KL001 (CAS: 309928-48-1) and KL044 (CAS: 1801856-93-8) were purchased from MedChemExpress (New Jersey, United States of America). Cell lysis buffer (P0013), BCA protein assay kit (P0012) and cAMP assay kit were purchased from Beyotime Biotechnology (Shanghai, China). α-MSH (M118985), ACTH (A118750), Forskolin (F127328) and IBMX (I106812) were obtained from Aladdin (Shanghai, China). Anti-Myosin Va antibody (sc-365986) were purchased from Santa Cruz Biotechnology (Santa Cruz, United States of America). Anti-CRY1 (ab54649), CRY2 (ab93802), tyrosinase (ab180753), TRP-1 (ab235447), TRP-2 (ab74073), Cdc42 (ab187643), Rab27a (ab55667), KIF 5b (ab167429), MITF (ab20663), PKA cat (ab216572), p-PKA cat (ab75991), CREB (ab32515), p-CREB (ab32096), and β-actin (ab8226) antibody were purchased from Abcam (Cambridge, UK). Masson-Fontana melanin staining solution was obtained from SenBeiJia Biological Technology (Nanjing, China).

B16F10 murine melanoma cells from National Collection of Authenticated Cell Culture (Shanghai, China) were maintained in DMEM medium (L110KJ, Basalmedia) containing 10% fetal bovine serum (FBS-S500, NEWZERUM) and penicillin-streptomycin solution (C0222, Beyotime) under 5% CO₂ at 37°C. B16 cells were transfected with siRNA against mouse CRY1 (sc-44835) and CRY2 (sc-44836) from Santa Cruz Biotechnology as the manufacturer’s instructions.

Normal human epidermal melanocytes (NHEMs) from Sciencell Research Laboratories (CA, United States of America) were maintained in human epidermal melanocyte complete medium (CM-H108, Procell).

Cell viability was determined using the MTT assay. Briefly, cells seeded in 96-well plates were treated with KL001 for 48 h. After washing with PBS 3 times, cells were treated with MTT solution (20 μL) for another 4 h. The absorbance was determined at 570 nm.

Intracellular melanin content was utilized as an important indicator of melanin synthesis (Lee et al., 2015; Lv et al., 2019). Briefly, cells seeded in 6-well plates were treated with different concentrations of KL001 in presence or absence of cAMP stimulators such as α-MSH, FSK, ACTH and IBMX. After 48 h, cell pellet was collected and dissolved in 100 μL of 1 mol/L NaOH working solution for 1 h at 80 °C. Melanin content was assessed by a microplate reader at 405 nm.

Samples were fixed and stained as previously reported (Gu et al., 2018). Briefly, slides were incubated in ammoniacal silver solution for 14 h at room temperature. After washing with double distilled water 3 times, slides were incubated in hypo solution for 6 min, followed by counterstain with neutral red stain for another 6 min. Then cell morphology and pigmentation were observed under a Nikon Ti2-U microscope.

B16F10 cells were fixed with glutaraldehyde fixing solution (4%) overnight, and post-fixed in 2% osmium tetraoxide in 0.1 M cacodylate for another 1 h at room temperature. Then, B16F10 cells were dehydrated using a graded series of ethanol and embedded in epoxy resin. The regions containing B16F10 cells were cut into ultrathin sections, stained with uranyl acetate and lead citrate, and visualized under TEM (Hitachi, H-7800).

B16F10 cells were grown on glass coverslips, fixed in glutaraldehyde fixing solution (4%) for 2 h and post-fixed in osmium tetroxide (1%), dehydrated with ethanol and finally dehydrated overnight with hexamethyldisilazane. Coverslips were gold-coated and visualized under SEM (Hitachi, Regulus-8100).

Cellular tyrosinase activity was measured following a previously described method (Lv et al., 2020b; Lv et al., 2021). Briefly, B16F10 cells were treated with different concentrations of KL001 (0, 5, 10, 20 μM) for 48 h. Cells were washed with cold PBS and lysed in lysis buffer. Then lysates were centrifuged to acquire the supernatant to determine tyrosinase activity and quantify protein levels. 100 μL PBS (0.1 M, pH 6.5) contenting 10 μg proteins mixed with 100 μL 0.1% L-DOPA. Optical absorbance was monitored at 475 nm using a microplate reader after incubation at 37°C for 1 h.

Cell-free tyrosinase activity was performed as the previous description (Lv et al., 2020b; Lv et al., 2021). Briefly, 100 μL of PBS (0.1 M, pH 6.5) containing different concentrations of KL001 (0, 5, 10, 20 μM) was mixed with mushroom tyrosinase (10 unit) and 50 μL of 0.03% L-tyrosine. Optical absorbance was monitored at 475 nm using a microplate reader after incubation at 37°C for 10 min.

B16F10 cells were pretreated with KL001 (or KL044) for 2 h and stimulated with FSK for 10 min in the presence of KL001 (or KL044). The cells were lysed with Triton X-100 (1%) at 4 °C for 1 h, and the lysates were clarified by centrifugation at 12,000 rpm for 20 min at 4 °C. The supernatant was used to assess the level of cAMP using cAMP ELISA Kit (E-EL-0056c, Elabscience Biotechnology), following the manufacturer’s instructions.

Western blot activity was performed following a previously described method (Lv et al., 2021). Briefly, protein samples were separated using SDS polyacrylamide gel electrophoresis, and then transferred using an electrophoretic transfer technique onto nitrocellulose filter membranes. After blocking with 3% BSA in TBST solution for 1.5 h at room temperature, cut horizontally to incubate with suitable primary antibodies for 12 h at 4°C. After washing with TBST 4 times, the membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature, and visualized using enhanced chemiluminescence. The membranes were subsequently stripped and reprobed with specific antibodies. Data reported here represent at least three independent replicates.

The animal experiment scheme in this work was approved by the animal care and use committee of Changzhou University (CZDX-2021009). Eight brown guinea pigs (∼300 g, 6 weeks) were obtained from the Institute of Laboratory Animal Science (Beijing, China). The animals were housed individually in rooms with constant temperature, humidity and a 12-h light and dark cycle. After adaptation, UVB-induced hyperpigmentation model was performed on the back of brown guinea pigs as previous description (Lee et al., 2013; Lv et al., 2020c; Lv et al., 2021). The control group was applied with vehicle (PEG400/EtOH = 7:3) and the treatment group was applied with KL001 (1%) on the hyperpigmented areas (20 μL per circle) twice a day for 4 weeks. The L-value was detected by spectrophotometer (YS3010, 3nh, Shenzhen, China) to calculate the ΔL-value, which was utilized to evaluate the degree of pigmentation. The calculation formula is shown below: ΔL = L (daily measured)-L (Day 0) (Lee et al., 2013; Lv et al., 2020b).

Values were expressed as mean ± SEM, and statistical comparisons were analyzed by GraphPad Prism using one-way ANOVA, followed by Turkey’s post hoc test for multiple comparison tests. When p < 0.05, the difference was statistically significant.

KL001 is identified as a small molecule activator of CRY (Hirota et al., 2012). To investigate whether CRY1 protein is activated after KL001 treatment in B16F10 cells, CRY1 protein level was determined by western blotting. As shown in Figure 1A, 20 μM KL001 markedly increase CRY1 protein expression in B16F10 cells. In addition, KL001 exerted no cytotoxicity up to 20 μM for 48 h (Supplementary Figure S1). These results revealed that CRY1 was regulated by KL001 treatment in B16F10 cells.

Next, the effect of KL001 on melanogenesis was determined by a melanin content assay and Masson-Fontana ammoniacal silver staining. As shown in Figure 1B, KL001 suppressed the basal melanogenesis and reversed α-MSH (100 nM)-induced melanin increase. Consistently, Fontana-Masson staining revealed that KL001 significantly reduced melanin concentration in dendrites and perinuclear region, as well as the increased dendrite number and length in presence or absence of α-MSH (Figure 1C).

Finally, the crucial role of CRY1 in KL001-induced anti-melanogenesis was demonstrated by effectively transfecting siRNA. CRY1 siRNA (or CRY2 siRNA) significantly reduced CRY1 (or CRY2) mRNA levels over 80% in the B16F10 cells (Figure S2). Silencing CRY2 did not reverse the effects of KL001, whereas KL001 treatment failed to inhibit melanogenesis in the absence of CRY1 (Figures 1D, E), reflecting that CRY1 activation by KL001 treatment suppressed melanogenesis in B16F10 cells.

Melanogenesis is closely related to the maturation of melanosomes (Raposo and Marks, 2007). To explore the effect of KL001 on melanosome maturation, melanosome stage was observed under TEM. As shown in Figure 2, stage III-IV melanosomes were significantly reduced in B16F10 cells after KL001 treatment, suggesting that KL001 inhibited the maturation of melanosomes.

Tyrosinase activity is necessary in melanogenesis (Lv et al., 2020a; Lv et al., 2021). L-DOPA oxidation and mushroom tyrosinase activity assay were used to determine the effect of KL001 on cellular and cell-free tyrosinase activity, respectively. Consistent with the anti-melanogenic effect, 20 μM KL001 significantly inhibited cellular activity of tyrosinase (Figure 3A). However, KL001 showed no effect on the enzymatic activities of mushroom tyrosinase (Figure 3B).

To determine whether the efficacy of KL001 was relative to tyrosinase, TRP-1 and TRP-2 protein, three key melanogenic enzymes, the protein expression levels were examined (Lv et al., 2019). As shown in Figure 3C, with α-MSH stimulation, KL001 significantly reduced the expression of tyrosinase, TRP-1 and TRP-2, indicating that the whitening effects of KL001 is mediated by the suppression of cellular tyrosinase activity and associated key melanogenic protein expression.

MITF is an essential transcription factor that induces gene expression of tyrosinase, TRP-1 and TRP-2 (Lv et al., 2020b), therefore the MITF dynamics following KL001 treatment were monitored. As shown in Figure 3D, following α-MSH stimulation, expression of MITF protein peaked at 2 h, and KL001 treatment significantly decreased it, which indicated that KL001 decreased α-MSH-induced tyrosinase, TRP-1 and TRP-2 expression through inhibiting MITF expression.

In melanocytes, melanin is produced in the cell body, transported to the dendrites, and finally to neighboring keratinocytes to complete distribution. As shown by Fontana-Masson staining, KL001 significantly decreased dendrite formation and the amount of melanin pigments in dendrites (Figure 1C). Furthermore, SEM results revealed that KL001 remarkedly inhibited melanocyte filopodia formation (Figure 4A).

To further elucidate the underlying mechanism, several key factors involved in melanosome distribution were determined. Firstly, Kinesin superfamily protein 5b (KIF5b) is contributed to the outward transport of melanosome along microtubules (Noguchi et al., 2014). While melanosomes transfer from microtubules to actin filaments, melanosomes are transported by the tripartite complex consisting of Rab27a-Mlph-myosin Va and then anchored to the plasma membrane by the Rab27a- Slp2-a complex (Ohbayashi and Fukuda, 2012; Ishida et al., 2014; Fukuda, 2021). In addition, Cdc42 contributes to dendrite extension and filopodia formation (Lv et al., 2020b). As shown in Figure 4B, Myosin Va, Rab27a and Cdc42 protein expression were significantly decreased after KL001 treatment, but not KIF5b, indicating that KL001 inhibited melanosome distribution by reducing Myosin Va, Rab27a and Cdc42 expression.

Recently, several studies have reported that the biological effects of CRY1 depend on the negative regulation of cAMP/PKA signaling pathway (Zhang et al., 2010; Narasimamurthy et al., 2012; Qin and Deng, 2015; Yamanaka et al., 2018; Huang et al., 2020). Therefore, it is worth investigating whether KL001 affects melanogenesis induced by other cAMP activators. Briefly, adrenocorticotropic hormone (ACTH) is an endogenous agonist of MC1R (Pillaiyar et al., 2017b), forskolin (FSK) is a direct stimulator of adenyl cyclase to produce cAMP, and 3-isobutyl-l-methylxanthine (IBMX) is a cAMP phosphodiesterase inhibitor that prevent the degradation of cAMP (Shin et al., 2015). As expected, KL001 treatment also inhibited ACTH (100 nM)-, FSK (10 μM)- and IBMX (100 μM)-induced melanogenesis in B16F10 cells (Figure S3).

Further, whether the cAMP/PKA pathway is activated after KL001 treatment in melanocytes is another key question that needs to be investigated. As shown in Figure 5A and Supplementary Figure S4, KL001 induced a decrease in the phosphorylation of CREB and PKA in presence of α-MSH. These results indicated that KL001 treatment can inhibit CREB-dependent MITF induction, which can influence melanogenic enzymes expression. In addition, the increased cellular cAMP level induced by FSK was also reduced after KL001 treatment (Figure 5B). Based on the above results, it can be speculated that KL001 treatment regulated the intracellular cAMP levels to regulate the subsequent downstream signaling pathway.

In addition, we investigated whether the whitening effects were shared by the other CRY activator. KL044 is a KL001 analogue with higher potency (Lee et al., 2015). In melanin content assay, KL044 also strongly suppressed melanin synthesis induced by α-MSH in a dose-dependent manner (Figure 5C). Expression level of tyrosinase and MITF was markedly decreased after KL044 treatment (Figure 5D). Mechanistically, KL044 strongly inhibited cellular cAMP production and subsequent activation of PKA/CREB signaling pathway (Figures 5E, F). These results confirmed that the activation of CRY inhibited melanogenesis through negative regulation of cAMP/PKA/CREB pathway.

The inhibitory efficacy of KL001 on melanogenesis was investigated in primary normal human epidermal melanocytes (NHEM). As shown in Figure 6A, KL001 treatment increased the expression of CRY1 protein in NHEM. Consistent with data from B16F10 cells, KL001 treatment also reduced α-MSH-induced melanin in dendrites and perinuclear region (Figures 6B, C). Western blotting results indicated that KL001 decreased the expression of tyrosinase, Cdc42, Myosin Va and Rab27a in NHEM (Figure 6D). Mechanistically, KL001 strongly inhibited the PKA/CREB signaling pathway (Figure 6E), confirming that the anti-melanogenic efficacy of KL001 in NHEM was mediated by PKA/CREB signaling pathway.

The anti-melanogenic efficacy of KL001 was also investigated in vivo in UVB-induced hyperpigmentation model in brown guinea pigs. Firstly, as shown by representative photographs, skin pigmentation was significantly suppressed after KL001 (1%) treatment (Figure 7A). To further illustrate the pigmentation degree, L value is calculated by Spectrophotometer and utilized as an index of brightness. As shown in Figure 7B, the ΔL value in KL001 group was significantly higher after 4 weeks, reflecting that KL001 reversed UVB-induced hyperpigmentation. Consistently, hyperpigmentation in the epidermal basal layer was visibly reduced after treatment with KL001 as shown by Fontana-Masson staining (Figure 7C). In addition, immunohistochemical staining of melanocyte marker protein S100 showed that the amounts of melanocyte were not affected by KL001 treatment (Figures 7D, E). Taking together, these observations indicated that KL001 has whitening efficacy on UVB-induced hyperpigmentation in vivo.