Elsevier

Free Radical Biology and Medicine

Volume 115, 1 February 2018, Pages 421-435
Free Radical Biology and Medicine

Original article
The 3-phenylcoumarin derivative 6,7-dihydroxy-3-[3′,4′-methylenedioxyphenyl]-coumarin downmodulates the FcγR- and CR-mediated oxidative metabolism and elastase release in human neutrophils: Possible mechanisms underlying inhibition of the formation and release of neutrophil extracellular traps

https://doi.org/10.1016/j.freeradbiomed.2017.12.012Get rights and content

Highlights

  • 3-PD 5 downmodulates neutrophil ROS generation, phagocytosis, and degranulation.

  • 3-PD 5 downmodulates the production and release of neutrophil extracellular traps.

  • 3-PD 5 downmodulates neutrophil chemotaxis towards fMLP but not FPR1 expression.

  • 3-PD 5 is not cytotoxic to neutrophils and does not interfere with microbial killing.

  • 3-PD 5 directly interacts with three catalytic residues of myeloperoxidase.

Abstract

In this study, we report the ability of a set of eight 3-phenylcoumarin derivatives bearing 6,7- or 5,7-dihydroxyl groups, free or acetylated, bound to the benzopyrone moiety, to modulate the effector functions of human neutrophils. In general, (i) 6,7-disubstituted compounds (5, 6, 19, 20) downmodulated the Fcγ receptor-mediated neutrophil oxidative metabolism more strongly than 5,7-disubstituted compounds (21, 22, 23, 24), and (ii) hydroxylated compounds (5, 19, 21, 23) downmodulated this neutrophil function more effectively than their acetylated counterparts (6, 20, 22, 24, respectively). Compounds 5 (6,7-dihydroxy-3-[3′,4′-methylenedioxyphenyl]-coumarin) and 19 (6,7-dihydroxy-3-[3′,4′-dihydroxyphenyl]-coumarin) effectively downmodulated the neutrophil oxidative metabolism elicited via Fcγ and/or complement receptors. Compound 5 also downmodulated the immune complex-stimulated phagocytosis, degranulation of elastase, and production and release of neutrophil extracellular traps, as well as the human neutrophil chemotaxis towards n-formyl-methionyl-leucyl-phenylalanine, without altering the expression level of formyl peptide receptor type 1. Both compounds 5 and 19 did not impair the neutrophil capacity to recognize and kill Candida albicans. Docking calculations revealed that compounds 5 and 19 directly interacted with three catalytic residues – Gln-91, His-95, and Arg-239 – inside the myeloperoxidase active site. Together, these findings indicate that (i) inhibition of reactive oxygen species generation and degranulation of elastase are closely associated with downmodulation of release of neutrophil extracellular traps; and (ii) compound 5 can be a prototype for the development of novel immunomodulating drugs to treat immune complex-mediated inflammatory diseases.

Introduction

Neutrophils are innate immune cells that represent about 70% of the circulating leukocytes in human, and play important roles in the onset and resolution of the inflammatory process. These cells use a framework of mechanisms to eliminate infectious agents, including phagocytosis, production of reactive oxygen species (ROS), release of antimicrobial components from their cytoplasmic granules, and production and release of neutrophil extracellular traps (NETs) and inflammatory mediators that modulate the response of other immune cells [1].

Several studies have sought to understand the relevance of neutrophils to the resolution of the inflammatory process, as well as to discover new targets and therapeutic approaches to minimize the harmful consequences of excessive infiltration and activation of these cells in the inflammation site [2]. One strategy is to search for molecules that modulate the effector functions of neutrophils without impairing the host immune response. Among the natural compounds, phenolic compounds seem to be promising prototypes for the development of novel anti-inflammatory and immune modulating drugs. Some flavonoids and coumarins selectively modulate the oxidative metabolism and other effector functions of neutrophils [3], [4], [5], [6].

Since the 1990's, our research team has been investigating the immune modulating action of phenolic compounds, in particular flavonoids, coumarins, and phenylcoumarin derivatives [7]. This study focuses on 3-phenylcoumarin derivatives (3-PDs), also known as 3-arylcoumarins or aryl-3-coumarins, which constitute a class of plant secondary metabolites with poorly described biological effects. Among the naturally occurring 3-PDs, 6,7-dihydroxy-3-phenylcoumarin and GU-7 (3-(3,4-dihydroxyphenyl)-8-hydroxymethyl-8-methyl-5-methoxy-6,7-dihydro-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one) display anticoagulant activity [8], [9], indicanine A (4-hydroxy-5-methoxy-3-(4′-methoxyphenyl)-2′′-(1-methylethenyl)dihydrofurano[4′′,5′′:6,7]coumarin) and indicanine B (4-hydroxy-3-(4′-hydroxyphenyl)-5-methoxy-2′′,2′′-dimethylpyrano [5′′,6′′:6,7] coumarin) display antimicrobial activity [10], [11], and 7,2′,4′-trihydroxy-3-arylcoumarin increases caspase-3 activity in H4IIE rat hepatoma cells [12].

The interest in chemical modification of the 3-phenylcoumarin moiety has grown in the last decade, and medicinal chemists have synthesized series of compounds with diverse biological effects, such as trypanocidal [13], [14], antidepressant [15], [16], antioxidant [14], [17], antiviral [18], and inhibitor of the activity of lipoxygenases [17], steroid-sulfatases [19], and protein kinase C (PKC) [20]. We have already reported the synthesis of a set of twenty 3-PDs (120) and screened their ability to scavenge free radicals, inhibit horseradish peroxidase activity, and modulate ROS generation in rabbit neutrophils [21], [22]. The qualitative and the two- and three-dimensional quantitative structure-activity relationship analyses have evidenced that the presence of the 3′,4′-dihydroxyl, 3′,4′-diacetoxyl or 3′,4′-methylenedioxyl group associated to at least one hydroxyl or acetoxyl group in the coumarin moiety are the structural requirements for the aforementioned biological effects of 3-PDs.

As the ultimate goal of our research is to find new prototypes for the development of drugs that can be applied in the treatment of human inflammatory diseases, we have conducted the subsequent experiments in human cells. We have selected a set of ten 3-PDs (1, 2, 5–10, 19, 20) to validate their effects in the Fcγ receptor (FcγR)-mediated oxidative metabolism of human neutrophils; the most active compounds (5, 6, 19, and 20) selectively modulate different steps of ROS production by inhibiting myeloperoxidase (MPO) activity without interfering with NADPH oxidase activity [23]. In this previous report, we have tested only di-substituted compounds bearing vicinal groups; in the present study, we addressed to what extent the presence of non-vicinal free or acetylated di-hydroxyl groups influences the immune modulating effect of 3-PDs. We compared the ability of the aforementioned four bioactive compounds with the ability of four new synthetic 3-PDs – named as 21, 22, 23, and 24 – to inhibit the human neutrophil oxidative metabolism elicited by immune complexes (IC), via FcγR. Next, to get first insights into the mechanisms by which 3-PDs act, we selected the most active compounds and examined whether they: (i) modulate the human neutrophil superoxide and total ROS generation elicited via FcγR and complement receptors (CR), either alone or in cooperation, as well as via PKC; (ii) modulate other effector functions of human neutrophils, including phagocytosis, degranulation, microbial killing, and formation and release of NETs; (iii) impair neutrophil chemotaxis; (iv) affect the expression levels of membrane receptors; and (v) inhibit the activity of MPO. We also performed docking calculations to examine how the 3-PDs interact with the catalytic site of MPO.

Section snippets

Chemicals

Bovine serum albumin, cytochalasin B, cytochalasin D, diphenyleneiodonium chloride (DPI), fluorescein isothiocyanate (FITC), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), lucigenin (bis-N-methylacridinium nitrate), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), ovalbumin, phorbol-12-myristate-13-acetate (PMA), Trypan Blue, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human leukocyte myeloperoxidase (MPO, EC 1.7.1.11), H2O2,

3-PDs modulate the human neutrophil oxidative metabolism elicited via FcγR and/or CR

First, we assessed whether the new 3-PDs 21–24 were cytotoxic towards human neutrophils. Compared with the untreated control, the four new 3-PDs did not significantly decrease neutrophil cell viability or increase LDH release, indicating that they were not cytotoxic, at least under the assessed conditions (Table 1). We have previously reported that the 3-PDs 5, 6, 19, and 20 are not cytotoxic towards human neutrophils, under the same experimental conditions [23].

Second, we used the CL-luc and

Discussion

This study is part of an ongoing investigation on the antioxidant and immune modulating properties of a series of synthetic 3-PDs bearing free and acetylated hydroxyl groups [21], [22], [23]. Here we examined the immune modulating effect of eight 3-PDs bearing the 3′,4′-dihydroxyl or 3′,4′-methylenedioxyl group associated with the 6,7- or 5,7-dihydroxyl group, as well as their respective acetylated analogues. In general, we found that changing the position of the dihydroxyl or diacetoxyl group

Concluding remarks

In the set of 3-PDs tested herein, compound 5 stood out as a promising candidate for the development of novel drugs that can be applied in the treatment of IC-mediated inflammatory diseases characterized by IC deposition, neutrophil activation, extracellular release of toxic molecules via frustrated phagocytosis, and production and release of NETs. This 3-PD lowers the neutrophil O2•- and total ROS levels by scavenging the oxidant species and suppressing MPO activity but not NADPH oxidase

Acknowledgments

The authors thank the colleagues from the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil, Dr. Larissa Fávaro Marchi and Prof. Marcelo Dias Baruffi, PhD, for scientific discussions, Mr. Alcides Silva Pereira and Mrs. Nadir Mazzucato for their helpful technical assistance, and Mrs. Fabiana Rossetto de Morais for helping us to perform the flow cytometry analyses.

This study was supported by the Brazilian funding agencies São Paulo Research Foundation (FAPESP,

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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