Melanosomal pH Research Articles

Modulating OCA2 Expression as a Promising Approach to Enhance Skin Brightness and Reduce Dark Spots

The oculocutaneous albinism II (OCA2) gene encodes a melanosomal transmembrane protein involved in melanogenesis. Recent genome-wide association studies have identified several single nucleotide polymorphisms within OCA2 genes that are involved in skin pigmentation. Nevertheless, there have been no attempts to modulate this gene to improve skin discoloration. Accordingly, our aim was to identify compounds that can reduce OCA2 expression and to develop a formula that can improve skin brightness and reduce hyperpigmented spots. In this study, we investigated the effects of OCA2 expression reduction on melanin levels, melanosome pH, and autophagy induction through siRNA knockdown. Additionally, we identified several bioactives that effectively reduce OCA2 expression. Ultimately, in a clinical trial, we demonstrated that topical application of those compounds significantly improved skin tone and dark spots compared to vitamin C, a typical brightening agent. These findings demonstrate that OCA2 is a promising target for the development of efficacious cosmetics and therapeutics designed to treat hyperpigmentation.

Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Identifying genetic defects in oculocutaneous albinism patients of West Bengal, Eastern India.

Oculocutaneous albinism (OCA) is a congenital heterogeneous group of autosomal recessive disorders characterized by the absence or loss of melanin in the skin, eyes and hair of the affected individuals. Based on the mutated gene, OCA has been classified into eight sub-types (OCA1-8) with overlapping clinical phenotypes. Mutations in the TYR gene cause OCA1, the most prevalent OCA worldwide including India. Mutations in OCA2 and SLC45A2, both of which regulate melanosomal pH that is critical to TYR activity, cause OCA2 and OCA4 respectively, the other common OCA subtypes in India. In the present study, we have included 54 OCA-affected cases from 41 unrelated families representing 16 different marriage/ethnic groups from 17 districts of West Bengal, India. We pursued a PCR-sequencing based approach followed by bioinformatic analysis to identify mutations in TYR, OCA2 and SLC45A2 genes. Mutations were detected in 27 of the 54 (50%) OCA patients from 18 unrelated families, representing 9 different marriage/ethnic groups from 11 districts of West Bengal. Three TYR variants: NM_000372.4: c.391 A > G, NP_000363.1: p. Lys131Glu; NM_000372.4: c.1037G > T; NP_000363.1: p. Gly346Val, NM_000372.4: c.715 C > T; NP_000363.1:p.Arg239Trp was identified for the first time in Eastern Indian OCA cases. A novel nonsense variant: NM_016180.5: c.389T > A, NP_057264.4: p. Leu130* and a novel synonymous variation NM_016180.5: c.1092 A > G; NP_057264.4: p.364E = were identified in SLC45A2. Additionally, NM_016180.5: c.904A > T; NP_057264.4: p. Thre302Ser was identified for the first time in any Eastern Indian OCA case. We identified 2 previously reported mutations in OCA2. In concordance with previous reports, NM_000372.4: c.832C > T, NP_000363.1: p. (Arg278*) was the commonest TYR mutation. The results of our study enrich the mutational spectrum of the known OCA causing genes in Eastern India, which would facilitate accurate diagnosis, familial screening, carrier detection and containment of the disease load.

Distinct cAMP Signaling Microdomains Differentially Regulate Melanosomal pH and Pigmentation

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways-the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway-affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype affects sAC-dependent regulation of melanin synthesis. Although sAC loss of function in wild-type human melanocytes stimulates melanin synthesis, sAC loss of function has no effect on melanin synthesis in MC1R nonfunctional human and mouse melanocytes or skin and hair melanin in e/e mice. Interestingly, activation of transmembrane adenylyl cyclases, which increases epidermal eumelanin synthesis in e/e mice, leads to enhanced production of eumelanin in sAC-knockout mice relative to that in sAC wild-type mice. Thus, MC1R- and sAC-dependent cAMP signaling pathways define distinct mechanisms that regulate melanosomal pH and pigmentation.

PH-dependent aggregation of the Pmel17 repeat domain characterized by NMR.

In studying amyloid-forming proteins, it is imperative to determine the monomer conformational dynamics that precede fibrillation. This study utilizes NMR and the paramagnetic relaxation enhancement (PRE) effect to observe monomer properties of the repeat domain (RPT) from a human functional amyloid, premelanosomal protein (Pmel17). RPT is generated through Pmel17 post-translational processing during melanosome maturation. The melanosome is an acidic organelle within specialized cells where melanin biogenesis occurs. At low melanosomal pH, RPT self-assembles into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report dynamics of the short (sRPT) isoform, which has been demonstrated to be a fibrillation nucleator. NMR experiments were performed to determine conformational differences in sRPT by comparing aggregation-prone vs. non-aggregating solution conditions at pH ranges from 4 to 6, respectively. We observed significant chemical shift perturbations localized to residues near the important singular tryptophan residue, demonstrating that the local chemical environment of the amyloid core region is sensitive to changes in pH even in the monomer form. Next, we introduced several cysteine point mutations in order to covalently attach PRE ligands to sRPT for observation of intramolecular interactions. Long-range PRE effects indicate potential contacts to residues on opposite ends of sRPT. These PRE effects might be an indication of initial molecular events to facilitate intermolecular interactions, which can go on to trigger fibrillation. These results also hint at a transient conformation that inhibits fibrillation. Thus, by raising or lowering solution pH, the relative population of this state would be modulated. Taken together, these results show that sRPT monomers adopt a conformation inconsistent with fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight the tight regulatory mechanisms that can affect fibrillation activity of proteins like RPT.

The potential impact of melanosomal pH and metabolism on melanoma.

Melanin is synthesized in melanocytes and is transferred into keratinocytes to block the effects of ultraviolet (UV) radiation and is important for preventing skin cancers including melanoma. However, it is known that after melanomagenesis and melanoma invasion or metastases, melanin synthesis still occurs. Since melanoma cells are no longer involved in the sun tanning process, it is unclear why melanocytes would maintain melanin synthesis after melanomagenesis has occurred. Aside from blocking UV-induced DNA mutation, melanin may provide other metabolic functions that could benefit melanoma. In addition, studies have suggested that there may be a selective advantage to melanin synthesis in melanoma; however, mechanisms regulating melanin synthesis outside the epidermis or hair follicle is unknown. We will discuss how melanosomal pH controls melanin synthesis in melanocytes and how melanosomal pH control of melanin synthesis might function in melanoma. We will also discuss potential reasons why melanin synthesis might be beneficial for melanoma cellular metabolism and provide a rationale for why melanin synthesis is not limited to benign melanocytes.

TRPA1 promotes UVB-induced skin pigmentation by regulating melanosome luminal pH.

Melanocytes stimulated by ultraviolet radiation (UVR) produce melanin and melanosomes, which causes skin pigmentation and acts as an important physiological defence process for photoprotection. Neutral luminal pH of melanosomes is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin synthesizing enzyme tyrosinase (TYR). As a major component of extraocular phototransduction pathway, transient receptor potential ankyrin1 (TRPA1) can be activated by ultraviolet B (UVB) and reported to be expressed in melanocytes. However, whether TRPA1 is involved in the regulation of melanogenesis remains unclear. Melanogenic activity of TRPA1 was evaluated in primary normal human epidermal melanocytes (HEMs) and murine B16-F10 cell cultures, and the effects of topical applications of TRPA1 specific agonist and antagonist on UVB-induced skin pigmentation were confirmed on in vivo guinea pig models. Calcium (Ca2+ ) imaging and pH imaging were performed to analyse the effects of TRPA1 on intracellular Ca2+ concentration ([Ca2+ ]ic ) and melanosome luminal pH. TRPA1 regulated melanin synthesis, UVB-induced Ca2+ influx and melanosome luminal pH in HEMs and B16-F10 cells. Topical treatment of TRPA1 specific agonist JT010 increased UVB-induced skin pigmentation in guinea pigs, while topical using of TRPA1 selective antagonist HC-030031 mitigated such pigmentation. Our results indicated that TRPA1 activated by UVB enhanced the skin pigmentation, most likely by regulating the [Ca2+ ]ic and the melanosomal pH, consequently influencing the enzymatic activity of TYR. Therefore, the results suggest TRPA1 as a potential therapeutic target in the treatment of skin pigmented disorders that are at high risk under UVB irradiation.

656 Effects of UV on melanosome pH and metabolism

Ultraviolet (UV) radiation is a cause of skin cancer and aging and has been shown to affect a variety of biological phenomena. Whereas UV radiation can induce local hormones that affect melanin synthesis, the mechanisms by which UV radiation directly affects melanocyte biology and melanin synthesis, independent of DNA mutation, remain poorly understood. In this study, we examined the effects of UV radiation on melanosomal pH and metabolism in melanocytes and melanoma cells in vitro. We demonstrate that UV radiation elevates melanosomal pH in a soluble adenylyl cyclase (sAC) and reactive oxygen species (ROS) dependent manner.

Ablation of Proton/Glucose Exporter SLC45A2 Enhances Melanosomal Glycolysis to Inhibit Melanin Biosynthesis and Promote Melanoma Metastasis

Sequence variation in SLC45A2 are responsible for oculocutaneous albinism type 4 in many species and are associated with melanoma susceptibility, but the molecular mechanism is unclear. In this study, we used Slc45a2-deficient melanocyte and mouse models to elucidate the roles of SLC45A2 in melanogenesis and melanoma metastasis. We found that the acidified cellular environment impairs the activity of key melanogenic enzyme tyrosinase in Slc45a2-deficient melanocytes. SLC45A2 is identified as a proton/glucose exporter in melanosomes, and its ablation increases the acidification of melanosomal pH through enhanced glycolysis. Intriguingly, 13C-glucose-labeled metabolic flux and biochemical assays show that melanosomes are active glucose-metabolizing organelles, indicating that elevated glycolysis mainly occurs in melanosomes owing to Slc45a2 deficiency. Moreover, Slc45a2 deficiency significantly upregulates the activities of glycolytic enzymes and phosphatidylinositol 3-kinase/protein kinase B signaling to promote glycolysis-dependent survival and metastasis of melanoma cells. Collectively, our study reveals that the proton/glucose exporter SLC45A2 mediates melanin synthesis and melanoma metastasis primarily by modulating melanosomal glucose metabolism.

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III–IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

Genetic Analysis of 28 Chinese Families With Tyrosinase-Positive Oculocutaneous Albinism.

BackgroundTyrosinase-positive oculocutaneous albinism (OCA, type II, OCA2) is an autosomal recessive genetic disease in which the biosynthesis of melanin decreases in the skin, hair, and eyes. OCA2 disease is caused by mutations in OCA2 gene. The gene product plays a role in regulating the pH of melanosomes. Up to now, hundreds of OCA2 mutations have been reported and novel variants are still being discovered.MethodsIn this study, we reviewed the records of OCA2 patients who had conducted albinism genetic testing, and then analyzed the clinical and genetic information of 28 OCA2 patients who had been genetically diagnosed by using Sanger sequencing and next-generation sequencing.ResultsIn this study, we reported 31 variants screened from 28 Chinese OCA2 families, and characterized the detailed molecular and clinical presentations. There were 12 novel variants among all detected variants, including 3 missense variants (p.G393V, p.T482A, and p.R720P), 4 frameshift variants (p.R53Gfs∗49, p.N279Kfs∗17, p.I469Lfs∗4, p.I655Nfs∗12), 2 splicing variants (c.1637-2A > G, c.1951 + 1G > C), 2 stopgain variants (p.L278X, p.W652X) and 1 insertion variants (p.P315LinsT). One potential cluster of missense variants was implicated indicating the important roles of the underlying domains in OCA2 pathogenesis.ConclusionOur results were beneficial for diagnosis and precision clinical management for OCA2-related disorder, and this study expanded the mutation spectrum of oculocutaneous albinism.

Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

541 Inhibition of soluble adenylyl cyclase (sAC) rescues defective melanosomal pH and pigmentation in oculocutaneous albinism type 2 (OCA2)

Oculocutaneous albinism type 2 (OCA2) results from a defective melanosomal anion channel leading to an acidic melanosomal pH, reduced tyrosinase activity and decreased melanin production. Reduced melanin production in OCA2 affected people leads to the development of skin cancers and other forms of damage due to ultraviolet radiation. Currently, pharmacological approaches to repair melanosomal pH and pigmentation do not exist. We have shown that inhibition of soluble adenylyl cyclase (sAC) in melanocytes alkalinizes melanosomal pH and enhances tyrosinase (TYR) activity. Therefore, we asked whether inhibition of sAC would restore wild type melanosomal pH and melanin production in OCA2 melanocytes and mice. OCA2 melanocytes had a more acidic melanosomal pH and sAC inhibitors restored wild type melanosomal pH and increased tyrosinase activity. We generated Oca2-/-, sAC floxed (f/f), Tyr-CRE-ERT2 mice and induced sAC knock out via post-natal (P2-4), topical application of 4-hydroxytamoxifen. The Oca2-/-; sAC-/- mice (n=23) had noticeably darker hair color at P28 as compared to OCA2-/-; sACf/f (n=14) littermates. HPLC analysis of the hair collected on P28 showed a significant increase in total melanin in Oca2-/-; sAC-/- mice corresponding to increased amounts of both eumelanin (PTCA) and pheomelanin (AHP). Individual hair strands of Oca2-/-; sAC -/- mice revealed distinctive banding patterns as compared to Oca2-/-; sACf/f littermates, and histology showed that the Oca2-/-; sAC -/- mice also had less overall hair follicles. Thus, loss of sAC in melanocytes may affect hair morphogenesis. In conclusion, our studies show that restoring melanosomal pH by inhibiting the sAC signaling pathway is a potential therapeutic approach for the treatment of OCA2 and related diseases.

Chemical and biochemical control of skin pigmentation with special emphasis on mixed melanogenesis.

Melanins are widely distributed in animals and plants; in vertebrates, most melanins are present on the body surface. The diversity of pigmentation in vertebrates is mainly attributed to the quantity and ratio of eumelanin and pheomelanin synthesis. Most natural melanin pigments in animals consist of both eumelanin and pheomelanin in varying ratios, and thus, their combined synthesis is called "mixed melanogenesis." Gene expression is an established mechanism for controlling melanin synthesis; however, there are multiple factors that affect melanin synthesis besides gene expression. Due to the differential sensitivity of the eumelanin and pheomelanin synthetic pathways to pH, melanosomal pH likely plays a major role in mixed melanogenesis. Here, we focused on various factors affecting mixed melanogenesis including (1) chemical regulation of melanin synthesis, (2) melanosomal pH regulation during normal melanogenesis and effect on mixed melanogenesis, and (3) mechanisms of melanosomal pH control (proton pumps, channels, transporters, and signaling pathways).

Measurement of Melanin Metabolism in Live Cells by [U-13C]-L-Tyrosine Fate Tracing Using Liquid Chromatography-Mass Spectrometry

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography (HPLC). Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation, and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-13C] tyrosine using liquid chromatography-mass spectrometry (LC-MS). Using this method, we confirmed previous reports of melanin synthesis differences between melanocytes derived from individuals with different skin color or MC1R genotype, and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-DOPA, an important metabolite of tyrosine, is differentially controlled by multiple factors. Because tyrosine fate tracing is compatible with untargeted LC-MS based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.

Single organelle measurements of melanosome pH using the novel ratiometric indicator RpHiMEL.

Melanocytes are specialized cells that produce melanin pigments responsible for skin, hair, and eye pigmentation. The synthesis and storage of melanin occurs in unique lysosome-related organelles called melanosomes, which regulate melanin production via complex regulatory mechanisms. Maintenance of the melanosome luminal ionic environment and pH is crucial for proper function of the main melanogenic enzymes. Defects in genes encoding pH-regulating melanosomal proteins result in oculocutaneous albinism, which is characterized by hypopigmentation, impaired vision, and increased susceptibility to skin and eye cancers. We recently uncovered several ion channels and transporters that modulate melanin synthesis by acidifying or neutralizing the luminal pH of melanosomes. However, our understanding of how melanosomes and other related organelles maintain their luminal pH is far from complete. The study of melanosome pH regulation requires robust imaging and quantification tools. Despite recent advances in the development of such methods, many limitations remain, particularly for quantitative analysis of individual organelle pH. In this chapter, we will provide an overview of the available methods used for melanosome pH determination, including their advantages, limitations, and challenges. To address the critical, unmet need for reliable melanosome pH quantification tools, we engineered a novel genetically encoded, ratiometric pH sensor for melanosomes that we named RpHiMEL. Here, we describe the design and optimization of RpHiMEL, and provide a pH quantification method for individual melanosomes in live cells. We demonstrate that RpHiMEL is a highly versatile tool with the potential to advance our understanding of pH regulation in melanosomes and related organelles.

SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation.

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type 4 (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with the cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic L374 variant. Our data suggest that SLC45A2 maintains melanosome neutralization that is initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common allelic variant imparts reduced activity due to protein instability.

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis.

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.

Ocular melanin binding of drugs: in vitro binding studies

AbstractCertain drugs accumulate into pigmented tissues due to their binding to melanin, a macromolecule inside pigmented cells. Melanin can affect the drug’s pharmacokinetics by acting as a drug reservoir. Binding of drugs to the ocular melanin might enable ocular drug targeting, prolonged duration of action and retinal delivery after non‐invasive drug administration (eye drops, tablet). Interestingly, chloroquine levels are maintained in the pigmented retinal and choroidal cells for months after discontinuation of dosing. The aim of the experimental part was to study melanin binding of a set of compounds in vitro with isolated melanin from the retinal pigment epithelium (RPE) and choroid of porcine eyes. The in vitro binding with melanin was studied at three different pH (i.e. 5, 6, 7.4) since melanosomal pH is unknown but expected to be acidic, so these range of pH will help us to understand the effect of pH on binding affinity and capacity. The compounds chosen for the study; papaverine, levofloxacin, terazosin, pazopanib, are small molecules with diverse physicochemical properties (octanol/water partitioning coefficient (log P), pKa, acid/base status). The binding parameters (Bmax and Kd) estimated from Sips isotherm where the actual distribution of binding energies is most likely continuous and unimodal. Binding parameters were calculated based on experimental data and terazosin is the most potent melanin binder by having K value of 13.30 (K = Bmax/Kdn ). In addition, pH has quite large effect on binding for example binding affinity and capacity of levofloxacin at pH 5 is almost as three times as pH 7.4. This study demonstrates the importance of melanosomal pH on melanin binding of drugs and a useful step toward building a bottom‐up pharmacokinetic model for ocular drug delivery.

A basic way to tan

Skin Biology Darker-skinned individuals have more melanin in their skin and a lower risk for skin cancers. The production of melanin in organelles called melanosomes is pH sensitive. Zhou et al. found that the enzymatic activity of soluble adenylyl cyclase (sAC) resulted in decreases in both melanosome pH and melanin synthesis. sAC deficiency or inhibitors increased melanosome pH and pigmentation in mice. This mechanism for rapidly regulating melanin synthesis could potentially be exploited to reduce skin cancer risk for fair-skinned individuals. Sci. Signal. 11 , eaau7987 (2018).