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. 2026 Oct 8;17(10):2121–2126. doi: 10.1021/acsmedchemlett.6c00034

Development of a Lactam-Bridged Peptide as Interleukin-11 Antagonist for Renal Fibrosis Treatment

Xu Yang 1, Na Zhao 1, Ranran Zheng 1, Jian Li 1, Yujie Wu 1, Zhenxin Ma 1, Ningning Pang 1, Xin Meng 1, Ruirui Liu 1, Xiaochun Xiong 1,*, Chunmei An 1,*
PMCID: PMC13656272  PMID: 42866003

Abstract

In recent years, the role of interleukin-11 (IL-11) in renal fibrosis has garnered considerable interest and is regarded as a promising therapeutic target for this condition. Herein, we explored the effects of replacing the oxidation-sensitive thioether moiety with a lactam bridge. By screening linker length and performing further optimization, we developed an analog, 4L6–16. This compound exhibited a high binding affinity for IL-11, with a KD value of 134 nM, along with enhanced antagonistic activity. Moreover, 4L6–16 demonstrated significant renoprotective effects in both in vitro and in vivo models of renal fibrosis, supporting its potential for further optimization to develop novel IL-11-targeted therapeutic agents.

Keywords: Interleukin-11, Renal fibrosis, Peptide, Structure−activity relationship

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Introduction

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) superfamily, which forms a hexametric complex with specific receptor subunit IL-11Rα and the common signal transducer GP130 to activate downstream signaling. , Traditionally, IL-11 has been recognized as a hematopoietic cytokine and has been employed clinically to treat patients with thrombocytopenia. In recent years, however, emerging evidence suggests that IL-11 plays a significant role in the pathogenesis of inflammatory and fibrotic diseases. Notably, Stuart A. Cook and colleagues have conducted pioneering research to elucidate and validate the pro-fibrotic functions of IL-11. , In light of these findings, IL-11 inhibition is regarded as an effective approach to treating fibrosis.

Antibodies against IL-11 and IL-11R have been firstly developed by the Cook group and have shown efficacy in alleviating fibrosis in multiple organs, including heart, liver, lungs, and kidneys. − However, their application may be limited by complex preparation processes, stability issues, and high costs. Additionally, IL-11 derived antagonists such as IL-11-6M and its long-acting analogs, and the IL-11-W148C dimer have also been developed. , Although these analogs exhibit beneficial effects against fibrosis, their stability and the potential toxicity associated with PEGylation still require further validation. Compared with biologic therapies, small-molecule drugs offer advantages, such as improved stability and the potential for oral administration. Currently, bazedoxifene is the only small molecule reported to inhibit the IL-11 pathway. Nevertheless, it does not act specifically on IL-11, as it also blocks IL-6 signaling. Therefore, there is a need to develop a novel IL-11 antagonist for fibrosis treatment.

Peptides have demonstrated considerable potential in antagonizing protein–protein interactions (PPIs). In particular, technologies such as phage display and mRNA display offer significant advantages for the efficient identification of candidates. , Lear et al. reported the first peptide antagonist of IL-11 through phage display. However, this peptide exhibited only weak inhibitory activity, with an IC50 of 70.9 μM in an IL-11-dependent reporter assay. In our earlier work, we employed the random nonstandard peptide integrated discovery (RaPID) system to identify the macrocyclic peptide 4L2-P13D. While 4L2-P13D shows high binding affinity, its poor solubility limits further modification for comprehensive structure–activity relationship (SAR) studies. Moreover, the presence of either a disulfide or a thioether bond in the aforementioned peptides could limit their development due to the inherent instability of the thiol group.

In this study, we focused on the optimization of peptide 4L6 discovered from our prior research. A key modification involved the substitution of its oxidation-labile thioether linkage with a lactam bridge. A series of analogues were synthesized, among which compound 4L6-1 displayed activity similar to that of the parent peptide 4L6. A follow-up alanine scan of 4L6-1 successfully generated a superior analogue 4L6-16. Importantly, the effects of 4L6-16 against renal fibrosis were confirmed in both cellular and animal models.

Results and Discussion

In our previous study, we obtained a macrocyclicpeptide 4L6, which demonstrates strong binding affinity for IL-11 (KD = 183 nM) and exhibits a moderate inhibitory activity with a IC50 value of 26.6 μM. However, the oxidation-sensitive nature of the thioether groups presents a challenge for further development. This is exemplified in the case of Zilucoplan, where the thioether moiety was replaced with a more stable lactam bridge. Inspired by this, this study focuses on exploring analogues that contain a lactam bridge.

The peptides were prepared by following the synthetic route outlined in Scheme . Key to the process was an orthogonal palladium-catalyzed deprotection, which liberated the free acid and amine functionalities to form the lactam bridge. Subsequent global deprotection with a cocktail reagent yielded the final target compounds.

1. Schematic Presentation of the Synthetic Route.

1

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With the analogues in hand, we proceeded to assess their antagonistic activities using a STATs-based luciferase reporter cell line (Figure S1). As shown in Figure , the length of the linkers significantly influences the activity. We substituted the cysteine at position 14 with glutamic acid or aspartic acid, followed by direct lactam bridge formation with the N-terminal amine (4L6-1 and 2). The results indicated that the Glu-substituted analogue exhibited better activity, likely due to its similar length compared to the thioether moiety. Then, we fixed position 14 with glutamic acid and investigated the effect of the linker length by modifying the N-terminus. Activity evaluation revealed that analogs 4L6-4 and 5 showed a significant loss in potency, except for 4L6-3, which exhibited activity comparable to that of 4L6. This suggests that increasing the ring size may reduce the antagonistic activity.

1.

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Antagonistic activities of the synthesized peptides in a STATs-based luciferase reporter cell line. IC50 values were the mean ± SD obtained from three independent experiments.

Next, we intended to determine the influence of each residue on the activity. To this end, we performed the alanine scan on 4L6-1 and measured the IC50 value for each (Table , Figure S1). As shown in Table , alanine substitution at positions 7, 8, 12, and 13 (peptides 4L6-6, 7, 11 and 12) had a negligible effect on potency, indicating that those positions might not be involved in crucial interactions. In addition, replacement of the leucine residue at position 3 (4L6-16) increased activity by 2.5-fold, displaying an IC50 of 9.3 ± 1.0 μM, which implies this position might participate in interactions with IL-11 (Figure A). In contrast, substitutions at the remaining positions (peptides 4L6-8, 9, 10, 13, 14, 15 and 17) led to a dramatic loss of activity, indicating that these sites are crucial for function and warrant further investigation in the future.

1. Antagonistic Activities of Analogs with Alanine Substitution at Positions 2 to 13 .

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a

IC50 values were mean ± SD obtained from three independent experiments.

2.

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(A) Antagonistic activity of 4L6-16 in a STATs-based luciferase reporter cell line. IC50 values were mean ± SD obtained from three independent experiments. (B) Binding affinity evaluation of 4L6-16 for IL-11.

We also evaluated the binding affinity of 4L6-16 to IL-11 using surface plasmon resonance (SPR). The results demonstrated that 4L6-16 binds to IL-11 with a stronger affinity (KD = 134 nM) compared to that of 4L6 (KD = 183 nM, Figure S5), which is consistent with the findings from our cell-based activity assay (Figure B). Next, we made further modifications on peptide 4L6-16. First, we replaced the Ala at position 3 with Ser, Phe, Gly and cyclohexylalanine to evaluate the effects of steric hindrance, aromaticity and hydrophobicity on activity (Figure S1 and Table S3). However, all resulting analogues showed reduced activities compared to that of 4L6‑16. Additionally, we also explored substitutions at position 1 (Figure S1 and Table S4). Most of these analogues exhibited significantly lower activities except the analogue with Trp replacement, which showed a 4-fold loss in activity. These findings highlight the need for determining the structure of the 4L6-16/IL-11 complex to better understand their interactions in the future. Taken together, 4L6-16 exhibited the highest activity and was therefore selected for subsequent evaluations.

We next evaluated the anti-renal fibrotic effect of 4L6‑16 in TGF-β1 stimulated HK-2 cells. First, the cell viability was assessed after treatment with various concentrations of 4L6‑16. As shown in Figure S2, 4L6‑16 did not exhibit significant cytotoxicity toward HK-2 cells at concentration up to 200 μM. Furthermore, 4L6‑16 dose-dependently suppressed the protein level of fibrotic markers, including fibronectin (FN1) and type I collagen (COL1A1), which were significantly upregulated following TGF-β1 treatment (Figure ).

3.

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Anti-fibrosis effects of 4L6-16 in TGF-β1 induced HK-2 cells. (A) The expression levels of FN1 and COL1A1. (B) The ratios of relative FN1/GAPDH and COL1A1/GAPDH. Statistical significance was determined by using a parametric t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the TGF-β1 treated group; ###P < 0.001,####P < 0.0001 compared with the control group; ns, no significance.

To evaluate the pharmacological effects of 4L6-16 in vivo, a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO) was employed. UUO mice were subcutaneously administrated of 4L6-16 daily at doses of 0.5 mg/kg and 2 mg/kg. PFD (50 mg/kg/d) was intraperitoneally injected as a positive control. After 7 days, the expression levels of fibrosis markers FN1 and α-SMA were significantly elevated in UUO mice, whereas they were markedly reduced in the 4L6-16 treated group. In particular, the group receiving 2 mg/kg of 4L6‑16 showed efficacy comparable to that of the PFD group (Figure A and B). Furthermore, 4L6-16 significantly downregulated the levels of inflammatory markers TGF-β and IL-6, which were notably increased in UUO mice (Figure C). Since the blood urea nitrogen (BUN) and creatinine are key biomarkers for renal function assessment, we measured their concentrations. As shown in Figure D, both BUN and creatinine levels were significantly elevated in UUO mice but were reduced following 4L6-16 treatment. Together, these cellular and animal findings demonstrate that 4L6-16 may serve as a potent anti-renal fibrosis agent.

4.

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Anti-renal fibrosis effects of 4L6-16 in UUO mice. (A) The expression levels of FN1 and α-SMA in kidney tissues. (B) The ratios of relative α-SMA/GAPDH, FN1/GAPDH in kidney tissues (n = 6/group) were analyzed. (C) The mRNA level of TGF-β and IL-6 was determined (n = 6/group). (D) Concentration of creatinine in mice blood and concentration of blood urea nitrogen (BUN) were measured. Statistical significance was determined by using a parametric t-test. *P < 0.05, **P < 0.01 ***P < 0.001, ****P < 0.0001 compared with the UUO group; #### P < 0.0001 compared with the control group; ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the vehicle group (n = 6); ### P <0.001, #### P <0.0001 compared with the sham group; ns, no significance (n = 6/group).

Conclusions

Antagonists targeting the IL-11 pathway have demonstrated promising anti-fibrotic efficacy across various organ models. In this study, we explored lactam-bridged analogs to circumvent potential issues associated with the thioether moiety in compound 4L6. Through optimization of the linker length, we obtained one analogue, peptide 4L6-1, with activity comparable to that of 4L6. Further extensive medicinal chemistry efforts led to the development of peptide 4L6-16, which exhibited improved activity and binding affinity. Moreover, 4L6-16 demonstrated remarkable anti-fibrotic efficacy in both in vitro and in vivo renal fibrosis models. Collectively, these findings underscore 4L6-16 as a promising candidate for further therapeutic development against renal fibrotic diseases.

Supplementary Material

ml6c00034_si_001.pdf (2.3MB, pdf)

Acknowledgments

The Public Experimental Research Center of Xuzhou Medical University is acknowledged.

Glossary

Abbreviations

hIL-11

human interleukin-11

CKD

chronic kidney disease

HK-2 cells

human proximal tubular epithelial cells

UUO

unilateral ureteral obstruction

α-SMA

α-smooth muscle actin

COL1Al

collagen I a1

FN1

fibronectin

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00034.

  • Yield and purity of synthesized peptides (Table S1); Primers used in qPCR (Table S2); Antagonistic activities of analogs with modifications at positions 1 and 3 of 4L6–16 (Table S3 and S4); In vitro antagonistic activities (Figure S1); Cytotoxicity evaluation of peptide 4L6–16 in HK-2 cells (Figure S2); Uncropped Western blot images (Figure S3 and S4); The binding affinity evaluation of 4L6 to IL-11 (Figure S5); LC and MS spectra of the synthetic peptides (PDF)

†.

X.Y., N.Z., and R.Z. contributed equally to this work. X.X. and C.A. conceived the work and designed the experiments. X.Y., N.Z., R.Z., J.L., Y.W., Z.M., N.P. X.M. and R.L. performed the experiments and analyzed the data. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was funded by the Scientific Research Foundation for Talented Scholars in Xuzhou Medical University (D2022002), Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (202310313023Z).

No unexpected or unusually high safety hazards were encountered throughout the experimental work conducted here.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml6c00034_si_001.pdf (2.3MB, pdf)

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