Tyrosinase is a key enzyme responsible for the formation of melanin (a natural skin pigment with ultraviolet-protection properties). However, some people experience melanin overproduction, so new, safe, and biocompatible enzyme inhibitors are sought. New tripeptide tyrosinase inhibitors were developed using molecular modeling. A combinatorial library of tripeptides was prepared and docked to the mushroom tyrosinase crystal structure and investigated with molecular dynamics. Based on the results of calculations and expert knowledge, the three potentially most active peptides (CSF, CSN, CVL) were selected. Their in vitro properties were examined, and they achieved half-maximal inhibitory concentration (IC 50) values of 136.04, 177.74, and 261.79 µM, respectively. These compounds attach to the binding pocket of tyrosinase mainly through hydrogen bonds and salt bridges. Molecular dynamics simulations demonstrated the stability of the peptid–tyrosinase complexes and highlighted the persistence of key interactions throughout the simulation period. The ability of these peptides to complex copper ions was also confirmed. The CSF peptide showed the highest chelating activity with copper. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed that none of the test tripeptides showed cytotoxicity toward the reconstructed human epidermis. Our results indicated that the developed tripeptides were non-toxic and effective tyrosinase inhibitors. They could be applied as raw materials in skin-brightening or anti-aging cosmetic products.
Skin is the outer barrier of the human body. It is responsible for the protection of the body from external factors, such as air pollution, toxic chemicals, pathogenic microorganisms, or ultraviolet (UV) radiation. The frequency of exposure to the sun can trigger oxidative stress and inflammation or lead to cell damage or tumor formation. One of the defense mechanisms of the body against UV radiation is the production of the dark pigment melanin. Melanin is formed in specialized cells called melanocytes, and its most common varieties are eumelanin and pheomelanin. The protective role of melanin is significant, but in some cases, the pigment is overproduced. The phenomenon is manifested by the formation of melanin clusters on the skin and the development of freckles or age spots, also known as “hyperpigmentation changes”. To deal with this problem, it is necessary to intervene in one of the stages of melanogenesis.
Tyrosinase is the key enzyme that manifests its activity, especially at the beginning of melanin synthesis. It is responsible for catalyzing the oxidation of tyrosine or levodopa (L-DOPA) to dopaquinone, which is then converted to melanin. The use of substances that bind to the active site of this enzyme will stop or slow down the formation of pigment and, thus, reduce the intensity of skin changes.
In the food and cosmetic industries, tyrosinase inhibitors of natural and synthetic origin are used, including resveratrol, arbutin, kojic acid, azelaic acid, or L-ascorbic acid. Mixtures of substances are also used, for example, in the form of plant extracts, with or without the isolation of individual compounds responsible for the inhibition. However, the adverse effects associated with their use (proven toxicity, mutagenic or carcinogenic potential, as well as causing secondary skin inflammation) have led to restrictions in their applications. Several countries have banned the use of hydroquinone for skin treatments. Some restrictions have been introduced in the use of kojic acid or arbutin. Therefore, it is necessary to develop non-toxic and biocompatible molecules with a skin-brightening effect. Among all tyrosinase inhibitors, peptides deserve special attention. Thanks to the multitude of possible combinations of amino acids, peptides can stimulate cells to synthesize biomolecules, increase transport of other ingredients to the skin, or modulate the action of enzymes. Peptides with tyrosinase-inhibitory properties have been identified but not described thoroughly. Thaha et al. indicated that peptides obtained from natural raw materials with the amino-acid sequence VWWW, IRW, and VKAGFAWTANQQLS could be antioxidants and substances preventing the darkening of products caused by tyrosinase-catalyzed oxidation. Feng et al. studied a tripeptide with the sequence FPY, present in Juglans regia L. (walnut), which is a tyrosinase inhibitor. Peptides with the sequences NYRRE and RHAKF obtained from Chinese quince have also been tested for tyrosinase-inhibiting activity. Active dipeptides with the sequences IR, LK, and VY have also been identified. Hsiao et al. developed new peptide inhibitors based on pharmacophore modeling, resulting in two cysteine-containing tripeptides: CRY and RCY. CRY was characterized as having the best tyrosinase-inhibitory properties among test compounds.
Attempts have also been made to modify peptide molecules with moieties that increase the stability of the peptide–protein complex based on non-peptide inhibitors. J.-M. Noh and colleagues described the amides of kojic acid and tripeptides obtained by solid-phase synthesis. They showed a stronger inhibitory effect than pure kojic acid (reference substance), similar to the combination of thiosemicarbazone with tripeptides with the sequences FFY, FWY, and FYY.
Often, test peptides are isolated from natural resources, but synthetic compounds have also been used. A thorough analysis encompassing all possible combinations of tripeptides and their potential inhibitory activity against tyrosinase has not been carried out. A method that permits the screening of large databases or combinatorial libraries is called “virtual screening”. It is used widely in the development of small molecule-active substances with a description of their interaction in the binding pocket of the enzyme. In silico methods significantly reduce the time needed to find potentially effective substances, eliminating the need for tedious syntheses and, thus, reducing costs. Molecular modeling allows for the initial characterization of compounds, exploring vast chemical space, and finding the molecular mechanisms of action.
In this work, new tripeptide tyrosinase inhibitors were studied. A virtual combinatorial library of tripeptides was generated and docked to the crystal structure of tyrosinase. All docked compounds were classified based on the presence and quality of interactions with the binding pocket, the value of the scoring function, and expert knowledge. The three most promising tripeptides were synthesized on demand, and their ability to reduce tyrosinase activity was determined experimentally. Additionally, the best peptides were subjected to a 1000 ns molecular dynamics simulation, including the mapping and timeline representation diagrams showing protein–ligand contacts.
Sequence alignment is a useful tool to identify protein fragments (including enzymes) that are similar at the structural level. The manipulation of human tyrosinase presents inherent challenges; its crystal structure is not available, so it is necessary to carry out homology modeling or use other enzymes that are most structurally similar. Entire sequences of four enzymes, from A. bisporus (2Y9X), M. domestics (6ELS), I. batatas (1BT1), and human tyrosinase, were aligned using Clustal Omega. Figure 1 illustrates the MSA of tyrosinases from different species only for selected segments relevant to the active site of tyrosinase to ensure better readability. Twenty-seven identical residues were identified (including six histidine residues), which were responsible for the coordination of copper ions in the active center (which is crucial for the catalytic ability of tyrosinases). A. bisporus (2Y9X), M. domestics (6ELS), and I. batatas (1BT1) showed 20.12%, 18.98%, and 19.72% identity with human tyrosinase, respectively.
Tyrosinase from A. bisporus (2Y9X) is used as a substitute for human tyrosinase in virtual screening. Among the aligned sequences, mushroom tyrosinase showed the highest similarity to human tyrosinase, so it was selected for molecular modeling. An additional advantage is the possibility of testing compounds on the same enzyme in in vitro inhibition tests and in silico studies. Nonetheless, it is important to note that sequence identity does not always equate to structural similarity. While the primary structure, or the amino acid sequence, may be similar, this does not necessarily imply resemblance in secondary, tertiary, or quaternary structures.
Virtual screening is a powerful and relatively inexpensive tool for selecting compounds with potential specific biological activity. Searching for tyrosinase inhibitors enables the rapid testing of thousands of potential tripeptide combinations, thereby leading to the identification and selection of the most promising candidates.
In the traditional approach (high-throughput screening), to make such a selection, it would be necessary to synthesize or purchase 8000 tripeptides an
