Development of New Anti-Wrinkle Peptide Using Cheminformatics-Assisted Peptidomimetic Design

Abstract

Peptides are recognized as multifunctional bioactive ingredients in cosmetic science, as they offer diverse beneficial effects such as skin rejuvenation, anti-aging, and skin barrier enhancement. In this study, we applied a cheminformatics-assisted peptidomimetic design platform to design novel peptides targeting heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone that is downregulated during skin aging. Using molecular fingerprint similarity-based peptide design and protein–peptide docking simulations, five candidate peptides were screened, among which ICP-1225 (TY) emerged as a potent stimulator of Hsp47 and collagen (COL1A1 and COL3A1) expression in dermal fibroblasts. To improve stability and skin penetration, fatty acid-conjugated derivatives of ICP-1225 were synthesized, and acetyl-TY (ICP-1236) demonstrated the most consistent upregulation of Hsp47 and collagen in vitro. Restoration of Hsp47 protein expression and dermal collagen levels in UVB-damaged ex vivo human skin explants was also observed. These findings highlight the potential of cheminformatics-assisted peptide design in the development of next-generation cosmetic actives. ICP-1236 represents a promising anti-wrinkle candidate through the modulation of Hsp47 and collagen pathways, warranting further clinical evaluation.

1. Introduction

Peptides are short chains of amino acids with a length and molecular weight ranging from 2 to 100 and from 0.5 to 10 kDa, respectively. In addition to their role in the structure of proteins, the biological roles of peptides are quite diverse, encompassing aspects such as the modulation of various enzyme activities, hormonal activity, antimicrobials, and neurotransmitters [1]. Based on these effects, peptides have been extensively investigated for their potential application as cosmetic ingredients with skin regeneration, anti-wrinkle, antioxidant, skin brightening, and skin-calming effects [2]. In addition to their wide-ranging applications, peptide ingredients have potential advantages over other bioactive ingredients, including increased selectivity, efficacy, safety, and lower toxicity and immunogenicity [3]. Peptides can be isolated from natural sources, including food products [4], marine organisms [5], and venom [6], and they can also be produced by biotechnical manufacturing processes [7] and chemical synthesis [8].

Recent advances in computational tools, driven by the increased availability of numerous peptide sequences and their functions [9], and the use of artificial intelligence (AI) in peptide research have sparked the development of advanced methodologies for the development of novel peptides for cosmetic applications [10]. While artificial intelligence (AI)-guided peptide design has recently emerged as a promising strategy for accelerating bioactive peptide discovery, its effectiveness is often limited by the scarcity of comprehensive structure–activity data. To address this, we developed a novel cheminformatics-assisted peptidomimetic design system for cosmetic applications capable of generating structured molecular datasets that support AI learning and the generalization of structure–function relationships. This is a custom-built, cheminformatics-driven screening tool that incorporates a molecular fingerprint-based structural similarity search algorithm developed using the RDKit cheminformatics toolkit [11,12]. This platform enables ultra-large-scale similarity-based screening across a virtual peptide library containing approximately 67 million unique sequences. Molecular fingerprints are vectorized representations of small molecules that encode their structural features and relevant biochemical properties. After primary candidates were screened using a chemical structure–similarity-based peptidomimetics design, which refers to peptides designed to mimic the chemical structure of reference ligands or agonists/antagonists of the target protein, the potential binding of the designed peptides against the target protein was predicted, and hypothetical binding affinities were calculated with a peptide–protein docking simulating program. As a result, lead peptides with the highest binding properties were selected, synthesized, and subjected to further biological testing (Figure 1). Cheminformatics provides the structured molecular data and descriptors that enable AI to learn and generalize structure–function relationships. Integrating both approaches represents a natural progression toward data-driven, predictive peptide discovery.

Heat shock proteins (Hsp) are a highly conserved family of proteins primarily produced in response to various forms of stress, such as increased temperature (heat shock), cold, ultraviolet light, inflammation, and hypoxia [13]. Hsp47, also known as Serpin H1, is a molecular chaperone required for the correct folding of procollagen in vertebrates. Decreased expression of Hsp47 in the skin of aged mice and increased expression and secretion of type I collagen in dermal fibroblasts by Hsp47 stimulation [14] suggest that the use of Hsp47 could be plausible in anti-wrinkle applications. We explored the applicability of the cheminformatics-assisted peptide design for developing new anti-wrinkle peptide ingredients with Hsp47-stimulating activity. Therefore, in this study, we aimed to design and validate novel peptide ingredients that stimulate Hsp47 in anti-wrinkle applications using our cheminformatics-assisted peptide design system.

2. Materials and Methods

2.1. Materials

In total, 5 peptides designed using the cheminformatics-assisted peptide design system (listed in Figure 3) and acyl-derivatives of ICP-1225 were synthesized using solid-phase peptide synthesis with fluorenylmethyloxycarbonyl chloride (fMOC) methods. After undergoing synthesis and cleavage, the compounds were purified with conventional preparative reverse-phase high-performance liquid chromatography (prep-RP-HPLC), and their purity was further confirmed using the Waters Alliance e2695 HPLC system (Waters Corp., Milford, MA, USA) equipped with an SQ Detector 2.

2.2. Ex Vivo Human Skin Explant Model Study

To investigate the changes in Hsp47 protein levels in human skin, we used an ex vivo human skin explant model. NativeSkin^® human skin tissues were purchased from GenoSkin S.A.S. (Toulouse, France). Skin tissues from the photo-protected site (abdomen) were obtained from healthy female donors with no history of allergies or dermatological disorders, according to the proprietary protocol developed by Genoskin [15]. Ethical compliance was enacted and authorized through official authorization from the French Ministry of Research (Protocol AC-2022-4863, 14 October 2022). All studies were performed in accordance with the principles of the Declaration of Helsinki.

After receiving the tissues, the skin models were stabilized in a 12-well culture plate containing 1 mL of maintenance medium (Genoskin) in a 5% CO₂ humidified chamber. After 2 h of stabilization, the tissues were harvested, embedded in paraffin, and sectioned to a thickness of 4 µm. To explore the effects of photodamage on Hsp47 expression, 50 mJ/cm² of UVB was irradiated on the epidermal surface of the skin tissue once a day for 2 consecutive days before peptide treatment was initiated. Peptides were topically applied after each irradiation. After 48 h of incubation, UVB-irradiated skin tissues were harvested and subjected to histological assessment. Using immunohistochemical staining, Hsp47 expression was observed, according to a previously reported procedure [15], with slight modifications. Rabbit anti-Hsp47 (Abcam, ab109117) was used for staining, and fluorescence intensity was analyzed under a fluorescence microscope (Eclipse Ni-U, Intenslight C-HGFI, DS-Ri2, Nikon, Tokyo, Japan) at 400× magnification. To observe dermal collagen, Masson’s trichrome staining was performed according to a previously described protocol [16]. All experiments were performed in duplicate, and histological analysis was performed by randomly selecting ten regions per slide. The expression levels of each protein were quantified by measuring the fluorescence intensity using the ImageJ 1.51s software (NIH, Bethesda, MD, USA).

2.3. Cheminformatics-Assisted Peptides Screening and Molecular Docking

The structural similarity between the reference molecule and the virtual peptide library of 67,000,000 peptides (2~6 amino acids), which was generated through combinatorial enumeration, was evaluated using the Tanimoto coefficient, which is a widely employed metric for molecular similarity assessment in cheminformatics. Before the screening, pre-filtering was applied based on physicochemical properties (molecular weight < 1 kDA, miLogP < 3, and net charge between −2 to +2) to ensure a tractable candidate. The canonical SMILES representation of the reference molecule (SMILES_R) and each virtual peptide (SMILES_n) were converted into molecular fingerprints (Fingerprint_R and Fingerprint_n, respectively). Pairwise similarity scores were then computed according to the following equation, as implemented in the RDKit cheminformatics toolkit [17]:

$$Similarity\left(n\right)=TanimotoSimilarity({Fingerprint}_R,{Fingerprint}_n)$$

The similarity value ranged from 0.0 to 1.0, with high scores indicating a greater structural resemblance to the reference molecule. The same procedure was systematically applied to all peptides in the virtual pool. After performing the similarity calculations, peptides with Tanimoto similarity ≥ 0.56 to the reference molecule (glucosyl–nicotinamide) were initially selected. The selected peptides were ranked in descending order, and high-priority peptide candidates were selected for protein–peptide molecular docking simulation using AutoDock (version 4.2) [18]. The crystal structure of human HSP47 (PDB ID: 7BDU) was retrieved from the Protein Data Bank (PDB). Protein preparation involved the removal of crystallized collagen and water molecules, the addition of polar hydrogen atoms, and the assignment of Kollman charges. Both the receptor and peptide ligands were converted to PDBQT format using AutoDockTools. The docking grid box was centered at X: −37.8250, Y: −34.5527, and Z: −19.2483, with dimensions of X: 85.7015 Å, Y: 85.4016 Å, and Z: 52.5783 Å, to sufficiently cover the entire putative binding region of the protein. The protein preparation steps have been outlined in detail (e.g., removal of water molecules, addition of polar hydrogens, and assignment of Kollman charges). Crucially, we also set the exhaustiveness parameter to 15 to ensure a thorough search of binding poses and enhance the accuracy of the affinity calculations.

2.4. In Vitro Efficacy Assessment

2.4.1. Cell Viability Assay

Normal human dermal fibroblasts (hDFs) (passages 5), human fibroblast expansion basal medium with a low serum growth supplement, and gentamicin/amphotericin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cells were cultured at 37 °C in a 5% CO₂ atmosphere. To measure cellular viability, 3000 cells/well of hDFs were seeded in 96-well plates and treated with the tested peptides at the indicated concentrations for 24 h. Cell viability was quantified using the Thiazolyl Blue Tetrazolium Bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). For comparison, non-treated cells served as the negative control group. Experiments were performed in triplicate, and the results are expressed as the arithmetic mean ± standard deviation.

2.4.2. Western Blot

The expression levels of Hsp47 and collagen 1A1, 3A1 proteins were measured using Western blotting. hDFs were seeded in 6-well plates (1 × 10⁵ cells/well) and cultured for 24 h. After removing the culture medium and washing, the cells were treated with the test peptides for 24 h and harvested. The cell lysates were then separated using SDS-PAGE, and the proteins were blotted onto nitrocellulose membranes. After blocking with a 0.5% bovine serum albumin solution containing 0.1% Tween-20, the membranes were incubated with anti-human Hsp47 antibody (ab109117, abcam), anti-COL1A1 (#39952, Cell Signaling Technology, Danvers, MA, USA), and anti-COL3A1 (#63034s, Cell Signaling Technology) overnight at 4 °C. Secondary antibodies conjugated with horseradish peroxidase were incubated for one hour at room temperature, and protein quantity was measured using an ECL detection reagent (Amersham Biosciences Corp., Little Chalfont, UK) using the ChemiDoc system (Alliance Mini HD 9, Uvitec, Cambridge, UK).

2.5. Statistical Analysis

Values are expressed as the arithmetic mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA with Tukey’s post hoc test (n = 3, p < 0.05 significance level). Significance indicators were added to figures and statistical analyses using SPSS ver. 20 (IBM, New York, NY, USA).

3. Results

3.1. Clinical Assessment of Hsp47 as a Potential Anti-Wrinkle Target

To explore whether Hsp47 could be a potential target for skin anti-aging, we investigated changes in Hsp47 protein expression with aging. In normal human skin tissues collected from individuals in their 20 s to 60 s, immunohistochemical staining against Hsp47 demonstrated pronounced immunoreactivity in the upper granular layer of the epidermis in younger skin samples. Interestingly, a progressive decline in staining intensity was observed in aged skin, with samples from older donors exhibiting markedly reduced immunoreactivity compared with samples from younger donors (Figure 2, upper panel). Considering that skin biopsies were taken from the same anatomical site of the abdomen, the age-dependent decrease in staining intensity suggests that there is a potential association between Hsp47 expression and chronological aging. Consistent with a previous study showing the attenuated expression of Hsp47 protein by UVA irradiation in cultured dermal fibroblasts [19], the significantly reduced expression of Hsp47 protein was detected in UVB (50 mJ/cm²)-irradiated tissues (Figure 2, lower panel). These findings suggest that Hsp47 is closely associated with skin aging, and Hsp47-modulating peptides could be used as anti-aging ingredients.

3.2. In Silico Design of Hsp47 Binding Peptide

To generate potential Hsp47 protein-modulating peptides using the cheminformatics-assisted peptidomimetic design system, reference molecules that potentially activate Hsp47 were designed. While several studies have reported potential modulators of heat shock proteins [20], few have reported on specific Hsp47 activators. Therefore, in this study, a virtual compound of glucosyl–nicotinamide was constructed to design potential Hsp47-modulating peptides. The rationale for selecting glucosyl–nicotinamide as the reference molecule was based on its dual functional relevance, as reported in previous studies. For instance, nicotinamide and its chemical derivatives have been reported to upregulate heat shock proteins [18], while glucose conjugation increases hydrogen-bonding capacity and potential affinity for Hsp47 [21]. Thus, glucosyl–nicotinamide (Figure 3a) was adopted as a representative small-molecule analog to guide virtual peptide screening within the cheminformatics-assisted design platform. Theoretical validation of the potential binding of glucosyl–nicotinamide on Hsp47 protein was confirmed by protein–peptide binding simulation (Figure 3b). Using glucosyl–nicotinamide as a reference molecule, peptides with structural similarities were screened using molecular fingerprint analysis, and 20 peptides were selected based on the Tanimoto coefficient. From the docking analysis of 20 candidate peptides, 5 peptides with the highest binding affinities were selected, and their interacting amino acid residues in Hsp47 were analyzed using BioVIA Discovery Studio (Figure 3b).

3.3. In Vitro Efficacy Assessment

To determine whether the cheminformatics-assisted peptidomimetic design system-generated peptides had the expected biological activities of Hsp47 stimulation and collagen synthesis, cultured human dermal fibroblasts were treated with the test peptides. No cytotoxicity was observed for any of the peptides up to a concentration of 100 µM, and subsequent experiments were performed under these conditions (Figure 4a). Among the five tested peptides, only ICP1222 and ICP-1225 showed stimulatory activity on Hsp47. Interestingly, only ICP-1225 upregulated both collagen 1A1 and 3A1 in dermal fibroblasts, suggesting that ICP-1225 may exert potential anti-wrinkle activity through Hsp47 modulation (Figure 4b). In contrast, biotinoyl hexapeptide-2, which demonstrated significant anti-wrinkle activity in a previous study [16], showed stimulatory effects only on collagen and not on Hsp47.

3.4. Synthesis and Efficacy Evaluation of Peptide Derivatives

While peptides have several advantages in cosmetic applications, such as higher biocompatibility, tunable bioactivities, and broad customizability, they have a few significant drawbacks, including lower stability and relatively unfavorable transcutaneous penetration profiles. To overcome these limitations, peptide derivatization is commonly used. Among the various strategies used for peptide derivatization, the present study employed a relatively simple fatty acid conjugation method, and based on in vitro studies, ICP-1225 was selected for further investigation. We chose three acyl chains (acetyl, hexanoyl, and palmitoyl) to explore the effects of the lipophilicity and chain length of the acyl group on the bioactivity of the peptides. Acetyl (ICP-1236), hexanoyl (ICP-1237), and palmitoyl derivatives (ICP-1238) of ICP-1225 were further synthesized, and their anti-wrinkle activities were examined. Among the three peptide derivatives, only ICP-1236 (acetyl-TY) showed a dose-dependent increase in both Hsp47 and collagen 1Aa proteins, while other derivatives showed noticeable stimulating activities only at lower concentrations (10 µM), but all proteins were downregulated at higher concentrations (100 µM) (Figure 5). From the preliminary studies, we confirmed that no cytotoxicity was observed up to a concentration of 100 µM for all compounds, and further investigation is warranted for these paradoxical effects at higher concentrations.

3.5. Ex Vivo Efficacy Testing

To further address the potential anti-aging effects of Hsp47 binding peptide derivatives, we employed an ex vivo human skin explant model. Normal human skin tissues were obtained from the abdomen through plastic surgery and maintained according to a previously reported protocol [16]. As a result, the attenuated expression of Hsp47 protein by UVB exposure was significantly restored in the epidermal layer (Figure 6, upper panel). Increased dermal collagen expression was also observed via Masson’s trichrome staining (Figure 6, lower panel).

4. Discussion

Recently, peptides have been increasingly recognized as potent and multifunctional bioactive ingredients for cosmetic applications. With the diverse specific activities that occur at the molecular and cellular levels, peptide ingredients can rejuvenate the skin, enhance the skin barrier, control pigmentation, balance the microbiome, and have anti-aging effects. The development of new peptide ingredients for cosmetic applications necessitates the identification of new targets, such as enzymes, ligands, receptors, or structural proteins, and tools for designing new molecules. As a potential solution, artificial intelligence (AI)-guided peptide design has been actively developed; however, its success depends on the availability of extensive peptide structure–activity data. In this study, we present a novel cheminformatics-assisted peptidomimetic design system for cosmetic applications, which can generate high-quality molecular data that enable AI to learn and generalize structure–function relationships.

Various types of noninvasive esthetic skincare devices that use electric and/or phonic energy have been developed. Among them, radiofrequency (RF)-based devices are known to be effective at stimulating immediate collagen structure changes, resulting in skin tightening and firming. Unlike other photothermolysis devices that target specific skin chromophores, RF devices induce local temperature increases within the area of application through skin tissue impedance [22] and stimulate the expression of stress-induced factors, including heat shock proteins. Heat shock proteins (HSPs) are a highly conserved family of proteins that are upregulated under various stress conditions. Under physiological conditions, HSPs facilitate protein folding, the removal of misfolded proteins, and chaperoning and trafficking proteins [13]. In human skin, HSPs are known to protect cells from external stressors, such as ultraviolet rays, physical strain, and thermal changes [23], and regulate wound-healing and repair processes [24]. Hsp47, encoded by the SerpinH1 gene, is a collagen-specific molecular chaperone. Unlike canonical Serpins, Hsp47 does not exhibit protease inhibitory activity but rather stabilizes the triple-helix structure of procollagen to achieve proper collagen folding [25]. Previous studies have also reported that heat shocks enhance the epidermal expression of Hsp47 proteins both ex vivo [26,27] and in the skin of aged animals [28], suggesting that Hsp47-binding molecules can upregulate collagen expression in the skin.

Due to the lack of Hsp47-specific ligands reported, in this study, we generated a hypothetical compound, glucosyl–nicotinamide, as a reference molecule for peptide design. To validate whether this hypothetical compound can interact with the target protein, in silico docking simulation was performed, and the results demonstrated that four amino acids of the Hsp47 protein possibly interacted with glucosyl–nicotinamide, with a binding affinity of −6.8. Using this compound as a reference molecule, peptides that were chemically similar were screened from the library. Among the 20 peptide candidates generated, five peptides showed higher binding affinity compared with glucosyl–nicotinamide, with lower affinity values. Due to the high number of hydroxyl groups and amide-bearing scaffolds in the reference molecule, most of the designed peptides contained threonine (T) and tyrosine (Y), reflecting the algorithmic bias of the chemical similarity search engine. Interestingly, ICP-1221 showed a slightly lower predicted binding free energy (−8.2 kcal/mol) compared with ICP-1225 (−7.1 kcal/mol). However, ICP-1225 demonstrated stronger experimental activity against Hsp47 and downstream collagen markers (COL1A1, COL3A1). Notably, the observed discrepancy between the predicted binding affinity and the experimentally measured biological activity of the peptides reflects an inherent limitation of the current cheminformatics-assisted peptidomimetics design platform. Because the current model relies primarily on static docking-based affinity estimation, it may not fully capture the conformational flexibility, solvent interactions, or cellular processes influencing peptide–protein interactions. This limitation is acknowledged as a weakness of this model, indicating that the platform should be regarded as hypothesis-generating rather than a fully predictive system; further refinement through experimental feedback and machine learning-based optimization is required. Also, this study was conducted with a limited number of peptide samples and thus serves as a preliminary proof-of-concept validation of the cheminformatics-assisted platform. The authors are currently conducting several additional projects using the same platform, targeting anti-wrinkle and skin-brightening peptides, which are expected to provide larger datasets for the quantitative evaluation of predictive performance of the platform.

While peptides have significant benefits as bioactive ingredients, they have a few important limitations, including relatively low stability and suboptimal transcutaneous delivery profiles. Fatty acid conjugation is a commonly used derivatization technique for enhancing penetration, bioavailability, and stability. As one of the most common derivatization methods for peptides, acetylation introduces a short, polar acyl moiety that can improve peptide stability and reduce enzymatic degradation without significantly altering solubility. In contrast, the medium-chain hexanoyl and long-chain palmitoyl groups increase overall lipophilicity, which may enhance skin penetration and facilitate interaction with the hydrophobic regions of dermal proteins. In this study, among the five fatty acid-conjugated forms of ICP-1225, the acetyl conjugate showed the highest bioactivity in vitro. This systematic derivatization allowed us to assess the balance between hydrophilic–lipophilic properties and biological efficacy, ultimately identifying the acetyl derivative (ICP-1236) as the most potent and functionally favorable compound. Further, an ex vivo human skin explant model confirmed the potential anti-aging activity of the acetyl derivative (ICP-1236). In addition to the chemical derivatization, the use of a “mirror image” protein and peptide composed of D-type amino acids has been reported as a potential strategy for improving the stability of the peptide [29]. Considering that the current platform was constructed exclusively with naturally occurring L-type amino acids, expanding the virtual library to include D-type amino acids could represent a plausible next step toward enhancing the platform’s performance and diversity.

While additional research on the potential effects of acetyl conjugates on other proteins should be performed, these results suggest that Hsp47-modulating compounds may provide potential anti-aging effects. However, it should be noted that the proposed mechanism of action remains hypothesis-driven, as direct experimental confirmation of peptide–Hsp47 interaction has not yet been achieved. The current interpretation is therefore based on in silico binding analysis and previously reported studies proposing that Hsp47 plays a role in dermal collagen maturation and remodeling. To address this limitation, additional studies, including co-immunoprecipitation assays and Hsp47-knockdown fibroblast experiments, are warranted. Accordingly, the current results represent a preliminary demonstration supporting the feasibility of the cheminformatics-assisted design approach rather than a conclusive mechanistic verification. Clinical efficacy testing to confirm the anti-aging effects is also needed, and we plan to carry this out.

5. Conclusions

This study demonstrates the feasibility of applying a cheminformatics-assisted peptidomimetic design system for discovering novel cosmetic peptides. Hsp47, a collagen-specific molecular chaperone implicated in skin aging, was selected as the target protein, and peptide candidates potentially binding to Hsp47 were experimentally designed. Among the synthesized candidates, ICP-1225 significantly upregulated Hsp47 and collagen expression in dermal fibroblasts. The discrepancy between computational affinity prediction and experimental bioactivity is recognized as a key limitation of the present platform, emphasizing that the cheminformatics-assisted system functions mainly as a hypothesis-generating framework that requires further improvement through experimental validation. Peptide derivatization was further performed to improve the physicochemical properties of the screened peptide. As a result, the acetyl derivative of ICP-1236 demonstrated potential anti-aging efficacy in both in vitro and ex vivo models. These results suggest that Hsp47-modulating peptides, particularly ICP-1236 and its acetyl form, can be developed as potential anti-wrinkle agents. Future studies should focus on evaluating the long-term safety, validating anti-wrinkle efficacy within clinical settings, and optimizing delivery strategies to translate these findings into practical cosmetic applications.

6. Patents

The compounds described in this study, including ICP-1236 (N-acetyl-L-threonine-L-tyrosine) and its derivatives, are the subject of a patent application that has been filed (patent application number KR 10-2025-0050599). Based on the preliminary freedom-to-operate analysis, there are no active claims that overlap with the designated compounds, ICP-1236 and its derivatives. Accordingly, these materials have been designated as pending patent.