Rational Design and Structure Validation of a Novel peptide Inhibitor


ucation, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, ... APC-Asef interaction based on the rational drug design and structural ...
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Rational Design and Structure Validation of a Novel peptide Inhibitor of the Adenomatous Polyposis Coli (APC)-Rho Guanine Nucleotide Exchange Factor 4 (Asef) Interaction Xiuyan Yang, Jie Zhong, Qiufen Zhang, Jinxing Qian, Kun Song, Cong Ruan, Jianrong Xu, Ke Ding, and Jian Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01112 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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Rational Design and Structure Validation of a Novel peptide Inhibitor of the Adenomatous Polyposis Coli (APC)-Rho Guanine Nucleotide Exchange Factor 4 (Asef) Interaction Xiuyan Yang,† Jie Zhong,† Qiufen Zhang,† Jinxing Qian,† Kun Song, † Cong Ruan, † Jianrong Xu,§ Ke Ding,|| and Jian Zhang*,†,‡ †

Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Ed-

ucation, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China ‡

Medicinal Bioinformatics Center, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China.

§

Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao-Tong University School of Medicine,

Shanghai 200025, China ||

School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China

Intramolecular Hydrogen Bond, Conformational Restriction, APC-Asef interaction, Drug Design

ABSTRACT: In colorectal cancer, adenomatous polyposis coli (APC) interacts with Rho guanine nucleotide exchange factor 4 (Asef), thereby stimulating aberrant colorectal cancer cell migration. Consequently, the APC-Asef interaction represents a promising therapeutic target to mitigate colorectal cancer migration. In this study, we adopted the rational design strategy involved introducing intramolecular hydrogen bonds and optimizing the lipophilic substituents to improve binding affinity of peptides, leading to the discovery of MAI-400, the best-known inhibitor to date of the APC-Asef interaction (Kd = 0.012 µM, IC50 = 0.25 µM). Comprehensive evaluation of MAI-400 by biochemical and biophysical assays revealed the formation and the effect of an intramolecular hydrogen bond. Cell-based assay showed MAI-400 efficiently blocked the APC–Asef interaction in a dose-dependent manner. Therefore, our study provides a best-in-class inhibitor MAI-400 that can effectively inhibit APC-Asef interaction based on the rational drug design and structural validation.

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INTRODUCTION Adenomatous polyposis coli (APC), a known tumor suppressor protein, is highly correlated with sporadic and familial colorectal tumorigenesis.1-3 The stimulation of APC through its receptor Asef (also known as Rho guanine nucleotide exchange factor 4) disrupts the auto-inhibited state of Asef and promotes constitutive Asef GEF activity,4-6 which regulates the activity of the downstream GTPase CDC42 to promote aberrant colorectal cancer cell migration.7-9 It has been reported that the proliferation of colorectal cancer cells decreased significantly in APC or Asef knockout mice.10 These characteristics make the interaction between APC and Asef a potential therapeutic target for mitigating the invasive migration of colorectal cancer.

A previous study on the APC-Asef complex revealed that the ARM domain of APC (APC-ARM) used a large surfaceexposed pocket, formed by the H3 helices of armadillo repeats 1 through 4 and the H1 helix of armadillo repeat 3, to interact with the ABR region of Asef (Asef-ABR).11 The APC-ARM pocket that bound to Asef-ABR was further confirmed by site-directed mutagenesis of key residues that were important for complex formation. In addition, structural and thermodynamic analyses of the APC-Asef interaction provided important potential hotspots that could be exploited for inhibitor design.

Based on the complex structure, we designed a series of peptides as novel inhibitors of the APC-Asef interaction using high-throughput screening of a peptide library and structure-based optimization. Among these peptides, MAI-203 was the most potent APC inhibitor with an IC50 of 0.57 µM, while MAI-150 was another promising analogue with an IC50 of 1.09 µM.9 Co-crystal structure of APC-MAI-150 shed light on the induced-fit effect of certain residues (e.g., R549) in the APC pocket, providing a crucial prerequisite for subsequent medicinal chemistry optimization.

With the goal of developing new inhibitors of APC-Asef interaction with improved binding affinity, in this study, we reported a rational design strategy that involved introducing intramolecular hydrogen bonds and optimizing the lipophilic substituents at the position 185 of MAI-150 to improve potency of APC inhibitors. Our efforts led to the discovery of a best-in-class inhibitor, MAI-400, that bound to APC with a Kd of 0.012 µM and an IC50 of 0.25 ± 0.01 µM. We further demonstrated the formation and the effect of the intramolecular hydrogen bond to the high-affinity peptide MAI-400 by Surface Plasmon Resonance, X-Ray Crystallographic Analysis, Intrinsic Tryptophan Fluorescence Assay, and Isothermal Titration Calorimetry. Using co-immunoprecipitation assay, we showed that MAI-400 effectively blocked the APC– Asef interaction in cell lysates in a dose-dependent manner. Taken together, our study provides a best-in-class inhibitor MAI-400 that can efficiently inhibit APC-Asef interaction based on the structure-based drug design.

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RESULTS Optimization of MAI-150 by introducing an intramolecular hydrogen bond. Drug discovery research has shown that the formation of intramolecular hydrogen bonds could be beneficial, as the corresponding ligands aligned more favorably with their protein pockets due to conformational restraint.12-16 Considering the flexible conformation of our linear peptide APC inhibitors, increased rigidity and stability of the peptide backbone introduced by proper intramolecular hydrogen bonds might improve the binding affinity of the compounds and better disrupt the APC-Asef interaction. Therefore, we firstly adopted an intramolecular hydrogen bond-based pseudo-cyclic peptide strategy for inhibitor optimization.

The probability of forming an intramolecular hydrogen bond correlated with the acceptor strength is in the order of C=O > heterocyclic N acceptor > S=O > alkoxy.17 Thus, the peptide backbone C=O group was preferred in our inhibitors design. According to the binding conformation of our previous inhibitor MAI-150 (181AGEALYE187) in the APC pocket (PDB ID: 5IZ6), the molecule exhibited a geometrical pattern of H———C=O with an angle of 120.9° and an interaction distance of 3.5 Å between the hydrogen atom of the A184 side chain and the L185 backbone oxygen atom which could be suitable to form an intramolecular hydrogen bond (Figure 1). We realized that if the A184 residue was replaced by amino acids containing hydrogen bond donors in their side chains, then an intramolecular hydrogen bond might be introduced between residues 184 and 185. Therefore, six derivatives of MAI-150, where residue 184 was Ser, Thr, Cys, Asp, Asn, or Tyr (namely, MAI-400, MAI-401, MAI-402, MAI-403, MAI-404, and MAI-405, respectively), were designed and subsequently synthesized.

The ability of the synthesized peptides to inhibit the APC-Asef interaction was quantified using a fluorescencepolarization (FP) competition assay as described previously.9 IC50 values were shown in Table 1 and the competitive binding curves were shown in Figure 2. Compared to MAI-150, replacement of Ala with an amino acid containing a polar side chain such as Ser (MAI-400) improved the binding affinity 4-fold (IC50 = 0.25 ± 0.01 µM). Binding affinity was similar to that of MAI-150 when Ala was substituted with Cys (MAI-402, IC50 = 1.51 ± 0.14 µM). However, changing Ala to Thr (MAI-401, IC50 = 2.01 ± 0.08 µM) decreased the binding affinity by 2-fold. In contrast, residues with a larger side chain such as Asp (MAI-403, IC50 = 6.35 ± 0.45 µM), Asn (MAI-404, IC50 = 5.19 ± 0.34 µM), or Tyr (MAI-405, IC50 = 5.46 ± 0.92 µM) reduced the binding affinity by approximately 5- or 6-fold. With improved binding affinity of MAI-400, an intramolecular hydrogen bond might be introduced between the L185 backbone carbonyl oxygen and the S184 side chain hydroxy group. In contrast, the introduction of other polar residues, such as Thr (MAI-401), Cys (MAI-402), Asp (MAI-403), Asn (MAI-404), or Tyr (MAI-405), at position 184 of MAI-150 could be in disfavor to form an intramolecular hydrogen bond. In detail, the formation of an intramolecular hydrogen bond by large side chains such as D184, N184,

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and Y184 in MAI-403, MAI-404, and MAI-405, respectively, may produce steric hindrance with the APC R549 residue (Supplementary Figure 1). Despite the lack of steric hindrance with APC residues, the intramolecular hydrogen bonds in MAI-401 and MAI-402 are disfavored due to the long distance (>3 Å) between the hydrogen bond donor and acceptor, which may not be accommodated by the conformational constrained APC pocket (Supplementary Figure 2). Taken together, the proper conformation and the steric constraints of the intramolecular hydrogen bond are critical and contributory to the binding affinity of the compound for the receptor.

Optimization of the position 185 of MAI-400. Our crystal structure of APC complexed with MAI-150 showed that the side chain of L185 located in the hydrophobic pocket formed by the F510, R463 and F458 of APC (Figure 1B).9 This hydrophobic interaction was critical to the high binding affinity. Previously we obtained MAI-203 that could efficiently block the APC–Asef interaction through optimization of the L185 side chain.9 To evaluate whether the binding affinity of MAI-400 to APC could be improved, we then adopted the same strategy to optimize the position 185 based on MAI-400 (Figure 1A).

Removal of the side chain of L185 in MAI-400 afforded MAI-437, which bound to APC with IC50 = 108.7±5.3 µM and was > 400-fold less potent than MAI-400 (Table 4). This dramatic loss in binding affinity was consistent with its lack of hydrophobic contacts. According to the result, we further investigated the shape, size and length of substituents at the position 185 for improving the binding affinity. More than 30 analogues with different aliphatic and aromatic hydrophobic groups were synthesized and evaluated as shown in Table 2-5.

We firstly investigated the effects of shape and size with various bioisosteres of the isobutyl group at the position 185 of MAI-400 (Table 2). Introducing cyclopentyl group at the position of R substituent resulted in a slightly less inhibitory analogue (MAI-412, IC50 = 0.62 ± 0.06 µM), compared to MAI-400. Our docking model showed that the cyclopentyl group was oriented into a hydrophobic pocket, and the small-modified structure of MAI-412 at the position led to a partial loss in binding affinity (Supplementary Figure 3A). Compared to isobutyl group, replacement of R with more branched group, such as tert-butyl (MAI-406, IC50 = 1.78 ± 0.22 µM), or larger groups such as cyclohexyl (MAI-413, IC50 = 1.3 ± 0.21 µM) and cycloheptyl (MAI-414, IC50 = 1.94 ± 0.33 µM), moderately decreased the binding affinity. Further docking studies revealed that the branched or larger groups at the position 185 of MAI-400 could be conflict with the side chain of R463 at the bottom of hydrophobic pocket and thus flip out of the hydrophobic pocket (Supplementary Figure 3B-D). In addition, we found that the binding affinities of analogues were also reduced when R was replaced by the moieties with a moderate size and more constrained conformation, such as propenyl (MAI-407, IC50 = 3.64 ± 0.22 µM), bromovinyl (MAI-408, IC50 = 1.82 ± 0.22 µM), cyclopropyl (MAI-410, IC50 = 1.26 ± 0.20 µM), and cyclobutyl (MAI-

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411, IC50 = 1.53 ± 0.21 µM). According to the docking model, the constrained side chain limited the orientation of substituent and that could explain the reduced affinities compared to MAI-400. Furthermore, binding affinity was also significantly reduced when the isobutyl group in MAI-400 was replaced by a linear moiety ethynyl (MAI-409, IC50 = 30.79 ± 1.64 µM), due to the diminished hydrophobic interaction at hydrophobic region.

We next explored the SAR of various aromatic groups at the position 185. All of analogues with five-membered heterocyclic rings (e.g. furanyl (MAI-420, IC50 = 6.95 ± 0.33 µM), thiophenyl (MAI-417, IC50 = 6.86 ± 0.27 µM; MAI-418, IC50 = 4.01 ± 0.16 µM), bromothiophenyl (MAI-419, IC50 = 5.00 ± 0.44 µM)) resulted in a loss of binding activities to APC. Meanwhile, binding affinities were significantly reduced when R was replaced by moieties that were similarly sized but less hydrophobic, such as thiazolyl (MAI-416, IC50 = 16.37 ± 0.58 µM) and imidazolyl (MAI-415, IC50 = 50.62 ± 1.09 µM). In addition, increase in the size of R substituents to phenyl (MAI-421, IC50 = 4.36 ± 0.28 µM; MAI-422, IC50 = 5.07 ± 0.31 µM), indolyl (MAI-423, IC50 = 10.51 ± 0.62 µM), and naphthyl (MAI-424, IC50 = 10.07 ± 0.53 µM; MAI-425, IC50 = 3.96 ± 0.20 µM) also reduced their binding affinities to APC. According to the docking models, the steric clash of the substituents in the analogues could occur with the R463 side chain of APC (Supplementary Figure 4). Therefore, fivemembered or six-membered aromatic groups based on MAI-400 could not be tolerable at the position 185 due to the conformational repulsion with the pocket residue of APC.

We further investigated the length of substituents that the hydrophobic pocket could accommodate at the position 185 based on MAI-400. As shown in Table 3, introduction of methylene unit at L185 side chain of MAI-400 (MAI-426, IC50 = 0.84 ± 0.19 µM) altered the activity slightly. However, introduction of sulfur atom (MAI-427, IC50 = 3.30 ± 0.41 µM) had a negative effect on activity potency to APC. In particular, oxidation to the sulfone (MAI-428, IC50 = 13.95 ± 0.57 µM; MAI-429, IC50 = 5.73 ± 0.3 µM) decreased the binding affinity by >10-fold. We then designed a longer R group as MAI-430, which also decreased the binding affinity (IC50 = 3.02 ± 0.27 µM). Docking studies showed that the long substituent in MAI-430 hardly accommodated into the hydrophobic pocket and thus flipped out of the pocket (Supplementary Figure 5A). On the other hand, introducing a shorter group at the position 185 of MAI-400 produced analogue MAI432, which also obviously decreased inhibitory activity (IC50 = 12.41 ± 0.52 µM) (Table 4). Similarly, other shorter groups like tert-butyl (MAI-433, IC50 = 31.47 ± 0.92 µM), sec-butyl (MAI-431, IC50 = 3.85 ± 0.21 µM), cyclopentyl (MAI434, IC50 = 4.07 ± 0.28 µM), cyclohexyl (MAI-435, IC50 = 2.95 ± 0.25 µM), and phenyl (MAI-436, IC50 = 4.29± 0.32µM) were observed to significantly weaken their binding affinities to APC. Docking analyses indicated MAI-433 and MAI-434 had similar binding conformation of MAI-400 (Supplementary Figure 5B-5E), but their substituents were too small to occupy the entire hydrophobic pocket. In addition, we also tried to optimize MAI-400 by other miscellaneous groups at the position 185 (MAI-438, IC50 = 10.35 ± 0.44 µM; MAI-439, IC50 = 128.1 ± 7.2 µM; MAI-440, IC50 = 25.59 ± 1.08 µM).

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Unfortunately, no one showed better binding potency compared to MAI-400 (Table 5), and our docking studies further confirmed the substituents could have steric conflicts with the hydrophobic pocket of APC (Supplementary Figure 6).

Collectively, the extensive SAR at the position 185 of MAI-400 demonstrated that the APC pocket for binding the side chain of 185 residue in MAI-400 was rigid and hard to induce a lot, indicating that the strategy at the position 185 from MAI-150 to MAI-203 may not be suitable for MAI-400. Therefore, the intramolecular hydrogen bond in MAI-400 provided an alternative way to optimize the affinity of MAI-150 to APC.

MAI-400 Binding Kinetic Analysis. To further explore the binding affinity of MAI-400 to APC, we measured the binding kinetics of MAI-400 using the Surface Plasmon Resonance (SPR) method on a CM5 sensor chip with immobilized APC protein. SPR data showed that both MAI-400 and MAI-150 interacted reversibly with APC in a dosedependent manner (Figure 3). As shown in Table 6, the binding of MAI-400 to APC reached an equilibrium, yielding a kon value of (3.05 ± 0.08) × 105 M-1 s-1 and a koff value of (6.7 ± 0.2) × 10-2 s-1. The parameters of max response (Rmax) and Chi2 for fitting evaluation in the SPR were shown in Supplementary Table 1. Compared to MAI-150, MAI-400 had a longer residence time (RT = 15 s) and a better kinetic KD value (koff/kon) of 0.22 ± 0.01 µM for binding to APC, which was in good agreement with the results obtained in the FP assays (Figure 2), indicating that the introduction of a hydroxy group at the Ala side chain improved binding affinity between MAI-400 and APC.

X-ray Crystallographic Analysis of the APC-MAI-400 Complex. To investigate the molecular basis for the improved binding affinity of MAI-400 for APC, we determined the crystal structure of MAI-400 bound to APC with a resolution of 1.79 Å. The structure showed unambiguous electron density for the bound MAI-400 in the APC-ARM pocket (Figure 4A), and the detailed parameters of data collection and refinement of the crystal structure were listed in Table 7. The structure showed that MAI-400 occupied one side of the APC-ARM pocket and made a curve around APC residue R549 (Figure 4B). As expected, MAI-400 contained an intramolecular hydrogen bond (distance = 2.7 Å, angle = 135.3°) formed by the hydroxy group of the S184 side chain and the carbonyl oxygen atom of the L185 backbone, leading to a pseudo-9-atom ring in the middle of MAI-400 (Figure 4C-4D). The strong intramolecular hydrogen bond fixed the flexible MAI-400 into a partially constrained conformation at the corner of the APC pocket and induced a local arrangement of the remaining atoms (CS184-CS184-NL185-CL185-CL185-OL185) in the ring. Overlapping the structure of APC-MAI-400 with APC-MAI-150 showed that the formation of the intramolecular hydrogen bond pushed the backbone atoms of S184 in MAI-400 to move toward N550 of APC, which resulted in improved hydrogen bonding between MAI-400 and APC with hydrogen bond distances of 2.5 Å and 2.7 Å compared to 2.7 Å and 2.9 Å in the MAI-150-APC complex (Figure 5A). In addition to the stronger hydrogen bonds, MAI-400 also maintained other key salt bridges, hydrogen bonds, and hydro-

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phobic interactions with APC (Figure 4C), as previously observed in MAI-150.9 The complex structure also showed that the constraint by the intramolecular hydrogen bond of MAI-400 induced a conformational change of W553 in APC, which could have a positive effect on stabilizing the conformation of APC (Figure 5B). To detect whether the intramolecular bond in MAI-400 exists before the peptide binds to APC, we performed a Hydrogen-Deuterium exchange assay by 1H Nuclear Magnetic Resonance (NMR), and the results showed that the hydrogen responses in NMR spectra were immediately disappeared when D2O was added (Supplementary Figure 7), indicating that the intramolecular bond in MAI-400 could not exist in solution without APC. The effect supported that APC can induce and stabilize the formation of the hydrogen bond in MAI-400 upon their binding. Therefore, this structure validated our intramolecular hydrogen bond strategy and showed that the conformational constraint induced by the intramolecular hydrogen bond tighten the binding with APC and stabilized the complex.

Intrinsic Tryptophan Fluorescence Assay. Biochemical and structural assays indicated that MAI-400 formed an intramolecular hydrogen bond that increased its binding affinity for APC compared to MAI-150 and changed the position of W553 of APC. Thus, we investigated the tryptophan fluorescence emission spectra of APC in the absence and presence of MAI-400 and MAI-150. The blue shift in fluorescence for the APC complexes with both peptides suggested their similar modes of binding (Figure 6). Meanwhile, the changes in the fluorescence maximum intensity were likely due to reposition of W553 of APC upon binding of MAI-400 and MAI-150, and the slightly higher fluorescence intensity of MAI400, in comparison with MAI-150, was likely due to a longer residence time of MAI-400 in complex with APC, which was in good agreement with the improved binding affinity of MAI-400 for APC as shown by the FP (Figure 2) and kinetic analyses (Figure 3).

Thermodynamic Analysis of the Binding Enthalpy and Entropy. The co-crystal structure and tryptophan fluorescence assay revealed that the formation of an intramolecular hydrogen bond in MAI-400 induced some additional conformational changes in the binding between APC and MAI-400. To furtherly explore how these changes impact on the binding affinity of MAI-400 for APC, we used an Isothermal Titration Calorimetry (ITC) assay to measure the energy contribution during binding of APC by MAI-400 and MAI-150 (Figure 7). The averaged dissociation constant (Kd), binding free energy (∆G), enthalpy (∆H), and entropy term (−T∆S) of three independent ITC experiments were listed in Table 8. The ITC results showed that the Kd value of MAI-400 was 17.5-fold better than that of MAI-150 (0.012 µM vs. 0.21 µM), due to the clear increase in enthalpic and decrease in entropic energies. Consistent with the conformational changes induced by compound binding, the increased enthalpic energy of MAI-400 might derive from the improved hydrogen bonding interactions between MAI-400 and APC and the corresponding conformational change in APC. Conversely, entropy loss was increased in the thermodynamic titration of MAI-400 to APC compared to MAI-150. Two fac-

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tors should be considered when calculating the entropy change associated with protein-ligand binding: conformational entropy change and desolvation entropy change. For MAI-400, the stable conformation maintained by an intramolecular hydrogen bond might contribute to more favorable entropy, but the entropic loss induced by the increased polar surface area of the S184 hydroxy group overcame the gain of entropic energy from the stable conformation, leading to the observed decreased entropic energy of MAI-400 compared to MAI-150. Collectively, the improved binding affinity of MAI400 was mainly derived from a favorable increase in enthalpic energy due to the introduction of an intramolecular hydrogen bond. We also performed the ITC experiments at 37 ºC. As expected, the binding of MAI-400 to APC became less favorable at body temperature (Supplementary Figure 8). Remarkably, MAI-400 (Kd = 0.21 µM) still had a significant advantage over MAI-150 (Kd = 1.8 µM) in terms of binding affinity.

Co-immunoprecipitation. To determine if MAI-400 disrupt the association of APC protein with Asef protein in cells, a co-immunoprecipitation (Co-IP) study was carried out on the lysates from HEK293T cells treated with DMSO, MAI150 (10 µM) or MAI-400 (2 µM, 10 µM). MAI-400 decreased the association of cellular APC protein with cellular Asef in a dose-dependent manner (Figure 8). Moreover, MAI-400 had more potency in the block of APC-Asef interaction compared to MAI-150 at 10 µM in cell lysate. To exclude the possibility of MAI-400 aggregation in the inhibition of proteinprotein interactions, MAI-400 spectra at different concentration were measured using 1H NMR. We did not observe any change of chemical shifts of hydrogens in MAI-400 even at the concentration of up to 10 mM (Supplementary Figure 9), suggesting that MAI-400 could not aggregate below the concentration. Actually, the maximal concentration of MAI-400 in our experiments was 0.4 mM and therefore it could not affect their binding to APC and their ability to inhibit proteinprotein interactions. All the results demonstrated that MAI-400 effectively inhibited the APC-Asef protein-protein interaction in the cell based co-IP assay.

DISCUSSION AND CONCLUSIONS The APC-Asef interaction is a promising novel therapeutic target for mitigating the invasive migration of colorectal cancer. Therefore, high affinity inhibitors of this interaction are urgently needed. Utilizing the co-crystal structure of MAI150 complexed with APC, we designed a series of peptides containing intramolecular hydrogen bond and various lipophilic substituents at the position 185. Through comprehensive structural and binding affinity evaluation by biochemical and biophysical assays, MAI-400 was determined to be a best-in-class inhibitor of the APC-Asef interaction.

A crystal structure of MAI-400 in complex with APC showed an intramolecular hydrogen bond between the L185 backbone carbonyl oxygen and the S184 side chain hydroxy group in MAI-400, which successfully improved the binding affinity of MAI-400 for APC, as expected. However, the changes at the position 185 of MAI-400 decreased the bind-

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ing affinity to APC, indicating that the strategy at the position 185 from MAI-150 to MAI-203 is not suitable for MAI-400. Therefore, the intramolecular hydrogen bond in MAI-400 provides an alternative novel way to optimize the affinity from MAI-150 to APC, leading to a better inhibitor MAI-400 compared to MAI-203.

The conformation constraint strategy successfully improved the binding efficiency, leading to MAI-400, which efficiently blocked the APC–Asef interaction in cell lysates in a dose-dependent manner. Actually, we tried our best to design several scaffolds of compounds through introduction a covalent bond in place of the intramolecular H-bond to form a 9-atom ring, and only MAI-441 has been synthesized (Supplementary Table 2). The FP assay showed that MAI-441 did not have the effective binding affinity to APC compared to MAI-400 (Figure 9). By overlapping MAI-441 into MAI400, we found that the rigid 9-atom ring in MAI-441 is hard to perfectly mimic the conformation of the intramolecular Hbond in MAI-400 (Supplementary Figure 10), and more efforts should be made for the design of such potential covalent scaffolds in future study.

In summary, we designed and synthesized a series of peptides that inhibited the of APC-Asef interaction using a rational design strategy, leading to a best-in-class inhibitor, MAI-400, with a Kd of 0.012 µM and an IC50 of 0.25 µM. Using a combination of X-ray crystal structure determination, tryptophan fluorescence measurements, and thermodynamic characterization, we demonstrated an enthalpy-driven improvement in the binding affinity of MAI-400 for APC due to the presence of an intramolecular hydrogen bond in MAI-400. This study demonstrates a useful approach for peptide optimization, resulting in the identification of a potent inhibitor of the APC-Asef interaction that warrants further study.

EXPERIMENTAL SECTION 1. Peptides Synthesis and Purification. The desired peptides were procured by coupling of the N-

Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids using O-Benzotriazole-N,N,N',N'-tetramethyl-uroniumhexafluorophosphate or Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexaflu as the coupling reagent and N,NDiisopropylethylamine as the base. Fmoc-protecting groups were removed using 20% piperidine in dimethylformamide (DMF) after each coupling step. After the peptides were assembled, the N-terminal amines were acylated with benzyl chloroformate in N,N-Diisopropylethylamine. The peptides were then cleaved from the resin and deprotected using a mixture of trifluoroacetic acid, water, and triisopropylsilane (95:2.5:2.5, v/v/v) for 2.5−3 h. The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and lyophilized to give a white powder. Purified peptides were analyzed by analytical RP-HPLC, and the integrity of the peptides was checked by negative ion electrospray ionization mass spectrometry (ESI-MS). All peptides were purified to ≥98% purity using RP-HPLC and the purity was verified using ESI-MS (as shown in the Supporting Information).

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Peptide MAI-400. Prepared as described above. ESI-MS (m/z): 899.30 [M - H]-, HPLC Relative purity: 99.55%. Peptide MAI-401. Prepared as described above. ESI-MS (m/z): 913.4 [M - H]-, HPLC Relative purity: 98.58%. Peptide MAI-402. Prepared as described above. ESI-MS (m/z): 915.2 [M - H]-, HPLC Relative purity: 98.03%. Peptide MAI-403. Prepared as described above. ESI-MS (m/z): 927.3 [M - H]-, HPLC Relative purity: 98.7%. Peptide MAI-404. Prepared as described above. ESI-MS (m/z): 926.3 [M - H]-, HPLC Relative purity: 98.35%. Peptide MAI-405. Prepared as described above. ESI-MS (m/z): 976.4 [M - H]-, HPLC Relative purity: 98.69%. Peptide MAI-406. Prepared as described above. ESI-MS (m/z): 913.3 [M - H]-, HPLC Relative purity: 99.06%. Peptide MAI-407. Prepared as described above. ESI-MS (m/z): 897.4 [M - H]-, HPLC Relative purity: 99.10%. Peptide MAI-408. Prepared as described above. ESI-MS (m/z): 963.1 [M - H]-, HPLC Relative purity: 98.2%. Peptide MAI-409. Prepared as described above. ESI-MS (m/z): 881.3 [M - H]-, HPLC Relative purity: 98.86%. Peptide MAI-410. Prepared as described above. ESI-MS (m/z): 897.35 [M - H]-, HPLC Relative purity: 98.87%. Peptide MAI-411. Prepared as described above. ESI-MS (m/z):911.30 [M - H]-, HPLC Relative purity: 99.41%. Peptide MAI-412. Prepared as described above. ESI-MS (m/z):925.75 [M - H]-, HPLC Relative purity: 98.31%. Peptide MAI-413. Prepared as described above. ESI-MS (m/z): 939.25 [M - H]-, HPLC Relative purity: 98.43%. Peptide MAI-414. Prepared as described above. ESI-MS (m/z): 953.55 [M - H]-, HPLC Relative purity: 98.44%. Peptide MAI-415. Prepared as described above. ESI-MS (m/z): 923.35 [M - H]-, HPLC Relative purity: 98.45%. Peptide MAI-416. Prepared as described above. ESI-MS (m/z): 940.15 [M - H]-, HPLC Relative purity: 98.68%. Peptide MAI-417. Prepared as described above. ESI-MS (m/z): 939.35 [M - H]-, HPLC Relative purity: 99.06%. Peptide MAI-418. Prepared as described above. ESI-MS (m/z): 939.15 [M - H]-, HPLC Relative purity: 98.89%. Peptide MAI-419. Prepared as described above. ESI-MS (m/z): 1018.9 [M - H]-, HPLC Relative purity: 98.47%.

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Peptide MAI-420. Prepared as described above. ESI-MS (m/z): 923.00 [M - H]-, HPLC Relative purity: 98.16%. Peptide MAI-421. Prepared as described above. ESI-MS (m/z): 933.40 [M - H]-, HPLC Relative purity: 98.35%. Peptide MAI-422. Prepared as described above. ESI-MS (m/z): 951.4 [M + H]+, HPLC Relative purity: 98.31%. Peptide MAI-423. Prepared as described above. ESI-MS (m/z): 974.5 [M + H]+, HPLC Relative purity: 98.06%. Peptide MAI-424. Prepared as described above. ESI-MS (m/z): 983.35 [M - H]-, HPLC Relative purity: 98.93%. Peptide MAI-425. Prepared as described above. ESI-MS (m/z): 983.20 [M - H]-, HPLC Relative purity: 98.62%. Peptide MAI-426. Prepared as described above. ESI-MS (m/z): 913.35 [M - H]-, HPLC Relative purity: 98.28%. Peptide MAI-427. Prepared as described above. ESI-MS (m/z): 917.45 [M - H]-, HPLC Relative purity: 98.51%. Peptide MAI-428. Prepared as described above. ESI-MS (m/z): 933.35 [M - H]-, HPLC Relative purity: 98.31%. Peptide MAI-429. Prepared as described above. ESI-MS (m/z): 949.30 [M - H]-, HPLC Relative purity: 98.95%. Peptide MAI-430. Prepared as described above. ESI-MS (m/z): 955.55 [M - H]-, HPLC Relative purity: 99.44%. Peptide MAI-431. Prepared as described above. ESI-MS (m/z): 901.5 [M + H]+, HPLC Relative purity: 98.30%. Peptide MAI-432. Prepared as described above. ESI-MS (m/z):887.4 [M + H]+, HPLC Relative purity: 98.34%. Peptide MAI-433. Prepared as described above. ESI-MS (m/z):899.30 [M - H]-, HPLC Relative purity: 99.00%. Peptide MAI-434. Prepared as described above. ESI-MS (m/z):911.25 [M - H]-, HPLC Relative purity: 98.74%. Peptide MAI-435. Prepared as described above. ESI-MS (m/z):925.35 [M - H]-, HPLC Relative purity: 98.45%. Peptide MAI-436. Prepared as described above. ESI-MS (m/z):919.20 [M - H]-, HPLC Relative purity: 98.14%. Peptide MAI-437. Prepared as described above. ESI-MS (m/z): 845.4 [M + H]+, HPLC Relative purity: 98.24%. Peptide MAI-438. Prepared as described above. ESI-MS (m/z): 925.5 [M - H]-, HPLC Relative purity: 98.17%. Peptide MAI-439. Prepared as described above. ESI-MS (m/z): 923.6 [M - H]-, HPLC Relative purity: 98.04%.

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Peptide MAI-440. Prepared as described above. ESI-MS (m/z): 923.6 [M - H]-, HPLC Relative purity: 98.28%. Peptide MAI-441. Prepared as described above. ESI-MS (m/z): 864.45 [M - H]-, HPLC Relative purity: 98.30%. 9

2. FP-Based Binding Assays. Procedures to determine the binding affinity have been previously reported. Assays

were performed in 96-well round-bottom plates using a Synergy H4 plate reader. A DMSO solution of the peptide (4 µL) at various concentrations and 91 µL of an APC protein that included residues 303–739 were mixed with the assay buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM DTT). After incubating the plates at room temperature with gentle shaking for 60 min, 5 µL of the probe was added and the plates were gently shaken for another 60 min. Three control wells including a blank (without protein and tracer), 100% inhibition (tracer only), and 0% inhibition (complex solution only) were included on each plate. Dose-response curves were constructed and IC50 values were calculated using GraphPad Prism 5.0 software. The corresponding IC50 values were calculated as previously described.9

3. Surface Plasmon Resonance. For SPR analysis, the His-APC protein was immobilized on a CM5 sensor chip (GE)

using an amine coupling reaction. Briefly, 0.2 M EDC and 0.05 M NHS were used to activate the surface, and then the APC protein (50 mM Hepes, 300 mM NaCl, 1 mM EDTA, pH 7.5) was injected for 420 s with a constant flow rate of 30 µL/min. The remaining activated groups were blocked with ethanolamine, pH 8.5. For a control surface, the upstream parallel flow cell was immobilized with BSA and the unreacted activated groups were blocked with 1 M ethanolamine. The binding assay was conducted in Hepes (0.05 M, pH 7.5) with the peptides (10 mM) injected into the flow system at a flow rate of 30 µL/min. Dissociation was conducted in Hepes (0.05 M, pH 7.5) for 6 min, and then the chip was regenerated with glycine-HCl (2 M, pH 2.0). The kinetic binding constants were analyzed via BIA T200 evaluation software using the 1:1 Langmuir binding model. 4. Crystallization and Structure Determination. Expression, purification and crystallization of the APC protein were

performed in a similar manner to our previous studies.9 The purity of MAI-400 was >95% as determined by HPLC. The APC protein (407–751) (15 mg/mL) and MAI-400 (final concentration, 2 mM) as well as three kits (SM4, IHT, and VV) were added to 96-well drop plates. Then, 0.5 µL of the mixture and 0.5 µL of the crystallization buffer in the sitting drop was equilibrated against a well of 50 µL of the crystallization buffer at 18 °C. The crystal was cryoprotected in 0.2 M ammonium sulfate, 0.1 M Tris pH 8.0, 25% (wt/vol) PEG 3350 and 15% glycerol and was then flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on beamline BL17U1 at Shanghai Synchrotron Radiation.18,19 The structure was solved, further refined and deposited into the PDB bank with code 5Z8H.

5. Tryptophan Fluorescence Measurements. Tryptophan fluorescence measurements (TFM) were performed using Synergy H4 (BioTek Instruments, Winooski, USA) in 96-well, round-bottom plates (Corning; #3650). TFM was per-

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formed by exciting the single tryptophan residue at 280 nm and scanning the emission fluorescence from 300 nm to 500 nm. The total volume of each sample was 50 µL. The buffer for TFM contained 50 mM Hepes pH 7.5, 300 mM NaCl, and 1 mM EDTA. Samples containing 5 µM of the APC protein alone, MAI-150 or MAI-400 at concentration of 35 µM were incubated at room temperature with gentle shaking for 30 min. Each experiment was performed in triplicate.

6. Thermodynamic Data Determination. ITC experiments were conducted in Hepes buffer (50 mM HEPES pH 7.5,

300 mM NaCl, and 1 mM EDTA) on a MicroCal iTC200 system (GE Healthcare) at 30 °C and 37 °C. Titrations were performed in a similar manner to those described in our previous studies.9 Titrations were repeated three times to ensure reproducibility. Finally, a single-site binding model with Origin for ITC version 7.0 (MicroCal)20,21 was employed and a nonlinear least-squares routine was used to obtain the stoichiometry (N), enthalpy of the reaction (∆H), and the association constant (K). 7. Co-immunoprecipitation. For exogenous Co-IP, human HEK293T cells were transfected with pBABE-Flag-APC

(303–876) and pBABE-HA-Asef (170–632) with Lipofectamine 2000 (Invitrogen; 11668019).22 Then, 24 h after transfection, cells were washed twice with cold PBS and lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche)) for 1 h on ice. The cell lysates were cleared by centrifugation at 20,000 g at 4 °C for 10 min. The supernatant was incubated with DMSO, MAI-150 (10 µM), MAI-400 (2 µM) or MAI-400 (10 µM) for 2 h at 4 °C. Then, 30 µL of anti-FLAG M2 Affinity Gel (Sigma-Aldrich; A2220) was added and incubated overnight at 4 °C on a vertical roller. Beads coated with protein were immunoprecipitated, washed four times with RIPA buffer, and applied to 10% SDS–polyacrylamide gels for western blotting analysis.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the internet at http://pubs.acs.org. Complementary experimental data, full biological data, docking information (2D diagrams), tables for macrocyclic analogs, and physical characteristics of peptides, analytical HPLC traces and ESI-MS spectra. Molecular formula strings and some data. APC−MAI-150 complex 3D coordinates 5IZ6.

AUTHOR INFORMATION Corresponding Author *J.Z.:phone, +86-21-63846590; email, [email protected]

Author Contributions

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J.Z. designed the research project. X.Y. carried out the peptide synthesis, purification and characterization. J.X., Q.Z., J.Z., performed the biological experiments and analyzed the data. K.S. solved the crystal structures. J.X. performed the SPR experiment. All authors contributed to the manuscript, with J.Z. assuming responsibility for the manuscript in its entirety.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was funded in part by grants from the National Basic Research Program of China (973 Program) (2015CB910403 to J.Z.), the National Natural Science Foundation of China (grant 91753117 to J.Z., 81322046 to J.Z., and 21702137 to X.Y.), Innovation Program of Shanghai Municipal Education Commission (2019 to J.Z.), and the Shanghai Sailing Program (grant 17YF1410600 to X.Y.).

ABBREVIATIONS APC

Adenomatous polyposis coli

Asef

Rho guanine nucleotide exchange factor 4

Cpa

(2S)-2-amino-3-cyclopentylpropanoic acid

FP

Fluorescence polarization

Fmoc

Fluorenylmethyloxycarbonyl

DMSO

Dimethyl sulfoxide

DMF

Dimethylformamide

DCM

Dichloromethane

EDTA

Ethylenediaminetetraacetic acid

TFA

Trifluoroacetic acid

DTT

Dithiothreitol

PBS

phosphate buffered saline

REFERENCES 1. Aoki, K.; Taketo, M. M. Adenomatous polyposis coli (APC): a multi-functional tumor suppressor gene. J.Cell Sci. 2007, 120, 3327-3335. 2. Akiyama, T.; Kawasaki, Y. Wnt signalling and the actin cytoskeleton. Oncogene 2006, 25, 7538-7544. 3. Schneikert, J.; Behrens, J. The canonical Wnt signalling pathway and its APC partner in colon cancer development. Gut 2007, 56, 417-425.

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4. Mitin, N.; Betts, L.; Yohe, M. E.; Der, C. J.; Sondek, J.; Rossman, K. L. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nat. Struct. Mol. Biol. 2007, 14, 814-823. 5. Kawasaki, Y.; Senda, T.; Ishidate, T.; Koyama, R.; Morishita, T.; Iwayama, Y.; Higuchi, O.; Akiyama, T. Asef, a link between the tumor suppressor APC and G-protein signaling. Science 2000, 289, 1194-1197. 6. Murayama, K.; Shirouzu, M.; Kawasaki, Y.; Kato-Murayama, M.; Hanawa-Suetsugu, K.; Sakamoto, A.; Katsura, Y.; Suenaga, A.; Toyama, M.; Terada, T.; Taiji, M.; Akiyama, T.; Yokoyama, S. Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism. J. Biol. Chem. 2007, 282, 4238-4242. 7. Gotthardt, K.; Ahmadian, M. R. Asef is a Cdc42-specific guanine nucleotide exchange factor. Biol. Chem. 2007, 388, 67-71. 8. Cheng, H. T.; Juang, I. P.; Chen, L. C.; Lin, L. Y.; Chao, C. H. Association of Asef and Cdc42 expression to tubular injury in diseased human kidney. J. Investig. Med. 2013, 61, 1097-1103. 9. Jiang, H.; Deng, R.; Yang, X.; Shang, J.; Lu, S.; Zhao, Y.; Song, K.; Liu, X.; Zhang, Q.; Chen, Y.; Chinn, Y. E.; Wu, G.; Li, J.; Chen, G.; Yu, J.; Zhang, J. Peptidomimetic inhibitors of APC-Asef interaction block colorectal cancer migration. Nat. Chem. Biol. 2017, 13, 994-1001. 10. Kawasaki, Y.; Sato, R.; Akiyama, T. Mutated APC and Asef are involved in the migration of colorectal tumour cells. Nat. Cell. Biol. 2003, 5, 211-215. 11. Zhang, Z.; Chen, L.; Gao, L.; Lin, K.; Zhu, L.; Lu, Y.; Shi, X.; Gao, Y.; Zhou, J.; Xu, P.; Zhang, J.; Wu, G. Structural basis for the recognition of Asef by adenomatous polyposis coli. Cell Res. 2012, 22, 372-386. 12. Nomura, M.; Kinoshita, S.; Satoh, H.; Maeda, T.; Murakami, K.; Tsunoda, M.; Miyachi, H.; Awano, K. (3-substituted benzyl)thiazolidine-2,4-diones as structurally new antihyperglycemic agents. Bioorganic & Medicinal Chemistry Letters 1999, 9, 533-538. 13. Harter, W. G.; Albrect, H.; Brady, K.; Caprathe, B.; Dunbar, J.; Gilmore, J.; Hays, S.; Kostlan, C. R.; Lunney, B.; Walker, N. The design and synthesis of sulfonamides as caspase-1 inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 809812. 14. Van Zandt, M. C.; Sibley, E. O.; McCann, E. E.; Combs, K. J.; Flam, B.; Sawicki, D. R.; Sabetta, A.; Carrington, A.; Sredy, J.; Howard, E.; Mitschler, A.; Podjarny, A. D. Design and synthesis of highly potent and selective (2arylcarbamoyl-phenoxy)-acetic acid inhibitors of aldose reductase for treatment of chronic diabetic complications. Bioorg. Med. Chem. 2004, 12, 5661-5675. 15. Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding in medicinal chemistry. J. Med.Chem. 2010, 53, 2601-2611.

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16. Giordanetto, F.; Tyrchan, C.; Ulander, J. Intramolecular hydrogen bond expectations in medicinal chemistry. Acs Med. Chem. Lett. 2017, 8, 139-142. 17. Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J. Y.; Renault, E. The pK(BHX) database: toward a better understanding of hydrogen-bond basicity for medicinal chemists. J. Med. Chem. 2009, 52, 4073-4086. 18. Zhao, Y.; Zhang, X.; Chen, Y.; Lu, S.; Peng, Y.; Wang, X.; Guo, C.; Zhou, A.; Zhang, J.; Luo, Y.; Shen, Q.; Ding, J.; Meng, L.; Zhang, J. Crystal structures of PI3Kα complexed with PI103 and its derivatives: new directions for inhibitors design. ACS Med. Chem. Lett. 2013, 5, 138-142. 19. Chen, S.; Hu, T.; Zhang, J.; Chen, J.; Chen, K.; Ding, J.; Jiang, H.; Shen, X. Mutation of Gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3Clike protease: crystal structure with molecular dynamics simulations. J. Biol. Chem. 2008, 283, 554-564. 20. Han, C.; Zhang, J.; Chen, L.; Chen, K.; Shen, X.; Jiang, H. Discovery of Helicobacter pylori shikimate kinase inhibitors: bioassay and molecular modeling. Bioorg. Med. Chem. 2007, 15, 656-662. 21. Lu, S.; Banerjee, A.; Jang, H.; Zhang, J.; Gaponenko, V.; Nussinov, R. GTP binding and oncogenic mutations may attenuate Hypervariable Region (HVR)-catalytic domain interactions in small GTPase K-Ras4B, exposing the effector binding site. J. Biol. Chem. 2015, 290, 28887-29000. 22. Liu, B.; Zheng, Y.; Wang, T.; Xu, H.; Xia, L.; Zhang, J.; Wu, Y.; Chen, G.; Wang, L. Proteomic identification of common SCF ubiquitin ligase FBXO6-interacting glycoproteins in three kinds of cells. J. Proteome Res. 2012, 11, 17731781.

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Table 1. Peptide inhibitors structures and APC binding affinities as measured by the FP-based assay.

Compd

R1

R2

IC50 ± SEM (µM)

MAI-400

0.25 ± 0.01

MAI-401

2.01 ± 0.08

MAI-402

1.51 ± 0.14

MAI-403

6.35 ± 0.45

MAI-404

5.19 ± 0.34

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a

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MAI-405

5.46 ± 0.92

MAI-203

0.57 ± 0.06

MAI-150

1.09 ± 0.06

a

Effects of APC peptide inhibitors on the fluorescence polarization (FP) competition assay were assessed as described in

the Materials and Methods section. IC50 values shown are the average of three independent experiments with typical variation of less than 20%.

Table 2. Structures and Binding Affinities of Peptides containing R with one methene unit.

Compd

IC50 ± SEM (µM)

R

MAI-406

1.78 ± 0.22

MAI-407

3.64 ± 0.22

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MAI-408

1.82 ± 0.19

MAI-409

30.79 ± 1.64

MAI-410

1.26 ± 0.20

MAI-411

1.53 ± 0.21

MAI-412

0.62 ± 0.06

MAI-413

1.30 ± 0.21

MAI-414

1.94 ± 0.33

MAI-415

50.62 ± 1.09

S

MAI-416

N

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16.37 ± 0.58

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MAI-417

6.86 ± 0.27

MAI-418

4.01 ± 0.16

MAI-419

5.00 ± 0.44

MAI-420

6.95 ± 0.33

MAI-421

4.36 ± 0.28

MAI-422

5.07 ± 0.31

MAI-423

10.51 ± 0.62

MAI-424

10.07 ± 0.53

MAI-425

3.96 ± 0.20

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a

Effects of APC peptide inhibitors on the fluorescence polarization (FP) competition assay were assessed as described in

the Materials and Methods section. IC50 values shown are the average of three independent experiments with typical variation of less than 20%.

Table 3. Structures and Binding Affinities of Peptides containing R with two methene units.

Compd

IC50 ± SEM (µM)

R

MAI-426

0.84 ± 0.19

MAI-427

3.30 ± 0.41

MAI-428

13.95 ± 0.57

MAI-429

5.73 ± 0.31

MAI-430

3.02 ± 0.27

a

a

Effects of APC peptide inhibitors on the fluorescence polarization (FP) competition assay were assessed as described in

the Materials and Methods section. IC50 values shown are the average of three independent experiments with typical variation of less than 20%.

Table 4. Structures and Binding Affinities of Peptides with R substituents.

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IC50 ± SEM (µM)

R

MAI-431

3.85 ± 0.21

MAI-432

12.41 ± 0.52

MAI-433

31.47 ± 0.92

MAI-434

4.07 ± 0.28

MAI-435

2.95 ± 0.25

MAI-436

4.29 ± 0.32

MAI-437

a

a

108.7 ±5.3

H

Effects of APC peptide inhibitors on the fluorescence polarization (FP) competition assay were assessed as described in

the Materials and Methods section. IC50 values shown are the average of three independent experiments with typical variation of less than 20%.

Table 5. Structures and Binding Affinities of Peptides with the modified Leu185.

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Compd

IC50 ± SEM (µM)

Xaa

MAI-438

10.35 ± 0.44

MAI-439

128.1 ± 7.2

MAI-440

25.59 ± 1.08

a

a

Effects of APC peptide inhibitors on the fluorescence polarization (FP) competition assay were assessed as described in

the Materials and Methods section. IC50 values shown are the average of three independent experiments with typical variation of less than 20%.

Table 6. Kinetic Parameters of the peptide inhibitors binding to APC as measured by SPR.

a

-

-

b

-

Compd

kon (M 1 s 1)

MAI-400

(3.05 ± 0.08) × 10

(6.7 ± 0.2) × 10

MAI-150

(1.92 ± 0.1) × 10

5

(8.2 ± 0.5) × 10

a

5

b

c

koff (s 1)

d

RT (s)

KD (µM)

-2

15 ± 0.9

0.22 ± 0.01

-2

12 ± 1.4

0.43 ± 0.04

c

d

kon ± SEM (n=3). koff ± SEM (n=3). RT = 1/koff, RT is expressed in seconds. KD = koff/kon.

Table 7. Crystallographic data collection and refinement statistics.

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APC-MAI-400*

Data collection

Space group

P21

Cell dimensions

a, b, c (Å)

51.486, 63.328, 52.974

α, β, γ (°)

90.00, 95.83, 90.00

Resolution (Å)

50–1.79 (1.82–1.79)

Rsym or Rmerge

0.064 (0.458)

I / σI

20.2 (2.6)

Completeness (%)

99.6 (99.9)

Redundancy

6.7 (6.5)

Refinement

Resolution (Å)

1.79

No. reflections

213857

Rwork / Rfree

0.154 / 0.211

No. atoms

3143

Protein

2728

Ligand

64

Solvent

18

Water

333

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B-factors

26.647

Protein

25.291

Ligand

35.943

Solvent

50.052

Water

34.700

R.m.s. deviations

Bond lengths (Å)

0.024

Bond angles (°)

2.123

*Highest-resolution shell is shown in parentheses.

Table 8. Thermodynamic data for peptide inhibitors binding to APC.

Compd

L/P ratio

Kd (µM)

∆G (kJ/mol)

∆H (kJ/mol)

-T∆S (kJ/mol)*

MAI-400

0.85

0.012

-60.22

-61.8

1.58

MAI-150

0.87

0.21

-39.58

-39.7

0.12

*All experiments were performed at 30 °C. The L/P ratio indicates the number of sites per APC. ∆H: change in enthalpy; T∆S: change in entropy; Kd: equilibrium dissociation constant determined by ITC.

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Figure 1. Crystal structure and the binding mode of MAI-150 complexed with APC (PDB code 5IZ6). (A) The chemical structure of MAI-150. (B) Crystal structure of APC complexed with MAI-150. APC is shown in cartoon form (gray), and MAI-150 is depicted by sticks (carbon atoms: yellow) with L185 and A184 highlighted (carbon atoms: blue). The red dashed lines represent the distance and the angle of the hydrogen bond.

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Figure 2. Competitive binding curves for newly designed peptides as determined by the FP assay. Data are presented as the mean ± s.d.; n = 3 wells from 3 independent experiments.

Figure 3. Representative SPR results for MAI-400 (A) and MAI-150 (B) binding to APC.

Figure 4. Characterization and co-crystal structure of MAI-400 complexed with APC (PDB code 5Z8H). (A) Electron density map for the bound MAI-400 in the APC-ARM APC is shown as a solvent-accessible surface (gray) and MAI-400 is depicted by sticks (carbon atoms: yellow). Electron density map was shown as bule mesh. (B) Crystal structure of APC in complex with

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MAI-400. APC is shown as a solvent-accessible surface (gray) and MAI-400 is depicted by sticks (carbon atoms: yellow). (C) Binding interactions between MAI-400 (carbon atom: blue) and APC (carbon atom: gray). The red dashed lines represent the intramolecular hydrogen bond and the yellow dashed lines represent the hydrogen bonds between MAI-400 and APC. (D) Perspective views showing the intramolecular hydrogen bond observed in the crystal structure of MAI-400. The red dashed lines represent the distance and the angle of the hydrogen bond.

Figure 5. Overlay co-crystal structure of MAI-400 complexed with APC (PDB code 5Z8H) and MAI-150 complexed with APC (PDB code 5IZ6). (A) Binding modes of MAI-400 (yellow) and MAI-150 (blue) complexed with APC resulting from the superimposition of the two structure complexes. The APC residue that interacts with the peptides is shown as sticks and is labeled. The red dashed lines represent hydrogen bonds between MAI-400 and APC, and the yellow dashed lines represent hydrogen bonds between MAI-150 and APC. (B) Comparison of the position of W553 of APC in complexes with MAI-400 (yellow) and MAI-150 (blue). W553 was shown as lines and APC was shown as cartoon.

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Figure 6. Tryptophan fluorescence spectra of APC alone or APC with MAI-400 or APC with MAI-150.

Figure 7. Representative ITC results and fitting curves of MAI-400 (A) and MAI-150 (B) binding to APC. N, the number of sites per APC; K, the binding constant; ∆H, heat change; ∆S, entropy change. The data shown are representative of three independent experiments.

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Figure 8. Co-IP in HEK293Tcells transfected with Flag–APC (303–876) and HA–Asef (170–632) and treated with DMSO, MAI-150 (10 µM), and MAIT-400 (2 µM and 10 µM) for 24 h. Western blots from a single experiment were developed with antiHA or anti-Flag.

Figure 9. Macrocyclic APC-Asef Inhibitor MAI-441 Derive from MAI-400

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Table of Contents graphic.

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Figure 1. Crystal structure and the binding mode of MAI-150 complexed with APC (PDB code 5IZ6). (A) The chemical struc-ture of MAI-150. (B) Crystal structure of APC complexed with MAI-150. APC is shown in cartoon form (gray), and MAI-150 is depicted by sticks (carbon atoms: yellow) with L185 and A184 highlighted (carbon atoms: blue). The red dashed lines represent the distance and the angle of the hydrogen bond. 87x120mm (300 x 300 DPI)

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Figure 2. Competitive binding curves for newly designed peptides as determined by the FP assay. Data are presented as the mean ± s.d.; n = 3 wells from 3 independent experiments. 88x74mm (300 x 300 DPI)

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Figure 3. Representative SPR results for MAI-400 (A) and MAI-150 (B) binding to APC. 177x58mm (300 x 300 DPI)

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Figure 4. Characterization and co-crystal structure of MAI-400 complexed with APC (PDB code 5Z8H). (A) Electron density map for the bound MAI-400 in the APC-ARM APC is shown as a solvent-accessible surface (gray) and MAI-400 is depicted by sticks (carbon atoms: yellow). Electron density map was shown as bule mesh. (B) Crystal structure of APC in complex with MAI-400. APC is shown as a solvent-accessible surface (gray) and MAI-400 is depicted by sticks (carbon atoms: yellow). (C) Binding interactions between MAI-400 (carbon atom: blue) and APC (carbon atom: gray). The red dashed lines represent the intramolecular hydrogen bond and the yellow dashed lines represent the hydrogen bonds between MAI-400 and APC. (D) Perspective views showing the intramolecular hydrogen bond observed in the crystal structure of MAI-400. The red dashed lines represent the distance and the angle of the hydrogen bond. 153x115mm (300 x 300 DPI)

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Figure 5. Overlay co-crystal structure of MAI-400 complexed with APC (PDB code 5Z8H) and MAI-150 complexed with APC (PDB code 5IZ6). (A) Binding modes of MAI-400 (yellow) and MAI-150 (blue) complexed with APC resulting from the supe-rimposition of the two structure complexes. The APC residue that interacts with the peptides is shown as sticks and is labeled. The red dashed lines represent hydrogen bonds between MAI-400 and APC, and the yellow dashed lines represent hydrogen bonds between MAI-150 and APC. (B) Comparison of the position of W553 of APC in complexes with MAI-400 (yellow) and MAI-150 (blue). W553 was shown as lines and APC was shown as cartoon. 174x77mm (300 x 300 DPI)

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Figure 6. Tryptophan fluorescence spectra of APC alone or APC with MAI-400 or APC with MAI-150. 88x92mm (300 x 300 DPI)

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Figure 7. Representative ITC results and fitting curves of MAI-400 (A) and MAI-150 (B) binding to APC. N, the number of sites per APC; K, the binding constant; ∆H, heat change; ∆S, entropy change. The data shown are representative of three independent experiments. 99x64mm (600 x 600 DPI)

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Figure 8. Co-IP in HEK293Tcells transfected with Flag–APC (303–876) and HA–Asef (170–632) and treated with DMSO, MAI-150 (10 µM), and MAIT-400 (2 µM and 10 µM) for 24 h. Western blots from a single experiment were developed with anti-HA or anti-Flag. 84x76mm (144 x 144 DPI)

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Figure 9. Macrocyclic APC-Asef Inhibitor MAI-441 Derive from MAI-400 38x16mm (600 x 600 DPI)

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Graphic abstract 44x59mm (300 x 300 DPI)

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