Exploring Probes & Inhibitors Designed to Engage hsTY: a Route to Melanogenesis In vivo Suppression – EPIDERMIS

Classical methods of medicinal chemistry were implemented, especially chemical synthesis and structure-activity relationships. Molecular docking experiments were undertaken for rationalizing molecule-enzyme interactions, using homology modelling. In addition, several biological and biochemical tests were conducted for in-depth evaluation of the developed molecules:

— Biochemical assays involving an enzymatic reaction between a substrate and human tyrosinase, with a colorimetric detection monitoring DOPAchrome production.

— Biochemical assays involving a similar reaction with human melanoma cell lysates, allowing a rapid response on a human system without the need for a purified enzyme, with a colorimetric detection monitoring the production of melanogenesis intermediates.

— Cellular assays on human melanoma whole cells, with a quantification of melanin production after 4 or 14 days.

— Cytotoxicity assays on human melanoma cells.

The project led to the synthesis and evaluation of approximately 150 compounds. Among these, 25 explored A-ring substitutions on the aurone scaffold, 15 investigated alternative C-ring structures, including extensions into other hemiindigoid scaffolds, and 35 focused on incorporating various potentially copper-chelating groups at the B-ring. The remaining compounds combined structural features inspired by these previously designed molecules.

The project progressed through three successive generations of inhibitors. The first generation was highly exploratory, targeting modifications at the A- and C-rings. This phase identified an initial hit capable of inhibiting isolated human tyrosinase with a Ki of 0.25 µM, comparable to the reference compound thiamidol. However, its efficacy against human melanoma whole cells remained limited (IC50 = 29 µM), highlighting an area for further optimization. A study detailing 38 first-generation compounds was published in 2023.

The second generation focused on diversifying the substituent at position 4 of the indanone scaffold, identified as promising in the first phase. This effort led to the development of novel 4-amino and 4-amido derivatives, which demonstrated strong activity in cell lysate assays (IC50 = 0.14 µM) and significant improvements in whole-cell assays (IC50 = 3.2 µM at 4 days, 0.77 µM at 14 days), closely matching thiamidol’s performance. A second study, featuring 18 second-generation compounds, was published in 2024. In both cases, molecular modeling provided insights into the binding geometry within the enzyme active site.

The third generation centered on B-ring modifications and the introduction of chemical groups capable of interacting with copper atoms. While promising results were obtained in cell lysate assays, increased cytotoxicity was observed in melanoma cells. Further studies are ongoing to evaluate the most effective compounds against isolated human tyrosinase. A publication presenting 46 third-generation compounds is currently in preparation.

The project opens significant prospects. On one hand, the best-performing molecules developed within the project will undergo further evaluation through tests on melanocytes and assessments on skin explants. The results will help determine the preclinical potential of these compounds and pave the way for future developments.

Additionally, since hemiindigoids are also an important class of photoswitches, promising opportunities in photopharmacology will be explored. The localization of tyrosinase in the skin makes it a prime target for light-controlled activity modulation, which the chosen scaffold enables. Initial trials have encountered challenges in designing a B-ring capable of both interacting with copper and facilitating photoswitching. However, ongoing efforts aim to overcome this hurdle, allowing for the expansion of the project into a more ambitious avenue of activity control.

B. Roulier, B. Pérès, R. Haudecoeur. Advances in the design of genuine human tyrosinase inhibitors for targeting melanogenesis and related pigmentations. J. Med. Chem. 2020, 63, 13428–13443

Human tyrosinase (hsTY) is a metalloenzyme involved in the synthesis of the main skin pigments, melanins, and especially in the two key steps of this biosynthetic pathway: the successive oxidations of L-tyrosine and L-DOPA. An uncontrolled production of melanins is associated with several pathologies, such as various hyperpigmentation-based diseases, and with the emergence of melanoma resistance to conventional anticancer therapies. The inhibition of tyrosinase is a well-established strategy for controlling melanin production in vivo, but almost all reported bioactive compounds were designed using non-relevant models such as mushroom tyrosinase, a readily commercially available enzyme that has only low homology with hsTY, and that displayed significantly different interaction patterns with inhibitors and substrates. Thus, almost all skin-whitening agents used worldwide, such as kojic acid, have shown very weak activities against hsTY, and are used in high concentrations in human-directed dermocosmetic applications, thereby leading to nocive adverse effects and carcinogenicity. By taking advantage of recent advances in hsTY structure knowledge and of novel large-scale hsTY production protocols, we propose to rely on the aurone scaffold, which has already demonstrated its potential against hsTY (one derivative is one of the most active inhibitors of the enzyme reported to date), for rationally conceiving new selective agents, active in a cellular context and in a preclinical assay, by getting rid of irrelevant tests in non-human medium. In addition, preliminary works already showcased the aurone scaffold as a promising platform for the development of fluorogenic probes for biomolecules. The, the use of fine-tuned analogues will allow a specific detection of hsTY in biological medium, by relying on a strategy of fluorescence “turn-on” upon contact with the target. The detection will be based on the specific structure of the protein, and not its catalytic activity, as it is the case for the totality of already reported tyrosinase probes, raising interference issues especially with ROS (Reactive Oxygen Species) and activity-related enzymes (e.g. tyrosine hydroxylase). Thus, these innovative biosensors will potentially lead to more accurate and reliable analytical tools for human-directed use, e.g. for the specific detection of hsTY in melanoma or Parkinson’s disease contexts.