Active Site Of Tyrosinase Research Articles

Biological, chemical and in silico fingerprints of Dianthus calocephalus Boiss.: A novel source for rutin

Extracts (methanol, ethyl acetate, and water) from Dianthus calocephalus Boiss. prepared by different extraction techniques (maceration, Soxhlet, and ultrasonication) were studied for possible inhibitory action against key enzymes (α-amylase, α-glucosidase, acetyl cholinesterase, butyryl cholinesterase, and tyrosinase). Antioxidant potential was established using a battery of assays and phenolic compounds profiled by RP-HPLC. Binding pose of tyrosinase with rutin was studied by means of molecular docking. Methanol extracts showed the highest phenolic (39.35–40.25 mgGAE/g) content and rich in rutin (61.38–72.07 mg/g extract). Ethyl acetate extracts of D. calocephalus were potent inhibitors of acetyl (1.45–1.48 mgGALAE/g) and butyryl (2.44–2.74 mgGALAE/g) cholinesterases. Docking studies showed that rutin interacts with the side chains of the key amino acid residues and to the copper atom found at the active site of tyrosinase. Methanol extracts showed highest antioxidant capacity. D. calocephalus showed interesting biological properties that could be further studied to manage diabetes, neurodegenerative diseases, Alzheimer's disease, and hyperpigmentation.

36H: A Novel Potent Inhibitor for Antimelanogenesis.

N-Hydroxycinnamoylphenalkylamides (36H) exhibited both antioxidation and antityrosinase abilities. The compound was studied for its antioxidative properties, using a 1,1-diphenyl-2-picrylhydrazul- (DPPH-) scavenging test, a ferric ion-reducing antioxidant power assay (FRAP) assessment, and a metal-chelating power assay. The results showed that 36H had antioxidative capabilities in the DPPH-scavenging and ferric-reducing power examinations but the chelating power assay did not demonstrate antioxidative capability. 36H was also measured for tyrosinase inhibitory activity applying various species platforms, including in vitro mushroom, B16F10 mouse melanoma, and human melanocyte cells. In terms of in vitro mushroom tyrosinase suppression, 36H restrained the melanogenesis processes. It is assumed that 36H blocked the tyrosinase active site as a competitive inhibitor for mushroom tyrosinase, hence not decreasing the human normal melanocyte cellular viability. A quantitative real-time polymerase chain reaction (qRT-PCR) and western blot discovered that 36H downregulated melanogenesis-related RNA and proteins, including pigment production (MITF, tyrosinase, TRP-1, and TRP-2), melanosome maturation (Rab27a), and melanosome transportation (Myo5a, MLPH and Mreg). Overall, 36H displayed the biofunctions of antioxidation and melanin suppression, so there was a possibility for its application as a food additive or a skin-whitening agent.

A novel anti-melanogenic agent, KDZ-001, inhibits tyrosinase enzymatic activity

BackgroundThe demand for anti-melanogenic agents is increasing due to the unwanted side effects of current treatments. To find an effective anti-melanogenic agent, we used zebrafish as a whole animal model for phenotype-based drug and cosmetic discovery screening. ObjectivesThe aim of this study was to identify and explore a small molecule that could be used for skin-whitening cosmetics. MethodsUsing zebrafish embryos, we examined the effects of 1000 compounds on zebrafish development and pigmentation. Pigmentation production was assessed by tyrosinase (TYR) enzymatic activity and melanin contents. Pigmentation marker expression in the human melanoma cell line HMV-II was analyzed by western blot. We also tested reconstituted human skin tissue and analyzed KDZ-001 with computational molecular modeling. ResultsWe identified three compounds that affected the pigmentation of developing melanophores in zebrafish. Among them, we identified KDZ-001, a novel anti-melanogenic agent, which strongly inhibits melanin synthesis in the developing melanophores of zebrafish, HMV-II cells, and reconstituted human skin with no toxicity. We found that KDZ-001 directly inhibits TYR enzymatic activity. Notably, computational molecular modeling of KDZ-001 suggested that its interaction with copper ions in the active site of TYR is essential for melanin synthesis, further demonstrating that KDZ-001 mainly acts as a TYR inhibitor to synthesize melanin. ConclusionKDZ-001 inhibits melanin synthesis and has a potential for use in skin-whitening cosmetics.

Molecular inhibitory mechanism of dihydromyricetin on mushroom tyrosinase

Tyrosinase is the rate-limiting enzyme for controlling the production of melanin in the human body, and overproduction of melanin can lead to a variety of skin disorders. In this paper, the inhibitory kinetics of Dihydromyricetin (DHM) on tyrosinase and their binding mechanism were determined using spectroscopy, molecular docking, antioxidant assays, and chromatography. The spectroscopic results indicate that DHM reversibly inhibits tyrosinase in a mixed-type manner through a multiphase kinetic process with the IC50 of 849.88 μM. It is shown that DHM has a strong ability to quench the intrinsic fluorescence of tyrosinase mainly through a static quenching procedure, suggesting that a stable DHM–tyrosinase complex is generated. Molecular docking results suggest that the dominant conformation of DHM does not directly bind to the active site of tyrosinase. Moreover, the antioxidant assays demonstrate that DHM has powerful antioxidant and reducing capacity but does not have the ability to reduce dopachrome to L-DOPA. Interestingly, the results of spectroscopy and chromatography indicate that DHM is a substrate of tyrosinase but not a suicide substrate. The possible inhibitory mechanism is proposed, which will be helpful to design and search for tyrosinase inhibitors.

Determination of the Bridging Ligand in the Active Site of Tyrosinase.

Tyrosinase is a type-3 copper enzyme that is widely distributed in plants, fungi, insects, and mammals. Developing high potent inhibitors against tyrosinase is of great interest in diverse fields including tobacco curing, food processing, bio-insecticides development, cosmetic development, and human healthcare-related research. In the crystal structure of Agaricus bisporus mushroom tyrosinase, there is an oxygen atom bridging the two copper ions in the active site. It is unclear whether the identity of this bridging oxygen is a water molecule or a hydroxide anion. In the present study, we theoretically determine the identity of this critical bridging oxygen by performing first-principles hybrid quantum mechanics/molecular mechanics/Poisson-Boltzmann-surface area (QM/MM-PBSA) calculations along with a thermodynamic cycle that aim to improve the accuracy. Our results show that the binding with water molecule is energy favored and the QM/MM-optimized structure is very close to the crystal structure, whereas the binding with hydroxide anions causes the increase of energy and significant structural changes of the active site, indicating that the identity of the bridging oxygen must be a water molecule rather than a hydroxide anion. The different binding behavior between water and hydroxide anions may explain why molecules with a carboxyl group or too many negative charges have lower inhibitory activity. In light of this, the design of high potent active inhibitors against tyrosinase should satisfy both the affinity to the copper ions and the charge neutrality of the entire molecule.

3D QSAR and molecular docking studies of 4-alkoxy- and 4-acyloxy –phenyl ethylene thiosemicarbazone derivatives as tyrosinase inhibitors

Three-dimensional quantitative structural–activity relationship studies were carried out to understand the factors affecting the activity of 4-alkoxy- and 4-acyloxy –phenyl ethylene thiosemicarbazone analogs. The quantitative structural activity relationship model has generated five points Pharmacophore features, having an excellent cross-validated correlation coefficient value (Q 2 = 0.8615), Pearson R (0.9577). This model also exhibited a high value of R 2 (0.9948), F-value (617.8), and lowest SD (0.089). The regression maps were developed on the basis of the best model and suggested aromatic electron withdrawing groups at the para position of phenyl ring which is crucial for Tyrosinase inhibitory activity. Toxicity and drug-likeness of these compounds were estimated by using OSIRIS molecular property explorer tool. The molecular modeling was performed by using Molegro Virtual Docker 2010.4.0. The binding pattern of the best active compound with Tyrosinase active site was analyzed and the residues HIS259, HIS85, His61, His263, VAL283, Glu322, Gly245, and Ala246 were found to have hydrophobic and hydrogen bond interactions.

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4′-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4′-hydroxylation and further decreased by 4′-methoxylation against mushroom tyrosinase. Surprisingly, 4′-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4′-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4′-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.

Tyrosinase inhibitory mechanism of betulinic acid from Dillenia indica

The fruit of Dillenia indica L. is extensively used as a food additive. Betulinic acid (BA) is the most prominent secondary metabolite present in D. indica. This study screened the bioassay guided isolation of BA from D. indica and explored its tyrosinase inhibitory mechanism. Half maximal inhibitory concentration (IC50) of BA were calculated as 13.93µM and 25.66µM for diphenolase and monophenolase. Enzyme kinetic analysis revealed that BA inhibited tyrosinase activity non-competitively. Further, conformational analysis of tyrosinase with BA was measured by fluorescence and circular dichroism spectroscopy. These results implied that diminish rigidity of enzyme might disturb the catalytic conformation of tyrosinase. Moreover, In-silico analysis confirmed probable binding polar and non-polar region on the active site of tyrosinase. Based on these findings, we suggest that BA from D. indica may be useful in preventing enzymatic browning reactions in food products.

The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation

Oxaloacetic acid (OA) is naturally found in organisms and well known as an intermediate of citric acid cycle producing ATP. We evaluated the effects of OA on tyrosinase activity and structure via integrating methods of enzyme kinetics and computational simulations. OA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type (IC50=17.5±0.5mM and Ki=6.03±1.36mM). Kinetic measurements by real-time interval assay showed that OA induced multi-phasic inactivation process composing with fast (k1) and slow (k2) phases. Spectrofluorimetry studies showed that OA mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational docking simulations further revealed that OA could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS259, HIS263, and VAL283. Our study provides insight into the mechanism by which energy producing intermediate such as OA inhibit tyrosinase and OA is a potential natural anti-pigmentation agent.

Inhibition of tyrosinase by fumaric acid: Integration of inhibition kinetics with computational docking simulations

Fumaric acid (FA), which is naturally found in organisms, is a well known intermediate of the citric acid cycle. We evaluated the effects of FA on tyrosinase activity and structure via enzyme kinetics and computational simulations. FA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type with IC50=13.7±0.25mM and Kislope=12.64±0.75mM. We newly established the equation for the dissociation constant (Kislope) for the parabolic inhibition type in this study. Kinetic measurements and spectrofluorimetry studies showed that FA induced regional changes in the active site of tyrosinase. One possible binding site for FA was identified under the condition without L-DOPA. The computational docking simulations further revealed that FA can interact with HIS263 and HIS85 at the active site. Furthermore, four important hydrogen bonds were found to be involved with the docking of FA on tyrosinase. Our study provides insight into the mechanism by which dicarboxylic acids such as FA inhibit tyrosinase. By inhibiting tyrosinase and its central role in pigment production, FA is a potential natural antipigmentation agent.

The effect of alpha-ketoglutaric acid on tyrosinase activity and conformation: Kinetics and molecular dynamics simulation study

Alpha-ketoglutaric acid (AKG) is naturally found in organisms and is a well-known intermediate in the production of ATP or GTP in the Krebs cycle. We elucidated the effects of AKG on tyrosinase activity and conformation via methods of inhibition kinetics integrated with molecular dynamics (MD) simulations. AKG was found to be a reversible inhibitor of tyrosinase (IC50=15±0.5mM) and induced parabolic slope mixed-type inhibition. Based on our newly established equation, the dissociation constant (Kislope) was determined to be 7.93±0.31mM. The spectrofluorimetry studies showed that AKG mainly induced regional changes in the active site of tyrosinase, which reflects the flexibility of the active site. The computational docking and molecular dynamics (MD) simulations further demonstrated that AKG could interact with several residues near the substrate-binding site located in the tyrosinase active site pocket. Our study provides insight into the mechanism by which energy-producing intermediates such as AKG inhibit tyrosinase through its ketone groups. Also, AKG could be a potential natural antipigmentation agent due to its non-toxic property.

Inhibitory mechanism of cardanols on tyrosinase

Abstract Cashew nut shell liquid (CNSL), extracted from cashew nut shell, is an abundant natural resource. Cardanols were the major phenolic components isolated from CNSL. In this research, we reported on the inhibitory mechanism of cardanols on tyrosinase for the first time. We studied the functions of cardanols and revealed the underlying mechanism of cardanols as tyrosinase inhibitors. Cardanol triene, cardanol diene and cardanol monoene could decrease the steady-state rate of the tyrosinase diphenolase activity efficiently. The IC50 values of three cardanol compounds were determined to be 40.5 ± 3.7, 52.5 ± 3.2 and 56.0 ± 3.6 μM (n = 3), respectively. Meanwhile, the kinetic analysis and the intrinsic/ANS-binding fluoresecence-quenching showed that one cardanol might enter into one tyrosinase. The characteristic values further revealed that cardanols could interact with tyrosinase. Besides, computational study with molecular docking implied that cardanols might affect the amino acid residues of the tyrosinase active site. Collectively, cardanols could moderate inhibitory activities on tyrosinase effectively.

Effect of Cd2+ on tyrosinase: Integration of inhibition kinetics with computational simulation

Cadmium ions (Cd2+) are a widespread and easily absorbed toxin to both humans and animals that can be spread via food, water, and air pollution. Tyrosinase (EC 1.14.18.1) is a multifunctional copper-containing enzyme that is ubiquitously expressed in animals and plays a critical role in melanin production. We evaluated the toxic effects of Cd2+ on tyrosinase activity and conformation by measuring kinetics and computationally simulating the interactions. We found Cd2+ to be a slope-hyperbolic noncompetitive-inhibition reversible inhibitor of tyrosinase, with an IC50 of 2.92±0.16mM and Ki of 0.23±0.02mM. Spectrofluorimetric measurements of intrinsic and ANS-binding fluorescence showed that Cd2+ did not induce significant changes to tyrosinase overall or to its regional active site conformations. Cd2+ showed its inactivation effect not by modulating apparent structural changes to tyrosinase, but by occupying binding sites. To gain further insight into the Cd2+/tyrosinase interaction, we performed computational docking and molecular dynamics simulations. The results consistently indicated that Cd2+ can interact with several residues near the tyrosinase active site, primarily HIS85 and ASN260. Our study provides insight into the mechanism of the toxic effects Cd2+ has on tyrosinase, which could affect the normal pigmentation pathway in animals.

Electrochemically reduced graphene and iridium oxide nanoparticles for inhibition-based angiotensin-converting enzyme inhibitor detection

In this work, a novel biosensor based on electrochemically reduced graphene oxide and iridium oxide nanoparticles for the detection of angiotensin-converting enzyme inhibitor drug, captopril, is presented. For the preparation of the biosensor, tyrosinase is immobilized onto screen printed electrode by using 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-Hydroxysuccinimide coupling reagents, in electrochemically reduced graphene oxide and iridium oxide nanoparticles matrix. Biosensor response is characterized towards catechol, in terms of graphene oxide concentration, number of cycles to reduce graphene oxide, volume of iridium oxide nanoparticles and tyrosinase solution. The designed biosensor is used to inhibit tyrosinase activity by Captopril, which is generally used to treat congestive heart failure. It is an angiotensin-converting enzyme inhibitor that operates via chelating copper at the active site of tyrosinase and thioquinone formation. The captopril detections using both inhibition ways are very sensitive with low limits of detection: 0.019µM and 0.008µM for chelating copper at the active site of tyrosinase and thioquinone formation, respectively. The proposed methods have been successfully applied in captopril determination in spiked human serum and pharmaceutical dosage forms with acceptable recovery values.

Highly potent tyrosinase inhibitor, neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC50 of 30nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC50=500nM), significantly. Another potent inhibitor 1 (IC50=2.9μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: Kiapp=1.48nM, k3=0.0033nM−1min−1 and k4=0.0049min−1. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95μM of IC50. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.

Effect of hydroxysafflor yellow A on tyrosinase: Integration of inhibition kinetics with computational simulation

Hydroxysafflor yellow A (HSYA) is found in the flower of Carthamus tinctorius. We evaluated the effects of hydroxysafflor yellow A (HSYA) on tyrosinase kinetics and computationally simulated the interaction between HSYA and tyrosinase. HSYA was found to be a mixed-type, reversible inhibitor of tyrosinase, with a Ki of 0.285±0.040mM. Kinetic measurements of intrinsic and ANS-binding fluorescence showed that HSYA induced changes in the tertiary structure of tyrosinase. HSYA showed a relatively strong binding affinity for tyrosinase and one possible binding site was identified. To gain further insight into the HSYA/tyrosinase interaction, we performed computational docking and molecular dynamics simulations. These experiments indicated that HSYA can interact with several residues near the tyrosinase active site. Our study provides insight into the mechanism by which chalcone compounds such as flavonoids inhibit tyrosinase. Since HSYA inhibits tyrosinase and is also an antioxidant, HSYA is a potential natural antipigmentation agent.

Inhibitory effects of cefotaxime on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, cefotaxime, a cephalosporin antibacterial drug, was tested as an inhibitor of tyrosinase. The results show that cefotaxime inhibits both the monophenolase and diphenolase activities of tyrosinase. For the monophenolase activity, cefotaxime increased the lag time and decreased the steady-state activity with an IC50 of 3.2 mM. For the diphenolase activity, the inhibition by cefotaxime is reversible and mix-I type with an IC50 of 0.14 mM. The inhibition constants (K(I) and K(IS)) were determined to be 0.14 and 0.36 mM, respectively. The molecular mechanism of inhibition of tyrosinase by cefotaxime was determined by fluorescence quenching and molecular docking. The results demonstrated that cefotaxime was a static quencher of tyrosinase and that cefotaxime could dock favorably in the active site of tyrosinase. This research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors in the future.

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 ± 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

Synthesis and Biological Evaluation of Kojic acid Derivatives as Tyrosinase Inhibitors

Tyrosinase 1 is a copper-containing enzyme which is widely distributed in nature. This enzyme is associated with the production of melanin for the protection of the skin from solar irradiation. However, overproduction of melanin in the skin is of particular concern to woman, for example, in the conditions melasma and lentigo. 2 Tyrosinase catalyzes the first two steps of melanin biosynthesis, namely the hydroxylation of L-tyrosine to L-dopa and the subsequent oxidation of L-dopa to dopaquinone. Tyrosinase is contained in vegetables and fruits and is responsible for the undesirable enzymatic browning that occurs upon long term storage of these. 3 Therefore, tyrosinase inhibitors have attracted attention as important depigmenting agents for the treatment of hyperpigmentation and as anti-browning agents in vegetables and fruits. Kojic acid 4 (1) is a well known tyrosinase inhibitor. However, its inhibitory activity is not potent enough for the above purposes. To overcome this drawback, many semisynthetic kojic acid derivatives have been synthesized, usually by modification of the C-2 hydroxyl group. 5 Recently, we synthesized kojic acid derivatives containing thioether 6 and ester 7 linkers and evaluated their tyrosinase inhibitory activities. Among them, 2-((cyclohexylthio)methyl)-5-hydroxy-4H-pyran-4-one (2b), showed the most potent inhibitory activity. Compound 2b is composed of three parts, including the kojic acid moiety, a thioether linker, and a hydrophobic cyclohexane group. We synthesized and evaluated additional compounds to get more structural insight into the tyrosinase inhibitory activity of the kojic acid derivatives. The crystallographic structure of tyrosinase has recently been established, enabling closer examination of its three-dimensional structure, which revealed the presence of a hydrophobic protein pocket adjoining the binuclear copper active site. 8 We also conducted a molecular docking study to elucidate the structural importance of the kojic acid derivatives in binding with the active site of tyrosinase. The synthetic pathways of the kojic acid derivatives are shown in Scheme 1. Thioether derivatives (2a-2e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of thiols. Ester derivatives (3a-3e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of acids. The inhibitory activities of the kojic acid derivatives on mushroom tyrosinase were investigated using kojic acid as a positive control. The results are summarized in Table 1.

Serendipitous discovery of short peptides from natural products as tyrosinase inhibitors.

Tyrosinase, which is the crucial copper-containing enzyme involved in melanin synthesis, is strongly associated with hyperpigmentation disorders, cancer, and neurodegenerative disease; thus, it has attracted considerable interest in the fields of medicine and cosmetics. The known tyrosinase inhibitors show numerous adverse side effects, and there is a lack of safety regulations governing their use. As a result, there is a need to develop novel inhibitors with no toxicity and long-term stability. In this study, we use molecular docking and pharmacophore modeling to construct a reasonable and reliable pharmacophore model, called Hypo 1, that could be used for identifying potent natural products with crucial complementary functional groups for mushroom tyrosinase inhibition. It was observed that, out of 47,263 natural compounds, A5 structurally resembles a dipeptide (WY) and natural compound B16 is the equivalent of a tripeptide (KFY), revealing that the C-terminus tyrosine residues play a key role in tyrosinase inhibition. Tripeptides RCY and CRY, which show high tyrosinase inhibitory potency, revealed a positional and functional preference for the cysteine residue at the N-terminus of the tripeptides, essentially determining the capacity of tyrosinase inhibition. CRY and RCY used the thiol group of cysteine residues to coordinate with the Cu ions in the active site of tyrosinase and showed reduced tyrosinase activity. We discovered the novel tripeptide CRY that shows the most striking inhibitory potency against mushroom tyrosinase (IC50 = 6.16 μM); this tripeptide is more potent than the known oligopeptides and comparable with kojic acid-tripeptides. Our study provides an insight into the structural and functional roles of key amino acids of tripeptides derived from the natural compound B16, and the results are expected to be useful for the development of tyrosinase inhibitors.