SUPPLEMENTARY INFORMATION

Comparative studies on the human serum albumin binding of EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187

Orsolya Dmtr, Karla Pelivan, Attila Borics, Bernhard. K. Keppler, ChristianR.Kowol, va A. Enyedy

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Table of contents

S1. Tables and figuresSI-2

S2. Binding stoichiometry estimations via Jobs methodSI-10

S3. Molecular docking with well-known site markersSI-11

S1. Tables and figures

Table S1 Molar absorbance values of EGFR inhibitors in aqueous solution determined on weight-in-volume basis at pH = 3.

EGFR inhibitor

max (nm)

(M-1cm-1)

ERL

342

18900

GEF

342

17800

AFA

350

18800

AFA-maleat

350

18700

OSI

372

30750

KP2187

392

10100

Table S2 Applied EX and EM values at spectrofluorometric measurements

Type of measurement

EX (nm)

EM (nm)

Quenching

295

305-400

WF displacement

310

320-500

DG, DS displacement

340

420-600

GEF, ERL (Job method)

330

360-580

KP2187 (Job method)

370

380-600

KP2187 (DG displacement)

400

405-650

Fig. S1. UVvis absorption spectra of KP2187 recorded at various pH values. Inset shows absorbance changes at 232 nm. {cKP2187 = 50 M; T = 25 C; I = 0.10 M (KCl); l = 1 cm}

Fig.S2. UVvis spectra of OSI recorded between pH 2.61 and 6.19. {cOSI = 10 M; l = 2 cm; T=25C; I = 0.10 M (KCl)}

Fig.S3. Normalized fluorescence emission spectra of ERL in n-hexane (black), benzene (green), n-octanol (red), and in the presence of 5 eq. HSA (violet) in aqueous solution at pH7.4. {cERL 1 M; EX=340nm; T=25C (in the organic solvents) or 37 C (with HSA)}

Fig.S4. Normalized fluorescence excitation spectra of ERL in n-hexane (black), benzene (green) and n-octanol (red) and its normalized UVvis spectra in n-hexane (gray dashed) and in water at pH 7.4 (blue dashed). {EM = 370 or 450 nm; T=25C}

Fig. S5. Emission spectra of KP2187 dissolved in various solvents. Inset shows the shift of max) in n-hexane in the presence of increasing amount of methanol; dotted line denotes peak maximum in pure methanol {clig = 1 M; EX = 370 nm; T = 25 C}.

Fig. S6. UV-Vis spectra of the HSAOSI (a) and HSAERL (c) systems and the OSI (b) and ERL (d) subtracted spectra of the same samples. {cHSA = 1 M; cOSI = 0, 1, 2, 6 and 15 M; cERL = 0, 1, 2, 5, 10 and 15 M; T = 37 C, pH = 7.40 (PBS)}

Fig. S7. Spectral overlap of the normalized emission band of HSA and normalized absorption band of (a) KP2187, (b) ERL (solid line), GEF (dashed line), (c) AFA and (d) OSI. All spectra are recorded at pH 7.40.

Fig. S8. Absorbance corrected (a-c) and non-corrected (d-f) 3D fluorescence spectra of HSA:ERL system at the sample compositions (M:M): 5:0 (a,d), 5:5 (b,e), 5:20 (c,f). ERL alone shows no significant fluorescence under these conditions {resolution = 5 nm; T = 37 C; pH = 7.40 (PBS)}.

Fig. S9. Fluorescence emission spectra of 1 M HSA titrated by ERL in the presence (black spectra) and in the absence (gray spectra) of 1 M WF. Inset shows intensity changes at 390nm. {cHSA = 1 M; cWF = 1 or 0 M; cERL = 0-18M; EX = 310 nm; T = 37 C; pH = 7.40 (PBS)}

The measured intensities of HSAWF samples decrease in the wavelength range 335350 nm, where the HSAERL (and HSAGEF) adduct is not fluorescent. Two sets of calculations were carried out: in the first case only the 335350 nm range of the spectra of the HSAWFERL ternary system were used, while the second set was dealing the whole spectra for both WF-free and WF containing samples and HSAERL adduct was also treated as emitting component in the data evaluation process. Both approaches gave similar displacement constants logK 4.27 0.01 and 4.36 0.01 respectively. The same procedure was utilized for GEF.

Fig. S10. Emission spectra of the HSAKP2187 system in the presence of increasing amounts of DG and the spectrum of HSADG sample. {cHSA = cKP2187 = 10 M; cDG = 0-150 M; EX = 400 nm; T = 37 C; pH = 7.40 (PBS)}

Fig. S11. Low-energy docked complexes of GEF and KP2187 in protonated and neutral forms in binding site II. Non-polar hydrogen atoms are omitted for clarity.

Fig. S12. Low-energy docked complexes of GEF and KP2187 in protonated and neutral forms in binding site III. Non-polar hydrogen atoms are omitted for clarity.

Fig. S13. Low-energy docked complexes of GEF and KP2187 in protonated and neutral forms in binding site I (as 2D ligand interaction diagrams). Non-polar hydrogen atoms are omitted for clarity.

S2. Binding stoichiometry estimations via Jobs method

A Job plot, otherwise known as the method of continuous variation or Job's method, is an approach used in analytical chemistry to determine the stoichiometry of a binding event. However, it is a useful tool to estimate stoichiometry of complex formation reactions, the applied concentration range and the (expected) binding affinities are serious limiting factors of this approach. Fig. S12 shows Job plots of four hypothetical systems where 1 or 2 binding sites are presented and binding constants are logK 4 or 6. It is noteworthy that at low affinities (logK 4) the curves for 1 or 2 binding sites are rather similar (Fig. S12/a). Even at logK 5 it is hard to estimate any binding stoichiometry (data not shown here). Job method requires the existence of relatively high affinity sites (logK 6) in order to get reliable results regarding the binding stoichiometry.

It should be noted that this affinity limit may decrease if higher concentrations are used (however it was not applicable in our case).

Fig. S14. Job plots calculated for the HSAcompound hypothetical systems owing one or two binding sites (with the same affinity) with logK = 4 (a) or 6 (b). {cHSA + ccomp = 10M}

S3. Molecular docking with well-known site markers

Re-docking of WF and CMPF to sites I and II of HSA, respectively, resulted in relative target-ligand orientations similar to those observed in the crystallographic structures (Fig. S13).[footnoteRef:1],[footnoteRef:2] [1: J. Ghuman,P. A. Zunszain,I. Petitpas,A. A. Bhattacharya,M. Otagiri,S. Curry, J.Mol.Biol. 353 (2005) 3852.] [2: P. A. Zunszain,J. Ghuman,A. F. Mcdonagh,S. Curry, J.Mol.Biol. 381 (2008) 394406.]

Fig. S15. Docked complexes (orange) of WF with site I (A), CMPF with site II (B) and bilirubin with site III (C) in comparison with ligand poses observed in the crystallographic structures (green).

Binding free energies of the WF and CMPF complexes were -11.25 kcal/mol and -12.39 kcal/mol, respectively. This corresponds to specific, high-affinity binding, although these ligands demonstrated lower affinity binding in vitro, with dissociation constants typically in the micromolar range.[footnoteRef:3] Nevertheless, binding poses of WF and CMPF in sites I and II of HSA, respectively, were sufficiently reproduced. Bilirubin, on the other hand, was not as deeply inserted in site III of HSA in the docked complexes as it was indicated by the corresponding crystallographic structure 2VUE (Fig. S13c). In this crystal structure HSA-bound bilirubin was shown to be held in the site III pocket by salt bridges formed with the sidechains of Arg117 and Arg186 and a 'polypeptide strap' formed by residues 110119.2 The binding mechanism of a relatively large molecule such as bilirubin may require the temporary displacement of this 'polypeptide strap', which is not modelled adequately by flexible docking. Despite the observed differences of binding poses, the calculated KD of bilirubin (28.4 nM) is in the range of reported values.[footnoteRef:4],[footnoteRef:5] Based on these results, docking calculations are more reliable for smaller molecules owing limited number of rotatable bonds like WF and CMPF or the investigated inhibitors GEF and KP2187. [3: G. Sudlow, D. J. Birkett, D. N. Wade, Mol. Pharmacol. 11 (1975) 824832.] [4: R. Brodersen, Physical chemistry of bilirubin: binding to macromolecules and membranes, in: Bilirubin, K.P.M. Heirwegh, S.B. Brown, (Eds.), Vol. 1, CRC Press, Florida, Boca Raton (1982) pp. 75123.] [5: O. Dmtr, C.G. Hartinger, A.K. Bytzek, T. Kiss, B.K. Keppler, .A. Enyedy, J. Biol. Inorg. Chem. 18 (2013) 917.]

SI-1

00.050.10.150.20.250.3

0.00.20.40.60.81.0270300330360

Normalizedintensity or absorbance

l/ nm

03 0006 0009 000380430480530580

Fluorescence intensity / a.u.

l

EM

/ nm

4104304500510

l

max

methanol / %(v/v)

n-octanolethanolmethanolwatern-hexane

0.000.010.020.030.040.05230280330380

DAbsorbancel/ nm

0.000.100.200.300.400.50230280330380

Absorbance

l / nm

15 eq.6eq.2 eq.1eq.HSA

(a) (b)

0.000.010.020.030.040.05230280330380

DAbsorbancel/ nm

0.000.100.200.300.400.50230280330380

Absorbance

l/ nm

(c) (d)

15 eq.10 eq.5 eq.2 eq.HSA1 eq.

(a) (b)(c) (d)

(a) (b) (c)(d) (e) (f)

0.01.02.0330380430480

Intensity 10

-3

/ a.u.

l

EM

/ nm

1.61.82.00.00.20.401020

c

ERL

/ mM

Int.Int.

01 0002 0003 0004 000410440470500530

Intensity/ a.u.

l

EM

/ nm

HSA-DG

10 mM:100 mM

site IGEF

+

GEF

0

KP2187

+

KP2187

0

0.00.20.40.60.81.01.21.400.20.40.60.81

Normalized intensity n

comp

/ n

(comp+HSA)

0.00.20.40.60.81.01.21.400.20.40.60.81

Normalized intensity n

comp

/ n

(comp+HSA)

(a) (b)

0.500.66onesitetwositesonesitetwosites

0.00.30.60.91.21.5200250300350400450500

Absorbance

l/ nm

0.10.20.30.40.5246810

Abs. at 323 nmpH

0.00.20.40.6250300350400450

Absorbance

l/nm

2.616.19

0.00.20.40.60.81.0350400450500550600

Normalized intensity

l

EM

/ nm