Human Myeloperoxidase-Driven Activation of Skin Sensitizing p-Phenylenediamine-related Aromatic Diamines and Phenylpropanoids – Insights from In Chemico and In Silico Approaches

Technical Summary: Human Myeloperoxidase-Driven Activation of Skin Sensitizing p-Phenylenediamine-related Aromatic Diamines and Phenylpropanoids

Problem Statement
Skin sensitization is initiated by the covalent binding of low-molecular-weight chemicals to skin proteins. For "prehaptens," this process requires oxidative activation to generate protein-reactive electrophiles. While abiotic oxidation is a well-established mechanism, the role of enzymatic oxidation within the skin remains poorly understood. Although cytochrome P450 enzymes are traditionally considered the primary candidates for enzymatic activation, their activity in human skin is generally low. Conversely, during the induction phase of skin sensitization, innate immune cells, specifically neutrophils, infiltrate the tissue and release the heme peroxidase myeloperoxidase (MPO). While MPO is known to modulate contact hypersensitivity and has been shown to oxidize specific compounds like eugenol, it is unclear whether human MPO contributes broadly to the oxidative activation of representative skin sensitizers, particularly aromatic diamines (e.g., p-phenylenediamine/PPD) and phenylpropanoids.

Methodology
The study employed an integrated in chemico and in silico approach to investigate the oxidative activation of six compounds: the PPD-related aromatic diamines (PPD, toluene-2,5-diamine/PTD, and 2-methoxymethyl-PPD/ME-PPD) and the phenylpropanoids (eugenol, dihydroeugenol, and cinnamyl alcohol).

1. Molecular Docking: Computational docking (Maestro/Schrödinger) was performed using the crystal structure of human MPO (PDB ID: 1CXP) to predict substrate orientation within the active site. Simulations were conducted at pH 7.4 and additional pH levels (5.0, 6.0, 7.0) to assess the impact of protonation states on binding poses.
2. In Chemico Kinetics: Oxidation reactions were conducted in closed vials with human MPO and hydrogen peroxide. Substrate depletion was quantified via HPLC-UV to determine kinetic parameters ($K_M$, $V_{max}$, $k_{cat}$, and catalytic efficiency $k_{cat}/K_M$) using Michaelis-Menten modeling.
3. Metabolite Profiling: High-resolution mass spectrometry (HRMS) coupled with UPLC was used to characterize oxidation products. Metabolites were identified based on accurate mass, isotopic distribution, and fragmentation patterns.
4. pH-Dependent Screening: UV-Vis spectroscopy monitored the formation of specific oxidation products (e.g., Bandrowski's Base for PPD, quinone methides for eugenol) across a pH range of 5.0 to 7.4.
5. Protein-Binding Prediction: The protein-binding potential of parent compounds and MPO-derived metabolites was assessed using the OECD QSAR Toolbox (version 4.8) to identify structural alerts associated with skin sensitization.

Key Results

• Substrate Selectivity and Binding Orientation: Molecular docking revealed that all tested compounds could bind to the MPO active site with similar scores. However, productive binding—defined by the orientation of the reactive functional group toward the catalytic heme—was observed for PPD, PTD, ME-PPD, eugenol, and dihydroeugenol. In contrast, cinnamyl alcohol adopted a non-productive orientation where the hydroxyl group was hydrogen-bonded to Glu102, positioning the putative oxidation site away from the heme (7.4 Å vs. 4.2–5.3 Å for other substrates).
• Catalytic Efficiency: In chemico experiments confirmed that MPO catalyzed the oxidation of the five susceptible substrates (PPD, PTD, ME-PPD, eugenol, dihydroeugenol) but not cinnamyl alcohol. Despite variations in individual kinetic parameters ($k_{cat}$ and $K_M$), all five convertible substrates exhibited comparable catalytic efficiencies ranging from $10$ to $25 \times 10^3 \text{ mM}^{-1} \times \text{min}^{-1}$.
• pH Dependence: The oxidation of PPD was strongly pH-dependent, with product formation (Bandrowski's Base) decreasing under acidic conditions. This was attributed to the protonation of PPD (pKa ~6.44) at lower pH, which reduced favorable electrostatic interactions within the active site. Conversely, eugenol oxidation remained constant across the pH range, consistent with its lack of protonation changes in this range (pKa ~9.8).
• Metabolite Profiles:
  • Aromatic Diamines: MPO oxidation produced mono-, di-, and trimeric species. PPD oxidation was dominated by the formation of Bandrowski's Base (BB). PTD and ME-PPD yielded more diverse patterns of oligomers.
  • Phenylpropanoids: Eugenol and dihydroeugenol oxidation resulted in monomeric quinone methides and dimeric coupling products. No higher oligomers were observed for these compounds.
• Protein-Binding Potential: QSAR analysis predicted that the parent compounds lacked structural alerts for protein binding. However, several MPO-derived metabolites, particularly those containing benzoquinone diimine or quinone methide motifs, were predicted to possess protein-binding potential.

Significance and Claims
The paper claims to identify a previously unrecognized role for neutrophil-derived MPO as an oxidative activation system during the induction phase of skin sensitization. The study demonstrates that:

1. MPO acts as a substrate-selective enzyme capable of generating protein-reactive metabolites from chemically susceptible sensitizers (aromatic diamines and phenylpropanoids).
2. Productive substrate orientation within the active site, rather than binding affinity alone, governs MPO-mediated oxidation.
3. MPO-mediated oxidation extends current concepts of oxidative activation beyond abiotic oxidation and constitutive epidermal metabolism (e.g., N-acetylation or P450 metabolism).
4. While MPO generates highly reactive intermediates (such as BB and quinone methides) predicted to bind proteins, the study modestly notes that catalytic efficiency alone does not predict overall skin sensitization potency (NESIL), as sensitization involves multiple downstream biological events.

The authors conclude that MPO may influence the local chemical fate of susceptible sensitizers at sites of chemical exposure, suggesting that the immune response itself (via neutrophil recruitment) can modulate the activation of sensitizers. Future work is suggested to define the quantitative contribution of this pathway using direct protein adduct analysis and complex human-relevant skin models.