ion cu e is he Compound A. By es ing he compounds in a single concen a ion es (Binding Check, Figu e 45) a 1 µM, no signi ican di e ence is egis e ed be ween he T ace 236 mobili y only bound o he p o ein (da k blue) and he T ace 236 egis e ed in he p esence o he Compounds B and C (pu ple and ed do s). -14 -12 -10 -8 -6 -4 810 815 820 ROCK-1 COMPETITION Log[inhibi o ] (M) T ace mobili y Compound A Compund B Compound C Compound B Compound C 100 Figu e 45 Binding Check wi h ROCK-1 in complex wi h T ace 236 (da k blue), he same complex wi h he addi ion o o hos e ic Compound A (ligh blue) and he allos e ic Compounds B and C (pu ple and ed). Binding Check P Value Signi ican ? ROCK-1 s. Compound A <0.0001 Yes (****) ROCK-1 s. Compound B 0.28 No ROCK-1 s. Compound C 0.95 No Compound A s. Compound B <0.0001 Yes (****) Compound A s. Compound C <0.0001 Yes (****) Compound B s. Compound C 0.51 No Howe e , in he compe i ion assay, he Compound B showed a sligh sigmoidal end, albei wi h a e y low R2 and Response Ampli ude, while he Compound C showed a endency o inc ease T ace mobili y a highe concen a ions. To e i y i he ends o he wo allos e ic compounds would be con i med in a eplica e and a highe concen a ions, bo h compound B and C we e addi ionally es ed wi h an ini ial i a ing concen a ion o 10 µM. [ROCK-1] 50 nM [INHIBITOR] 10 µM – 2.4 nM + CONTROL POINTS [TRACER 236] 15 nM TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01% Tween 20 TEMPERATURE 22°C 101 Figu e 46 Compa ison be ween wo compe i ion assays wi h Compound B, one s a ing om 1 µM (pu ple) and he second s a ing om 10 µM (yellow). pIC50 IC50 (nM) Hill slope Response Ampli ude R2 COMPOUND B (1 µM) ~ 9.6 ~ 0.2 1.50 1.5 0.34 COMPOUND B (10 µM) ~ 6.7 ~ 218.0 0.53 2.1 0.53 The poin ends o Compound B in bo h es ed condi ions a e no obus enough o conclude ha T ace 236 displacemen is occu ing (Figu e 46). Howe e , on he basis o he sligh end o he poin s owa d lowe T ace mobili y alues, i is no possible o comple ely exclude he possibili y ha inc eased Compound B concen a ions could impac he T ace 236-ROCK-1 in e ac ion o ha e some non-quan i iable dis al e ec on he ATP binding si e. Figu e 47 Compa ison be ween wo compe i ion assays wi h Compound C, one s a ing om 1 µM ( ed) and he second s a ing om 10 µM (ligh pink). -14 -12 -10 -8 -6 -4 815 816 817 818 819 820 COMPOUND B Log[inhibi o ] (M) T ace mobili y Compund B (10 M) Compound B (1 M) -14 -12 -10 -8 -6 -4 816 818 820 822 824 COMPOUND C Log[inhibi o ] (M) T ace mobili y Compound C (10 M) Compound C (1 M) 102 pIC50 IC50 (nM) Hill slope Response Ampli ude R2 COMPOUND C (1 µM) - - - - - COMPOUND C (10 µM) ~ 4.9 ~ 1263 1.08 6.10 0.65 Also o Compound C no obus i ing is occu ing in he compe i ion assay. A 10 µM an inc eased T ace 236 mobili y is egis e ed (Figu e 47). Since he T ace is mo ing in he opposi e di ec ion o he ee-T ace mobili y ( egis e ed a high concen a ions o Compound A), i is no possible o assume ha a displacemen is aking place, he e o e wo o he hypo heses can be made on his si ua ion: - The highes concen a ions poin s o Compound C induce an abe an mo emen o he p o ein-T ace complex due o i s closeness o he solubili y limi and o he po en ial o ma ion o agg ega es. - Compound C a highe concen a ions is able o bind he p o ein in ano he si e wi h espec o he T ace 236 bu also o induce a con o ma ional change esponsible o he di e en mobili y o he e na y complex ROCK-1-T ace 236-Compound C. In summa y, om he compe i ion expe imen al se i is possible o conclude ha Compound B and Compound C ha e a di e en binding mode han he o hos e ic Compound A. 4.2.2 DIRECT BINDING ASSAYS ROCK-1 COVALENT LABELING AND BINDING COMPETENCE TEST To pe o m a di ec binding assay he p o ein ROCK-1 needed o be luo escen ly labeled. The labeling p o ocol chosen o his pu pose is he Monoli h P o ein Labeling Ki RED-NHS 2nd Gene a ion (NanoTempe ) and, a e he labeling eac ion, a binding es wi h Compound A was pe o med o e i y he binding compe ence o he p o ein. Two labeling condi ions we e es ed: 1. Labeling in he p esence o a low po en o hos e ic inhibi o (Compound X, pIC50 < 7) o p o ec he ATP binding pocke . 2. Labeling in he absence o any ligand (wi h he only addi ion o DMSO a he same pe cen age o he condi ion o he ac i e si e p o ec ion). 103 The eason o his compa ison is o e i y he possible in e e ence o he co alen label wi h he binding compe ence o he p o ein. LABELING #1 (w/ ac i e si e p o ec ion) LABELING #2 [ROCK-1] 10 µM Dilu ion and labeling bu e Labeling Bu e NHS (NanoTempe ) 130 mM NaHCO3, 50 mM NaCl, pH 8.2-8.3 P e-incuba ion (ac i e si e p o ec ion) Compound X (pIC50 < 7) 100 µM DMSO 1 % Time o p e-incuba ion 20 min Dye RED-NHS 2nd Gene a ion (NanoTempe ) [Dye] 30 µM Incuba ion 30’ in he da k a 25°C Pu i ica ion sys em PD SpinT ap G-25 (Cy i a) Pu i ica ion bu e 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT A e he labeling eac ion he p o eins concen a ion and he Deg ee o Labeling (DOL) was measu ed h ough he acquisi ion o a UV spec a (Figu e 48). Figu e 48 UV spec a o wo labeled p o ein: on he le ROCK-1 labeled in he p esence o Compound X o p o ec he ac i e si e, while on he igh ROCK-1 labeled in he absence o ac i e si e p o ec o . LABELING #1 (w/ ac i e si e p o ec ion) LABELING #2 [ROCK-1 LB] 1.36 µM 1.19 µM DOL 0.28 0.34 The wo di e en labeling eac ions led o simila esul s in e ms o p o ein concen a ion and DOL. The main di e ence is he p esence o an abso p ion peak a 375 nm, p obably due o an incomple e 300 400 500 600 700 0.00 0.02 0.04 0.06 0.08 0.10 LABELING #1 (w/ ac i e si e p o ec ion) Wa elengh (nm) Abso bance 300 400 500 600 700 0.00 0.02 0.04 0.06 0.08 0.10 LABELING #2 Wa elengh (nm) Abso bance 104 emo al o Compound X om he sys em. The low a ini y o Compound X is a necessa y ea u e o acili a e i s emo al du ing he pu i ica ion phase, bu he use o a spin column o his phase p obably educed he e iciency o he p ocess compa ed o a slowe G a i y column (e.g. PD MiniT ap G-25, Cy i a). I necessa y, i has been demons a ed ha an addi ional pu i ica ion s ep in a size exclusion spin column can be pe o med o be e emo e Compound X. Howe e , he esidual p esence o he compound seems no o in e e e in he subsequen in e ac ion assay wi h he e e ence Compound A, p obably due o Compound X low po ency and he u he dilu ion o he binding assay. Indeed, he ROCK-1 LB #1 demons a ed a obus in e ac ion p o ile wi h Compound A a e he di ec binding assay was pe o med o con i m he binding compe ence o he labeled p o eins. On he con a y, he ROCK-1 LB #2 seemed no o be able o yield eliable binding in o ma ion. This can be due o in e e ence om he luo escen label in he in e ac ion be ween he p o ein and he e e ence Compound A (Figu e 49). [ROCK-1 LB] 20 nM [INHIBITOR] 1 µM – 0.24 nM + CONTROL POINTS TITRATION 1:3, 8 POINTS IN DUPLICATE DILUTION BUFFER 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT, 0.01% Tween 20 TEMPERATURE 22°C Figu e 49 Di ec binding assay wi h Compound A and ROCK-1 labeled in he p esence o an ac i e si e p o ec o (on he le ) and in he absence o an ac i e si e p o ec o (on he igh ). -14 -12 -10 -8 -6 -4 834 836 838 840 842 ROCK-1 LB #1 (w/ ac i e si e p o ec ion) + COMPOUND A Log[inhibi o ] (M) FNo m 5s -14 -12 -10 -8 -6 -4 806 808 810 ROCK-1 LB #2 + COMPOUND A Log[inhibi o ] (M) FNo m 5s 105 LABELING #1 (w/ ac i e si e p o ec ion) LABELING #2 pIC50 8.75 ~9.65 IC50 (nM) 1.8 0.2 R2 0.96 0.54 HILL COEFF 1.28 9.61 RESPONSE AMPLITUDE 5.03 2.29 MST-on TIME 5 s 5 s These da a sugges ha p o ec ion o he binding si e wi h an o hos e ic compound migh be a alid choice in o de o obse e and quan i a i ely desc ibe he in e ac ion wi h o hos e ic inhibi o s and con i m he p o ein binding compe ence a e labeling. The p esence o a ligand du ing he labeling phase p obably induces a educ ion in p o ein mobili y and a closu e o i s s uc u e, a oiding he labeling o no mally bu ied esidues ha a e possibly in ol ed in he o ma ion o binding pocke s. To e i y he obus ness and ep oducibili y o he labeling p ocedu e, an addi ional labeling unde he same condi ions as Labeling #1 has been pe o med, and he esul s a e epo ed in Figu e 50. Figu e 50 UV spec a o wo labeled p o ein: on he le he al eady epo ed ROCK-1 labeled in he p esence o Compound X o p o ec he ac i e si e, and on he igh a eplica e in he same expe imen al condi ions. LABELING #1 (w/ ac i e si e p o ec ion) LABELING #3 (w/ ac i e si e p o ec ion) [ROCK-1 LB] 1.36 µM 1.20 µM DOL 0.28 0.28 300 400 500 600 700 0.00 0.02 0.04 0.06 0.08 0.10 LABELING #1 (w/ ac i e si e p o ec ion) Wa elengh (nm) Abso bance 300 400 500 600 700 0.00 0.02 0.04 0.06 0.08 0.10 LABELING #3 (w/ ac i e si e p o ec ion) Wa elengh (nm) Abso bance 112 om he pe spec i es o bo h he inhibi o -p o ein complex and he p o ein alone con o ma ional changes o e ime. Fu he mo e, s oichiome y analysis wi h Ge i inib (Type I) and Lapa inib (Type II) e ealed di e en p o ein s a es as well as a di e en p opensi y o inhibi o s o bind hem. The s udy o i e e sible binding wi h Osime inib enabled hen he use o MST o de elop an al e na i e app oach o Jump Dilu ion o he di e en ia ion o e e sible and i e e sible inhibi o s, a po en ially use ul ool o he disco e y o unknown co alen d ugs. Finally, ROCK sys em pe mi ed o apply MST o he di e en ia ion o o hos e ic and allos e ic binde s, leading o aluable complemen a y in o ma ion h ough he combina ion o compe i ion and di ec binding assays. Fu he mo e, an in es iga ion o he bes labeling condi ions ound disc epancies be ween he wo es ed allos e ic compounds, e ealing a possible dis inc in e ac ion mechanism due o hei di e en sensi i i y o he dye posi ion on he p o ein. In conclusion, Mic oScale The mopho esis eme ged as a aluable ool o he cha ac e iza ion o se e al aspec s o p o ein in e ac ions, in addi ion o he simple de e mina ion o po ency; hus, his p ojec helped o be e unde s and MST po en iali y and applica ions h ough he es ablishmen o benchma ks o se e al binding modes, and he acqui ed knowledge could po en ially be applied o he u u e in es iga ion o new chemical en i y mechanisms, om he ea ly o he la e s ages o D ug Disco e y pipeline. 113 6. BIBLIOGRAPHY [1] X. 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