Triazine-based tyrosinase inhibitors identified by chemical genetic screening

As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long-term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged-triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC50 values in the range of 10 microM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with L-3,4-dihydroxyphenylalanine (L-DOPA) for binding to the DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 muM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation.     

Distal upstream tyrosinase S/MAR‐containing sequence has regulatory properties specific to subsets of melanocytes

A distal upstream regulatory element of the mouse tyrosinase gene has locus control region (LCR)‐like activity and is required for position‐independent expression of linked genes. It consists of a DNAse I hypersensitive site, which has enhancer activity in neural crest‐derived melanocytes, embedded within a scaffold/matrix attachment region (S/MAR), both of which are necessary for LCR activity. To address the role of the S/MAR in position‐independent expression, we assessed the ability of a fragment containing most of the S/MAR to insulate a transgene from position effects. The S/MAR sequence showed a striking cell type specificity in its function in all six multicopy transgenic lines, dampening position effects considerably in cutaneous melanocytes while allowing no expression in other neural crest‐derived melanocytes, and causing elevated expression in ocular melanocytes derived from the neural tube. The specificity of transgene expression in the eye suggested the presence of both positive and negative regulatory elements in this enhancer/S/MAR region, which was confirmed by transient transfection analyses. This is the first known regulatory element to exhibit different activities in melanocytes of different developmental origins. Dev. Genet. 25:40–48, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of acetylcholinesterase inhibitors using homogenous cell‐based assays in quantitative high‐throughput screening platforms

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell‐based assays in high‐throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10‐acetyl‐3,7‐dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH‐SY5Y). An enzyme‐based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high‐throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.     

Structure‐based design of combinatorial mutagenesis libraries

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high‐throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general‐purpose method, called “Structure‐based Optimization of Combinatorial Mutagenesis ” (SOCoM ), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library‐averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β‐lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure‐based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large‐scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure‐based assessments, such as the energy gap between alternative conformational or bound states.     

D‐tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity

Although L‐tyrosine is well known for its melanogenic effect, the contribution of D‐tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L‐tyrosine, D‐tyrosine dose‐dependently reduced the melanin contents of human MNT‐1 melanoma cells and primary human melanocytes. In addition, 500 μM of D‐tyrosine completely inhibited 10 μM L‐tyrosine‐induced melanogenesis, and both in vitro assays and L‐DOPA staining MNT‐1 cells showed that tyrosinase activity is reduced by D‐tyrosine treatment. Thus, D‐tyrosine appears to inhibit L‐tyrosine‐mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D‐tyrosine inhibited melanogenesis induced by α‐MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D‐tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D‐tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte‐derived cells.     

The TYRP1‐mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains

Tyrosinases are melanocyte‐specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra‐melanosomal domains of tyrosinase, TYRtr (19–469), and tyrosinase‐related protein 1, TYRP1tr (25–472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero‐oligomeric complexes to maintain the protective function.     

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Predicting binding modes of reversible peptide‐based inhibitors of falcipain‐2 consistent with structure–activity relationships

Falcipain‐2 (FP‐2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide‐based inhibitors of FP‐2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP‐2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near‐native ligand conformations when applied to a validation set of protein‐ligand structures. Overall, we proposed substrate‐like binding modes of the studied compounds fulfilling the structural requirements for FP‐2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666–1683. © 2017 Wiley Periodicals, Inc.     

Synthesis,molecular docking and kinetic studies of novel quinolinyl based acyl thioureas as mushroom tyrosinase inhibitors and free radical scavengers

The enzyme tyrosinase plays a vital role in melanin biosynthesis and enzymatic browning of vegetables and fruits. A series of novel quinolinyl thiourea analogues (11a-j) were synthesized by reaction of 3-aminoquinoline and corresponding isothiocyanates, in moderate to excellent yields with different substitutions and their inhibitory effect on mushroom tyrosinase and free radical scavenging activity were evaluated. The compound N-(quinolin-3-ylcarbamothioyl)hexanamide (11c) exhibited the maximum tyrosinase inhibitory effect (IC₅₀ = 0.0070 ± 0.0098 µM) compared to other derivatives and the reference Kojic acid (IC₅₀ = 16.8320 ± 0.0621 µM). The docking studies were carried out and the compound (11c) showed most negative estimated free energy of −7.2 kcal/mol in mushroom tyrosinase active site. The kinetic analysis revealed that the compound (11c) inhibits the enzyme tyrosinase non-competitively to form the complex of enzyme and inhibitor. The results revealed that 11c could be identified as putative lead compound for the design of efficient tyrosinase inhibitors.     

Analysis of major elements in pigmented melanocytic chicken skin using laser‐induced breakdown spectroscopy

The concentration difference of major elements in melanocytic skin with respect to pigmentation level is analysed by laser‐induced breakdown spectroscopy (LIBS) to investigate the applicability of LIBS as an in situ feedback tool for selective and complete laser removal of melanocytic skin tissue like nevus. The skin of black silkie chicken which had a characteristic darkly pigmented perifollicular skin surrounded by lightly pigmented extrafollicular skin was used as the sample. The results showed higher LIBS signal intensities of Ca²⁺ and Mg²⁺ but lower intensities of Na⁺, Cl^– and K⁺ in the perifollicular skin than in the extrafollicular skin, which demonstrated the feasibility to use LIBS as a reliable method to distinguish skin tissues with difference in pigmentation level.

Plasma emission of biochemical elements generated with a laser irradiation on melanocytic skin lesion.     

Paving the way towards effective plant-based inhibitors of hyaluronidase and tyrosinase: a critical review on a structure–activity relationship

Human has used plants to treat many civilisation diseases for thousands of years. Examples include reserpine (hypertension therapy), digoxin (myocardial diseases), vinblastine and vincristine (cancers), and opioids (palliative treatment). Plants are a rich source of natural metabolites with multiple biological activities, and the use of modern approaches and tools allowed finally for more effective bioprospecting. The new phytochemicals are hyaluronidase (Hyal) inhibitors, which could serve as anti-cancer drugs, male contraceptives, and an antidote against venoms. In turn, tyrosinase inhibitors can be used in cosmetics/pharmaceuticals as whitening agents and to treat skin pigmentation disorders. However, the activity of these inhibitors is stricte dependent on their structure and the presence of the chemical groups, e.g. carbonyl or hydroxyl. This review aims to provide comprehensive and in-depth evidence related to the anti-tyrosinase and anti-Hyal activity of phytochemicals as well as confirming their efficiency and future perspectives.     

Zebrafish as a new model for phenotype‐based screening of melanogenic regulatory compounds

Although many hypo‐pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo‐pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole‐animal model for phenotype‐based screening of melanogenic inhibitors or stimulators. We used both the well‐known melanogenic inhibitors (1‐phenyl‐2‐thiourea, arbutin, kojic acid, 2‐mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost‐effectiveness, and physiological relevance.     

The fibroblast growth factor‐2 is not essential for melanoma formation in a transgenic mouse model

Fibroblast growth factor 2 (FGF2) has been assigned a role in melanocyte proliferation and in development of human cutaneous melanoma. We have used a transgenic mouse melanoma model in combination with mice lacking mouse FGF2 to analyse the possible implication of FGF2 in melanomagenesis. Tyr::N‐ras^Q61K transgenic mice which are deficient for FGF2 and the tumor suppressors p16^INK4a and p19^ARF are hyperpigmented and develop cutaneous metastasizing melanoma, with no difference to mice wildtype for FGF2. We conclude from our data, that FGF2 is not essential for melanoma progression and metastasis.     

Optimization of norbornyl‐based carbocyclic nucleoside analogs as cyclin‐dependent kinase 2 inhibitors

We report on the discovery of norbornyl moiety as a novel structural motif for cyclin‐dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC₅₀ of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics‐based scoring was used to rationalize the affinities. In conclusion, the discovered 9‐hydroxymethylnorbornyl moiety was shown by joint experimental‐theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.     

Biological responses in Balb/c mice after long‐term parenteral administration of the light subunit of mushroom tyrosinase

Light subunit of mushroom tyrosinase (LSMT) is a protein of unknown function from mushroom Agaricus bisporus that has been demonstrated to permeate through rat intestine ex vivo. Thus, it can be absorbed in the intestine, thereby holding a promise as a drug carrier for oral administration, similar to HA‐33 protein from botulinum, one of the closest structural homologs of LSMT. However, the safety of LSMT should be ensured prior to its use. Here, we described biological response of LSMT upon weekly intraperitoneal administration of 50 μg/day to the Balb/c mice for 12 weeks. Motoric and behavior profiles, as well as the index of main organs (liver, spleen, lung, heart, and kidney), and body weight, were not significantly changed as compared with the control group. Also, no IgG was detected in the serum. The results suggest that LSMT is safe for further development.     

Genetic variation in IRF4 expression modulates growth characteristics,tyrosinase expression and interferon‐gamma response in melanocytic cells

A SNP within intron4 of the interferon regulatory factor4 (IRF4) gene, rs12203592*C/T, has been independently associated with pigmentation and age‐specific effects on naevus count in European‐derived populations. We have characterized the cis‐regulatory activity of this intronic region and using human foreskin‐derived melanoblast strains, we have explored the correlation between IRF4 rs12203592 homozygous C/C and T/T genotypes with TYR enzyme activity, supporting its association with pigmentation traits. Further, higher IRF4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following UVR, an etiological factor in melanoma development. Since UVR, and accompanying IFNγ‐mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of IRF4 status. Manipulation of IRF4 levels followed by IFNγ treatment revealed a subset of chemokines and immuno‐evasive molecules that are sensitive to IRF4 expression level and genotype including CTLA4 and PD‐L1.