Resorcinol-based hemiindigoid derivatives as human tyrosinase inhibitors and melanogenesis suppressors in human melanoma cells

Highlights

• Thirty-eight resorcinol compounds are screened against melanoma cell lysates.

• Two hemiindigoids are identified as nanomolar inhibitors of human tyrosinase.

• The proposed binding mode is reminiscent of those of natural substrate l-tyrosine.

• An improvement of cellular activity is recorded compared to the parent compound.

Abstract

Human tyrosinase (hsTYR) catalyzes the key steps of melanogenesis, making it a privileged target for reducing melanin production in vivo. However, very few hsTYR inhibitors have been reported so far in the literature, whereas thousands of mushroom tyrosinase (abTYR) inhibitors are known. Yet, as these enzymes are actually very different, including at their active sites, there is an urgent need for new true hsTYR inhibitors in order to enable human-directed pharmacological and dermocosmetic applications without encountering the inefficiency and toxicity issues currently triggered by kojic acid or hydroquinone. Starting from the two most active compounds reported to date, i.e. a 2-hydroxypyridine-embedded aurone and thiamidol, we combined herein key structural elements and developed new nanomolar hsTYR inhibitors with cell-based activity. From a complete series of thirty-eight synthesized derivatives, excellent inhibition values were obtained for two compounds in both human melanoma cell lysates and purified hsTYR assays, and a promising improvement was observed in whole cell experiments.

Introduction

Human tyrosinase (hsTYR, EC 1.14.18.1) is a type-3 copper-containing metalloenzyme characterized by a coupled binuclear active site that performs both the ortho-hydroxylation of monophenols and the oxidation of catechols [1]. Especially, hsTYR carries out the hydroxylation of l-tyrosine to l-DOPA and the subsequent oxidation of l-DOPA to dopaquinone, thereby initiating the spontaneous synthesis of melanin pigments in specialized skin cells, i.e. the melanocytes [2]. Melanin plays a crucial role in cell protection against UV radiations and free radicals. Nonetheless, the production of these pigments, i.e. melanogenesis, can also induce abnormal levels of melanin accumulation, leading to hyperpigmentation disorders, melasma or skin lesions, and ultimately causing health issues and disfigurements, sometimes severe and profoundly affecting the quality of life [3,4]. In addition, melanoma is closely related to an uncontrolled synthesis of melanins, which promotes the emergence of resistance to almost all traditional anti-cancer therapies [5,6]. As a consequence, melanoma is the most deadly skin cancer (more than 60% of the deaths related to skin cancers), while its incidence rate is low (1% of the skin cancers) [7]. This multiresistance could be bypassed by the administration of inhibitors as adjuvants in immuno-, chemo- or radiotherapy for sensitizing melanoma, thereby improving the prognosis of the disease [8,9]. Overall, the inhibition of hsTYR is a well-established strategy for lowering or suppressing melanogenesis in vivo, and numerous inhibitors have been developed in the past decade for therapeutic and/or dermocosmetic purposes (Fig. S7) [[10], [11], [12]]. However, the production of properly matured hsTYR in high amounts is a challenge, due to its multiple specific N-glycosylations unavailable in simple organisms such as E. coli for recombinant production, mostly jeopardizing large-scale inhibitor screenings.¹³ Hence, most studies only relied on in vitro inhibition assays using the cheap and readily-available mushroom tyrosinase from Agaricus bisporus (abTYR), despite a low homology between these two enzymes (22% identity) and very distinct interaction patterns [13,14]. As a consequence, even the most widely used compound in human-directed dermocosmetic applications, i.e. kojic acid (KA), is a hundred-fold less active against hsTYR than abTYR (K_i = 350 μM vs. 4.3 μM) [15], resulting in the use of high concentrations, thereby promoting associated adverse effects, such as dermatitis or erythema, as well as carcinogenicity. Generally, such activity gaps were observed for almost all TYR reference inhibitors, including hydroquinone, arbutin, benzoic acid and several simple resorcinol derivatives.¹³ Therefore, a development process involving an appropriate array of tests performed using human models appears as a requirement for producing melanogenesis suppressors that could act efficiently and safely in a human context.

In the recent years we have developed aurones as a new class of TYR effectors. Aurones are naturally occurring flavonoids with a promising medicinal potential [16]. They provide bright yellow flower pigmentation to some of the most advanced taxa in the plant kingdom, among other still little-known roles [17]. Some derivatives from this subclass were found initially as melanogenesis suppressors in cell-based assays [18]. A broader study showed that aurone analogues can actually adopt versatile behaviors, acting either as inhibitors, activators or alternative substrates of several isolated mushroom and bacterial TYRs, depending on their substitution pattern at the B-ring [[19], [20], [21]]. In addition, we showed recently that the aurone scaffold is particularly suited for building interactions with active site residues located relatively far from the dicopper center in hsTYR, yielding compound 1 as one of the best hsTYR inhibitors reported to date (K_i = 0.35 μM), but with membrane penetration and water solubility issues probably due to the presence of a 2-hydroxypyridine-N-oxide (HOPNO) moiety [22]. Thus, we propose to dismiss the HOPNO chelating group. In our previous studies, aurones bearing a resorcinol moiety, i.e. 2′,4′-dihydroxyaurones, were always associated with an inhibitor behavior, regardless of the TYR source. This observation was consistent with the known beneficial effect of resorcinol moiety in the frame of TYR inhibition, as witnessed by the number of reported inhibitors containing this chemical group[[23], [24], [25], [26], [27], [28]]. Indeed, if some resorcinols were previously found to undergo a slow oxidation process in the presence of TYR and additives [29,30], they act as true inhibitors in physiological conditions.¹⁴ Some of them are actually used for treating human hyperpigmentation disorders, such as 4-n-butylresorcinol (i.e. rucinol, marketed as Iklen®, Fig. 1) [25,26]. The resorcinol-based thiamidol (Fig. 1) seems especially very promising, regarding both in vitro (K_i = 0.25 μM against hsTYR),¹⁴ and clinical contexts (facial hyperpigmentation improvement in 96% of treated women with melasma after 12 weeks of treatment) [31]. Altogether, the introduction of a resorcinol moiety on an aurone scaffold, or on a related hemiindigoid core, could combine an efficient copper-chelating resorcinol head with a hsTYR-tailored hemiindigoid backbone, able to interact with the second coordination sphere of the enzyme.

Thus, we report herein a series of thirty-eight molecules designed starting from compound 1 and including a 2′,4′-resorcinol moiety, or close analogues, at the B-ring. The best derivatives, identified through a preliminary screening using hsTYR-containing human melanoma MNT-1 cell lysates, were evaluated both on the isolated enzyme expressed in a mammalian-cell system, thus insuring the expression of a mammalian glycosylation profile, and against melanogenesis in a human whole-cell system. Potential cytotoxicity issues were finally examined, and a binding geometry was proposed through molecular modelling, using a recent homology model of hsTYR.