A Fusion Protein of the p53 Transaction Domain and the p53-Binding

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A Fusion Protein of the p53 Transaction Domain and the p53 Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors Rong Chen, Jingjing Zhou, Lingyun Qin, Yao Chen, Yongqi Huang, Huili Liu, and Zhengding Su Biochemistry, Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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Biochemistry

A Fusion Protein of the p53 Transaction Domain and the p53 Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors Rong Chen1, Jingjing Zhou1, Lingyun Qin1, Yao Chen1, Yongqi Huang1,*, Huili Liu2,* and Zhengding Su1,* 1

Institute of Biomedical and Pharmaceutical Sciences and Key Laboratory of Industrial

Fermentation (Ministry of Education), Hubei University of Technology, Wuhan, Hubei 430068 China; 2

National Center for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic

Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, Hubei, 430071 China

*

Corresponding Authors: [email protected], [email protected] or

[email protected]

Author Contributions: R.C., and J.Z. carried out the mutagenesis, protein preparation, fluorescence measurements and high-throughput screening; L.Q., and H.L. conducted the NMR analysis; L.Q., and Y. C. performed the ITC assays; Y.H analyzed the binding model; R.C. and Y.C. prepared the compound library; Y.H., H.L., and Z.S. designed the experiments and wrote the paper. Z.S. conceived of the project. 1

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Abbreviations:

MdmX, murine double minute X; Mdm2, murine double minute 2; p53p, MdmX/Mdm2 binding peptide from p53 transactivation domain; HSQC, heteronuclear single quantum correlation; NMR, nuclear magnetic resonance; FP, fluorescence polarization; ITC, isothermal titration calorimetry; SAR, quantitative structure-activity relationship; QSAR, quantitative structureactivity relationship; HTS, high-throughput screening; FLU, fluorescence measurement.

TOC:

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Abstract In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homolog MdmX, demanding efficient therapeutics to disrupt this aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already got into clinical investigations, searching for MdmX-specific inhibitors has become very attracting, requiring more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (NMdmX) through a flexible amino acid linker, where the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of ligand within 65 µM. The fusion protein was further evaluated for its feasibility of high-throughput screening using a model compound library including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

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Both Mdm2 and MdmX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor p53 protein, accounting for the occurrence of nearly half of all cancers.(1-3) Pharmacological intervention of the p53-Mdm2/MdmX interactions to restore p53 activity in the tumors provides a fundamentally therapeutic strategy against many types of cancers, aiming at a targeted and non-toxic cancer treatment.(3,

4)

Thanks to advanced

combinatorial chemistry, many small molecule inhibitors of Mdm2 with distinct scaffolds mimicking α-helical structure have been discovered.(5, 6) Among these inhibitors, nutlin-3a is the golden standard in studying the p53-Mdm2/MdmX interactions and its derivative (RG7112) is currently under clinical trial for the treatment of hematological neoplasms and solid tumors.(7, 8) Although MdmX and Mdm2 are highly similar to each other regarding their amino acid sequences and three-dimensional conformations,(9) Currently-found Mdm2-specific inhibitors exhibit weak affinity binding to MdmX.(6) For example, the binding affinity of nutlin-3a for MdmX is 500 ~ 1000-fold less than that for Mdm2.(7, 10) So far, only few potent MdmX-specific inhibitors have been identified including SJ172550 (11) and XI-006 (12). Pre-clinical investigations on the interaction of p53-Mdm2/MdmX in cancer cells indicate that inhibiting both Mdm2 and MdmX could activate p53 more efficiently than agents that only antagonizing Mdm2 activity.(1315)

The sole use of Mdm2 inhibitor in clinical trials has been found to cause drug-resistant by

unexpected overexpression of MdmX.(16) On the other hand, more and more cancer cases have been identified to associate solely with the overexpression of the oncoprotein MdmX.(3, 17-20) Therefore, it is urgent to design MdmX-specific inhibitors and/or Mdm2/MdmX dual inhibitors as anti-cancer therapeutics. (17, 21)

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Tremendous progresses were made in search of Mdm2 and MdmX inhibitors in past dozen years, which were largely attributed to the application of target-based biochemical and SAR assays, and visual fragment screening.(22) More significantly, the application of scaffoldhopping approach on reported Mdm2 inhibitors leaded to design potent dual Mdm2/MdmX inhibitors.(23) The structural information on the complexes between Mdm2 and various ligands facilitated to generate an epitope-type Mdm2 inhibitor.(24, 25) Molecule fusion of isopropyletherpyrrolidone and alpha-phenylethylamine-pyrrolidone derived potent Mdm2 inhibitors.(26) The incorporation of affinity-based screening with fluorescent polarization assay identified several dual Mdm2/MdmX inhibitors.(27) On the other hand, the combination of similarity searching and docking identified chemically-tractable scaffolds that bound to the p53-binding site of Mdm2.(28) A robust fragment-based QSAR model built up with piperidinone-derived compounds exhibited satisfactory statistical parameters for the experimentally reported dataset.(29) An ultrafast shape recognition strategy enabled to accelerate the visual screening of DrugBank database for searching novel Mdm2/MdmX inhibitors.(30) The visual screening on NCI and SPECS databases using structure-based pharmacophore strategy in combination with docking successfully discovered potent Mdm2/MdmX inhibitors, which could serve as lead compounds for further optimization.(31) These efforts were mainly focused on designing of Mdm2 inhibitors, but should be applicable for MdmX inhibitors. Previous investigations suggested that the ligand-binding pocket on MdmX was much more dynamic than that on Mdm2, even in complex with nutlin analogs.(32)(33, 34) These work suggested that a dynamic-restricted or a rigid protein model was required for designing of MdmX-specific inhibitors and for high-throughput screening of compound library in search of new scaffolds as well.

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In this work we attempted to design a fusion protein model to use the tryptophan of p53p as an intrinsic fluorescence probe to assay the SAR of MdmX inhibitors and for screening of compound library in search of novel scaffolds of MdmX inhibitors. The rationale was based on previous structural and biochemical studies that the Trp23 in the p53p was not only a key residue to determine the high affinity of the p53p for MdmX and Mdm2, but also simultaneously a sole intrinsic fluorescence probe in the interaction of p53p with the p53-binding domains of MdmX and Mdm2 (shortened as N-MdmX and N-Mdm2, respectively)(35, 36). Thus, we constructed a fusion protein of p53p and N-MdmX which provided many advantages over other protein models as no external fluorescence probe was required. We evaluated this fusion protein model with numerous Mdm2 inhibitors with fluorescence spectroscopy and found that the newlyconstructed fusion protein was a simple and efficient tool for high-throughput screening of compound libraries to search for MdmX specific inhibitor.

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MATERIALS and METHODS

Molecular Cloning and Protein Preparation The genes encoding the p53-binding domain of human MdmX (N-MdmX, amino acids 22–110) and human Mdm2 (N-Mdm2, amino acids 22–110) were synthesized with E. coli preference codons and sub-cloned into pHG1 vector, a modified version of pET28 plasmid (Novagen, USA) in which the thrombin cleavage site was replaced with a TEV protease site. To construct the p53p-N-MdmX fusion protein, a synthetic gene encoding p53p, a linker and NMdmX were subcloned into pET28b, and the resultant plasmid was defined as pHG1-p53p-NMdmX. A similar version of plasmid for the expression of the p53p-N-MdmX fusion protein was constructed and defined as pHG1-p53p-N-Mdm2. All of the constructs were confirmed with DNA sequencing. Recombinant proteins were expressed using E. coli BL21 (DE3) cells. Cells were grown in LB media, and protein expression was induced with 0.4 mM IPTG at 20°C for 8 ~12 h. For heteronuclear NMR experiments, protein samples were uniformly labeled with MOPS medium with BME vitamins (Sigma, USA) containing 2 g/L

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N and

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C in

NH4Cl and 1 g/L[13C6]-

glucose as the sole sources of nitrogen and carbon, respectively. Cells were harvested by centrifugation at 6000 xg for 30 min, resuspended in a lysis buffer, and subjected to a short period of sonication for 1 min, followed by homogenization at 4 °C. The lysates were cleared by spinning at 20,000 rpm for 30 min, and the supernatant was loaded onto a 10 ml Ni-NTA agarose column (Qiagen, USA). The elute was concentrated to 10 ~ 12 ml, followed by purification with a Sephedex75 gel filtration column. Peak fractions were combined and concentrated to 1 mg/ml,

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except that the p53p-N-MdmX fusion protein was concentrated to 5 mg/ml. The protein samples were flashly frozen with liquid nitrogen, and sored at -80°C. Chemicals and Mdm2 Inhibitor Library Commercially-available Mdm2 inhibitors including nutlin-3a, RG7112, AMG232, MI773, JNJ-26854165 and AMG232 were purchased from APExBIO (Boston, USA) or SigmaAldrich (St Louis, USA). Other compounds were prepared by ABI Biotech (Wuhan, China) according to the protocols published previously (see Supplementary Information). The p53p peptide (amino acids 15–29) labeled with fluorescein isothiocyanate at the N-terminus for fluorescence polarization (FP) experiments was synthesized by PuAo Biotech (Shanghai, China). NMR Measurements All NMR spectra were collected at 25°C on a Bruker Avance 800 MHz spectrometer equipped with a triple-resonance pulse-field gradient probe.

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N-1H HSQC NMR spectra were

recorded in the echo-antiecho mode for quadrature detection. All the NMR samples were prepared in a buffer containing 20 mM sodium phosphate (pH 6.8), 200 mM NaCl, 0.1% NaN3, 10% D2O and 2 mM DTT. The final protein concentration was approximately 0.4 ~ 1.0 mM. The NMR sample subjected to TEV protease cleavage analysis was prepared with the same protein except containing 0.5 mM EDAT at pH8.0. All datasets were acquired with 2048 complex points in t2 and 256 complex points in t1. Data were processed using Topspin. Tryptophan Fluorescence Measurements The interaction between ligand and fusion protein was analyzed using fluorescence measurements. The assay mixture usually contained 0.1 µM fusion protein in a cuvette being continuously stirred with or without adding ligand in the buffer containing 20 mM phosphate 8


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(pH6.8), 200 mM NaCl and 1 mM DTT, and incubated for 1 min at 25 °C. The fluorescence spectrum was recorded from 300 to 500 nm with an excitation slit width of 5 nm at 278 nm using a Hitachi F-2500 fluorescence spectrophotometer. The fluorescence emission intensity at the emission maximum of 324 nm was determined from each spectrum, and the final value was obtained by averaging the values from three scans of the same sample. Control titration experiments were performed by adding the same volume of buffer instead of ligand. FP-based Competitive Binding Assay FP assays were conducted in assay buffer containing 20 mM phosphate (pH 6.8), 200 mM NaCl and 1 mM DTT. The FP assays were performed by pre-incubating 75 nM fluoresceinp53p with 1.5 µM N-MdmX for 30 min in 96-well black microplates (Corning, USA). Compound was then added and incubated for another 30 min. The FP assays were analyzed using a BioTek H1 multiplate reader with a 555 nm excitation filter and a 632 nm static and polarized filter. The unlabeled p53p peptide and nutlin-3a were used as positive controls. Calculation of Inhibitory Constants The Ki values for the competitive binding of a ligand to the p53p-N-MdmX fusion protein were determined by fitting the fluorescence titration data to a simple bimolecular association model as described by Lepre, C. et al.(37) The p53p-N-MdmX fusion protein exist as an equilibrium between an open conformation (PO) and a closed conformation (PC) with an equilibrium constant Kex. The ligand (L) is only able to binding the open conformation, since in the closed conformation the ligand binding site is occupied by p53p. Thus, the binding process can be described by the following equation:

+ ↔ ∙ 9


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the fluorescence intensity change (fi) is related to the conformational exchange constant (Kex) of p53p-N-MdmX and the inhibitory constant (Ki) of ligand as follows.

=

[] [] = [] + (1 + 1⁄ ) [] +

where [L] is the concentration of ligand L. Kiapp is the apparent inhibitory constant of ligand L for N-MdmX. This kind of treatment should be benefit to HTS processing. Fitting of the data was carried out using MicroCal Origin 8.0 (Northampton, MA). Average Kiapp values were determined from multiple independent measurements. Isothermal Titration Calorimetry (ITC) ITC assay of the protein-ligand interactions was characterized using ITC200 calorimeter (GE Healthcare, USA) or Nano-ITC (TA, USA) at 25°C. A typical experiment included the injection of 19 aliquots (2.0 µL each) containing approximately 0.2 mM ligand solution into a protein solution of 10 ~ 20 µM in the ITC cell (volume ~ 200 µL). An additional set of injections was run in a separate experiment with buffer in the cell instead of the protein solution as a control. Before data analysis, the control values were subtracted from the experimental data. The binding isotherms were integrated to give the enthalpy change (∆H) plotted as a function of the molar ratio of the ligand. When necessary, prior to the integration procedures, the baseline was manually adjusted to minimize the background noise. The initial titration point might be discarded if its heat was too small. The Origin 7.0-based software provided by GE/MicroCal or the software from TA was used for data analysis, and the one set of sites model was used. The association constant Ka (1/Kd) was determined from the slope of the central linear part of the fractional saturation curve. The Gibbs free energy change (∆G) and the entropy

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change (∆S) were calculated based on the following equations: ∆G = −RTlnKa = ∆H − T∆S, where ∆H was derived from the original ∆H/molar ratio plots. Fluorescence-based High-throughput Screening (HTS) For the HTS analysis of Mdm2 inhibitors, the reaction mixture contained 0.5 µM of p53p-N-MdmX fusion protein with or without a compound, in 20 mM sodium phosphate buffer (pH 6.8), 200 mM NaCl, and 2% DMSO. After an incubation at 25 °C for 10 min, the fluorescence intensity of tryptophan was measured with an excitation wavelength of 278 nm, an emission wavelength of 324 nm and a cutoff of 300 nm, using a Perkin Elemr LS55 (Waltham, MA) in a 96-well black plate. The fluorescence intensity of the same plate was measured one more time with an excitation wavelength of 278 nm, an emission wavelength of 380 nm and a cutoff of 380 nm to exclude the effect of the fluorescence signals caused by compounds. A model compound library contained 33 currently-available Mdm2 inhibitors including controls. MdmX inhibitors SJ172550

(11)

and XI-006

(12)

were used as positive controls and DMSO and a

p53p mutant where the three key residues (F19′, W23′ and L26′) were replaced with alanine (defined as AAA-p53p) as negative controls. In our HTS implementation, we also used guanidine hydrochloride (GdmCl) as a control to completely release p53p from N-MdmX.

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RESULTS

Structural Rationale to Design a Fusion Protein of p53p and N-MdmX The p53 binding to the N- MdmX or N-Mdm2 only required a small transactivation peptide domain (p53p) containing fifteen amino acid residues. Once the p53p bound to the ligand-binding pocket of N-MdmX, this peptide folded into a helical structure, as revealed by an X-ray crystal structure of N-MdmX in complex with p53p (Figure 1a). The complex structure also indicated that the C-terminus of p53p was adjacent to the N-terminus of N-MdmX. Thus, it was feasible to connect the two components to form one single polypeptide chain. A single chain of fusion protein could not only ensure a stable complex of p53p and N-MdmX, but also guarantee the interaction of p53p with N-MdmX in equal molar ratio. Based on the distance between the C-terminus of p53p and the N-terminus of N-MdmX, a flexible linker of thirteen amino acid residues was designed to tether p53p to N-MdmX (Figure 1b). The resultant fusion protein was designated as p53p-N-MdmX and its amino acid sequence was shown in Figure 1c. We also designed a similar version of fusion protein by tethering p53p to N-Mdm2 (Figure 1c). It is notable that each fusion protein contains only one tryptophan residue (W23' from the p53p segment). This tryptophan residue not only plays a key role in the interaction between p53p and N-MdmX or N-Mdm2, but also serves as an intrinsic fluorescence probe to monitor the binding process. Alteration of its fluorescence signal caused by any competitive inhibitor should directly reflect the changes bewteen p53p and N-MdmX. For example, a competitive inhibitor that binds to N-MdmX will change the signal of the intrinsic fluorescence probe, as described in Figure 1d.

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Figure 1. A rationale to construct a fusion protein of p53p and N-MdmX for measuring the interaction bewteen N-MdmX with small molecule compounds. a). A three-dimensional structure of the N-MdmX in complex with p53p (PDB ID: 3dab). Three key residues (i.e., F19′, W23′ and L26′) in p53p are depicted in sticks. b). A fusion protein model of p53p/N-MdmX with a flexible linker. The W23' residue on the p53p segment in the fusion protein is deeply buried in the binding pocket. c). The designed p53p-N-MdmX fusion protein is composed of the p53p segment (underlined), an amino acid linker and N-MdmX segment. A similar version of p53p-N-Mdm2 fusion protein was also constructed. Both amino acid sequences are individually numbered

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according to the translated products of their cDNAs. d). A model illustrating the influence of competitive inhibitor on the intrinsic fluorescence of p53p.

Preparation and Characterization of p53p-N-MdmX Fusion Protein A gene encoding the fusion protein of p53p and N-MdmX were synthesized in E. coli preference codons and sub-cloned in a modified version of pET28b protein expression vector for protein expression as described in detail in MATERIALS and METHODS. As shown in Figure 2a, SDS-PAGE results indicated that the fusion protein was overexpressed at 20 °C and almost half of expressed protein was in soluble form. To confirm that the fusion protein adopted a corrected folded structure,

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N-1H HSQC NMR measurement was performed. As shown in

Figure 2b, the resonance peaks of the 15N-1H HSQC NMR spectrum of the fusion protein were well dispersed, indicating that the fusion protein had a well-folded structure. Through 3D NMR experiments, we were able to assign the backbone resonances of the fusion protein, except the resonance peak of the L25' on p53p.

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Figure 2. Characterization of the fusion protein with NMR spectrometry and fluorescence spectroscopy. a). The expression and purification of the p53p-N-MdmX 15


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fusion protein in E coli BL21(DE3) strain was evaluated with SDS-PAGE electrophoresis. M: Molecule weight marker; Lanes 1 & 2: The soluble and insoluble fractions of the E coli strain without IPTG induction, respectively; Lanes 3 & 4: The soluble and insoluble fractions of the E coli strain with IPTG induction, respectively. Lanes 5 & 6: The purified fusion protein was treated with or without 2 mM β-mecaptolethanol, respectively. b). The

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N-1H HSQC NMR spectrum of the fusion protein was assigned with 3D NMR

experiments. The resonance peaks of N-MdmX are labeled in Red, and those of p53p in blue, and linker in green. c). The absorption spectrum (black) and the fluorescence emission spectrum (red) of the p53p-N-MdmX.

To investigate whether the inserted linker affected the binding of p53p to N-MdmX in the p53p-N-MdmX fusion protein, a TEV proteinase cleavage site was included in the linker (Figure 1c), which was used to release the p53p from the fusion protein. As shown in Figure S1, most resonance peaks in the

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N-1H HSQC NMR spectrum were not altered when the fusion protein

was treated by TEV protease. Importantly, the backbone resonance peaks of the three key residues from p53p (i.e., F19′, W23′ and L26′) as well as the side chain resonance peak of W23′ were unaffected by the treatment with TEV protease. Thus, our results indicated that the designed linker was flexible and it did not affect the interaction of p53p segment and N-MdmX. We then used

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N-1H HSQC NMR titration experiment to monitor the p53p releasing from its

binding-pocket on N-MdmX upon addition of nutlin-3a. As shown in Figure S2, the p53p residues including F19′, W23′ and L26′, were significantly perturbed with titrated with nutlin-3a in a molar ratio of 1:3, indicating the release of the p53p segment upon nutlin-3a-binding to N16


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MdmX. It is noted that the resonance peaks of the N-MdmX domain within the fusion protein were also perturbed as the interaction of nutlin-3a and N-MdmX is different from the interactions between p53p and N-MdmX. Before using the tryptophan residue W23′ to monitor the interaction bewteen p53p segment and N-MdmX in the fusion protein, the fluorescence spectrum of the fusion protein was initially scanned from 200 nm to 300 nm with a fixed emission wavelength at 340 nm to determine a maximum excitation wavelength. As shown in Figure 2c, a maximum excitation wavelength was detected at 278 nm. Thereafter, the fluorescence spectrum of the p53p-N-MdmX fusion protein was scanned from 300 nm to 500 nm with the maximum excitation wavelength, resulting in a maximum emission wavelength of 324 nm. This maximum emission wavelength suggested that the W23′ was deeply buried in a hydrophobic core, in totally compliance with the structure of p53p/N-MdmX complexes (Figure 1b).(35) Therefore, the alteration of the W23' fluorescence signal should be directly relevant to the association of p53p with N-MdmX, as proposed in Figure 1d. Quantitative Correlation of the Intrinsic Fluorescence Signal with Ligand-binding Affinity Next we examined how the alteration of the W23' fluorescence intensity in the fusion protein was correlated with the titration of MdmX-binding ligands. Although few relativelyhigh-affinity MdmX inhibitors including SJ172550 and XI-006 were available, Mdm2 inhibitors also exhibited binding with N-MdmX. One of the most thoroughly-studied Mdm2 inhibitors was nutlin-3a which was a promising scaffold template to design MdmX inhibitors.(38) Previous structural investigation revealed that nutlin-3a was not only able to mimic p53p and bind to the p53-binding pocket on Mdm2 (Figure 3a),(7) but also able to bind the p53-binding pocket on 17


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MdmX.(32) Nutlin-3a bound to Mdm2 with a high-affinity with the Kd value of 23 nM and nutlin3a exhibited certain selectivity for N-MdmX with the Kd value of 11 µM via ITC (Figure 3b & Figure S3). Furthermore, the structure of N-MdmX was highly homologue to that of N-Mdm2 (Figure 3c). Taken together, it was feasible to use nutlin-3a as a model compound to compete the interaction of p53p with N-MdmX in our designed fusion protein.

Figure 3. The fluorescence signal attenuation of the fusion protein as a function of nutlin-3a concentration. a). Nutlin-3a can mimic p53p peptide in the binding pocket of N-Mdm2, which is illustrated with superimposition of nutlin 3a (purple) over the three key residues of p53p (cyan). b). The binding affinities of p53p and nutlin-3a for N-Mdm2 18


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Biochemistry

and N-MdmX obtained by ITC. c). The structure of N-MdmX is highly homologue to that of N-Mdm2. N-MdmX, magenta; N-Mdm2, green. d). The fluorescence spectra of p53pN-MdmX were recorded at different concentration of nutlin-3a (from black to orange: 0, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80 and 100 µM, respectively). e). The fluorescence spectra of the free p53p were recorded at an excitation wavelength of 278 nm under various concentrations. 0 µM (black); 8.76 µM (red) and 40 µM (blue). f). The fluorescence spectra of nutlin-3a were recorded at an excitation wavelength of 278 nm under various concentrations. 0 µM (black); 5 µM (red) and 40 µM (blue). g). The binding isotherm of nutlin-3a to the fusion protein was obtained by monitoring the fluorescence changes of W23′ at 324 nm.

As shown in Figure 3d, the fluorescence spectra of the p53p-N-MdmX fusion protein were recorded from 300 nm to 500 nm with an excitation wavelength at 278 nm when the fusion protein was titrated with nutlin-3a. It was notable that the alteration of the fluorescence intensity of W23' at 324 nm was significantly decreased with increasing the concentration of nutlin-3a. From the viewpoint of ligand replacement, the fluorescence change upon the titration of nutlin3a indicated that nutlin-3a molecule displaced the p53p segment from the binding site of NMdmX. Such displacement exposed the p53p including its W23' residue into a solvent environment, resulting in the attenuation of fluorescence intensity at 324 nm and a red shift of the maximum wavelength to 350 nm. We also observed that a shoulder peak with a maximum emission wavelength at 380 nm appeared during the titration of nutlin 3a (Figure 3d). The intensity of the shoulder peaks

19


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increased with the increasing nutlin-3a concentration. To test whether this shoulder peak was caused by the tryptophan residue from the displaced p53p peptide or by nutlin-3a, we recorded the fluorescence spectra of free p53p peptide (Figure 3e) and nutlin-3a (Figure 3f) with the same excitation wavelength as that used for the p53p-N-MdmX fusion protein. By comparison, it was obvious that the shoulder peak was originated from nutlin-3a. Nevertheless, this shoulder peak had very subtle influence on the fluorescence emission peak of the W23' residue within the range of nutlin-3a concentration we tested (≤ 65 µM). The organic solvent DMSO which was used to dissolve nutlin-3a also had very subtle effects on our fluorescence spectroscopic experiment (Figure S4). In the above fluorescence titration experiments, we found that the attenuation of fluorescence intensity at 324 nm was specific for the binding of nutlin-3a to p53p-N-MdmX, as the maximum emission wavelength of the displaced p53p segment (350 nm) was different from that of the N-MdmX bound p53p segment (324 nm). Thus, the titration of nutlin-3a yielded a dose-dependent decrease of the W23' fluorescence intensity at 324 nm (Figure 3g). Theoretically, such fluorescence changes observed in the p53p-N-MdmX/nutlin-3a interactions could not exclude the possibilities of partial binding modes where one of two other key residues (i.e., F19′ or L26′ ) of the p53p peptide still bound to N-MdmX, whereas the W23′ became free. In practice, such complicated effects were negligible for our p53p-N-MdmX/nutlin-3a system, as the mutation analysis on the three key residues of p53p (i.e., F19′, W23′ and L26′) suggested that the interactions of the three key residues with the binding sites on N-MdmX followed a synergic mode.(36)

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Therefore, the titration curve shown in Figure 3g could be best fitted to a simple bimolecular equilibrium binding model to obtain an apparent inhibitory constant Kiapp which embeds a conformational exchange constant (Kex) between the closed and open states of the fusion protein (see MATERIALS AND METHODS). A resulting Ki

app

value was estimated to be

16.7 ± 4.8 µM. The interaction of nutlin-3a with N-MdmX was also assayed with monitoring its displacement of Fluorescein-labeled p53p complexed with N-MdmX using FP method, which yielded a Ki value of 15 ± 3.6 µM. Those inhibitory constants of nutlin-3a were compatible to those previously obtained with different biophysical methods (Table S1). As a control, we prepared the same version of fusion protein for p53p and N-Mdm2 (p53p-N-Mdm2). The p53pN-Mdm2 fusion protein was also used to examine the binding affinity of nutlin-3a for N-Mdm2 (Figure S5). As listed in Table S1, the inhibitory constant of nutlin-3a binding to N-MdmX obtained using the current fluorescence method was in consistent with those obtained using FP and ITC. Next we extended the strategy to examine the binding of fusion protein with other available Mdm2 inhibitors. The currently-published Mdm2 inhibitors contained a variety of scaffolds (Figure 4), such as imidazole,(7) spirooxindole,(39) benzodiazepine,(40) and imidazoindole scaffold.(9) Through either commercial sources or in-house synthesis, we prepared a small compound library containing 33 Mdm2 inhibitors (Table S2). The binding affinities of 33 compounds with N-MdmX were determined using the p53p-N-MdmX fusion protein fluorescence measurement (FLU) as well as FP assay (Figure 5). As shown in Figure 5a, all compounds exhibited weak affinity for N-MdmX within the range from low molar concentration (e.g., Compound 1) to high micromolar concentration (e.g., Compound 6). Five compounds including Compounds 1, 11, 14, 21 and 25 exhibited relatively higher affinity for N-MdmX than 21


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others. Although it was not necessary for the Kiapp values of these tested compounds measured from FLU matched with the Ki values obtained with FP, we found a good liner correlation between two Ki datasets. The correlation coefficient was 0.97 when ploting Kiapp from FLU against Ki from FP (Figure 5b). The good correlation between Kiapp values from FLU and Ki values from FP suggested that our strategy using the fusion protein in combination with fluorescence spectroscopy was a compatible approach to other biophysical technologies to determine the binding affinity of ligand with N-MdmX. This approach is sample and efficient, just requires conventional fluorescence instrumentation.

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Figure 4. A small combinatorial library of Mdm2 inhibitors. The currently-available Mdm2 inhibitors contain four kinds of scaffolds (A, B, C and D) with four different functional groups (R1, R2, R3 and R4). The annotation of each functional group originated from the nutlin-3a structure.

Figure 5. Comparison of inhibitory constants obtained with two methods. a). The Kiapp values obtained with FLU were in parallel compared with those by FP. The data were repeated three times and standard derivations were used. b). The correlation bewteen the Kiapp values from FLU and Ki values from FP.

Feasibility of the Fusion Protein for High-throughput Screening To test whether our fluorescence assay was suitable for identifying small molecule inhibitors of MdmX-p53 interaction in a high-throughput mode as a proof of concept, we further 23


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included positive and negative control compounds into our aforementioned model compound library. MdmX inhibitors SJ172550,(11) XI-006

(12)

and GdmCl were used as positive controls,

and DMSO and AAA-p53p were used as negative controls. Considering that the binding affinity of p53p for N-MdmX was about 0.5 µM,

(35)

we arbitrarily chose an empirical concentration of

each compound for screening experiment at 20xKi (=10 µM). These kind of concentration were commonly used for initial high-throughput screening. In our HTS implementation, we also used GdmCl (a final concentration of 1 M) as a control to completely release p53p from N-MdmX. After adaption of this fusion protein approach in HTS format, we performed an initial screen using the model compound library. As shown in Figure 6, except Compounds 19, 26, 28, 29, XI006 and DMSO, all compounds were able to attenuated fluorescent intensity in different extend. The Z'-factor of this HTS assay was determined to be 0.56 (Figure 6), indicating a suitable and reliable HTS screen approach.(41) Interestingly, we found that XI-006 did no bind to the fusion protein, indicating that this MdmX inhibitor interacted with sites on MdmX different from that p53p interacted with. Furthermore, in order to identify a suitable scaffold of Mdm2, we set the fluorescence intensity change caused by 100 µM of nutlin-3a to 100%. Accordingly we classified these compounds into four groups based on the fluorescence intensity attenuation each compound promoted (Group I: 0 – 25%; Group II: 25 – 50%; Group III: 50 – 75% and Group IV: 75 – 100%). As indicated in Figure S7, there were nineteen compounds in Group I, eight compounds in Group II, four compounds (Compounds 1, 11, 21 and 25) in Group III and one compound (Compound 14) in Group IV (Table S2). The structure features of the compounds from Top 5 compounds (Groups III and IV) indicated that an imidazo-indole scaffold

(9)

was more suitable

for template-based design of MdmX-inhibitors than other scaffold (i.e., nutlin compounds(32)). 24


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Figure 6. Z-score calculation with fluorescence data from the model compound library.

Interestingly, we also found that Compound 19 (an oxidized nutlin-3a) enhanced the fluorescence signal of W23’ in the p53p-N-MdmX fusion protein, indicating that Compound 19 allosterically bound to the fusion protein and enhanced the W23’ binding to N-MdmX. This enhancement was also observed during titration experiment at low concentration (data not

25


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shown). Nevertheless, the compounds in the Group III and IV should be further considered as potential templates for design of MdmX inhibitor. CONCLUSION AND DISCUSSION In summary, we constructed a fusion protein by fusing p53p to N-MdmX via a flexible linker. The fusion protein contained an intrinsic fluorescence tryptophan residue, which was also a key residue to determine the binding affinity of p53p for N-MdmX. Thus, the fusion protein provided many advantages over N-MdmX alone for screening MdmX-inhibitors as no external fluorescence probe was needed. More importantly, the molar ratio of N-MdmX and p53p in the fusion protein was equal so that the replacement of the bound p53p peptide from the surface of the fusion protein by a competitive ligand was readily identified and quantitatively ranked. For a proof of concept, we used nutlin-3a (a weak MdmX binding ligand) to examine the fusion protein. Our results demonstrated that the change of the intrinsic fluorescence readout was correlated with the titration of nutlin-3a. Using NMR titration experiments, we found that the p53p segment was significantly perturbed in a ratio of 1:3 (protein:nutlin-3a). It is notable that the ratio of protein and compound usually much higher in a typical HTS approach such as FPHTS. Furthermore, this strategy was then expanded to test with thirty-three Mdm2 inhibitors. The distribution of apparent binding constants obtained with our current intrinsic fluorescence assay was in a good correlation with those obtained with fluorescence polarization approach. Next the fluorescence approach was evaluated in a small size of compound library including 33 Mdm2 inhibitors, 2 positive controls, 2 negative controls and 1 protein denaturant control. With these known compounds, we were able to confirm SJ172550 as a relatively-strong MdmX inhibitor, while XI-006 was not an inhibitor targeting the interface of p53p-MdmX interaction, which XI-006 might interact with other functional part on MdmX protein. Although 26


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the size of the model library was small, its Z-score indicated our fluorescence approach should be a reliable one for screening compound library in a high-throughput format, comparable to other methods such as FP-based HTS. Thus, when this approach is applied to library, the controls described in this work are recommended so that any false positive or false negative should be readily avoided. Although the methods described in this work was tested on a small library with known compounds, our NMR data, FP data as well as fluorescence results strongly suggested that this method should be implementable as reliable as FP-based HTS method. On the other hand, this method provided in this work should more convenient than others as no external probe is needed. On the other hand, it was the first evaluation of Mdm2 inhibitors for binding MdmX systematically. We also demonstrated that the fusion protein was feasible for ranking the binding affinities of our collected Mdm2 inhibitors for N-MdmX via high-throughput screening by use of fluorescence spectroscopy. Regarding their affinities for N-MdmX, these compounds could be classified into four groups and five compounds exhibited potential as templates for designing MdmX inhibitors. The structure features of these five compounds suggested that imidazo-indole scaffold(9) was more suitable for template-based design of MdmX-inhibitors than others. Through this fusion protein strategy, we also found that one compound (i.e., an oxidized nutlin3a) could enhance the binding of p53p to N-MdmX rather than the replacing of the peptide from the N-MdmX at a low concentration. Thus, our work provides a convenient and efficient protein model for high-throughput screening of MdmX inhibitors. This strategy is currently extended to screen novel inhibitors of Mdm2 and dual inhibitors of MdmX/Mdm2 using natural compound library. We believe that the fusion protein strategy described in this work also can be applicable to other important proteins for drug discovery with or without further modifications. 27


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Acknowledgments This research was supported by Wuhan Natural Science Foundation (ZDS, 201506101010033), State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (ZDS and HLL, T152604). This work was also partially supported by NSFC (YC and ZDS, 31500132) and (YQH, 210603121).

Competing financial interests The authors declare no competing financial interests. Supporting Information Available: Supplementary Experimental Procedures, Results and Discussions are available in the online version of this paper. References associated with these sections appear only in the online version.

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