Towards Predictable Transmembrane Transport: QSAR Analysis of the Anion Binding and Anion Transport Properties of Thioureas
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The transport of anions across biological membranes by small molecules is a growing research field due to the potential therapeutic benefits of these compounds. However, little is known about the exact mechanism by which these drug-like molecules work and which molecular features make a good transporter. An extended series of 1-hexyl-3-phenylthioureas were synthesized, fully characterized (NMR, mass spectrometry, IR and single crystal diffraction) and their anion binding and anion transport properties were assessed using 1H NMR titration techniques and a variety of vesicle-based experiments. Quantitative structure-activity relationship (QSAR) analysis revealed that the anion binding abilities of the mono-thioureas are dominated by the (hydrogen bond) acidity of the thiourea NH function. Furthermore, mathematical models show that the experimental transmembrane anion transport ability is mainly dependent on the lipophilicity of the transporter (partitioning into the membrane), but smaller contributions of molecular size (diffusion) and hydrogen bond acidity (anion binding) were also present. Finally, we provide the first step towards predictable anion transport by employing the QSAR equations to estimate the transmembrane transport ability of four new compounds.
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Towards Predictable Transmembrane Transport: QSAR Analysis of the Anion Binding and Anion Transport Properties of Thioureas
- 1. Towards Predictable Transmembrane Transport: QSAR Analysis of the Anion Binding and Anion Transport Properties of Thioureas Philip A. Gale N. Busschaert, S.J. Bradberry, M. Wenzel, C.J.E. Haynes, J.R. Hiscock, I.L. Kirby, L.E. Karagiannidis, S.J. Moore, N.J. Wells, J. Herniman, G.J. Langley, P.N. Horton, M.E. Light, V. Félix, J.G. Frey, P. A. Gale, Chemical Science 2013, 4, 3036-3045 .
- 3. Tools from medicinal chemistry • Hansch analysis: used in medicinal chemistry to examine quantitative structure-activity relationships across a series of molecules. The log(1/IC50) value is correlated to a linear combination of predictable molecular properties, usually logP (water:octanol partition coefficient) and/ or Hammett constants using multiple regression techniques: log(1/IC50) = k1π + k2σ + k3 where IC50 = molar concentration required for a standard response π is related to lipophilicity (logP) σ = Hammett coefficient (related to binding strength) C. Hansch, A. Leo and R. W. Taft, Chem. Rev., 1991, 91, 165.
- 4. Transporter design R S N H variation of hydrogen bond donor strength (σ) and lipophilicity 1 R = Br 2 R = CF3 3 R = Cl 4 R = CN 5 R = COCF3 6 R = COMe 7 R = COOMe 8R=F 9R=H 10 R = I 11 R = NO2 thiourea anion binding site N H 12 13 14 15 16 17 18 19 20 21 22 R R R R R R R R R R R = = = = = = = = = = = O(CO)Me OCF3 OEt OMe SMe SO2Me CH3 CH2CH3 (CH2)2CH3 (CH2)3CH3 (CH2)4CH3
- 5. Anion transport at 2% loading NO3- Cl- Chloride efflux promoted by a selection of compounds 1-22 (2 mol% thiourea to lipid) from unilamellar POPC vesicles loaded with 489 mM NaCl buffered to pH 7.2 with 5 mM sodium phosphate salts. The vesicles were dispersed in 489 mM NaNO3 buffered to pH 7.2 with 5 mM sodium phosphate salts. At the end of the experiment, detergent was added to lyse the vesicles and calibrate the ISE to 100 % chloride efflux. Each point represents the average of at least 9 trials. DMSO was used as control.
- 6. Anion transport trends Br Hill analysis S ! E = EmaxCn/(EC50n+Cn) N N ! H H E is the magnitude of an observed effect Emax is the maximum value of this effect (in this case Emax = 100 % chloride efflux) C is the concentration of carrier n is the Hill coefficient of sigmoidality EC50 is the effective concentration of carrier required to mediate 50 % of the maximum response EC50 = 0.80 mol% w.r.t. lipid A. V. Hill, Biochem. J., 1913, 7, 471.
- 7. Transporter design R S N H variation of hydrogen bond donor strength (σ) and lipophilicity 1 R = Br 2 R = CF3 3 R = Cl 4 R = CN 5 R = COCF3 6 R = COMe 7 R = COOMe 8R=F 9R=H 10 R = I 11 R = NO2 thiourea anion binding site N H 12 13 14 15 16 17 18 19 20 21 22 R R R R R R R R R R R = = = = = = = = = = = O(CO)Me OCF3 OEt OMe SMe SO2Me CH3 CH2CH3 (CH2)2CH3 (CH2)3CH3 (CH2)4CH3
- 8. Transporter design R S N H variation of hydrogen bond donor strength (σ) and lipophilicity 2 3 4 5 R R R R = = = = CF3 Cl CN COCF3 7 R = COOMe 8R=F 9R=H 10 R = I 11 R = NO2 thiourea anion binding site N H 12 R = O(CO)Me 13 R = OCF3 15 16 17 18 19 R R R R R = = = = = OMe SMe SO2Me CH3 CH2CH3 21 R = (CH2)3CH3 22 R = (CH2)4CH3
- 9. Hansch analysis ! log(1/EC50) = k1A + k2B + k3C + k4 N. Busschaert, S.J. Bradberry, M. Wenzel, C.J.E. Haynes, J.R. Hiscock, I.L. Kirby, L.E. Karagiannidis, S.J. Moore, N.J. Wells, J. Herniman, G.J. Langley, P.N. Horton, M.E. Light, V. Félix, J.G. Frey, P. A. Gale, Chemical Science 2013, 4, 3036-3045 .
- 10. Mr (g/mol) PSA solvent acc surface area volume Surface Area Molecular Volume Vsmax Parachor Index of refraction Bp TPSA Vsmin PI Molar refractivity Molecular volume logD (pH1,7) logD (pH4,6) logD (pH6,5) logD (pH7,4) logD (pH8) pKa (arom NH) pKa (alkyl NH) pKa2 (NH) logS (pH1,7) Vx logPS Vd SCBO SMTI ARR SMTIV GMTIV logS (pH6,5) logS (pH7,4) logS (pH8) polarizability MW AMW Sv Se RBN GMTI RBF ZM2 ZM2V Qindex Xu SPI W WA Har Har2 QW Whetv Whete PW2 Whetp PW3 J JhetZ Jhetm Jhetv Jhete Jhetp MAXDN PW4 PW5 PJI2 CSI ECC AECC DECC GGI1 GGI2 GGI3 GGI4 GGI5 GGI6 GGI7 GGI8 GGI9 GGI10 JGI1 JGI2 JGI3 JGI4 JGI5 JGI6 JGI7 JGI8 W3D J3D H3D AGDD DDI ADDD G1 G2 RGyr SPAM SPH ASP FDI DISPm QXXm QYYm QZZm DISPv QZZv DISPe QXXe QYYe QZZe DISPp QXXp QYYp QZZp G2u QXXv QYYv logS (pH4,6) ZM1 ZM1V Sp Ss Mv SNar TI2 Me HNar Mp GNar Xt HyDp RHyDpw MDDD UNIP ww MAXDP CENT VAR BAC Ms Dz Ram Pol Rww D/D Wap WhetZ Whetm DELS TIE S0K S1K Lop ICR LPRS S2K S3K VDA MSD PHI D/Dr06 JGI9 JGI10 JGT PJI3 G3u L/Bw SEig E1u HOMA RCI AROM HOMT E2u E3u L1m L2m L3m L1p L2p L3p L1u L2u L3u P1u P2u G1u E2m E3m L1v L2v L3v P1v P2v G1v G2v G3v E1v E2v E3v L1e L2e L3e P1e P2e G1e G2e G3e E1e E2e E3e G1p G2p G3p E1p E2p E3p L1s L2s L3s P1s P2s G1s G2s G3s E1s E2s E3s Tu Tm Tv Te Tp Ts Au Am Av Ae Ap As Gu Gm Gs Ku Km Kv Ke Kp Ks Du Dm Dv De Dp Ds Vu Vm Vv Ve BLTF96 BLTD48 BLTA96 P1m P2m P1p G1m G2m G3m E1m P2p Molecular parameters http://www.vcclab.org/lab/indexhlp/dragon_descr.html Vp Vs BLI nHAcc Ui Hy AMR TPSA(NO) TPSA(Tot)
- 11. QSAR analysis of anion binding logKa(H2PO4-) = 0.85(±0.06)*σp + 2.38(±0.02) logKa(Cl-) = 0.55(±0.03)*σp + 1.17(±0.01) N = 18, R² = 0.96, R²adj = 0.96, RMSE = 0.04, F = 424 logKa(HCO3-) = 0.88(±0.10)*σp + 2.40(±0.04) N = 17, R² = 0.92, R²adj = 0.91, RMSE = 0.09, F = 167 N = 16, R² = 0.84, R²adj = 0.83, RMSE = 0.13, F = 73 Graphical representation of the correlation between anion binding (logKa) and the Hammett constant σp for compounds 1-22 (excluding 1, 6, 14 and 20). Linear fits are represented by a blue line. (a). interaction with Cl- vs. Hammett constant; (b) interaction with H2PO4 vs. Hammett constant-; (c) interaction with HCO3- vs. Hammett constant. M. J. Hynes, J. Chem. Soc. Dalton Trans., 1993, 311; Hammett constants for substituents in para-position taken from: A. Hansch et al., Chem. Rev., 1991,91, 165.
- 12. Lipophilicity The logP of a compound can be correlated to the retention time in a reverse phase HPLC experiment (elution with water:methanol gradient). The retention times were correlated with calculated logP values to identify the best model. R2 = 0.955 ClogP calculated using Daylight v4.73 R2 = 0.719 ClogP calculated using VCC labs website C. Giaginis et al., J. Liq. Chromatogr. Relat. Technol., 2008, 31, 79; (b) R. Quesada and co-workers, Chem. Commun., 2012, 48, 5274. www.vcclabs.org; I. Moriguchi et al., Chem. Pharm. Bull., 1992, 40, 127.
- 13. QSAR analysis of anion binding logKa(H2PO4-) = 0.85(±0.06)*σp + 2.38(±0.02) logKa(Cl-) = 0.55(±0.03)*σp + 1.17(±0.01) N = 18, R² = 0.96, R²adj = 0.96, RMSE = 0.04, F = 424 logKa(HCO3-) = 0.88(±0.10)*σp + 2.40(±0.04) N = 17, R² = 0.92, R²adj = 0.91, RMSE = 0.09, F = 167 N = 16, R² = 0.84, R²adj = 0.83, RMSE = 0.13, F = 73 Graphical representation of the correlation between anion binding (logKa) and the Hammett constant σp for compounds 1-22 (excluding 1, 6, 14 and 20). Linear fits are represented by a blue line. (a). interaction with Cl- vs. Hammett constant; (b) interaction with H2PO4 vs. Hammett constant-; (c) interaction with HCO3- vs. Hammett constant. M. J. Hynes, J. Chem. Soc. Dalton Trans., 1993, 311; Hammett constants for substituents in para position taken from: A. Hansch et al., Chem. Rev., 1991,91, 165.
- 14. Mr (g/mol) PSA solvent acc surface area volume Surface Area Molecular Volume Vsmax Parachor Index of refraction Bp TPSA Vsmin PI Molar refractivity Molecular volume logD (pH1,7) logD (pH4,6) logD (pH6,5) logD (pH7,4) logD (pH8) pKa (arom NH) pKa (alkyl NH) pKa2 (NH) logS (pH1,7) Vx logPS Vd SCBO SMTI ARR SMTIV GMTIV logS (pH6,5) logS (pH7,4) logS (pH8) polarizability MW AMW Sv Se RBN GMTI RBF ZM2 ZM2V Qindex Xu SPI W WA Har Har2 QW Whetv Whete PW2 Whetp PW3 J JhetZ Jhetm Jhetv Jhete Jhetp MAXDN PW4 PW5 PJI2 CSI ECC AECC DECC GGI1 GGI2 GGI3 GGI4 GGI5 GGI6 GGI7 GGI8 GGI9 GGI10 JGI1 JGI2 JGI3 JGI4 JGI5 JGI6 JGI7 JGI8 W3D J3D H3D AGDD DDI ADDD G1 G2 RGyr SPAM SPH ASP FDI DISPm QXXm QYYm QZZm DISPv QZZv DISPe QXXe QYYe QZZe DISPp QXXp QYYp QZZp G2u QXXv QYYv logS (pH4,6) ZM1 ZM1V Sp Ss Mv SNar TI2 Me HNar Mp GNar Xt HyDp RHyDpw MDDD UNIP ww MAXDP CENT VAR BAC Ms Dz Ram Pol Rww D/D Wap WhetZ Whetm DELS TIE S0K S1K Lop ICR LPRS S2K S3K VDA MSD PHI D/Dr06 JGI9 JGI10 JGT PJI3 G3u L/Bw SEig E1u HOMA RCI AROM HOMT E2u E3u L1m L2m L3m L1p L2p L3p L1u L2u L3u P1u P2u G1u E2m E3m L1v L2v L3v P1v P2v G1v G2v G3v E1v E2v E3v L1e L2e L3e P1e P2e G1e G2e G3e E1e E2e E3e G1p G2p G3p E1p E2p E3p L1s L2s L3s P1s P2s G1s G2s G3s E1s E2s E3s Tu Tm Tv Te Tp Ts Au Am Av Ae Ap As Gu Gm Gs Ku Km Kv Ke Kp Ks Du Dm Dv De Dp Ds Vu Vm Vv Ve BLTF96 BLTD48 BLTA96 P1m P2m P1p G1m G2m G3m E1m P2p Molecular parameters http://www.vcclab.org/lab/indexhlp/dragon_descr.html Vp Vs BLI nHAcc Ui Hy AMR TPSA(NO) TPSA(Tot)
- 15. Final model: relative parameters R S N H 1 R = Br 2 R = CF3 3 R = Cl 4 R = CN 5 R = COCF3 6 R = COMe 7 R = COOMe 8R=F 9R=H 10 R = I 11 R = NO2 N H 12 R = O(CO)Me 13 R = OCF3 14 R = OEt 15 R = OMe 16 R = SMe 17 R = SO2Me 18 R = CH3 19 R = CH2CH3 20 R = (CH2)2CH3 21 R = (CH2)3CH3 22 R = (CH2)4CH3 NO3- Cl- ! log(1/EC50) = 0.82(±0.08)*π + 0.66(±0.18)*σp – 0.26(±0.07)*ΔSPAN – 0.43 ! ! ! ! ! Calculated using JMP® 9.0.0 N = 18, R² = 0.89, R²adj = 0.87, RMSE = 0.21, F = 42 N. Busschaert, S.J. Bradberry, M. Wenzel, C.J.E. Haynes, J.R. Hiscock, I.L. Kirby, L.E. Karagiannidis, S.J. Moore, N.J. Wells, J. Herniman, G.J. Langley, P.N. Horton, M.E. Light, V. Félix, J.G. Frey, P. A. Gale, Chemical Science 2013, 4, 3036-3045 .
- 16. Linear combinations of parameters Graphical depiction of the values of the coefficients in the equation when the descriptor values are scaled to have a mean of zero and a range of two using JMP 9.0.0. This shows that lipophilicity (RT or logP) has the greatest effect on anion transport. ! log(1/EC50) = 0.82(±0.08)*π + 0.66(±0.18)*σp – 0.26(±0.07)*ΔSPAN – 0.43 ! ! N = 18, R² = 0.89, R²adj = 0.87, RMSE = 0.21, F = 42 Calculated using JMP® 9.0.0 N. Busschaert, S.J. Bradberry, M. Wenzel, C.J.E. Haynes, J.R. Hiscock, I.L. Kirby, L.E. Karagiannidis, S.J. Moore, N.J. Wells, J. Herniman, G.J. Langley, P.N. Horton, M.E. Light, V. Félix, J.G. Frey, P. A. Gale, Chemical Science 2013, 4, 3036-3045 .
- 17. Predictions N. Busschaert, S.J. Bradberry, M. Wenzel, C.J.E. Haynes, J.R. Hiscock, I.L. Kirby, L.E. Karagiannidis, S.J. Moore, N.J. Wells, J. Herniman, G.J. Langley, P.N. Horton, M.E. Light, V. Félix, J.G. Frey, P. A. Gale, Chemical Science 2013, 4, 3036-3045 .
- 18. Conclusions ! ! Hansch analysis of anion transport results may reveal the molecular parameters that we should optimise in order to design an efficient anion transporter. For the thioureas studied these are logP, affinity and molecular size. ! ! ! ! ! Chemical Science www.rsc.org/chemicalscience Volume 4 | Number 8 | August 2013 | Pages 2979–3348 ISSN 2041-6520 EDGE ARTICLE Philip A. Gale et al. Towards predictable transmembrane transport: QSAR analysis of anion binding and transport “Anion transporters and biological systems” P.A. Gale, R. Pérez-Tomás and R. Quesada, Acc. Chem. Res. 2013, DOI: 10.1021/ ar400019p. 2041-6520(2013)4:8;1-N “From anion receptors to transporters” “Small molecule lipid bilayer anion transporters for biological applications” N. Busschaert and P.A. Gale, P.A. Gale, Acc. Chem. Res. 2011, 44, 216-226. Angew. Chem. Int. Ed., 2013, 52, 1374-1382.
- 19. Current group: Former group members: ! ! Dr Jenny Hiscock Dr Wim van Rossom Dr Nathalie Busschaert ! Isabelle Kirby Louise Karagiannides Francesca Piana Stuart Berry Xin Wu Michael Spooner ! ! ! ! Dr Christine C. Tong Dr Korakot Navakhun Dr Joachim Garric Dr Claudia Caltagirone Dr Gareth W. Bates Dr Marco Wenzel Dr Stephen Moore Sam Bradberry Dr Cally Haynes Research support and collaborations in Southampton: ! Dr. Mark E. Light Dr John Langley Dr Neil Wells Julie Herniman Prof. Jonathan Essex Prof. Jeremy Frey
- 20. Prof. Jeff Davis Dr William Harrell Jr. ! Prof Janez Plavec Dr Damjan Makuc Prof Kate Jolliffe ! Prof Tony Davis Dr Hennie Valkenier ! Dr Roberto Quesada ! Prof. Ricardo Pérez-Tomás ! Prof. Vítor Félix ! !