Antibacterial, Anti-Inflammatory and Anti-Melanogenic Effects of a Novel Peptide

Abstract

Background

Acne-induced post-inflammatory hyperpigmentation (PIH) is driven by Cutibacterium acnes–mediated inflammation and dysregulated melanogenesis; however, effective treatments targeting both processes remain limited.

Objective

This study aimed to evaluate the anti-bacterial, anti-inflammatory, and anti-melanogenic effects of a newly made 2-isopropyl-5-methylphenol-conjugated valine–tryptophan dipeptide (2IP5MP-VW).

Methods

IPMP-VW conjugates were synthesized and screened for antibacterial activity against C. acnes. The effects of 2IP5MP-VW on inflammation and melanogenesis were assessed using Raw 264.7 macrophages and Mel-ab cells, and ex-vivo human skin.

Results

Among the synthesized candidates, 2IP5MP-VW showed the strongest antibacterial activity against C. acnes. 2IP5MP-VW suppressed lipopolysaccharide-induced nitric oxide (NO) production via downregulation of inducible NO synthase without cytotoxicity. It also reduced melanin content in both unstimulated and forskolin-stimulated melanocytes. In ex vivo human skin, 2IP5MP-VW showed a decreasing trend in ultraviolet B-induced melanin accumulation.

Conclusion

2IP5MP-VW exhibits multifunctional anti-bacterial, anti-inflammatory, and anti-melanogenic activities, suggesting its potential as a candidate for the management of acne-induced PIH.

INTRODUCTION

Cutibacterium acnes, a gram-positive anaerobic bacterium, is a major component of the normal skin microbiota and preferentially colonizes sebaceous-rich areas¹. Dysregulated proliferation of C. acnes induces inflammatory acne, which is frequently followed by pigmentary sequelae such as post-inflammatory hyperpigmentation (PIH) and post-inflammatory erythema¹,²,³. These sequelae are particularly prevalent and persistent in individuals with darker and PIH-prone skin types, posing both therapeutic and psychosocial challenges²,³.

Acne-induced PIH results from inflammatory damage to the epidermal layer caused by C. acnes–mediated immune responses and external aggravating factors including ultraviolet (UV) and visible light exposure³,⁴. Repeated inflammation disrupts epidermal integrity and an increased epidermal melanin can deposit within the dermis, leading to prolonged hyperpigmentation in a mouse model⁵.

Melanogenesis is a tightly regulated biological process in melanocytes in the basal layer of the epidermis. While melanin plays a protective role against UV radiation and oxidative stress, excessive or dysregulated melanogenesis contributes to pathological hyperpigmentation⁶,⁷. Inflammatory mediators such as nitric oxide (NO), produced by keratinocytes following microbial or UV stimulation, further enhance melanogenic signaling⁷. Key enzymes involved in melanin synthesis include tyrosinase (TYR), tyrosinase-related protein 1 (Tyrp-1) and dopachrome tautomerase (DCT), whose expression is regulated by signaling pathways involving microphthalmia-associated transcription factor (MITF) and protein kinase C⁸,⁹,¹⁰.

Current therapeutic approaches for acne-induced PIH primarily focus on pigment-lightening agents¹⁰,¹¹; however, these strategies often fail to address the underlying inflammation or bacterial growth³, resulting in incomplete or delayed clinical improvement. Therefore, there is a growing need for multifunctional topical agents that simultaneously target viability of C. acnes, inflammation and exaggerated melanogenesis.

2-Isopropyl-5-methylphenol (IPMP), a natural monoterpenoid derived from Thymus vulgaris, exhibits antimicrobial, anti-inflammatory, and antioxidant properties⁹. To enhance its efficacy, we designed three peptide–compound conjugates by conjugating IPMP to a dipeptide backbone composed of valine and tryptophan (VW). Among the synthesized candidates, 2IP5MP-VW demonstrated the strongest antibacterial activity against C. acnes.

Therefore, in this study, we investigated the multifunctional effects of 2IP5MP-VW, focusing on its inhibition on inflammatory process and hyperpigmentation.

MATERIALS AND METHODS

Chemicals and reagents

Forskolin (FSK) was purchased from MCE (Monmouth Junction, NJ, USA) and Kojic acid was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Unless otherwise indicated, FSK and Kojic acid were used at concentrations of 10 μM and 200 μM, respectively. The synthetic and analytical procedures for the 2IP5MP-VW compounds are described in the Supplementary Data 1.

Cell cultures

Mel-ab cells, a spontaneously immortalized melanocyte cell line derived from mice, were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; WELGENE, Kyungsan, Korea), 1% antibiotic–antimycotic solution (AA; Thermo Scientific, Rockford, IL, USA), 1 nM cholera toxin, and 100 nM 12-O-tetradecanoylphorbol-13-acetate as described previously¹²,¹³. Raw 264.7 cells were obtained from the Cell Line Bank of Korea (Seoul, Korea), and maintained in DMEM supplemented with 10% FBS and 1% AA. All cells were maintained at 37°C in a humidified environment with 5% CO₂.

Cell viability assay

Mel-ab or RAW 264.7 cells were seeded in 12-well plates at a density of 1.5×10⁵ cells/well and cultured for 24 hours. To evaluate the cytotoxicity of 2IP5MP-VW, cells were treated with various concentrations of the compound for 24 or 72 hours. Cell viability was then determined using an MTT assay (Duchefa Biochemie, Haarlem, Netherlands). Briefly, the culture medium was replaced with MTT solution (2.5 mg/ml in phosphate-buffered saline [PBS]), and cells were incubated at 37°C for 1 hour. After removing the solution, the resulting formazan crystals were dissolved in 1 ml of dimethyl sulfoxide. The absorbance was measured at 565 nm using a BioTek microplate reader (Winooski, VT, USA). Viability was displayed as a percentage relative to the vehicle-treated control (100%).

Bacterial culture and anti-bacterial activity assay

C. acnes was obtained from the Korean Collection for Type Cultures (KCTC 3314, Seoul, Korea), and cultured in an anaerobic state in the reinforced clostridium medium at 37°C for 72 hours. The bacterial suspension was adjusted to a density of 1.5×10⁶ CFU/ml and treated with the peptide for additional 72 hours, following with minor modification of the previously described method¹⁰.

Melanin content and cellular TYR activity

Mel-Ab cells were treated with 2IP5MP-VW, 4IP3MP-VW, or 5IP2MP-VW, either alone or 1 hour prior to stimulation with 10 μM FSK. After 72 hours of incubation, the cells were lysed in 1 N NaOH and heated at 95°C for 30 minutes to solubilize the melanin. To determine the melanin content, the absorbance of the supernatant was measured at 405 nm using a BioTek microplate reader. The values were normalized to the total protein concentration determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Melanin content is expressed as a percentage relative to the vehicle-treated control (100%).

Intracellular TYR activity was assessed by measuring the rate of dopachrome formation from L-3,4-dihydroxyphenylalanine using cellular lysates. Mel-Ab cells were incubated with the vehicle, 2IP5MP-VW alone, or in combination with FSK for 72 hours. The cells were then lysed in TYR lysis buffer (0.1 M phosphate buffer, pH 6.8, containing 1% Triton X-100) via multiple freeze-thaw cycles. TYR activity was determined by measuring the absorbance at 475 nm. The activity is expressed as a percentage of the control cells, with 200 μM kojic acid serving as a positive control.

Messenger RNA (mRNA) isolation and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)

Mel-Ab cells were incubated with the vehicle, 2IP5MP-VW alone, or in combination with FSK as indicated in the figures and total RNA was extracted using the FavorPrep™ Total RNA Purification Mini Kit (Favorgen Biotech, Ping-Tung, Taiwan) following the manufacturer’s instructions. First-stranded complementary DNA (cDNA) was synthesized using total RNA extracted using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using Fast SYBR™ Green Master Mix (Toyobo, Osaka, Japan) with a LightCycler 480 Instrument II (Roche Applied Science, Indianapolis, IN, USA). Gene-specific primers are listed in Table 1 and glyceraldehyde 3-phosphate dehydrogenase was used as an internal reference gene to normalize the relative levels of each transcript.

Table 1. Primer sequence list.

Primer	Forward	Reverse
mGAPDH	5'-CATCACTGCCACCCAGAAGACTG-3'	5'-ATGCCAGTGAGCTTCCCGTTCAG-3'
mTRP1	5'-TCACTGATGCGGTCTTTGAC-3'	5'-CTGACCTGGCCATTGAACTT-3'
mTyr	5'-TTATGCGATGGAACACCTGA-3'	5'-GAGCGGTATGAAAGGAACCA-3'
mDCT	5'-CTTTGCAACCGGGAAGAACG-3'	5'-CCGACTAATCAGCGTTGGGT-3'

mGAPDH: mouse glyceraldehyde-3-phosphate dehydrogenase, mTRP1: mouse tyrosinase-related protein 1, mTyr: mouse tyrosinase, mDCT: mouse dopachrome tautomerase.

Western blotting

Cells were washed with ice-cold PBS and lysed in 1% sodium dodecyl sulfate (SDS) lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM ethylenediaminetetraacetic acid, and 1% SDS). The lysates were boiled at 100°C for 10 minutes for protein denaturation. Equal amounts of protein were separated by 8%–15% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham, Marlborough, MA, USA). The membranes were blocked and then incubated with primary antibodies against inducible nitric oxide synthase (iNOS), DCT, TYR, Tyrp-1 (Abcam, Cambridge, UK), and MITF (Cell Signaling Technology, Danvers, MA, USA). HSP90α/β (Santa Cruz Biotechnology, Dallas, TX, USA) was used as an internal loading control. Protein bands were visualized using an ECL solution (Cyanogen, Bologna, Italy) and detected with a ChemiDoc imaging system (Lugen Science, Bucheon, Korea).

Ex vivo human skin tissue culture

Human abdominal skin tissues were obtained with informed consent from patients undergoing abdominoplasty at Asan Medical Center (Seoul, Korea), in accordance with the guidelines of the Institutional Review Board (IRB No. 2020-0091). The skin specimens were placed onto Transwell inserts with the epidermis facing upward and the dermis in contact with DMEM supplemented with 10% FBS and 10% AA. To induce melanogenesis, the skin explants were exposed to 200 mJ/cm² UVB radiation. Subsequently, the explants were topically treated with either the vehicle or 50 μM 2IP5MP-VW (dissolved in 30% propylene glycol and 70% ethanol) every 48 hours. After 72 hours of incubation, the tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and subjected to Fontana–Masson staining for histological analysis. The melanin index was quantified using ImageJ software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) as previously described¹⁴.

Measurement of NO production

RAW 264.7 cells were treated with 1 μg/ml of lipopolysaccharide (LPS), either alone or in combination with various concentrations of 2IP5MP-VW, and incubated at 37°C under light-protected conditions. After incubation, 100 μl of the culture supernatant was collected and reacted with 150 μl of Griess reagent, consisting of 0.2% naphthyl ethylenediamine dihydrochloride and 0.2% sulfanilamide in 10% phosphoric acid (H₃PO₄). NO production was quantified by measuring the absorbance of the mixture at 570 nm using a microplate reader.

Statistics

All data were expressed as the mean ± standard error of the mean. All statistical analysis was performed using GraphPad Prism (version 8; GraphPad Software, San Diego, CA, USA). Unpaired analysis of variance (ANOVA) was used for the comparison of two groups, and one-way ANOVA with Tukey's test for post hoc analysis for the comparison of more than two groups. The p-values <0.05, <0.01, and <0.001 were considered statistically significant.

RESULTS

Synthesis and antibacterial activities of IPMPs-dipeptides conjugates

To identify structural modifications that may enhance the anti-C. acnes and anti-inflammatory activities of IPMP, we synthesized a series of derivatives, including 2IP5MP, 4IP3MP, and 5IP2MP. Building on studies of non-cytotoxic, hydrophobic dipeptides as melanogenesis inhibitors (Fig. 1A)¹⁵,¹⁶, we hypothesized that a molecular hybrid linking IPMP with an anti-melanogenic dipeptide could exert synergistic effects by simultaneously addressing microbial infection and hyperpigmentation. This rational design led to the successful development of IPMP-VW conjugates as potent multi-target agents for PIH treatment (Fig. 1B). While the VW peptide alone did not affect the viability of C. acnes, all three IPMP-VW conjugates significantly reduced bacterial viability at 100 μM. Notably, the 2IP5MP-VW conjugate (hereafter referred to as 2IP5MP-VW) exhibited a clear dose-dependent inhibitory response (Fig. 1C).

Fig. 1. Chemical structures and antibacterial activities of VW and its IPMP-conjugated derivatives. (A) Chemical structure of VW. (B) Chemical structures of IPMP-VW conjugates: 2IP5MP-VW, 4IP3MP-VW, and 5IP2MP-VW. (C) Antibacterial effects of VW and IPMP-VW conjugates against C. acnes.

VW: valine–tryptophan, IPMP: 2-isopropyl-5-methylphenol, 2IP5MP-VW: 2-isopropyl-5-methylphenol-conjugated valine–tryptophan, 4IP3MP-VW: 4-isopropyl-3-methylphenoxyacetyl-conjugated valine–tryptophan, 5IP2MP-VW: 5-isopropyl-2-methylphenoxyacetyl-conjugated valine–tryptophan.

Anti-melanogenic effect of 2IP5MP-VW in Mel-ab cells

Given that the 2IP5MP-VW conjugate exhibited superior antibacterial activity against C. acnes, we further investigated its anti-melanogenic potential, starting with an assessment of its effect on melanocyte viability. Treatment with 2IP5MP-VW at concentrations up to 100 μM showed no significant cytotoxicity in Mel-ab cells (Fig. 2A). Subsequently, the anti-melanogenic efficacy of 2IP5MP-VW was evaluated under both basal and FSK-stimulated conditions, the latter of which induces melanogenesis via the cAMP signaling pathway. 2IP5MP-VW significantly reduced melanin content in a dose-dependent manner in both conditions. At a concentration of 100 μM, it achieved a maximal reduction in melanin content of 37% and 70% under basal and FSK-stimulated conditions, respectively (Fig. 2B). To investigate the underlying mechanisms of 2IP5MP-VW-mediated melanogenesis suppression, we examined whether it affects the activity of TYR, the rate-limiting enzyme in melanin biosynthesis. Treatment with 50 μM 2IP5MP–VW for 72 hours significantly inhibited cellular TYR activity in both basal and FSK-stimulated Mel-ab cells (Fig. 2C).

Fig. 2. Inhibitory effects of 2IP5MP-VW on melanogenesis in Mel-ab cells. (A) Effect of 2IP5MP-VW on the viability of Mel-ab cells, determined by MTT assay. (B) Effect of 2IP5MP-VW (50 μM) on melanin content in Mel-ab cells under basal and FSK-stimulated conditions following 72 hours of treatment. (C) Effect of 2IP5MP-VW (50 μM, 72 hours) on cellular tyrosinase activity in Mel-ab cells. Kojic acid (200 μM) was used as a positive Ctrl. Data are presented as the mean ± standard error of the mean.

2IP5MP-VW: 2-isopropyl-5-methylphenol-conjugated valine–tryptophan, FSK: forskolin, Ctrl: control, DMSO: dimethyl sulfoxide.

Statistical significance is indicated as follows: ⁺p<0.05, ⁺⁺p<0.01, ⁺⁺⁺p<0.001 vs. Ctrl; ^***p<0.001 vs. FSK-treated group.

Downregulation of melanin biosynthesis related gene expression by 2IP5MP-VW

To further elucidate the mechanisms underlying the suppression of melanogenesis and TYR activity by 2IP5MP-VW, we investigated its effect on the expression of key melanogenic genes, including TYR, Tyrp-1, and DCT. Treatment with 50 μM 2IP5MP-VW for 24–72 hours downregulated the mRNA levels of TYR and DCT under both basal and FSK-stimulated conditions in Mel-ab cells, whereas Tyrp-1 expression remained unaffected (Fig. 3A). Consistently, 72 hours of treatment with 50 μM 2IP5MP-VW reduced the protein levels of TYR and DCT in both basal and FSK-stimulated cells, as well as the upstream regulator MITF (Fig. 3B).

Fig. 3. Inhibitory effects of 2IP5MP-VW on the expression levels of melanogenic genes. (A) Effects of 50 μM 2IP5MP-VW on the mRNA levels of Tyrp-1, tyrosinase, and DCT in basal and FSK-stimulated Mel-ab cells at 24, 48, and 72 hours post-treatment. (B) Effects of 50 μM 2IP5MP-VW on the protein expression levels of MITF, tyrosinase, Tyrp-1, and DCT in basal and FSK-stimulated Mel-ab cells. HSP90 was used as an internal loading control. Data are presented as the mean ± standard error of the mean.

2IP5MP-VW: 2-isopropyl-5-methylphenol-conjugated valine–tryptophan, mRNA: messenger RNA, Tyrp-1: tyrosinase-related protein 1, DCT: dopachrome tautomerase, FSK: forskolin, MITF: microphthalmia-associated transcription factor, Ctrl: control.

Statistical significance is indicated as follows: ⁺⁺⁺p<0.001 vs. control; ^**p<0.01, ^***p<0.001 vs. FSK-treated group.

Reduction of melanin accumulation in ex vivo human skin tissues by 2IP5MP-VW

To evaluate the clinical relevance of 2IP5MP-VW’s anti-melanogenic activity, we employed an organotypic human skin explant model. Fontana–Masson staining revealed that UVB irradiation of abdominal human skin explants significantly increased epidermal melanin content compared to the vehicle-treated control. Subsequent topical application of 2IP5MP-VW led to a reduction in UVB-induced epidermal melanin accumulation (Fig. 4A). Quantitative analysis using the melanin index demonstrated that UV irradiation increased melanin content by 40% compared to the vehicle-treated control, while treatment with 2IP5MP-VW significantly reduced this UV-stimulated accumulation in the human epidermis by 36% (Fig. 4B).

Fig. 4. Inhibitory effect of 2IP5MP-VW on UVB-induced pigmentation in ex vivo human skin. (A) Fontana–Masson staining of ex vivo human abdominal skin tissues treated with UVB (200 mJ/cm²) and either topical application of vehicle or 2IP5MP-VW for 5 days. (B) Melanin index analysis showing a significant reduction in pigmentation by 2IP5MP-VW compared to vehicle-treated groups. Data are presented as the mean ± standard error of the mean.

2IP5MP-VW: 2-isopropyl-5-methylphenol-conjugated valine–tryptophan, UVB: ultraviolet B, Ctrl: control.

Statistical significance is indicated as follows: ⁺⁺p<0.01 vs. control; ^***p<0.001 vs. UVB-treated group.

Anti-inflammatory effects of 2IP5MP-VW via suppression of iNOS

To investigate the underlying anti-inflammatory mechanisms of 2IP5MP-VW, we employed an in vitro model using RAW 264.7 macrophages. Treatment with 2IP5MP-VW at concentrations up to 250 μM showed no significant cytotoxicity (Fig. 5A), confirming that the subsequent observed effects were not due to compromised cell viability. NO is a critical inflammatory mediator produced by activated macrophages, and its excessive production via iNOS induction contributes to inflammatory tissue damage¹⁷. LPS stimulation significantly increased NO production by approximately 14-fold compared to the untreated control. However, 2IP5MP-VW treatment (3.91–31.25 μM) resulted in a dose-dependent reduction in LPS-induced NO levels, achieving up to 50% inhibition at 31.25 μM (Fig. 5B). Furthermore, iNOS protein expression, which was markedly upregulated by LPS, was dramatically attenuated by 2IP5MP-VW. These findings suggest that 2IP5MP-VW exerts potent anti-inflammatory effects primarily by suppressing the iNOS-mediated NO signaling pathway in LPS-stimulated RAW 264.7 cells (Fig. 5C).

Fig. 5. Anti-inflammatory effects of 2IP5MP-VW in LPS-stimulated RAW 264.7 cells. (A) Effect of 2IP5MP-VW on the viability of RAW 264.7 cells, determined by MTT assay. (B) Inhibitory effect of 2IP5MP-VW on NO production in LPS-stimulated RAW 264.7 cells. (C) Inhibitory effect of 2IP5MP-VW on iNOS protein expression in LPS-stimulated RAW 264.7 cells. β-actin was used as an internal loading control. Data are presented as the mean ± standard error of the mean.

2IP5MP-VW: 2-isopropyl-5-methylphenol-conjugated valine–tryptophan, LPS: lipopolysaccharide, NO: nitric oxide, iNOS: inducible nitric oxide synthase, Ctrl: control.

Statistical significance is indicated as follows: ⁺⁺⁺p<0.001 vs. control; ^***p<0.001 vs. LPS-treated group.

DISCUSSION

In the present study, we demonstrated that a novel peptide, 2IP5MP-VW, an IPMP-conjugated VW dipeptide, exerts multifaceted biological activities which may be applicable to management of acne-induced PIH¹,²,³. Among the three newly made peptides, 2IP5MP-VW was based on antibacterial activity against C. acnes. 2IP5MP-VW also suppressed inflammatory responses in LPS-induced macrophage models which are, in part, involved in the development of PIH⁴,⁵,⁷. Furthermore, 2IP5MP-VW reduced melanin synthesis in melanocytes and ex vivo human skin.

C. acnes is a key driver of inflammatory acne lesions and is known to induce the production of pro-inflammatory mediators and NO¹,²,³. To examine the anti-inflammatory effects of 2IP5MP-VW, we used LPS-stimulated Raw 264.7 macrophages¹⁷,¹⁸,¹⁹. The elevated levels of NO were significantly suppressed following treatment with 2IP5MP-VW through reduced iNOS expression through nonspecific cytotoxic effects. The LPS model shares similar inflammatory signaling mechanisms with C. acnes-induced inflammation¹⁷,¹⁸,¹⁹. LPS, a major component of Gram-negative bacterial cell walls, primarily activates immune cells via Toll-like receptor 4 (TLR4), thereby stimulating nuclear factor-κB (NF-κB) pathway and inducing inflammatory responses¹⁹. Likewise, C. acnes activates TLR2 and TLR4, which subsequently stimulate the NF-κB pathway to promote the production of cytokines and inflammatory mediators, including NO¹⁸,¹⁹. C. acnes-induced inflammation model may be required in future studies for direct evaluation of acne-induced PIH. PIH is a common sequela of skin inflammation, characterized by hyperpigmentary changes⁴,⁵,⁶,⁷. Melanocytes are critical for melanin production, and their activity is influenced by inflammatory mediators and interactions with other cells including keratinocytes, fibroblasts, macrophages and immune cells²⁰,²¹,²².

Excessive NO production contributes not only to inflammatory tissue damage but also to PIH¹⁷,¹⁸,¹⁹,²⁰,²¹,²². Importantly, NO has been implicated as a key mediator linking inflammation to melanogenesis²¹. NO produced by keratinocytes and immune cells can act in a paracrine manner on melanocytes, enhancing cellular TYR activity and melanin synthesis²². Therefore, the inhibition of NO production by 2IP5MP-VW may contribute not only to its anti-inflammatory effects but also to the observed reduction in melanin content via paracrine manner.

The ability of this model to fully capture the intricate inflammatory processes of acne. Therefore, future research should consider more comprehensive models that incorporate effects on other inflammatory cytokines involved in the pathogenesis of acne and Th17 immune-inflammatory pathways¹⁷,¹⁸,²³. Unlike conventional depigmenting agents that primarily compete with TYR⁸,⁹,¹⁰,¹¹,²⁴ 2IP5MP-VW reduced MITF expression and its downstream targets. Consistent with this mechanism²⁵, 2IP5MP-VW reduced melanin content in both unstimulated and FSK-stimulated melanocytes and showed a decreasing trend in UVB-induced melanin accumulation in ex-vivo human skin. Additional in vivo studies are required to confirm its efficacy.

Taken together, our findings suggest that newly synthesized peptide, 2IP5MP-VW may provide a multifunctional advantages targeting acne or other inflammation-associated hyperpigmentation disorders.

SUPPLEMENTARY MATERIAL

Supplementary Data 1

Synthetic procedures