Structure-based design and evaluation of tyrosinase inhibitors targeting both human and mushroom isozymes

Abstract

Tyrosinase inhibition represents an attractive challenge to fight skin hyperpigmentation for medicinal and cosmeceutical application. We have previously provided insights for the development of novel compounds with a specific shape and functional groups interacting with tyrosinases from distinct sources. We chose to employ the Agaricus bisporus tyrosinase (AbTYR) isoform as a cost-effective and rapid screening method prior to carrying out the assay toward human tyrosinase (hTYR). Through this approach, the inhibitor [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3) has been identified as an effective inhibitor against both TYRs with potency comparable to that of the marketed inhibitor Thiamidol. Continuing our efforts, in this work we designed a focused small series of MehT-3 derivatives that were in silico predicted for their ability to occupy the cavity of AbTYR and hTYR; subsequently, we proceeded with the execution of a very simple and efficient synthetic procedure to obtain the designed compounds. As a result, we obtained potent AbTYR and hTYR inhibitors with affinity values ranging from 5.3 to 40.7 μM. Notably, compounds 2 and 3 emerged as the most promising candidates; they exhibited superior activity against hTYR and demonstrated low toxicity as effective antioxidant agents and sunscreen products. Overall, these achievements further strengthened our computational protocol, which could be effectively applied to develop newer tyrosinase inhibitors.

Fig. 1. Chemical structure of [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3) and reference compound isobutylamido thiazolyl resorcinol (Thiamidol).

Fig. 2. Chemical structure of designed compounds 1–5 inspired by [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3).

Fig. 3. Superimposition of the ligands with MehT-3 (cyan stick) on the AbTYR X-RAY structure, pale-cyan cartoon (PDB 2Y9X) (A) and hTYR homology model, light-pink cartoon (B). The ligands are shown as sticks: 1, yellow; 2, pink; 3, gray; 4, purple; and 5, orange. The copper ions are depicted as brown spheres. The images were created with PyMOL (PyMOL Molecular Graphics System, Version 3.0, Schrödinger, LLC).

Fig. 4. The RMSD of AbTYR protein over a 100 ns MD trajectory for its complexes with derivatives 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E).

Fig. 5. The RMSD of homology modelling of hTYR protein over a 100 ns MD trajectory for its complexes with derivatives 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E).

Scheme 1. Synthesis of 1–5. Reagents and conditions: (a) DMF, r.t., 15 h; (b) DMF, HBTU, TEA, r.t., 15 h; (c) DMF, r.t., 2 h; (d) THF, r.t., 2 h; (e) DMF, DIPEA, r.t. 2.5 h.

Fig. 6. Cell viability of HaCaT cell line after treatment with varying concentrations of compounds 2 and 3, ranging from 0 (NT) to 50 μM.

Fig. 7. Inhibition of H₂O₂-induced ROS generation by compounds 2 and 3 on HaCaT cells. NT, non-treated cells; T, cells treated with H₂O₂ only.