Exploiting the 1-(4-fluorobenzyl)piperazine fragment for the development of novel tyrosinase inhibitors as anti-melanogenic agents: Design, synthesis, structural insights and biological profile.

The development of Tyrosinase inhibitors (TYRIs) could represent an efficacious strategy for pharmacological intervention on skin pathologies related to aberrant production of melanin. Based on in silico studies we designed and tested a library of twenty-four compounds bearing the 4-(4-fluorobenzyl)piperazin-1-yl]-fragment. As result, we identified several compounds with excellent inhibit effects at low micromolar concentration against TYR from Agaricus bisporus (TyM). Among them, compound 25 (IC50 = 0.96 µM) proved to be ∼20-fold more potent than the reference compound kojic acid (IC50 = 17.76 µM) having wide applications in the cosmetics and pharmaceutical industries. The mode of interaction of active inhibitor 25 was deciphered by means of crystallography as well as molecular docking and these results were consistent with kinetic experiments. Moreover, the identified compound 25 exhibited no considerable cytotoxicity and showed anti-melanogenic effects on B16F10 melanoma cells. Therefore, a combination of computational and biochemical approaches could represent a rational guidelines for further structural modification of this class of compounds as future anti-melanogenic agents.

Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments.

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.

Altering 2-Hydroxybiphenyl 3-Monooxygenase Regioselectivity by Protein Engineering for the Production of a New Antioxidant.

2-Hydroxybiphenyl 3-monooxygenase is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing 3-hydroxybiphenyl for the first time, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity to the well-studied piceatannol. The crystal structure of M321A was determined (2.78 Å), and molecular docking of the 3-substituted phenol provided a rational explanation for the altered regioselectivity. Furthermore, HbpA was found to possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas variant M321F exhibited improved enantioselectivity. Based on the biochemical characterization of several mutants, it was suggested that Trp97 stabilized the substrate in the active site, Met223 was involved in NADH entrance or binding to the active site, and Pro320 might facilitate FAD movement.

The unravelling of the complex pattern of tyrosinase inhibition.

Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor.

A crystal structure of 2-hydroxybiphenyl 3-monooxygenase with bound substrate provides insights into the enzymatic mechanism.

2-Hydroxybiphenyl 3-monooxygenase (HbpA) is an FAD dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. We have determined the structure of HbpA from the soil bacterium Pseudomonas azelaica HBP1 with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5Å to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. The entrance to the active site was confirmed by generating variant G255F which exhibited only 7% of the wild-type's specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications.