High-Throughput Screening for Discovering Small Molecules Capable of Increasing the Wnt/β-Catenin Signaling Pathway to Promote Hair and Nail Growth

doi:10.21203/rs.3.rs-4370130/v1

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High-Throughput Screening for Discovering Small Molecules Capable of Increasing the Wnt/β-Catenin Signaling Pathway to Promote Hair and Nail Growth

https://doi.org/10.21203/rs.3.rs-4370130/v1

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Hair and nails serving protective roles differ in structure. Recent bioinformatics research has found that Wnt signaling is crucial for their growth. However, they show unique expression patterns of specific elements such as R-spondin, LGR receptors, and BMP.

To identify small molecules that can enhance the Wnt/β-catenin pathway, assess their effects on RSPO, LGR, and BMP expression, and determine their influence on hair and nail growth.

FDA-approved drugs and natural compounds (n = 5,170) were screened using HEK293 cells with TCF/LEF luciferase gene by measuring luciferase activity and cell viability. Selected drugs were tested with human dermal papilla cells to observe Wnt signaling gene expression. Three top candidates were further tested with C57BL/6 mice for hair and nail growth effects.

Nine drugs were identified as significant activators of Wnt signal and categorized into antivirals (Imidocarb, Proflavine, Aminoacridine), anticancer drugs (Entinostat, Tucidinostat, Enzastaurin, Abemaciclib), and GSK-3β inhibitors (CP21R7, BIO). RT-PCR revealed that Aminoacridine and Proflavine notably increased Wnt-related genes RSPO3 and RSPO4. Aminoacridine also significantly reduced the Wnt inhibitor WIF1 gene. In animal experiments, Aminoacridine, especially combined with Minoxidil, resulted in better hair growth than other drugs and Minoxidil alone. Imidocarb and Proflavine also significantly increased toenail length.

Six new drugs were discovered, with Aminoacridine and Aminoacridine combined with Minoxidil showing high potential for hair and nail regeneration via the Wnt/β-catenin pathway, underscoring the need for extensive clinical trials to verify these drugs' safety and efficacy, offering hope for effective hair and nail loss treatments.

Biological sciences/Drug discovery

Biological sciences/Biochemistry

Wnt/β-catenin signaling

High-throughput screening

R-spondin

hair

nail

A study of hair follicle (HF) and nail unit has revealed intriguing similarities and differences from both phenotypic and genotypic perspectives, reflecting the complexity and specificity of human integumentary structures¹. Phenotypically, both hair and nails primarily serve a protective function, with hair shielding the scalp and nails safeguarding fingertips. This protective role is largely facilitated by the production of hard keratin, a commonality hinting at a shared evolutionary pathway^2,3. However, they show significant differences in structure, with hair characterized by long, twisted fibers offering flexibility and nails distinguished by their flat layers of keratin, conferring rigidity⁴.

In the case of the second genotype, similarities and differences in gene expression related to hair and nails offer a fascinating area of study. Both hair follicles and nails are primarily regulated by the Wnt/β-catenin signaling pathway. However, expression levels of specific genes within this pathway show considerable variations, reflecting their distinct morphological characteristics⁵. For instance, the R-spondin family of proteins, particularly RSPO3 and RSPO4, play a significant role in these structures. RSPO3 is more associated with hair follicle development, while RSPO4 shows a stronger link with nail growth^6,7.

Such differential expression suggests a fine-tuning of the Wnt pathway, where certain genes are upregulated in one structure but not the other, leading to unique phenotypic traits of hair and nails. In previous studies, single-cell analysis of hair follicle-related genes and spatial transcriptome analysis of tissues surrounding hair follicles were helpful in discovering targets for drug development and studying mechanisms⁷. For example, genes such as LGR5 and LGR6 have been implicated in hair follicle stem cell maintenance and activation, whereas their role in nail physiology is less clear. Similarly, specific keratin genes exhibit distinct expression patterns in hair and nails, contributing to their differing structural properties⁸.

Understanding these genetic nuances provides critical insights into the molecular basis of skin appendage development and offers potential therapeutic targets for conditions affecting hair and nails. Recently, advances in hair restoration research have led to the development of techniques focused on the collection, culture, and proliferation of follicular dermal papilla (DP) cells. These DP cells play a critical role in hair follicle formation and growth. Cultured DP cells can be used to create hair germ, an early differentiated form of hair follicles, which can then be transplanted with the aim of promoting hair growth. This approach represents a promising avenue in treating hair loss and baldness^9-11.

However, challenges remain in this process. Notably, it has been observed that expression levels of activation genes in dermal papilla cells are significantly decreased when these cells are grown in cell culture conditions¹². Furthermore, as these cells undergo successive passages in culture, there is a progressive decline in the expression of these critical activation genes^13,14. This reduction in gene expression correlates with a decreased efficacy of establishing hair follicles after transplantation. In other words, the longer the DP cells are cultured and the more they are passaged, the less effective they become in generating functional hair follicles post-transplantation¹⁵.

This phenomenon highlights a key hurdle in hair follicle bioengineering: maintaining intrinsic hair-inducing properties of dermal papilla cells throughout an in vitro culture process. Overcoming this challenge is essential for improving the success rate of hair follicle transplantation and offers a significant area for further research and development in the field of regenerative medicine for hair loss.

Building on the foundation of previous genetic analysis in hair follicle and nail unit bioinformatics, our research team has embarked on a dual-faceted screening process using High-Throughput Screening (HTS)technology. This approach is designed to identify potential Wnt agonists and antagonists, focusing on compounds from a comprehensive library of 5,170 natural products and FDA-approved drugs. Our primary objective is to find molecules capable of activating the canonical Wnt/β-catenin signaling pathway, which is central to the development and growth of hair follicles and nails.

On the one hand, we have focused on identifying Wnt agonists that can simultaneously activate canonical Wnt signals and the R-Spondins/LGR pathway. This could potentially lead to more effective treatments for hair and nail growth by harnessing synergistic effects of these pathways. On the other hand, our research also aims to systematically discover Wnt antagonists that can modulate the Wnt/β-catenin signaling pathway, thus influencing gene expression relevant to hair and nail growth.

Further evaluations of the efficacy of these compounds were conducted through both in vitro and in vivo tests. In vitro tests allow us to analyze molecular and cellular responses to these compounds in a controlled environment. Subsequently, we plan to conduct animal experiments to observe direct effects of these Wnt modulators (both agonists and antagonists) on hair and nail development. These in vivo studies are crucial for understanding the physiological impact of Wnt modulation in complex biological systems. Through this comprehensive approach, integrating HTS, in vitro, and in vivo methodologies, we aim to unravel the complex genetic and molecular underpinnings of hair and nail biology, potentially leading to innovative treatments for various conditions related to these integumentary structures.

1. High-throughput screening for Wnt agonist discovery and assessment of cytotoxicity

Our HTS process focused on evaluating Wnt-stimulating effects and cytotoxicities of 5,170 FDA-approved drugs and natural products using the TCF/LEF promoter assay. Identified Wnt agonists that were effective in three or more repeated experiments were primarily classified into three categories: antibacterial agents (as shown in Figs. 2a, 2b, and 2c), anticancer agents (Figs. 2d, 2e, 2f, and 2g), and GSK3beta inhibitors (Figs. 2h and 2i).

Among the nine effective drugs, CP21R7 and BIO, both GSK3 beta inhibitors, are already known for their Wnt signal activation effects^16,17.

Entinostat, an HDAC inhibitor, has also been recently reported to activate the Wnt signal¹⁸. This HTS led to the discovery of six new drugs as potential Wnt agonists.

Most drugs demonstrated maximum luciferase expression at concentrations between 1 µM and 10 µM. Notably, Enzastaurin showed the most significant effect at a higher concentration of 100 µM (Fig. 2f). Regarding the maximum effect of each drug, five drugs - Aminoacridine, Entinostat, Tucidinostat, Enzastaurin, and CP21R7 - exhibited a more potent effect than 10 mM concentration of LiCl, a positive control. In particular, CP21R7 displayed an activity level ten times higher than that of 10 mM LiCl (Fig. 2j).

In terms of cytotoxicity, there was a noticeable variation depending on the drug class. Antibacterial drugs (Figs. 3a, 3b, and 3c) showed minimal toxicities, even at high concentrations of 100 µM or more. However, anticancer drugs (Figs. 3f and 3g) and GSK3beta inhibitors (Figs. 3h and 3i) exhibited strong toxicities at concentrations of 100 µM or higher. Among anticancer drugs, HDAC inhibitors (Figs. 3d and 3e) were observed to be relatively less toxic.

2. Effects of candidate drugs on expression levels of Wnt-related RNA in human dermal papilla cells

To evaluate the in vitro efficacy of the nine selected drugs, real-time RT-PCR was conducted to observe their effects on expression levels of Wnt pathway-related genes in hair follicle cells.

R-spondins genes: For RSPO3 (Fig. 4a) and RSPO4 (Fig. 4b) genes, proflavine and abemaciclib demonstrated overall excellent effects. Aminoacridine specifically increased the expression of RSPO3, while enzastaurin uniquely boosted RSPO4 expression.

Wnt Inhibitors - WIF1 and DKK1: Effects of Wnt inhibitors on WIF1 (Fig. 4c) and DKK1 (Fig. 4d) were notable. Proflavine increased WIF1 expression to 7.7- fold, the highest among all drugs, but Proflavine significantly decreased DKK1 expression to 1/0.27 (approximately 3.7 -fold), the lowest observed.

Wnt Promoters - TCF7 and LEF1 Genes: Proflavine, enzastaurin, and CP21R7 substantially increased the expression of TCF7 (Fig. 4e) and LEF1 (Fig. 4i) genes, key promoters of the Wnt signaling pathway, with a higher increase noted for LEF1. These drugs also significantly increased the expression of downstream genes of the TCF/LEF promoter, such as SERP2 (Fig. 4f), RUNX1 (Fig. 4g), and RUNX2 (Fig. 4h). Expression levels of genes considered to be hair germ activation genes, such as LEF1 (Fig. 4i), RGS2 (Fig. 4j), ALPL (Fig. 4k), and NOG (Fig. 4l), were maintained at relatively high levels after treatment with proflavine, enzastaurin, and CP21R7.

LGR Receptors: In the case of LGR receptors of R-spondins, LGR4 (Fig. 4m) showed a modest increase after treatment with Wnt agonists, whereas LGR6 (Fig. 4o) had a significant increase, showing a 4.7-fold increase after treatment with proflavine. LGR5 (Fig. 4p) was exceptionally increased by 150-fold after treatment with proflavine, indicating a strong enhancement in LGR5 expression among LGRs.

Bone morphogenetic proteins: Contrary to expectations, BMP genes, which usually show opposing effects to Wnt signaling, exhibited increases after treatment with Wnt agonist drugs. Notably, BMP2 (Fig. 4p) and BMP4 (Fig. 4q) were increased approximately 100 times or more after treatment with CP21R7 and over 30 times after treatment with proflavine. Additionally, proflavine increased expression levels of BMP5 (Fig. 4r) and BMP7 (Fig. 4s) by 5.4 times and 14.6 times, respectively.

These results demonstrate varied and complex effects of candidate drugs on the expression of genes involved in the Wnt signaling pathway and related processes in hair follicle cells, providing valuable insights into their potential as therapeutic agents for hair growth.

3. Effect of various drugs on hair and nail germ length

In our study, a comparative analysis was conducted to evaluate effects of various drugs on hair and nail germ length. Notably, treatment with Imidocarb showed the most significant impact, resulting in the highest average hair germ length change of approximately 334.7 ± 35.64 μm. This suggests a strong potential for Imidocarb in promoting hair germ growth. Other drugs, including Enzastaurin, MXD (Minoxidil), and Abemaciclib, also considerably increased hair germ length, with average changes recorded at approximately 308.89 ± 25.32 μm, 308.12 ± 33.14 μm and 300.54 ± 34.85μm, respectively, as shown in Fig. 5. Similarly, in the evaluation of nail germ length, Imidocarb treatment again resulted in the most significant growth, with an average increase of approximately 324.99 ± 45.20 μm. Enzastaurin, MXD, and Abemaciclib also notably increased nail germ lengths with average changes of approximately 306.36 ± 63.54 μm, 308.13 ± 45.60 μm, and 277.30 ± 15.85μm, respectively, as illustrated in Fig. 5d. These findings highlight the potential of these drugs, especially Imidocarb, in enhancing the growth of both hair and nail germs, warranting further investigation into their mechanisms of action and therapeutic implications.

4. Animal model and surgical procedures

4.1 Animal model for nail growth

In our study, we concentrated on assessing the impact of various drugs on nail growth by measuring changes in toenail length over a 14-day period. Results from our comparative analysis were quite revealing. MXD treatment emerged as the most effective one, leading to the highest average increase in toenail length of approximately 0.66 mm. This significant growth indicates MXD's potential as an effective promoter of toenail growth. In addition to MXD, other drugs such as Imidocarb and Proflavine also showed promising results. Imidocarb, in particular, demonstrated an impressive average increase in toenail length of about 0.74 mm, while Proflavine exhibited an increase of approximately 0.59 mm. These findings, detailed in Fig. 6e, highlight the potential of both Imidocarb and Proflavine as promising agents for enhancing toenail growth. This study thus sheds light on possible therapeutic applications of these drugs for conditions related to nail growth and health, offering valuable insights for further research and development in this area.

4.2 Anagen induction assay of Wnt agonists in C57BL/6 mice

In our anagen induction assay with C57BL/6N mice, we aimed to investigate effects of Aminoacridine drugs on mouse hair regrowth, using Minoxidil as a positive control. The dorsal skin of each 8-week-old C57BL/6N male mouse was shaved for this purpose. For treatments including Vehicle, Aminoacridine, Aminoacridine with Minoxidil, Imidocarb, and Proflavine, each drug was topically applied to mouse's dorsal skin daily for 28 days. Mice were then observed for morphological changes at weekly intervals for 4 weeks, as depicted in Fig. 7.

Observations revealed distinct patterns of hair regrowth across different treatment groups. After one week, Aminoacridine with Minoxidil group uniquely exhibited a darkening of the mouse back skin (Fig. 7d), indicating early signs of hair regrowth. By week 2, both Aminoacridine and Aminoacridine with Minoxidil groups showed early hair growth. Specifically, the Aminoacridine with Minoxidil group had a hair regrowth area score of 2.78 ± 0.53, translating to a 29.8% average regrowth area percentage. The Minoxidil group, serving as the positive control, also developed darkened skin by this time (Fig. 7b). By week 3, the Minoxidil group's hair regrowth area score was 2.71 ± 0.56 (26% average regrowth area percentage), whereas the Aminoacridine with Minoxidil group exhibited a significantly higher score of 3.14 ± 0.50, corresponding to a 49% average regrowth area percentage. The Aminoacridine group had a regrowth area score of 2.40 ± 0.40, which was about 22.5% average regrowth area percentage. By the fourth week, both the Minoxidil and Aminoacridine with Minoxidil groups showed significant hair regrowth, with scores of 3.34 ± 1.86 (54% average regrowth area percentage) and 4.14 ± 0.89 (78% average regrowth area percentage), respectively. Notably, by the end of the fourth week, all experimental groups, including those treated with Aminoacridine, Imidocarb, and Proflavine, demonstrated more pronounced hair growth effects than vehicle control groups.

4.3 In vivo fluorescence imaging analysis with C57BL/6 mice

In our study, we utilized in vivo fluorescence imaging analysis with the IVIS spectrum system to evaluate skin permeability of Aminoacridine in mice. Results are shown in Supplementary Figure 1 (Fig. S1). Results indicated that Aminoacridine was consistently absorbed into the skin, irrespective of alcohol concentration in the solution, ranging from 30% to 60%. Notably, around 70% of the drug was absorbed within the first 60 minutes post-application. This absorption rate increased over time, reaching over 90% after 360 minutes.

Our study delves into phenotypic and genotypic intricacies of hair follicles and nail units, shedding light on their similarities and differences which are pivotal for understanding human integumentary structures. The challenge in treating alopecia has long been the inability of drugs and hair transplantation techniques to regenerate hair follicles. This has made hair follicle neogenesis a pivotal strategy in alopecia treatment, as highlighted in previous studies^19,20. Despite the recognized role of Wnt signaling in hair follicle biology, few Wnt agonists have made the transition to clinical use for hair loss treatment^21,22. To address this gap, our research focused on identifying new drugs, with a particular emphasis on safe, side effect-free Wnt agonists suitable for broad human application, leveraging both natural products and FDA-approved drugs.

In this study, a high-throughput screening strategy involved treatment of HEK293 cells with TCF/LEF engineered luciferase using 5170 drugs. It measured the binding degree of introduced β-catenin to the promoter within the nucleus, indicating Wnt pathway signaling activation. This comprehensive screening revealed nine effective drugs: three antiviral, four anticancer, and two GSK-3 inhibitors^23-25. Among these nine drugs, CP21R7 and BIO, which are GSK3 beta inhibitors, have been reported to have Wnt signal activation effects. Additionally, Entinostat, an HDAC inhibitor, has been recently reported to activate the Wnt signal ^26,27.

As a result, 9 types of effective drugs were identified, including 3 antiviral drugs, 4 anticancer drugs, and 2 GSK-3 inhibitors ^28-30 . Notably, CP21R7 and BIO, GSK3 beta inhibitors, are known for their Wnt activation effects, and Entinostat, an HDAC inhibitor, has also been reported to activate the Wnt signal^15,31. Our HTS process uniquely identified six new drugs acting as Wnt agonists, marking the first extensive investigation in the world with a focus on identifying treatments for hair loss and nail issues that could stimulate the Wnt/β-catenin signaling pathway.

RT-PCR measurements were conducted to assess effects of these nine drugs on expression level of Wnt pathway-related genes in hair follicle cells. Aminoacridine, Abemaciclib, and Proflavine notably increased expression levels of RSPO3 and RSPO4 genes, with Aminoacridine notably enhancing RSPO3 levels by 19-fold, the highest among tested drugs. The importance of RSPOs in enhancing canonical Wnt/β-catenin signaling and their roles in stem cell biology have been well established^32,33. Moreover, the human genome harbors four genes that encode related RSPO proteins^34-36. Based on their physical interactions, RSPOs were ﬁrst described as Wnt signaling agonists³⁷. RSPOs collaborate with Wnt proteins to enhance the activity of the canonical Wnt/β-catenin signaling pathway. Notably, LGR5, identified as a stem cell marker, alongside two closely related proteins, LGR4 and LGR6, are specific receptors for RSPOs. Extensive research has significantly advanced our understanding of the functional roles of RSPO/LGR and Wnt signaling in stem cell biology^38,39.

We have previously shown that Wnt /β-catenin activation in the onychodermis contributes to the proliferation of the nail matrix epithelium and hair matrix via RSPO4-mediated signaling⁴⁰. Our findings also highlighted high expression of RSPO4 and LGR6 in the DP, suggesting their roles in the proliferation of mesenchymal and epithelial progenitors during hair follicle regeneration⁴¹.

Comparing Wnt-stimulating effects of these drugs, CP21R7 exhibited the highest luciferase activity, surpassing lithium chloride, the positive control, by ten-fold. On the other hand, proflavine displayed the lowest effect. It showed only 20% of the positive control's increase in Wnt stimulation. Interestingly, proflavine, potentially a lead compound, was not initially qualified based solely on signal activation from a promoter study. However, leveraging a gene bank derived from bioinformatics studies provided deeper insights into genes influencing target cells. Employing this approach, which involved secondary screening of hits identified in the promoter assay, propelled us closer to developing disease-specific drugs by scrutinizing target cell responses and gene expression.

Furthermore, the 3D spheroid structure formed by combining fibroblasts and keratinocytes initially presented as a mixed configuration akin to the arrangement of skin cells in first row of Figure 4a. Subsequently, these two cell types segregated into distinct regions while coexisting. Notably, only dermal papilla cells exhibited a germ-like structure, wherein spherical fibroblasts and keratinocytes elongated in a unidirectional manner. This structure serves as a widely used method to evaluate drug-induced hair growth effects by observing length changes⁴².

In this study, spheroids were created using identical skin keratinocytes and fibroblasts fromvarious origins (skin, hair, and nail). The spheroid shape was influenced by the fibroblast's source, as depicted in Figure 4a.

Interestingly, nail cells demonstrated the creation of a similar germ-like structure resembling hair cells. Minoxidil, a well-known hair growth drug, increased lengths of both nail and hair germs. Both nail and hair germs exhibited analogous responses to drug treatments. Among the nine drugs we tested, Imidocarb and Proflavine were notable for significantly increasing the expression of all four types of fresh DP markers and hair germ markers identified by Ohyama⁴³. However, only Imidocarb was effective in altering lengths of both hair and nail germs. This finding highlights Imidocarb's unique capability. Contrary to Proflavine, which solely upregulated gene markers, Imidocarb also manifested these genetic changes into tangible morphological alterations in hair and nail growth. This dual functionality of Imidocarb highlights its potential as a valuable agent in treatments focused on hair and nail regeneration, suggesting a broader impact than merely genetic upregulation.

In our animal study, we intentionally excluded drugs with known adverse effects, specifically four chemotherapeutic agents (Entinostat, Tucidinostat, Enzastaurin, Abemaciclib) known for their toxicities. Additionally, we excluded non-tonic formulations, specifically drugs that could not be dissolved in a tonic solvent composed of water and ethanol, such as CP21R7 and BIO. This approach allowed us to focus on the efficacy of new compounds. Aminoacridine, Imidocarb, and Proflavine were selected for their potential benefits in promoting hair and nail growth. These compounds were assessed through rigorous animal experiments, applying the same criteria for exclusion to ensure consistency across our study focused on both hair and nail growth. Notably, a combination of Aminoacridine and Minoxidil exhibited higher hair growth effects than Minoxidil alone, which served as a positive control.

A striking observation in the Aminoacridine with Minoxidil group was the blackening of the skin on the mice's backs within a week, a change associated with the onset of the anagen phase of hair growth. This effect was not observed in the Minoxidil-only group, suggesting a unique action of Aminoacridine. In vitro tests revealed that Aminoacridine significantly increased RSPO3 RNA levels, indicating its potential role in activating this gene. This finding aligned with Niida et al.’s research, which confirmed that RSPO3 could induce a proliferative effect through standard Wnt signaling, suggesting a possible mechanism for Aminoacridine's effects on hair growth. However, despite Aminoacridine showing the highest level of RSPO3 expression, there was a lack of consistency in results between animal experiments and hair germ experiments. This discrepancy, despite Aminoacridine's apparent effectiveness, indicates a complex interaction at play and necessitates further investigation to fully understand the drug's mechanisms and effects in different biological contexts.

In our assessment of effects of various drugs on toenail length, including a positive control, we observed that collected drugs generally stimulated fingernail and toenail growth. However, it was Imidocarb that stood out, showing statistically significant effects across the board. This finding is consistent with observed changes in nail germ length. Imidocarb's distinct efficacy in promoting nail growth at a statistically significant level highlights its potential as a targeted treatment for nail growth enhancement. The consistency of its effects in both toenail growth and changes in nail germ length underscores the reliability and potential applicability of Imidocarb in clinical settings for nail-related conditions.

Furthermore, our in vivo fluorescence permeability imaging tests showed that Aminoacridine had a good skin permeability, which was not adversely affected by increased alcohol concentrations. This characteristic is crucial for its effectiveness as a topical treatment.

In summary, our extensive screening of 5,170 drugs using HTS techniques led to the discovery of six new drugs, among which Aminoacridine, Abemaciclib, and the combination of Aminoacridine with Minoxidil showed promising results in animal models. These drugs demonstrate potential as effective therapeutic agents for hair regeneration by activating Wnt/β-catenin signaling pathways.

However, our study has some limitations. First, we only tested three drugs in animal models. Further research involving more drugs is necessary to determine optimal concentrations and assess their safety and effectiveness comprehensively. Additionally, large-scale clinical trials are essential to confirm the safety and efficacy of these potential Wnt/β-catenin activators. Such trials could pave the way for the development of effective treatments for hair loss and nail health issues.

1.Primary high-throughput screening

For primary high-throughput screening, HEK293 cells stably integrated with the firefly luciferase gene under the control of TCF/LEF response elements (Cat. #60501, BPS Bioscience, San Diego, CA, USA) were utilized. These cells served as a model to evaluate the activation of Wnt signaling. The screening targeted two major libraries: a FDA-approved drug library containing 2,570 compounds (Cat. #L1300, Selleckchem, Houston, TX, USA) and a natural product drug library comprising 2,600 compounds (Cat. #L1400, Selleckchem, Houston, TX, USA).

HEK293 cells were cultured in MEM medium (Cat. #SH30024.01, Hyclone) supplemented with 10% Fetal Bovine Serum (FBS) (Cat. #S1480, Biowest), 1% non-essential amino acids (Cat. #SH30238.01, Hyclone), 1 mM sodium pyruvate (Cat. #SH30239.01, Hyclone), and 1% Penicillin/Streptomycin (Cat. #SV30010.01, Hyclone). These cells were maintained in a humidified atmosphere containing 5% CO₂at 37°C.

For the screening, 3x10³ cells per well were seeded in a 384-well plate. After an overnight incubation to allow cell attachment, each compound from the libraries was diluted with the medium to achieve a final concentration of 5 µM in the well, using Dimethyl sulfoxide (DMSO) as the solvent. Compounds were treated in duplicate for 24 hours per drug. Lithium chloride (LiCl) at 10 mM concentration was employed as a positive control.

To assess both cell viability and luciferase activity post-drug treatment, the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Cat. # E7120, Promega) was used following the manufacturer's instructions. A Mithras multi-reader (Cat. #LB940, Berthold Technologies, Bad Wildbad, Germany) was utilized for measurements. Drugs were selected based on the criterion that the average of two luciferase measurement values per drug was more than five times higher than that of the vehicle group treated with 0.1% DMSO. From this screening, seven drugs that showed activity consistently on three or more separate occasions were chosen for further analysis.

2. Secondary biological in vitro assay

2.1 RT-PCR

Human hair follicle dermal papilla cells (Promocell, Heidelberg, Germany, used at passages 2-5) were seeded at 3x10⁴ cells/well in 60 mm dishes with DMEM high glucose serum medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Cells were treated with seven candidate drugs at 5 to 10 uM, which is a high concentration of b-catenin in the nucleus, and cultured for 24 hours.

Total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA). Template cDNAs were obtained by reverse transcription of total RNAs using an oligo (dT) primer and a PrimeScript™ RT reagent Kit(TAKARA, Japan). Amplification was carried out using SYBR® Green Realtime PCR Master Mix (Toyobo, Japan). The sequences of real-time reverse transcription-polymerase chain reaction (RT-PCR) primers used are in Supplementary Table 1.

They were designed following a previous study. Expression levels of target genes were analyzed by the number of copies per copy of b-actin.

2.2 Hair and Nail Germ Formation and Elongation Assay

For the formation and elongation assay of hair and nail germs, primary skin fibroblasts, skin keratinocytes, dermal papillae, and nail fibroblasts were obtained from patients under ethical considerations. Cells from only 1 to 5 passages were utilized in all experiments. This part of the study received approval from the Institutional Review Board (IRB) of Samsung Medical Center, ensuring compliance with ethical research standards (IRB number:2021-12-134) Informed consent was obtained from all participating patients, adhering to ethical research guidelines.

In the experimental setup, spheroids were created using a precise count of 2,000 fibroblasts and 2,000 keratinocytes for each spheroid. These cells were mixed with 60 µl of DMEM supplemented with 10% FBS. The cell mixture was then placed in U-Shaped-Bottom Microplates (Thermo Fisher, Waltham, MA, USA) designed for spheroid formation. Drug compounds for the assay were sourced from Selleckchem (Houston, TX, USA), with each being used at a final concentration of 5 µM.

Spheroids were initially cultured and maintained for a period of 2 days to establish their form. Following this incubation period, drug compounds were administered to these spheroids, which were then further cultured for an additional 5 to 7 days. This duration was chosen to allow sufficient time for drugs to exert their effects on spheroids.

To quantify the elongation of each spheroid, images were obtained using an inverted microscope (CKX53, Olympus, Shinjuku, Tokyo, Japan). Obtained images were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For statistical robustness, more than 10 spheroids per experimental group were measured. To ensure the reliability and reproducibility of results, the entire experiment was repeated twice. This methodical approach allowed for a comprehensive assessment of the impact of drug compounds on hair and nail germ formation and elongation.

3. Animal model and surgical procedures

3.1 Anagen induction assay with C57BL/6 mice

For the anagen induction assay, forty-eight male C57BL/6 mice aged 7 weeks and weighing between 20–22g were procured from Orient BIO (Seoul, Korea). Prior to the commencement of experiments, these mice underwent an acclimatization period of one week under laboratory conditions. The ethical aspect of this study was rigorously adhered to, with all mouse experiments receiving approval from the Samsung Medical Center Institutional Animal Care and Use Committee (Approval code: 20210617001). Procedures were conducted in accordance with the AAALAC International Guidelines and the United States Animal Welfare Act to ensure humane and ethical treatment of animals.

Mice were housed under controlled conditions, including a 12-hour light/dark cycle, regulated temperature, and a humidity range of 35–60%. They were provided with standard mouse food and water. After the one-week adaptation period, 8-week-old mice were ready for experimental procedures.

For the assay, all mice were anesthetized using isoflurane. Their dorsal areas, measuring approximately 2 cm in width and 4 cm in length, were shaved using hair clippers to prepare the skin for treatment application. These mice were then randomly divided into six groups (eight mice per group): a Vehicle group, a Minoxidil group, an Aminoacridine group, an Aminoacridine with Minoxidil group, an Imidocarb group, and a Proflavine group. The Vehicle group received a Phosphate Buffered saline (PBS) solution without any active treatment.

For drug preparation, Aminoacridine was dissolved in a mixture of propylene glycol (10%, v/v) and ethanol (60%, v/v) to achieve a concentration of 1% (w/v). Minoxidil was similarly dissolved in a mixture of propylene glycol and ethanol to a concentration of 0.3% (w/v). Each group was treated with 200 µL of the respective solution, which was applied topically to shaved areas of mice once daily for 28 days^15,23.

At the conclusion of the 28-day treatment period, the efficacy of each treatment was assessed and mice were humanely euthanized using CO2 gas. This protocol allowed for a comprehensive evaluation of the potential of each compound to induce anagen phase in hair follicles, providing valuable insights into their effectiveness for hair growth promotion.

3.2 Nail growth assay with C57BL/6 mice

In the nail growth assay conducted with C57BL/6 mice, careful preparation and measurement techniques were employed to assess the efficacy of various drug treatments on toenail growth. A day before drug application and CT scan, toenails were cut to prevent bone damage and digit tip regeneration, as shown in Fig. 5a.

After CT scanning cut nails, mice were immersed in a solution containing 0.3% (w/v) of each drug in 30% ethanol for 10 seconds, 5 times a week for 14 days. CT scans were performed both at the beginning and after the 14-day treatment period to monitor changes in toenail length.

For precise measurement in CT image analysis, the tip of the toenail was marked by adjusting the Hounsfield Unit (HU) value to a Window Level (WL) of 287 and a Window Width (WW) of 332. The first toe joint was marked by adjusting the WL to 693 and WW to 436. This precise calibration allowed for accurate and consistent measurements across all samples. This analysis was conducted on middle three toes of both left and right feet of mice, with four mice in each group being tested. This methodical approach allowed for accurate assessment of drug effects on toenail growth over a two-week period.

3.2 Image Analysis

To assess the efficacy of treatments in promoting hair growth, systematic image analysis was conducted for hair loss sites of mice. Photographs of these sites were captured at specific intervals: 1, 2, 3, and 4 weeks following the commencement of each treatment. These time points were chosen to provide a comprehensive view of the hair regrowth process over the course of treatment.

For the analysis of hair regrowth, a scoring system was implemented. This scoring was based on the percentage of hair regrowth observed in the treated area, allowing for a quantifiable and standardized assessment of the treatment's effectiveness. Scoring criteria were as follows: Score 0, No hair regrowth observed; Score 1, 0-20% hair regrowth; Score 2, 20–40% hair regrowth; Score 3, 40–60% hair regrowth; Score 4, 60–80% hair regrowth; and Score 5, 80–100% hair regrowth.

3.3 In vivo fluorescence imaging Analysis

For in vivo fluorescence imaging analysis, we utilized an IVIS spectrum system (Caliper Life Sciences), a sophisticated tool for capturing fluorescence images of live subjects. This method was applied to evaluate the distribution and penetration of the medication applied topically to mice. In each case, a dose of 100 μl of the medication was applied to the skin on the back of a mouse. To monitor dynamics of the medication's absorption and distribution over time, fluorescence transmittance was assessed at multiple time points: 5, 60, 120, and 360 minutes post-application. These intervals were strategically chosen to provide a comprehensive view of the medication's behavior within the first few hours after application.

To ensure consistency and accuracy in the imaging process, all images were acquired under identical field conditions. This uniformity in imaging conditions is crucial for reliable comparison and analysis of the data. Additionally, visibility of the region of interest (ROI) in the fluorescence image was automatically normalized using the Living Image software. This software is integral to the IVIS system, allowing for precise quantification and analysis of fluorescence signals.

Author Contributions

Gulimila Abudureyimu: Data analysis and writing of the manuscript

Hyung-Suk Jang: Data analysis and writing of the manuscript

Joonho Shim: Idea formation

MinHee Kim: In vitro, In vitro testing

Minjeong Kang: In vitro, In vitro testing

Acknowledgements

This work was supported by the Ministry of Health and Welfare Republic of Korea. (HI21C0694, Development of treatment for intractable skin diseases using low-molecular compounds with enhanced Wnt/beta-catenin).

Financial Disclosure Statement: The authors have the following to disclose support provided by the Ministry of Health and Welfare (HI21C0694, Development of treatment for intractable skin diseases using low-molecular compounds with enhanced Wnt/beta-catenin), Republic of Korea.

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High-Throughput Screening for Discovering Small Molecules Capable of Increasing the Wnt/β-Catenin Signaling Pathway to Promote Hair and Nail Growth

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