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Comment on “Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc” Ferenc Zsila J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08431 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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Comment on “Multiple independent binding sites for small-molecule inhibitors on the oncoprotein c-Myc”

Ferenc Zsila* Biomolecular Self-Assembly Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, POB 286, H-1519, Budapest, Hungary

Corresponding author: Ferenc Zsila POB 289, H-1519, Budapest, Hungary Email: [email protected]

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Metallo and his co-workers examined the non-covalent association of six small molecule inhibitors to the intrinsically disordered c-Myc transcription factor overexpressed in a variety of human cancers.1 Employing NMR, fluorescence and circular dichroism (CD) spectroscopic methods, binding of the inhibitors to various truncated segments of the basic helix-loop-helix leucine zipper domain of c-Myc (353-437 residues) was assessed including the determination of binding location and affinity constant as well as binding induced secondary structural modification of the peptides. Upon interaction with the inhibitors, far-UV CD spectra of c-Myc peptides exhibited significant changes. The intense negative ellipticity peak around 205 nm indicative to the disordered, random coil conformation of the free peptides was greatly diminished. Besides this alteration, peptide association of the inhibitor named 10074-A4 produced a so called extrinsic or induced CD spectroscopic signal: between 235 and 250 nm where the chiroptical contribution of the peptides is close to zero, a strong negative band appeared allied to the respective absorption band of the ligand (Fig. 1)1. 10074-A4 consists of a carbazole and a thiazolidinedione unit connected by a propyl chain bearing a chiral center and it was used in a racemic form. Apart from some general notes about the induced CD (ICD) phenomenon, however, the underlying mechanism responsible for the extrinsic CD signal and its correlation with other structural data remained unclear in the paper. To obtain insight into possible mechanisms of ligand binding induced CD, the molar circular dichroic absorption coefficient (∆ε) of the extrinsic ellipticity band has to be determined.2 To estimate this parameter, the exact molar concentrations of the interacting partners are required. These were not specified in the paper1 but did in the Ph.D. dissertation of one co-author3 showing that the inhibitor was used in a five-fold molar excess (100 µM) in relation to the c-Myc peptides (20 µM). Thus, it can be assumed that the single binding site of the peptide is saturated with the inhibitor molecules. Furthermore, the ICD signal is attributed solely to the peptidebound fraction of 10074-A4. Taking these into consideration, conversion of the peptide residue based molar ellipticity data results in a surprisingly large ∆εmax value (Fig. 1). It is to be noted that optically pure chiral carbazole derivatives display a substantially weaker CD activity (typically |∆εmax| < 10).4-8 In analogy with small molecule-protein adducts, ligand-peptide binding interactions can induce CD activity by two principal mechanisms.9 The conjugated πsystem of the ligand may acquire optical activity by a right- or a left-handed helical twist provoked by its conformational adjustment to the asymmetric binding environment. In contrast 1 ACS Paragon Plus Environment

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to the chromophoric system of e.g., congo red,10 cibacron blue,11 bromphenol blue,12 and quercetin13 owing some degree of steric freedom, the planar carbazole ring of 10074-A4 prevents structural accommodation. On the other hand, combination of a ligand with the chiral peptide sequence may result in dipole-dipole or exciton coupling. This involves coulombic interaction between the electronically allowed π-π* transition moments of the chromophoric unit(s) of the guest compound and proximal aromatic side chains.14-18 This type of coupling is referred as nondegenerate since the coupled transitions are different in energy. The carbazole chromophore shows a strong absorption band around 250 nm (1Ba, εmax ~ 28,000 M-1 cm-1).19,20 Two opposite ICD signals appear in the CD spectrum corresponding to the absorption band of the ligand and the interacting residues, respectively. The latter one could not be observed due to the strong masking effect of the intrinsic CD activity of the peptide/protein at lower wavelengths. The sign of the exciton CD effect depends on the helicity of the coupled dipole moments (left- or righthanded) while its magnitude is determined by the distance, dipole strength, relative orientation, and energy separation of the transition moments.18,21,22 The high amplitude of the ICD peak in Fig. 1 predicts a relatively short aromatic residue-carbazole distance (~5 Å). Theoretically, intramolecular exciton splitting may also occur between the π-π* transitions of the carbazole and the thiazolidinedione moiety but the dipole strength of the latter is too low to produce such an intense extrinsic CD signal (εmax ~ 3000 at 228 nm)23. It should be stressed that for this kind of CD induction no asymmetric center in the guest molecule is required. The heterocyclic β-carboline alkaloid norharman is similar to that of the carbazole moiety of 10074-A4 but bears no chiral center. Upon its binding to human serum albumin (HSA), a positive ICD band appears, the spectral position and magnitude of which are reminiscent to that obtained with 10074-A4 (Fig. 2).17 Similarly, ICD spectrum of the carbazole derivative drug (±)-carprofen added to HSA also exhibits intense peaks due to ligand-aromatic residue exciton coupling. However, these data per se are inconclusive regarding the binding stereoselectivity of carprofen.24 By analogy, the ICD band of 10074-A4 cannot be considered either to prove or disprove of its enantioselective c-Myc association. To clarify this issue, analytical methods (e.g., equilibrium dialysis, ultrafiltration) combined with chiral separation techniques or comparative measurements with the pure antipodes should be applied.6,24 The results of the NMR spectroscopic studies are in line with the mechanism proposed above for the ICD response, i.e. the chiral exciton coupling between the aromatic side chains and the 2 ACS Paragon Plus Environment

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carbazole nucleus of 10074-A4. According to the NMR data, the binding site of 10074-A4 on cMyc370-409 involves 12 residues spanning from Phe374 to Glu385 (Phe-Phe-Ala-Leu-Arg-AspGln-Ile-Pro-Glu-Leu-Glu).1 This sequence encompasses two Phe side chains which exhibit a lower (1La, εmax ~ 10,000, λmax ~ 210 nm) and a higher energy (Ba/Bb, εmax ~ 60,000, λmax ~180 nm) π-π* transition suitable for exciton coupling with the inhibitor. Importantly, other peptides lacking these Phe residues failed to induce any CD band that was observed only with c-Myc fragments of 370-409, 353-405, and 353-439, respectively.1 The lack of the ICD activity obtained with c-Myc363-381 can be ascribed to the truncated binding sequence. Overall, here it is shown that careful analysis and comparison of the ICD data with other experimental results can provide more insight into the structural details of drug-peptide interactions. Besides the missing discussion of the CD spectroscopic results, some additional weak points of this work should be mentioned. The UV absorption curves of the inhibitor-peptide mixtures were not shown.1 In most instances, consideration of the absorption spectroscopic data is obligatory for proper interpretation of the CD spectrum including mutual comparison of number, shape, spectral position, and intensity of the CD and absorption bands. Furthermore, the whole spectral region has to be scanned where the sample exhibits light absorption. The carbazole unit of 10074-A4 is UV active also above 250 nm. Since CD effects occur in the absorption bands of the chromophore, longer-wavelength extrinsic signals of 10074-A4 lost by stopping the scan at 250 nm. The effect of 10074-A4 on the far-UV CD spectrum, i.e. the secondary structure of some c-Myc peptides seems to be misinterpreted. In contrast to the authors’ claim, addition of the inhibitor to c-Myc400-439 did markedly change the CD spectrum displaying and intense positive ellipticity peak below 200 nm which could do not be observed under drug-free condition (Fig. 2F in Supporting Information1). Moreover, not a “little change” but substantial transformation of the CD profile of c-Myc380-439 was provoked by the addition of 10074-A4 (Fig. 2D in Supporting Information1). The single negative extremum (λmax ~ 225 nm) was replaced by two, well resolved minima around 209 and 223 nm, suggesting significant increase of the α-helical content. Unfortunately, quantitative secondary structure estimations were not performed either for free- or drug-loaded forms of the peptides.

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The inhibitors were added to PBS samples (pH 7.4) of c-Myc peptides from 10 mM ethanolic stock solutions. However, neither the volume fraction of EtOH in the inhibitor-peptide mixtures nor its effect on the secondary structure of the peptides were reported.

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References

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binding sites for small-molecule inhibitors on the oncoprotein c-Myc. J. Am. Chem. Soc. 2009, 131, 7390-7401. (2)

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immunosuppressant drug sirolimus. RSC Adv. 2015, 5, 84523-84525. (3)

Viacava Follis, A. G. G. Exploiting structural disorder to enhance small molecule

inhibition of the oncoprotein c-Myc dimerization with its partner Max. Ph.D. Thesis, Georgetown University, 2009. (4)

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D. M. Carbazole alkaloids from the stems of Clausena lansium. J. Nat. Prod. 2012, 75, 677-682. (8)

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bridged dinuclear zinc(II) complex formation: metal-assisted π-association and -dissociation processes. Chem. Eur. J. 2014, 20, 15159-15168. (9)

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human serum albumin by circular dichroism, electronic absorption spectroscopy and molecular modelling methods. Biochem. Pharmacol. 2003, 65, 447-456. (14)

Zsila, F.; Kámán, J.; Bogányi, B.; Józsvai, D. Binding of alkaloids into the S1 specificity

pocket of alpha-chymotrypsin: evidence from induced circular dichroism spectra. Org. Biomol. Chem. 2011, 9, 4127-4137. (15)

Zsila, F.; Iwao, Y. The drug binding site of human α1-acid glycoprotein: insight from

induced circular dichroism and electronic absorption spectra. Biochim. Biophys. Acta 2007, 1770, 797-809. (16)

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Figure 1. Effect of the addition of (±)-10074-A4 (100 µM) on the CD spectrum of 20 µM c-Myc370-409 peptide (modified version of Fig. 5a adapted from ref. 1). CD data are plotted in mean residue molar ellipticity units ([Θ]) calculated by the following equation: [Θ] = Θ/(10ncl), where Θ is the measured ellipticity as a function of wavelength (nm), n is the number of residues in the peptide (40), c is the molar concentration of the peptide, and l is the optical path length (0.1 cm). The [Θ]max value of the ICD peak around 242 nm (≈ -6,000) was converted into ∆ε unit (M-1 cm1

) by using the formalism of ∆ε = Θ/(32982cl), where ‘Θ’ is the ellipticity at 242 nm (-4.8

millidegree). Here ‘c’ is the molar concentration of the CD active, peptide-bound form of the inhibitor which was considered to be 20 µM due to the high molar excess of 10074-A4. Sequence of the c-Myc peptide is shown (one-letter amino acid code).

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Figure 2. Difference CD and UV absorption spectra of norharman and (±)-carprofen added in high molar excess to 5 µM HSA solution (10 mM potassium phosphate buffer, pH 7.3, 0.05 M Na2SO4). Spectra were acquired in 0.1 cm cuvette at 25 °C on a JASCO J-715 spectropolarimeter. ∆ε values were calculated on the basis of the molar concentration of the HSA-bound form of the ligands which was considered to be the same as the protein concentration of the sample (5 µM). For (±)-carprofen that binds to HSA in 2:1 stoichiometry23 10 µM was used. 9 ACS Paragon Plus Environment