Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy.

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT-mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

Persister state-directed transitioning and vulnerability in melanoma.

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition.

Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration [I]0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoiding making interpolations between different values of iD. The analytical expression of the IC50, for the different types of inhibition, are related to the apparent inhibition constant (KIapp). Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC50, the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC50 value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in iD and IC50 with substrate concentration. The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition (iD does not depend on [D]0) and the rest. In the case of competitive inhibition, this dependence of iD on [D]0 leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of KI1app is carried out from the IC50 expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of iD vs. n, with n=[D]0/KmD, allows us to distinguish between them. A hyperbolic iD vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of KI2app is immediate from the IC50 value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), KI1app and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), KI2app are obtained from the dependence of iD vs. n, and the results obtained must comply with the IC50 value.

Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase.

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A-ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 µM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.

MITF induces escape from innate immunity in melanoma.

BACKGROUND: The application of immune-based therapies has revolutionized cancer treatment. Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood. In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair. Whether MITF also controls the immune response is unknown. METHODS: By using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response. ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene. Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing. RESULTS: Here we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells. By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system. Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance. CONCLUSION: Our results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.

PRMT1-dependent methylation of BRCA1 contributes to the epigenetic defense of breast cancer cells against ionizing radiation.

The therapeutic effect of irradiation is thought to come from DNA damage that affects rapidly proliferating cancer cells; however, resistant cells rapidly initiate mechanisms to repair such damage. While DNA repair mechanisms responsible for cancer cell survival following DNA damage are understood, less is known about the epigenetic mechanisms resulting in resistance to radiotherapy. Although changes in DNA methylation are related to mechanisms of long-term resistance, it is more likely that the methylation state of a series of proteins could be responsible for the first-line of defense of cancer cells against irradiation. In this study, we observed that irradiation of breast cancer cells was accompanied by an overproduction in S-adenosylmethionine, which increases the activity of cellular methylases. We found that by activating PRMT1, irradiation triggers a BRCA1-dependent program that results in efficient DNA repair and inhibition of apoptosis. Depletion of PRMT1 in irradiated cells resulted in a switch of BRCA1 functions from repair and survival in the nucleus to activation of cell death signals in the cytoplasm. We conclude that by modulating the cellular localization of BRCA1, PRMT1 is an important regulator of the oncogenic functions of BRCA1, contributing to the epigenetic defense of breast cancer cells against ionizing radiation.

Rigid π-Extended Boron Difluoride Complex with Mega-Stokes Shift for Bioimaging.

The rational design of a rigid π-extended ligand, suitable for the formation of four-coordinate boron complexes, has led to the synthesis of the fused hexacyclic structure of carbazolo[2,1-c]phenanthridine. The photophysical characterization of the novel fluorophore revealed a significant Stokes shift whose intramolecular charge transfer origin has been corroborated by computational calculations. The usefulness of the reported N,N-difluoroboryl complex as fluorescent probe with large Stokes shift has been demonstrated for cancer cells imaging.

Acriflavine, a Potent Inhibitor of HIF-1α, Disturbs Glucose Metabolism and Suppresses ATF4-Protective Pathways in Melanoma under Non-Hypoxic Conditions.

Hypoxia-inducible factor (HIF)-1α is constitutively expressed in melanoma cells under normoxic conditions and its elevated expression correlates with the aggressiveness of melanoma tumors. Here, we used acriflavine, a potent inhibitor of HIF-1α dimerization, as a tool to investigate whether HIF-1α-regulated pathways contribute to the growth of melanoma cells under normoxia. We observed that acriflavine differentially modulated HIF-1α-regulated targets in melanoma under normoxic conditions, although acriflavine treatment resulted in over-expression of vascular endothelial growth factor (VEGF), its action clearly downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1), a well-known target of HIF-1α. Consequently, downregulation of PDK1 by acrifavine resulted in reduced glucose availability and suppression of the Warburg effect in melanoma cells. In addition, by inhibiting the AKT and RSK2 phosphorylation, acriflavine also avoided protective pathways necessary for survival under conditions of oxidative stress. Interestingly, we show that acriflavine targets activating transcription factor 4 (ATF4) for proteasomal degradation while suppressing the expression of microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and a melanoma oncogene. Since acriflavine treatment results in the consistent death of melanoma cells, our results suggest that inhibition of HIF-1α function in melanoma could open new avenues for the treatment of this deadly disease regardless of the hypoxic condition of the tumor.

Macrovipecetin, a C-type lectin from Macrovipera lebetina venom, inhibits proliferation migration and invasion of SK-MEL-28 human melanoma cells and enhances their sensitivity to cisplatin.

BACKGROUND: The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. METHODS: Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. RESULTS: Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced the expression of TRADD, p53, Bax, Bim and Bad and down-regulated the Bcl-2 expression and ROS levels in SK-MEL-28 cells. Interestingly, these treatments impaired SK-MEL-28 cell adhesion, migration and invasion through modulating the function and expression of αvß3 integrin along with regulating E-cadherin, vimentin, ß-catenin, c-Src and RhoA expression. In silico study suggested that only the α chain of macrovipecetin interacts with a region overlapping the RGD motif binding site on this integrin. CONCLUSIONS: We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells. GENERAL SIGNIFICANCE: The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.

Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and suicide inactivation.

Different mechanisms for inhibiting tyrosinase can be designed to avoid postharvest quality losses of fruits and vegetables. The action of tyrosinase on caffeic acid and its n-nonyl ester (n-nonyl caffeate) was characterized kinetically in this work. The results lead us to propose that both compounds are suicide substrates of tyrosinase, for which we establish the catalytic and inactivation efficiencies. The ester is more potent as inactivator than the caffeic acid and the number of turnovers made by one molecule of the enzyme before its inactivation (r) is lower for the ester. We proposed that the anti-browning and antibacterial properties may be due to suicide inactivation processes.

Structural and kinetic considerations on the catalysis of deoxyarbutin by tyrosinase.

Deoxyarbutin, a potent inhibitor of tyrosinase, could act as substrate of the enzyme. Oxytyrosinase is able to hydroxylate deoxyarbutin and finishes the catalytic cycle by oxidizing the formed o-diphenol to quinone, while the enzyme becomes deoxytyrosinase, which evolves to oxytyrosinase in the presence of oxygen. This compound is the only one described that does not release o-diphenol after the hydroxylation step. Oxytyrosinase hydroxylates the deoxyarbutin in ortho position of the phenolic hydroxyl group by means of an aromatic electrophilic substitution. As the oxygen orbitals and the copper atoms are not coplanar, but in axial/equatorial position, the concerted oxidation/reduction cannot occur and the release of a copper atom to bind again in coplanar position, enabling the oxidation/reduction or release of the o-diphenol from the active site to the medium. In the case of deoxyarbutin, the o-diphenol formed is repulsed by the water due to its hydrophobicity, and so can bind correctly and be oxidized to a quinone before being released. Deoxyarbutin has been characterized with: [Formula: see text] = 1.95 ± 0.06 s-1 and [Formula: see text] = 33 ± 4 µM. Computational simulations of the interaction of ß-arbutin, deoxyarbutin and their o-diphenol products with tyrosinase show how these ligands bind at the copper centre of tyrosinase. The presence of an energy barrier in the release of the o-diphenol product of deoxyarbutin, which is not present in the case of ß-arbutin, together with the differences in polarity and, consequently differences in their interaction with water help understand the differences in the kinetic behaviour of both compounds. Therefore, it is proposed that the release of the o-diphenol product of deoxyarbutin from the active site might be slower than in the case of ß-arbutin, contributing to its oxidation to a quinone before being released from the protein into the water phase.

Action of tyrosinase on alpha and beta-arbutin: A kinetic study.

The known derivatives from hydroquinone, α and ß-arbutin, are used as depigmenting agents. In this work, we demonstrate that the oxy form of tyrosinase (oxytyrosinase) hydroxylates α and ß-arbutin in ortho position of the phenolic hydroxyl group, giving rise to a complex formed by met-tyrosinase with the hydroxylated α or ß-arbutin. This complex could evolve in two ways: by oxidizing the originated o-diphenol to o-quinone and deoxy-tyrosinase, or by delivering the o-diphenol and met-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the o-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of o-quinone, generating o-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize α and ß-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 ± 0.58 mM and 3 ± 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for ß-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 ± 0.33 s-1 and 3.7 ± 0.29 s-1 for α and ß-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and ß-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.

Action of 2,2',4,4'-tetrahydroxybenzophenone in the biosynthesis pathway of melanin.

2,2',4,4'-tetrahydroxybenzophenone (Uvinul D50), a sunscreen used in cosmetics, has two effects in the melanin biosynthesis pathway. On the one hand, it acts a weak inhibitor of tyrosinase and on the other, it accelerates the conversion of dopachrome to melanin. Uvinul D50 was seen to behave as a weak competitive inhibitor: apparent constant inhibition=2.02±0.09mM and IC50=3.82±0.39mM established in this work. These values are higher than those in the bibliography, which tend to be undersetimated. This discrepancy could be explained by the reaction of Uvinul D50 with the dopachrome produced from l-tyrosine or l-dopa, which would interfere in the measurement. Based on studies of its docking to tyrosinase, it seems that Uvinul D50 interacts with the active site of the enzyme (oxytyrosinase) both in its protonated and deprotonated forms (pKa=7). However, it cannot be hydroxylated, meaning that it acts as a weak inhibitor, not as an alternative substrate, despite its resorcinol structure. Uvinul D50 can be used as sunscreen, in low concentrations without significant adverse effects on melanogenesis.

Characterization of the action of tyrosinase on resorcinols.

The action of tyrosinase on resorcinol and some derivatives (4-ethylresorcinol, 2-methylresorcinol and 4-methylresorcinol) was investigated. If the catalytic cycle is completed with a reductant such as ascorbic acid or an o-diphenol such as 4-tert-butylcatechol, these compounds act as substrates of tyrosinase in all cases. The reaction can also be carried out, adding hydrogen peroxide to the medium. All the above compounds were characterized as substrates of the enzyme and their kinetic constants, KM (Michaelis constant) and kcat (catalytic constant) were determined. Measurement of the activity of the enzyme after pre-incubation with resorcinol, 4-ethylresorcinol or 4-methylresorcinol points to an apparent loss of activity at short times, which could correspond to an enzymatic inactivation process. However, if the measurements are extended over longer times, a burst is observed and the enzymatic activity is recovered, demonstrating that these compounds are not suicide substrates of the enzyme. These effects are not observed with 2-methylresorcinol. The docking results indicate that the binding of met-tyrosinase with these resorcinols occurs in the same way, but not with 2-methylresorcinol, due to steric hindrance.

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells.

Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.

Tyrosinase-Catalyzed Hydroxylation of 4-Hexylresorcinol, an Antibrowning and Depigmenting Agent: A Kinetic Study.

4-Hexylresorcinol (HR) is a compound used in the food and cosmetic industries as an antibrowning and lightening agent. Its use is mainly attributed to its inhibitory effect on the enzyme tyrosinase. However, the enzyme hydroxylates HR to an o-diphenol, which it then oxidizes to an o-quinone, which rapidly isomerizes to p-quinone. For tyrosinase to act in this way, the Eox form (oxy-tyrosinase) must be present in the reaction medium, which can be brought about by (a) hydrogen peroxide, (b) ascorbic acid, or (c) catalytic concentrations of o-diphenol and a reductant (NADH) to maintain it constant. This work demonstrates that HR is a substrate of tyrosinase and proposes a mechanism for its action. Its kinetic characterization provides a catalytic constant of 0.85 ± 0.04 s(-1) and a Michaelis constant of 60.31 ± 6.73 µM.

Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma.

Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p=0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p=0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients.