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Lead optimization yields high affinity Frizzled 7-targeting peptides that modulate Clostridium difficile toxin B pathogenicity in epithelial cells Simon Hansen, Aaron Nile, Shrenik Mehta, Jakob Fuhrmann, and Rami N. Hannoush J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00500 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Journal of Medicinal Chemistry

Lead optimization yields high affinity Frizzled 7-targeting peptides that modulate Clostridium difficile toxin B pathogenicity in epithelial cells Simon Hansen1¶, Aaron H. Nile1, 3¶, Shrenik C. Mehta2, Jakob Fuhrmann1 and Rami N. Hannoush1* 1Department of Early Discovery Biochemistry, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA 2Pharma Technical Development, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA ¶ Equal contribution 3Current address: Calico Life Sciences, LLC, 1130 Veterans Blvd, South San Francisco, CA, 94080, USA * --Corresponding author: TEL: +1(650) 467-3696. Address: 1 DNA Way, South San Francisco, California, 94080, USA. Email: [email protected] Keywords Frizzled, FZD1, FZD2, FZD7, Wnt, intestinal stem cells, peptide, drug, lead, C. difficile Abstract Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B and FZD receptors, and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide proof-of-concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from C. difficile toxin B pathogenicity.

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Introduction Wnt proteins are secreted fatty acylated glycoproteins that modulate a variety of biological activities ranging from differentiation and development to stem cell regeneration1-2. At the same time, mutations in the Wnt pathway lead to misregulation of Wnt signaling and have been associated with a variety of human cancers3. Wnt/β-catenin signaling is initiated upon binding of Wnt proteins to the extracellular cysteine rich domain (CRD) of Frizzled (FZD) receptors at the plasma membrane2, 4. Recent data indicate that Wnt ligands may induce dimerization of FZD receptors5-10, and therefore rapidly amplify the assembly of ternary complexes with other coreceptors including the low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5/6). In general, Wnt signaling could be propagated via distinct molecular mechanisms, and Wnt/βcatenin signaling is the best understood pathway to date. It is characterized by stabilization of intracellular β-catenin and its translocation to the nucleus, leading to activation of downstream Wnt gene expression. There are ten Frizzled receptors that are encoded in the human genome and they are divided into four subclasses based on their sequence homology: FZD1/2/7, FZD5/8, FZD3/6, FZD4/9/105, 11. It was demonstrated that the FZD7 receptor subclass is important for intestinal stem cell self-renewal12-14, indicating a potential for pharmacological targeting of FZD7 in certain disease contexts. On the other hand, FZD5 is upregulated in pancreatic ductal adenocarcinomas and required for their growth in xenograft mouse models15. Moreover, antibodies directed against FZD1/2/7/5/8 have been used in the clinic for treating solid tumors16 but on-target safety adverse events including bone fractures were observed, arguing about the need for more selective agents in the development of Wnt-targeting therapeutics. More recently, studies utilizing CRISPR/Cas9-mediated genome-wide screens identified a role for FZD1/2/7 receptors in mediating C. difficile toxin B (TcdB) pathogenicity in the mouse colonic epithelium17-18. C. difficile is a gram-positive bacterium that remains a major health threat; it infects the human colon leading to a significant number of deaths per year in the United States19. 2 ACS Paragon Plus Environment

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TcdB is a critical virulence factor causing life-threatening diseases associated with C. difficile infection20-21. It was demonstrated that FZD1/2/7 triple-knockout cells are highly resistant to TcdB. Consistent with these observations, the colonic epithelium in FZD7 KO mice seems to be less susceptible to TcdB-induced tissue damage in vivo, highlighting the dependency of TcdBinduced pathogenicity on FZD7 receptors. However, pharmacological strategies to block pathogenesis via directly targeting FZD7 receptors have not been explored. Phage display approaches have led to the identification of the first peptide antagonist that targets a specific subset of FZD receptors13. Peptide dFz7-21 comprises a disulfide-bridged dimer ((Ac-LPSDDLEFWCHVMY-NH2)2; 1). It binds selectively to FZD7 receptor sub-class, inhibits Wnt/β-catenin signaling and disrupts stem cell integrity in intestinal organoids13, phenocopying the effects of genetic ablation of FZD7 proteins14. Structural studies demonstrated that peptide 1 stabilized human FZD7 (hFZD7) CRD in a non-functional dimer conformation via binding at the CRD alpha-helical dimer interface, thereby leading to disruption of Wnt - FZD ternary complex formation with Lrp613. However, the effect of peptide 1 on C. difficile infections in cells has not been studied. Moreover, its affinity could be further improved to enable its utility in studying the role of FZD7 in disease models. In this work, we sought to improve the affinity of peptide 1 and to investigate whether direct targeting of FZD7 receptors could be a viable pharmacological approach for blocking C. difficile toxin B pathogenesis. We describe an integrated approach for lead optimization, which encompasses structure-based rational design and phage display-based affinity maturation (Figure 1a), leading to the discovery of peptides with picomolar affinities against FZD7-class CRDs. The obtained peptides block TcdB interaction with FZD7 and protect epithelial cells from TcdB-mediated cell rounding. Our proof of principle study demonstrates how systematic optimization of a peptide hit could yield new molecules with favorable biophysical properties for further therapeutic development.



Results Peptide lead optimization via rational design. Our hit-to-lead chemistry optimization effort involved structure-based rational design and was also directed towards understanding structureactivity relationships (SAR). We varied specific amino acid positions of dFz7-21 located within the peptide-protein interface including Ser3, Leu6, Phe8, Val12 and Tyr14 (Figure 1b-d). Each of the newly designed peptides was then generated by solid phase synthesis, oxidized to a dimer, purified to >95% purity by RP-HPLC and its identity was confirmed by LC-MS (SI Fig. 1). The binding kinetics and affinities of all synthesized peptides to FZD7 CRD were then measured by surface plasmon resonance (SPR). We first determined the binding constant KD of the initial hit peptide 1 to be 1.2 nM (ka = 48 × 105 M–1 s–1; kd = 5.6 × 10–3 s–1; Table 1 and Figure 1e), in agreement with an earlier reported value of 2.7 nM (ka = 22 × 105 M–1 s–1; kd = 5.9 × 10–3 s–1)13. The 2-fold difference is likely due to different lots of FZD7 CRD protein used in the experiments. Also, in this study, the binding constants were derived from single cycle kinetics to increase experimental throughput (Figure 1e). Nonetheless, the newly measured KD value in this study served as a reference point for comparing the binding constants of the different peptide analogues generated. To illustrate the importance of the peptide dimer architecture in FZD7 CRD recognition, we generated peptide Fz7-21S (2), containing a Cys10 to Ser substitution which did not show detectable binding to hFZD7 CRD by SPR (Table 1), consistent with structural observations supporting the role of Cys10 in peptide dimer formation (Figure 1c). The structural data indicated that the two N-terminal residues of peptide 1 were solvent exposed and did not directly interact with the protein target (Figure 1c). Therefore, we deleted residues Leu1 and Pro2, and the newly generated peptide dFz7-21Δ2 (3) displayed a 3-fold improvement in binding affinity, primarily due to a faster on-rate and slower off-rate compared to peptide dFz7-21 (KD = 0.4 nM; ka = 62 × 105 M–1 s–1; kd = 2.3 × 10–3 s–1; Table 1 and Figure 1f). Given this observed enhancement in binding affinity, we selected peptide dFz7-21Δ2 (3) as the 4 ACS Paragon Plus Environment

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framework for conducting further structure-guided rational design work and for understanding structure-activity relationships (SAR). In light of the structural data highlighting an essential role of the disulfide bond in peptide dimer integrity and binding (Figure 1b,c and Table 1), we wondered whether it could be replaced with a more redox stable linkage without impacting peptide binding. Computational modeling suggested that a diselenide bond, which has a lower reduction potential than a disulfide bond, would be tolerated. Replacement of the disulfide linkage with a diselenide bond led to ~2-fold improvement in binding affinity (Table 1, peptide dFz7-21Δ2.C10Sec, 4), which seems to be primarily driven by a slower off-rate. Another position we examined is Ser3. Replacement of Ser3 with Thr led to a 2-fold improvement in binding affinity, primarily due to a slower off-rate (5; Table 1). The introduction of a methyl group within Ser residue was predicted based on molecular modeling to enable potential van der Waals contacts with Phe140 on FZD7 CRD, while still maintaining the hydrogen bonding between the hydroxyl group and backbone carbonyl of Phe138 (Figure 1g).

Table 1. Binding kinetics of dFz7-21 analogues against human FZD7 CRD-Fc as measured by

Peptide

#

ka (M-1s-1) × 105

kd (s-1) × 10-3

KD (nM)

dFz7-21

1

47.5 ± 3.6

5.6 ± 0.7

1.18 ± 0.10

dFz7-21S

2

n.b.

n.b.

n.b.

dFz7-21Δ2

3

61.9 ± 16.1

2.3 ± 0.6

0.37 ± 0.02

dFz7-21Δ2.C10Sec

4

52.7 ± 1.4

1.0 ± 0.1

0.19 ± 0.02

dFz7-21Δ2.S3T

5

51.5 ± 3.2

0.72 ± 0.07

0.14 ± 0.01

surface plasmon resonance.

n.b.; no binding detected



Design and characterization of peptide analogues with Leu6 substitutions. The peptide residue Leu6 is involved in both inter- and intramolecular interactions. It is proximal to a hydrophobic cleft formed at the FZD7 CRD dimer interface and comprising a conserved FGF motif (Phe138, Gly139 and Phe140). It also forms stabilizing intramolecular interactions with Tyr14 on dFz7-21 (Figure 1h). Alanine replacement of Leu6 led to ~800-fold reduction in cellular potency13, underscoring the important role that Leu6 exhibits in mediating peptide interaction with FGF motif on FZD7 CRD. To explore the SAR around residue Leu6, we designed a series of natural and unnatural hydrophobic side chains that were meant to sample the interaction space with FGF motif on FZD7 CRD. Strategies geared at extending the side chain of Leu6, such as its replacement with L-homoleucine (Hol; 6), L-homophenylalanine (Hof; 7), L-phenylalanine (Phe; 8), L-norleucine (Nle; 9) and branched side chain (biVal, 10), led to a reduction in binding affinity (Table 2). We also systematically investigated a series of cyclic aliphatic side chains of different sizes. Replacement of Leu6 with cyclopentyl-L-alanine (Cpa; 11) and cyclobutyl-L-alanine (Cba, 12) led to a gradual decrease in binding affinity, respectively (Table 2). However, peptide dFz721Δ2.Leu6Tba containing a tert-butyl-L-alanine (Tba; 14) displayed a 1.7-fold improvement in KD over its parent molecule (3) (Tables 1-2 and Figure 1h). Additional analogues with larger ring size such as cyclohexyl-L-alanine (Cha; 13) showed reduction in binding affinity.


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Table 2. Binding kinetics of designed dFz7-21 Leu6 analogues against human FZD7 CRD-Fc as measured by surface plasmon resonance.

#

ka (M-1s-1) × 105

kd (s-1) × 10-3

KD (nM)

Hol

6

10.9 ± 2.2

123.5 ± 14.6

115.0 ± 16.6

Hof

7

69.6 ± 23.5

9.1 ± 0.9

1.4 ± 0.3

Phe

8

5.3 ± 1.6

62.8 ± 11.6

121.6 ± 21.0

Nle

9

42.8 ± 8.7

23.1 ± 3.5

5.4 ± 0.3

biVal

10

73.6 ± 24.3

29.2 ± 11.2

3.9 ± 0.3

Cpa

11

46.5 ± 12.8

9.9 ± 3.3

2.1 ± 0.2

Cba

12

4.6 ± 1.8

9.7 ± 4.8

20.3 ± 2.1

Cha

13

56.7 ± 11.1

75.1 ± 9.1

13.7 ± 4.3

dFz721Δ2.L6X

Structure



Tba

14

44.0 ± 0.9

0.95 ± 0.03

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0.22 ± 0.01

Design and characterization of peptide analogues with substitutions at Phe8 and Val12. Phe8 of dFz7-21 forms multiple hydrophobic interactions with residues Leu81, His84 and Glu82 of FZD7 CRD, and intramolecular interactions with Val12 (Figure 1i). Interestingly, alanine replacement of Phe8 was observed to be tolerated without affecting cellular activity13, suggesting that this position could tolerate amino acid substitutions. Therefore, we sought to expand the peptide contacts into the hydrophobic patch created by Leu81. Based on molecular modeling, we generated two phenylalanine derivatives with chlorinated substitutions at the meta (15) and para positions (16) on its aromatic ring (Figure 1i). Peptide 15 displayed a 3.7-fold improvement in KD compared to peptide 3, primarily originating from a slower off-rate, which is likely due to increased hydrophobic interactions at Leu81 and van der Waals interactions at His84 (Figure 1i and Table 3). However, the F(4-Cl) substitution was not favorable in this position, although F8L substitution (17) led to a 4.6-fold improvement in binding affinity over peptide 3, due to a slower off-rate (Table 3). Collectively, these results indicate that additional gains in binding energy could be achieved from modifications introduced within this region of the peptide. Similar to Leu6, peptide residue Val12 exhibits intramolecular interactions with Phe8 and multiple intermolecular interactions with FZD7 CRD (Figure 1j). It is situated within ~ 3.3 Å distance from a hydrophobic patch on FZD7 CRD that is formed by Leu81, and is in close proximity to the γ-carbon on Gln85. We generated peptide analogues with Val12 substitutions in order to improve interactions with FZD7 CRD at Leu81, His84 and Gln85 as well as intramolecular contacts (Figure 1j). Replacement of Val12 with Leu (dFz7-21Δ2.Val12Leu, 18) or Ile (dFz721Δ2.Val12Ile, 19) led to ~1.3 - 2.1-fold improvement in KD, suggesting that the introduction of more hydrophobic side chains is favorable at this position (Table 3 and Figure 1j). However, 8 ACS Paragon Plus Environment

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peptide dFz7-21Δ2.Val12Tba, containing a tert-butyl-L-alanine residue (20), did not confer any added binding benefit (Table 3). Further exploration of the interaction space of Val12 was systematically investigated with a series of cyclic aliphatic side chain analogues. A peptide analogue containing cyclopropyl-L-alanine (dFz7-21Δ2.Val12Cpra, 21) showed a ~ 2.3-fold improvement in binding affinity compared to peptide 3. Other aliphatic cyclic side chains with larger ring size did not confer any further advantage in binding affinity. For instance, cyclobuty-Lalanine (dFz7-21Δ2.Val12Cba, 22) showed a similar affinity as peptide 3, whereas cyclopentyl-Lalanine (dFz7-21Δ2.Val12Cpa, 23) exhibited a dramatic reduction in affinity. In sum, these data indicate that both Ile and cyclopropyl-L-alanine are favorable substitutions in lieu of Val residue.



Figure 1. Peptide dFz7-21 binds to FZD7 CRD at its dimer interface. (a) Scheme of the lead optimization approach combining rational design and phage display. SPR, surface plasmon resonance. (b) X-ray crystal structure of hFZD7 CRD dimer (surface representation) in complex with dFz7-21 (ribbon representation; PDB ID: 5WBS). (c) Zoomed-in view of dFz7-21 highlighting residues that interact with hFZD7 CRD (stick representation), with residues chosen for structureactivity analysis highlighted (orange). (d) Chemical structure of dFz7-21, highlighting in color amino acid positions that were varied. (e, f) Kinetic titration experiment with surface plasmon resonance for determining the binding affinity of dFz7-21 (e) or dFz7-21Δ2 (f) to FZD7 CRD (black) overlaid with fit to 1:1 binding model (red). (g-j) Zoomed-in view of dFz7-21 (green, stick and ribbon representation) or rationally designed peptide analogues highlighting inter and intramolecular (gray, hFZD7 CRD; ribbon and stick representation) interactions for (g) Ser3, (h) Leu6, (i) Phe8 or (j) Val12 positions. Distances are shown as dashed lines in Å. FZD7 CRD is depicted as a surface representation (green, hydrophobic; magenta, hydrophilic). Description of structures and acronyms are found in Tables 1-5. All models were generated using the X-ray crystal structure of Fz7-21 bound to hFZD7 CRD (PDB ID: 5WBS) as the starting point for molecular modeling.

Table 3. Binding kinetics of designed dFz7-21 Phe8 and Val12 analogues against human FZD7 CRD-Fc as measured by surface plasmon resonance.


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#

ka (M-1s-1) ×105

kd (s-1) × 10-3

KD (nM)

F(3-Cl)

15

66.3 ± 22.7

0.63 ± 0.10

0.10 ± 0.02

F(4-Cl)

16

16.5 ± 1.1

1.71 ± 0.04

1.04 ± 0.07

Leu

17

76.5 ± 7.2

0.60 ± 0.01

0.079 ± 0.007

#

ka (M-1s-1) ×105

kd (s-1) × 10-3

KD (nM)

Leu

18

67.2 ± 3.1

1.90 ± 0.28

0.28 ± 0.03

Ile

19

55.1 ± 14.9

0.96 ± 0.18

0.18 ± 0.03

Tba

20

68.8 ± 11.2

2.16 ± 0.09

0.32 ± 0.05

Cpra

21

80.4 ± 17.6

1.26 ± 0.14

0.16 ± 0.02

Cba

22

53.0 ± 5.8

2.13 ± 0.18

0.40 ± 0.03

dFz7-21Δ2.F8X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dFz7-21Δ2.V12X

Structure

Structure



Cpa

23

30.4 ± 14.1

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44.1 ± 20.5

14.5 ± 0.6

Design and characterization of peptide analogues with substitutions at Tyr14. Tyr14 residue is proximal to Phe140 on FZD7 CRD and also forms intramolecular interactions with Leu6 on peptide dFz7-21 (Figure 1c). Therefore, we generated derivatives containing substitutions at the phenyl ring, such as Phe(4-Cl) (24), Phe(4-Me) (25), Phe(4-CF3) (26), Phe(2,4-Cl) (27) and Phe(3,4-Cl) (28), in order to improve the hydrophobic interactions and expand into the FGF hydrophobic cleft on FZD7 CRD. However, all the peptides tested displayed weaker binding affinities (Table 4), suggesting space constraints to accommodate these modifications. All in all, our structure-based hit-to-lead optimization studies identified favorable modifications at specific positions, such as Δ2 deletion (3), S3T (5), F8F(3-Cl) (15), F8L (17) and V12I (19), that led to the identification of high affinity ligands exhibiting 2-10-fold improvement over the initial peptide 1 hit. Finally, combining F8F(3-Cl) and V12I mutations had a favorable effect, yielding peptide 29 (dFz721Δ2.F8F(3-Cl).V12I, Table 5) with 83 pM affinity (ka = 68 × 105 M–1 s–1; kd = 0.6 × 10–3 s–1; Table 5) and ~ 14-fold improvement in binding constant over peptide 1.


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Table 4. Binding kinetics of designed dFz7-21 Tyr14 analogues against human FZD7 CRD-Fc as determined by surface plasmon resonance.

#

ka (M-1s-1) × 105

kd (s-1) × 10-3

KD (nM)

F(4-Cl)

24

9.3 ± 0.4

0.82 ± 0.06

0.88 ± 0.10

F(4-Me)

25

10.2 ± 0.5

0.98 ± 0.05

0.97 ± 0.05

F(4-CF3)

26

1.45 ± 0.05

1.90 ± 0.19

13.1 ± 1.4

27

1.29 ± 0.19

2.89 ± 0.38

22.5 ± 0.7

28

1.40 ± 0.04

0.90 ± 0.19

6.4 ± 1.2

dFz721Δ2.Y14X

Structure

Cl

O

F(2,4-Cl)

OH Cl

F(3,4-Cl)

NH2

Affinity maturation of peptide dFz7-21 via phage display. As a non-biased alternative approach to rational design, we carried out affinity maturation by using directed evolution. We displayed peptide 3 on the surface of bacteriophage M13 and generated a soft randomization library spanning variations at each amino acid position (50% wild-type residue, 50% remaining 19 amino acid residues). After four rounds of selection against hFZD7 CRD-Fc fusion, two variants became enriched, namely E7K and F8L (Figure 2a). Although residue Glu7 is solvent exposed and does not seem to exhibit direct interactions with FZD7 CRD, in silico analysis indicates that replacing Glu with Lys introduces potential intramolecular polar interactions with Asp4 (Figure 2b), and hence may promote alpha-helix stabilization and indirectly enhance peptide binding affinity to FZD7 CRD. Consistent with these predictions, biophysical measurements demonstrated 13 ACS Paragon Plus Environment


that peptide 30, containing E7K mutation, displayed ~ 2.4-fold improvement in binding affinity over the wild-type (Table 5). Although it’s not clear why the combined E7K.F8L variant 31 (dFz721Δ2.E7K.F8L, Table 5) showed a similar binding affinity to E7K single mutant, peptide 31 displayed an improved binding kinetics profile, in particular a longer residence time due to a 2.2fold reduction in off-rate compared to E7K single point mutant (kd = 0.58 × 10–3 s–1 vs. 1.3 × 10–3 s–1, Table 5).

Figure 2. Affinity maturation of dFz7-21Δ2 peptide identified E7K and F8L as favorable substitutions. (a) Web logo showing residue conservation at each position after four rounds of panning. Number of unique sequences = 57. The wild-type sequence is shown in bold underneath the X-axis. (b) Zoomed-in view of dFz7-21 highlighting E7K on dFz7-21 (green or purple, ribbon and stick representation) and the formation of a hydrogen bond between E7K and D4 after energy minimization.

Finally, we combined the top variants from both the rational design and library display lead optimization studies to generate peptides containing double, triple and quadruple amino acid substitutions such as F8F(3-Cl).V12I.S3T (32), F8L.V12I (33), F8L.V12I.S3T (34) and E7K.F8L.V12I.S3T (35). All these analogues exhibited high affinity in the 39- 45 pM range (Table 5). The most potent peptides from this series, dFz7-21Δ2.F8F(3-Cl).V12I.S3T (32) and dFz721Δ2.F8L.V12I.S3T (34), displayed 39 pM affinity with fast on-rate and slow off-rate kinetics profile (ka = 139 - 166 × 105 M–1 s–1; kd = 0.54 - 0.64 × 10–3 s–1; Table 5 and SI Figure 2a) as 14 ACS Paragon Plus Environment

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measured by SPR. They also maintained a high binding affinity and exquisite selectivity to FZD1/2/7 CRDs, with no detectable binding to other members of the FZD receptor family (Table 6 and SI Figure 2b).

Table 5. Binding kinetics of dFz7-21 analogues containing substitutions from both rational design and phage affinity maturation approaches to human FZD7 CRD-Fc.

dFz7-21Δ2.X Peptide F8F(3-Cl).V12I E7K E7K.F8L F8F(3-Cl).V12I.S3T F8L.V12I F8L.V12I.S3T E7K.F8L.V12I.S3T

# 29 30 31 32 33 34 35

ka (M-1s-1) × 105 68.4 ± 5.9 85.6 ± 13.6 55.6 ± 50.9 139.1 ± 13.7 127.9 ± 9.6 165.6 ± 8.7 102.7 ± 1.3

kd (s-1) × 10-3 0.57 ± 0.23 1.33 ± 0.06 0.58 ± 0.19 0.54 ± 0.01 0.57 ± 0.03 0.64 ± 0.06 0.46 ± 0.10

KD (nM) 0.083 ± 0.028 0.157 ± 0.018 0.142 ± 0.065 0.039 ± 0.005 0.044 ± 0.005 0.039 ± 0.005 0.045 ± 0.010

Table 6. Binding affinities of dFz7-21 analogues against FZD CRD-Fc family members.

FZD CRD

KD (nM) dFz7-21 (1)

dFz7-21Δ2 (3)

dFz7-21Δ2 F8F(3-Cl). V12I.S3T (32)

1

2.42 ± 0.23

1.12 ± 0.10

0.34 ± 0.04

2

1.46 ± 0.15

0.99 ± 0.09

0.32 ±0.03

7

1.18 ± 0.1

0.37 ± 0.02

0.039 ± 0.005

5

n.b.

n.b.

n.b.

8

n.b.

n.b.

n.b.

4

n.b.

n.b.

n.b.

9

n.b.

n.b.

n.b.

10

n.b.

n.b.

n.b.

n.b.; no binding detected 15 ACS Paragon Plus Environment


Lead peptides are potent antagonists of Wnt/β-catenin signaling. To assess whether the newly generated peptides show improved potency in inhibiting Wnt signaling, we compared the activity of the parent peptide (1) as well as the four lead peptides displaying the highest affinities in a Wnt reporter cell-based assay. We observed that peptide 1 blocked Wnt signaling in HEK293 cells that were transfected with Wnt3a with an IC50 of 32 ± 10 nM, in agreement with a previously reported value13. Notably, peptides 33, 34 and 35 all exhibited improved IC50’s of 7.3 ± 2.0, 9.2 ± 2.7 and 4.8 ± 2.6 nM, respectively (SI Fig 3), consistent with their improved binding affinities, whereas peptide 32 inhibited Wnt signaling with an IC50 of 53 ± 27 nM. The weaker cellular potency is likely a result of poor solubility resulting from the presence of chlorinated residues, which can promote aggregation at high concentrations in aqueous solutions. In general, the discrepancy between cellular potency and the binding affinity of peptides could be due to several reasons including, but not limited to, differences in the assay readouts between SPR and luciferase reporter, potential serum protein binding, FZD receptor expression levels, oligomeric state and FZD dimer architecture on the cell surface, and peptide stability in the extracellular environment.

Assessment of peptide stability. Typically, peptides are prone to suffer from poor metabolic stability due to proteolytic hydrolysis and in the case of disulfide linkages the potential reduction of this redox labile bond. Therefore, to further understand the factors affecting the potency of the peptides in the cellular assays and lay the ground work for future optimization, we evaluated the stability of the lead peptides in cell culture medium containing 10% FBS at 37 ºC over 24 h period, which covers the time frame employed in the cellular assays in this study. Peptide 4 containing a diselenide bond exhibited better stability at earlier time points (6 h) compared to the parent peptide 3 which contained a disulfide bond (SI Fig. 4a). Moreover, peptide 34 containing three residue substitutions (F8L, V12I, S3T) was observed to be stable under the same conditions, suggesting 16 ACS Paragon Plus Environment

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that the amino acid composition in the alpha-helix contributes to peptide stability. To further explore the stability of the disulfide bond within the peptide dimer, we generated an analogue of peptide 34 that contained a thioether bond (hereafter named peptide 36). Peptide 36 displayed a 10-fold increase in binding constant (KD = 0.37 ± 0.01 nM; ka = 1.7 ± 1.1 × 107 M–1 s–1; kd = 6.3 ± 4.1 × 10–3 s–1) and lower stability in cell culture medium compared to peptide 34 (SI. Fig.4a). These results indicate that replacement of the disulfide bond with thioether affects both the bioactivity and stability of the peptide. On the other hand, all peptides tested demonstrated good stability in mouse serum over a 24 h incubation period at 37 ºC (SI Fig. 4b), with minimal chemical degradation observed. Moreover, we assessed the stability of these peptides under reducing conditions (SI Fig. 5). Peptide 36 was the most stable with no detectable disulfide reduction upon incubation with TCEP or DTT over 40 min at 37 ºC, as expected due to the presence of a thioether bond. Incubation with DTT only led to ~30% reduction of peptide 4, whereas peptides 3 and 34 underwent complete reduction. These findings indicate that the diselenide-containing peptide was more resistant to DTT reduction compared to disulfide (compare peptides 4 and 3). However, all peptides tested (3, 4, and 34) underwent complete reduction upon incubation with increasing concentration of TCEP (SI. Fig 5). It is noteworthy that lower concentrations of TCEP were required for complete peptide reduction compared to DTT. Overall, the above studies highlight differences in peptide stability that vary depending on the nature of the assay used and might be a result from a combination of several factors including proteolytic susceptibility as well as disulfide bond cleavage.

Peptides block TcdB interaction with FZD7 receptors. It was demonstrated that FZD7 receptors are physiologically relevant targets for C. difficile toxin B (TcdB) and are critical for mediating pathogen endocytosis and cell killing in the colonic intestinal epithelium18. Therefore, we sought to investigate whether the FZD-targeting high affinity peptides developed in this study could disrupt the interaction between FZD and TcdB. First, an enzyme-linked immunosorbent 17 ACS Paragon Plus Environment


assay (ELISA) was developed to validate TcdB binding to different members of the FZD7 receptor family. Consistent with prior data18, we observed that TcdB bound to only members of the FZD7 subclass, with no detectable binding to other FZD CRDs (Figure 3a). The addition of peptides effectively blocked the interaction of TcdB with FZD7 and FZD2 CRDs in a concentrationdependent manner (Figure 3b) with IC50 values in the nM range. The newly designed peptide analogues displayed ~ 50-100-fold improvement in their potency compared to the initial dFz7-21 peptide hit (1). Superimposition of the TcdB-FZD2 CRD complex structure with the dFz7-21-FZD7 CRD complex suggests that steric hindrance could be a plausible mechanism for peptidemediated inhibition of TcdB binding to FZD CRDs. TcdB binds to a FZD CRD monomer17, whereas peptide dFz7-21 induces a FZD dimer configuration, as observed by X-ray crystallography and biochemistry data13, and is therefore predicted to occlude TcdB binding to FZD CRD due to steric clashes (Figure 3c). Consistent with this proposed mechanism of action, SPR measurements demonstrated that peptide dFz7-21Δ2.F8F(3-Cl).V12I.S3T (32), with KD values of 0.035 nM and 0.32 nM against FZD7 and FZD2 CRDs, respectively (Table 6), effectively blocked the interaction of TcdB with FZD7 or FZD2 CRDs (Figure 3d), in agreement with ELISA results above (Figure 3b). In these studies, we observed that the interaction between TcdB and FZD7 or FZD2 CRDs exhibited low nanomolar affinities (3.3 and 1.1 nM, respectively), as previously reported17. It is also remarkable that blockade of the TcdB – FZD interaction could be achieved likely via altering the conformation of FZD7 receptor.


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Figure 3. Peptides disrupt interaction of TcdB with FZD receptors. (a) TcdB binds specifically to FZD1/2/7 subclass as shown by ELISA. (b) Competition ELISA demonstrating concentrationdependent inhibition of FZD7 and FZD2 CRD interaction with TcdB by dFz7-21 peptide derivatives. The experiment was performed three independent times, each with technical triplicates. Representative example of one experiment is shown. Values represent the mean of three technical replicates with standard deviation. (c) Structural superposition of TcdB-FBD:FZD2 CRD (PDB ID: 6C0B) and FZD7 CRD:dFz7-21 (PDB ID: 5WBS) complexes reveals steric clashes between TcdB and dFz7-21-bound FZD CRD dimer. FBD, Frizzled binding domain. (d) dFz7-21 and dFz7-21Δ2.F8F(3-Cl).V12I.S3T (32) peptides block binding of TcdB to FZD7 and FZD2 CRDs as measured by SPR.

Peptides block TcdB pathogenesis in cells. We assessed whether TcdB effect on cells could be modulated via pharmacological targeting of FZD7 receptors. Two cells lines were studied,



Caco-2 cells which have been widely used as an in vitro model of the intestinal epithelial barrier and are originally derived from epithelial colorectal adenocarcinoma, and Chinese hamster ovary (CHO-K1) cells. Caco-2 cells express FZD receptors22 but not chondroitin sulfate proteoglycan 4 (CSPG4) receptor18, which has been shown to also mediate TcdB entry into cells. CHO-K1 cells express low levels of FZD receptors but are sensitive to TcdB pathogenicity18. Treatment of both cell lines with TcdB led to a cell rounding phenotype, which could be quantified by measuring the average cell area revealed by using phalloidin conjugated to AlexaFluor488 dye to mark the actin cytoskleleton. Peptide 34 was used in these assays because it displayed good solubility among the most affinity-improved peptide analogues. Treatment of Caco-2 cells with peptide 34 showed a concentration-dependent protective effect against TcdB-induced cell rounding (Figure 4a). The protective effect of peptide 34 varied between 34% and 69% depending on the concentration of TcdB used (0.1 – 1 ng/ml), with lower TcdB concentrations resulting in maximal protective effects of the peptide (Figure 4a). To evaluate the range of TcdB concentrations in which peptide 34 shows a protective effect, we incubated Caco-2 cells with varying TcdB concentrations (0.015 to 4 ng/ml) in the absence and presence of peptide 34 (50 µM). The highest protective effect of the peptide was observed at TcdB concentrations between 0.25 and 0.5 ng/ml, whereas no protective effect was observed at the higher TcdB concentration of 4ng/ml. It’s noteworthy that the cell rounding phenotype was not observed at TcdB concentrations below 0.031 ng/ml (Figure 4b). As a control experiment, peptides incorporating specific point mutations such as dFz7-21S (2), which showed no binding to FZD7 CRD by SPR, and dFz7-21.L6A, which showed ca. 800 fold less activity in blocking canonical Wnt signaling13, failed to exhibit any protective effect against TcdB-induced cell rounding in Caco-2 cells, similar to the DMSO vehicle control (Figure 4c, d). We also observed protective effects when using control recombinant FZD CRD proteins which presumably function as protein ‘traps’ to sequester TcdB in the extracellular milieu. Caco2 cells were completely protected from TcdB-induced cell rounding upon treatment with FZD1/2/7 20 ACS Paragon Plus Environment

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CRDs, but not FZD4/8 CRDs (Figure 4e, f)18, consistent with the binding preference of TcdB for FZD7-class CRDs as observed by ELISA (Figure 3a). In contrast, a minimal protective effect against TcdB-induced cell rounding was observed in CHO-K1 cells that were treated with either peptide 34 or FZD1/2/7 CRDs (Figure 4g-i). The FZD4/8 CRDs and IgG-Fc controls also showed no effect. While the reasons for these observations remain unclear, the lack of observed protective effect by the peptides or FZD7 CRD in CHO-K1 cells is likely due to the expression of other receptors that mediate TcdB-induced cell rounding, such as chondroitin sulfate proteoglycan 4 (CSPG4)18, and / or low levels of expression of FZD receptors in CHO-K1 cells. A truncated construct of TcdB that is defective in binding to CSPG4 displays weaker cell killing activity in CHOK1 compared to wild-type TcdB18. It is noteworthy that CSPG4 is not expressed in Caco-2 cells, which makes TcdB-induced cell rounding dependent on FZD receptors which are robustly expressed in this cell type, as demonstrated by the protective effect of peptide 34 and FZD CRDs (Figure 4a, e). We can also rule out the possibility that the peptide is not functional in CHO-K1 cells due to sequence differences in FZD species. The peptide binding epitope is strictly conserved across human, mouse, golden and Chinese hamster FZD2; it is also conserved between human and golden hamster FZDs1/7, suggesting that the peptide still targets FZD7 receptor sub-class in CHO-K1 cells. Altogether, our findings demonstrate that peptide 34 blocks TcdB-induced Caco-2 cell rounding via disrupting its interaction with FZD receptors, either by inducing receptor dimerization and / or receptor internalization. Our results also suggest that targeting FZD receptors as an approach to protect epithelial cells from C. difficile TcdB pathogenicity warrants further investigations. Finally, the use of FZD7-targeting peptides underscores the FZD-dependency of TcdB and reveals the sensitivity of different cell lines to TcdB-mediated cell rounding. The discrepancy between the measured binding affinity and cellular potency of the peptide in toxininduced cell rounding assays is not understood at the time being. The SPR assay measures direct binding between two purified components. In contrast, the toxin cellular assays measure cell 21 ACS Paragon Plus Environment


rounding as a readout, which is downstream of TcdB – FZD interaction at the cell surface, and low concentrations of TcdB that enter cells could still be effective at inducing cell rounding in this assay. These differences likely explain, in part, the high IC50 observed in cellular toxin assays compared to SPR.


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Figure 4. Lead peptide dFz7-21Δ2.F8L.V12I.S3T (34) protects Caco-2 cells against TcdBinduced cell rounding. (a) Normalized average cell size of Caco-2 cells treated with different TcdB and peptide 34 concentrations for 24h. (b) Normalized average cell size of Caco-2 cells treated with peptide 34 (50 µM) or DMSO vehicle control and a range of TcdB concentrations (ns: p>0.05, *: p

Lead optimization yields high affinity Frizzled 7 ... - ACS Publications

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