CO NAM ŘEKNE KAPILÁRNI ELEKTROFORÉZA OHLEDNĚ INTERAKCÍ BIOMOLEKUL Lenka Michalcová INTERAKCE VS. CHEMICKÁ REAKCE 2 3 TYPY VAZEB • Kovalentní - sdílí elektrony • Polární vs. nepolární • Silné / pevné • Ireverzibilní nebo jen velmi obtížně reverzibilní • 150 - 450 kJ/mol • Alkylující cytostatika, organofosfáty • Nekovalentní - nevytváří společný elektronový obal • Slabé interakce • Reverzibilní interakce - typické pro farmaka • Účastní se dějů - rozpouštění, krystalizace sublimace, desublimace, vypařování • iont-iont, dipól-iont, dipól-dipól, Wan der Waals, Coulombické int., H-můstky, hydrofóbní interakce SILA VAZBY Intramolekulární vs. intermolekulární 3 - 50 kJ/mol — B.Vuľ&a van der Waalsovymi silám 1- ch, / :h, ch, .Xcŕ Óh,4-5' \ :h, ch, j \ i i / - 1. ch; . ' i nd Like v an é nnnmontálnl, fluktuující dipóly 0,1 - 10 kJ/mol Hydrogen Bonding in Biological Systems generic (acceptor) ^||,........., [_|_ p (donor] DNA Q llllllllllll] |-| — -i N\ DNA (bass pairing', \ IM iimiimiiii H — // N\ protein ia-helix, -sheet) ^^=0 ■■■■■■■■■■■■■■ i—i— cellulose \-\ (crystatUne assemblies) \ 0......mull H — / o 2 kJ/mol H-můstek 10 - 40 kJ/mol 4 LULLMANN, Heinz, Klaus MOHR a Lutz HEIN. Barevný atlas farmakologie. Vyd. 4., české. Praha: Grada, 2012. ISBN 978-80-247-3908-3. Nature 405, 681 (2000) 7 BIOMAKROMOLEKULY • Struktura všech biomolekul je dána kombinací kovalentních a nekovalentních vazeb • Kovalentní vazby - statické, málo ovlivněny prostředím • Nekovalentní vazby (hydrofobní interakce, vodíková vazba a elektrostatické přitahování) - dynamická rovnováha, ovlivnitelné faktory jako je teplota, obsah iontů a pH • Biomolekuly musí být - „flexibilní a tuhé" pro dosažení řádného fungování, a proto síly, které drží molekulární tvar, musí dosahovat rovnováhy s prostředím BIOMOLEKULY -SLABÉ INTERAKCE 8 • Rozpoznání • Struktura • Funkce BIOMOLEKULARNI INTERAKCE Typy látek Protein - ligand Protein - Protein Protein - NK NK- ligand atd. • • • • Sacharidy, lipidy, proteiny, NK 9 10 INTERAKCE RECEPTOR-LIGAND kon koff P + D ^PD koff [PD] kon 1 b [P][D] koff Kt d AG° = -RTlnKb= RTlnK( d AG° = AH°-TAS° AG < 0 exergonické AH < 0 exothermické AG > 0 endergonické AH > 0 endothermické 11 KB VS. KD VS. AG° Kd (M) Kb AG° (kJ/mol) (kcal/mol) No affinity (high millimolar) >10-1 <101 >(-5.9) >(-1.4) Very weak affinity (low millimolar) 10-3 - 10-1 101 - 103 (-18)-(-5.9) (-4.3)-(-1.4) Low affinity (high micromolar) 10-5 - 10-3 103 - 105 (-30)-(-18) (-7.1)-(-4.3) Moderate affinity (low micromolar) 10-6 - 10-5 105 - 106 (-36-(-30) (-8.5)-(-7.1) High affinity (nanomolar) 10-9 - 10-6 106 - 109 (-53)-(-36) (-13)-(-8.5) Very high affinity (pico/femtomolar) 10-14- 10-9 109 - 1014 (-83)-(-53) (-20)-(-13) Effectively irreversible (low femtomolar) <10-14 >1014 <(-83) <(-20) 12 PROČ STUDOVAT INTERAKCE? Analýza povahy intermolekulárních interakcí - typ interakce (hydrofobní, H-můstky,...) • Pochopeni biologických procesu - dochází k interakci? jak silná? co vše jí ovlivňuje? • Aplikace poznatků - ve vědě / lékařství • Vývoj léčiv • Biotechnologie • Objev podstaty biologické děje / onemocnění Fyzikálni chemie Biologie 14 TECHNIKY MERENI Fyzikálně-chemické vlastnosti látek a prostředí Rychlost analýz /mÉS^ĚĚk f*% Studovaný systém VBĚZ^^^N Dostupnost Komplementarita „In„ Moderní vs. klasický přístup INFORMACE MERENI Kvalitativní Semi-kvantitativní Kvantitativní Metody Separační Bez separace Ultracentrifugace Ultrafiltrace Rovnovážná dialýza LC, CE Spektrofotometrické metody Kalorimetrické metody SPR, AFM, MST 16 Databáze Virtualní screening Molekulární dokování N 17 Technique High affinity intermediate Low affinity Low sample Online Solution Fast Label Insensitive to (nM-pM) affinity Kq tmM-M) amount detection based analysis free contaminants KD{\Q*-iffm (^Oug) (min-h) Surface plasmon resonance s X X t InmnMjíii [Dt«] : plateau height. |IJf,=] : plateau height [D(r„] : peak area Change in u of the injected compound [DtMiui]: peak area 1 Df:c=] : peak area Change in ct of one of the species Main Advantages anil Drawbacks - slow and fast kinetics systems: analysis possible - multiple equilibria easily studied - stoichiometric information - binding percentage easily obtained - slow kinetics systems (K.> If/NT1) required - multiple equilibria easily studied - stoichiometric information - binding percentage easily obtained - highly purified samples not required - knowledge of the exact [D] not needed - highly purified samples not required - enantiomeric separation -[P]»[D] - stoichiometric information not obtainable - multiple equilibria : difficult Hi deal with - multiple equilibria easily studied - stoichiometric information - binding percentage easily obtained - highly purified samples not required - require higher amount of P and D than other CE set-ups - HGK absorption too low : poor sensitivity - BGE absorption loo strong : detector saturation - binding percentage easily obtained by VP but not by VACE - highly purified samples not required Vuignier, K., et al.: Drug-protein binding: a critical review of analytical tools, Anal. Bioanal. Chem. (2010) 398, 53-66 VACE Setup Measured peak area, [LfreJ migration time, parameters A u peak area, [Lbouild] peak area, [Lfree] migration time, An 35 Informa- K„ n,,K2, n2 K( tion estimated K,. n„ K2, n, K,. n,, K2, n2 S-vacancy:K, Conditions ^l jt Us, uSL uSL * u.L H*. = Us m,. = us L-vacancy: K,. n„ K2, n2 S-vacancy: Nevidalovä, H., et al.: Electrophoresis, v tisku L-vacancy: 36 CHARAKTERISTICKÉ ELEKTROFOREGRAMY Michalcová,L., Glatz, Z., Chem.Listy 110, 249-257 (2016) t; □ Q- : = 3 O í d T -n H S 3 O S 00 38 JAKA METODA NA CO? FA, HD, VP, ACE, VACE - , - , - , ACE, VACE FA, HD, VP, ACE, VACE FA, HD, VP, - , VACE - , - , - , - , VACE Busch, M.H.A. et al., J.Chromatogr.A 777, 329-353 (1997) 39 JAKÁ METODA NA CO? FA, HD, VP, ACE, VACE - , - , - , ACE, VACE FA, HD, VP, ACE, VACE FA, HD, VP, - , VACE - , - , - , - , VACE Busch, M.H.A. et al., J.Chromatogr.A 777, 329-353 (1997) 40 FA, HD, VP, - , VACE - , - , - , - , VACE JAKA METODA NA CO? ßp — ßc ± ßü ßp ± ßc — ßü ßp — ßc ± ßü \ip±\ic± \iD ßp ± ßc — ßü FA, HD, VP, ACE, VACE - , - , - , ACE, VACE FA, HD, VP, - , VACE D detekovatelné 1 P detekovatelné ßp — ßc ± ßü \ip±\ic± \iĽ ßp ± ßc — ßü í Komplex 1:1 FA, HD, VP, - , VACE - , - , - , ACE, VACE FA, HD, VP, ACE, VACE PDn ßplSgßD Busch, M.H.A. et al., J.Chromatogr.A 777, 329-353 (1997) 41 ACE Jeden z interakčních partnerů v BGE Vzorek - druhý interakční partner Podmínky - různé mobility + bez purifikace + enantiomery + bez kalibrace stechiometrie 1:1 enantiomery 42 ACE Interakce BSA-diklofenak -6,00E+03 £ -8,00E+03 -1,00E+04 -1,20E+04 <■—> <■—> % ) -1,40E+04 a. o -1,60E+04 §< -1,80E+04 -2,00E+04 -0,2 -0,15 -0,1 M (D,P) - M (D) -0,05 0 43 Literatura - CD, kovy, kompetice, multi-assay.... 44 CE-FA BGE bez interagujících partnerů Vzorek - oba interakční partneři, dávkování široké zóny vzorku Podmínky - „stejná" mobilita komplex a jeden z partnerů + bez purifikace - kalibrace + % vazby + stechiometrie +nízká spotřeba 45 CE-FA A) < CD TO o TO 350 300 250 200 150 100 50 0 0 50 |J M BSA + 500jM SA 500 JM SA Experimental conditions: BGE (borate buffer, pH 8.5), 58.5/50.0 cm (ltot/leff), 25°C, hydrodynamic injection (40 s, 35 mbar (3.5 kPa)), separation voltage: 24 kV, X = 214 nm, samples: 500 M SA (HS: plateau height of free drug), and mixture of 50 M BSA with 500 M SA (HF: plateau height of free drug). (•) indicates the BSA-SA complex and free BSA; (♦) free SA ♦ 3 4 migration time [min] Michalcova, L., Glatz, Z.: Comparison of various capillary electrophoretic approaches for the study of drug-protein interaction with emphasis on minimal consumption of protein sample and possibility of automation, J. Sep. Sci. (2015) 38, 325-331. 1 2 5 6 7 46 CE-FA Interakce BSA-SA to ■<ď 90 80 70 60 50 40 30 20 10 0 y = 0,0886x + 0,5244 R2 = 0,999 200 400 600 concentration (SA) 800 1000 900 SA 700 SA [300 SA / \ 100 SA ... . 1 \ 50 SA 25 5A , ...... 10 SA . . . 0 47 CE-FA Interakce BSA-SA 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 200 400 600 concentration free SA 14 0 48 TERMODYNAMIKA Jaký druh vazeb hraje roli??? Table 2. Binding constants Kbl number of binding sites n-u and thermodynamic parameters of BSA-SA system at different temperatures T{K) (ATb ± SD) 10 3 (Umol) n\±SD R1 AG^kJ/mol) A/^lkJ/mol) AS°(J/mol-K) R1 293.15 12.20 ± 2.28 0.93 ± 0.29 0.9646 -22.9 -130.3 -366 0.9969 298.15 5.60 ± 0.28 1.85 ± 0.40 0.9855 -21.3 303.15 2.10 ± 0.29 0.86 ± 0.30 0.9838 -19.3 All data were obtained by CE-FA. Interakce je spontální H-můstky Van der Waals vazby 49 RUZNE CE METODY Podmínka: malá spotřeba vzorku (proteinu) ACE CE-FA HD VP I VACE Table 1. Summary ot binding parameters obtained by different methods Hi ±SD (Kb ±SD}10-3 (Umol) CE-FA 1.79 ± 0.31 5.66 ± 0.19 HD-ex 1.71 ± 0.27 3.55 ± 1.34 HD-in 2.22 ± 0.35 5.26 ± 1.74 ED 5.32 ±1.11 3.72 ± 0.82 Data are expressed as mean values of triplicate measurements. Michalcova, L., Glatz, Z.: Comparison of various capillary electrophoretic approaches for the study of drug-protein interaction with emphasis on minimal consumption of protein sample and possibility of automation, J. Sep. Sci. (2015) 38, 325-331. 50 CE-FA A JINE METODY Interakce HSA-DCF 15 20 30 35 Log Kb 4.41 ±0.01 Kb (2.56±0.07)-104 L/mol Experimental conditions: BGE (borate buffer, pH 8.5), 58.5/50.0 cm (ltot/leff), 25°C, hydrodynamic injection (40 s, 35 mbar (3.5 kPa)), separation voltage: 14 kV, X = 276 nm, samples: mixture of 75u M HSA with 50-800 uM DCF. (a - 5s, b - 10s, c - 20s, d - 30s, e - 40s, f -60s, g - 80s, h - 120s) 6 5 4 r 3 2 1 0 0 50 100 150 200 250 300 350 400 450 500 free diclofenac [|jmol/L] h d b a 51 4 METODY metoda léčivo n Kb Log Kb CE-FA DCF 5,00±0,12 (2,56±0,07)E+04 4,41 ±0,01 ITC DCF 2,00±0,11 (1,22±0,09)E+04 4,09±0,03 CD DCF (7,91±0,12)E+04 4,90±0,01 ED DCF 5,05±0,04 (3,55±0,26)E+04 4,55±0,03 52 CITLIVOST: HSA / GHSA Cl H,N S II s JL .UJ H H Carbutamide "CH3 CH, H H Chlorpropamide H3C S " H H ° CD °° s Ji H G8 Acetohexamide CH3 Tolbutamide f 5 2C0 \__5 s 4 s 3 s 1.5 2 25 3 Time [rrirg Experimental conditions: BGE (67 mM phosphate buffer, pH 7.4, ) 48.5/8.5 cm (ltot/leff), 37°C, hydrodynamic injection (5 s, 35 mbar (3.5 kPa)), separation voltage: -10 kV, X = 214 nm, samples: mixture of 75 M HSA with 500 M drug Table 1. Summary of the validation parameters Parameters Tolbutamide Chlorpropamide Acetohexamide Carbutamide Detection wavelength (nu) 214 200 250 250 Linearity range3* (|ulM) 20-&O0 50-800 20-800 50-800 Regression equation y = 0.0&35x -0.3148 Y = 0.1SS3x + 1.4402 y = 0.0874 x - 0.1300 y = C.1105x - 1.8760 Correlation coefficient 0.9997 0.9991 0.9999 0.9989 Repeatability of plateau height - intradayb) |%) <3.37 <2.56 < 3.53 < 3.08 Repeatability of plateau height - inteiday1' |%) <3.06 <3.08 < 2.41 < 3.06 LOD (|jlM) 5.4 11.2 5.2 11.7 LOQ(|iM] 16.3 33.9 15.7 35.5 a) All points were measured in triplicate (n b) RSDforintraday (n = 3}. c) RSD for interday [n = 6). 3!. 9:0 4:10 ICO 0 0 53 CITLIVOST: HSA / GHSA 3 2.5 2 i- 1.5 1 0.5 0 ♦ x 200 300 400 500 600 Free chlorpropamide |jM] 1. BC 2. BC 3. BC 4. BC 200 400 600 Free acetohexamide [ |iM] X1.BC • 2.BC " 1 -3.BC •4.BC 0.5 0 » X1.BC 2. BC 3. BC 4. BC 100 200 300 400 500 600 700 Free tolbutamide |JM] 1. BC 2. BC 3. BC 4. BC 200 400 600 Free carbutamide [ uM] Repeatability of binding curves (BC) obtained for A) chlorpropamide-HSA, B) tolbutamide-HSA, C) acetohexamide-HSA, D) carbutamide-HSA systems with the CE-FA method. 2 5 A) B) 0 100 700 0 2.5 2.5 0.5 0.5 0 0 800 0 800 Table 2. Summary of binding parameters for HSA and gHSA HSA gHSA Kb ± SD (L/mol) n-, ± SD Kb ± SD (L/mol) rij ± SD Tolbutamide Chlorpropamide Acetohexamide Carbutamide (1.34 ±0.36) 104 (9.71 ± 2.42) 103 (4.18 ± 0.16) 104 (9.53 ± 1.07) 103 2.22 ±0.17 2.47 ± 0.43 1.78 ±0.09 1.54 ±0.27 (4.84 ± 1.59) 103 (2.93 ± 1.06) 103 (1.95 ± 0.59) 104 (2.27 ± 0.06) 103 2.60 ± 0.42 2.84 ± 0.41 1.26 ±0.07 3.08 ± 0.50 54 KOMPETICE léčivo Log Kb DCF 4,41 ±0,01 TRP 3,95±0,01 55 Feng-Qin Wang' Qiao-Qiao Li1 Qian Zhang1 Yin-Zhen Wang1 Yuan-Jia Hu2 Peng Li2 Jian-Bo Wan2 Feng-Qing Yang1 Zhi-Ning Xia1 1 School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, R R. China 2StaTe Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, R R. China Received July 27, 2016 Revised November 14, 2016 Accepted December?, 2016 A NENÍ TO JEN O MOLEKULÁCH... Electrophoresis 2017, 38, 938-941 Short Communication Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACF), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACF (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACH results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiornetry (ri = 5xl09) and binding constant (Kj, = lxlO4 L/mol) of the interaction between RAW264.7 and naringin. Keywords: Affinity interaction / Cell aggregation / Frontal analysis capillary electrophoresis/ RAW264.7 macrophage DOI 10.1002/elps.201 600345 Chromatoo.raphia<2018) 81:509-516 https://doi.Org/10.1007/sl 0337-018-3476-6 Evaluation of the Interactions Between Platelets and Alkaloids by Frontal Analysis Capillary Electrophoresis Using Polyvinyl Alcohol-Coated Capillary Qiao-Qiao Li1 ■ Su-Ying Li2 - Feng-Qin Wang1 - Hua Chen1 - Yuan-Jia Hu3 ■ Zhi-Ning Xia1 ■ Feng-Qing Yang1 Received: 15 August 2017/ Revised: 25 December 2017/Accepted: 15 January 2018 / Published online: 27 January 2018 © Springer-Verlacj GmbH Germany part of Springer Nature 2018 Abstract Capillary electrophoresis (CE) has been widely used in the study of The interactions between targets (biological macromol-ecules) and ligands due to its good biocompatibility. However, the separation and analysis of platelets, which play a pivotal role in thrombotic diseases, were limited owing to platelets adhesion in inner wall of capillary during CE analysis. In this paper, polyvinyl alcohol-coated capillaries were simply prepared to prevent the adhesion of platelet. Then, a frontal analysis CE (FACE) method was developed to evaluate the interactions between platelets and eight alkaloids include glaucine. magnoflorine, dehydrocorydaline. palmarine, berberine. isorhynchophylline, rhynchophylline; and brucine. The binding constants and stoichiometries of the interactions were calculated by Scatchard equation tor strong and specific interaction, and the non-specific binding equation for weak interaction. The results indicated that glaucine. dehydrocorydaline. and isorhynchophylline showed relatively high affinity interaction with platelets. The developed FACE method may be further applied in the evaluation of interactions between platelets and other small molecular compounds. 56 FACCE BGE bez interagujících partnerů Vzorek - oba interakční partneři, kontinuální dávkování Podmínky - „stejná" mobilita komplexu a jeden z partnerů, druhý partner (ligand) má mobilitu větší + % vazby - kalibrace + stechiometrie - vyšší spotřeba vzorku +nízká spotřeba - LOD 58 KINETICKÉ MODY CE Prof. Krylov - 2002 Několik variant KCE Kb' Kď korv koff NECEEM - Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures 60 KCE 61 PROC CE RESP. CE-FA? 1. Nevyžaduje vysoce purifikované vzorky, značení ani imobilizaci 2. Nízká spotřeba vzorku (nL) a dalších reagentů 3. Rychlost analýz 4. Možná simulace různých prostředí 5. Velmi robustní metoda - díky platu 6. Využitelnost metody pro systémy s rychlou i pomalou kinetikou 7. Možnost fitování dat s využitím vícevazných modelů a charakterizace vazebné stechiometrie 8. Separace volného ligandu a jeho analýza je prováděna v jednom kroku - integrované řešení 9. Přímé stanovení volného ligandu ve vzorku 62 TAKE HOME MESSAGE Výběr z množství technik - různé principy, požadavky na vzorky, detekční limity, ... Neexistuje jediný ideální způsob Znalost metod je zásadní pro dosažení nejlepších výsledků Důležité je metody kombinovat