Small-Molecule Host-Defense Peptide Mimetic Antibacterial and Antifungal Agents Activate Human and Mouse Mast Cells via Mas-Related GPCRs

Abstract

Host-defense peptides (HDPs) have an important therapeutic potential against microbial infections but their metabolic instability and cellular cytotoxicity have limited their utility. To overcome these limitations, we utilized five small-molecule, nonpeptide HDP mimetics (smHDPMs) and tested their effects on cytotoxicity, antimicrobial activity, and mast cell (MC) degranulation. None of the smHDPMs displayed cytotoxicity against mouse 3T3 fibroblasts or human transformed liver HepG2 cells. However, one compound had both antifungal and antibacterial activity. Surprisingly, all five compounds induced degranulation in a human MC line, LAD2, and this response was substantially reduced in Mas-related G protein-coupled receptor (GPCR)-X2 (MRGPRX2)-silenced cells. Furthermore, all five compounds induced degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was abolished in cells expressing naturally occurring loss-of-function missense variants G165E (rs141744602) and D184H (rs372988289). Mrgprb2 is the likely mouse ortholog of human MRGPRX2, which is expressed in connective tissue MCs (CTMCs) such as cutaneous and peritoneal MCs (PMCs). All five smHDPMs induced degranulation in wild-type PMCs but not in cells derived from Mrgprb2⁻/⁻ mice. These findings suggest that smHDPMs could serve as novel targets for the treatment of drug-resistant fungal and bacterial infections because of their ability to harness CTMCs' host defense functions.

Keywords: Host-defense peptide mimetics; MRGPRX2; Mast cells; Mrgprb2.

Figure 1

Structure and antifungal activity of three smHDPMs compounds (Cpd). (A) Structure of Cpd 1, Cpd 2, and Cpd 3. (B) Minimum inhibitory concentration (MIC) values (μg/mL) of smHDPMs against C. albicans, A. fumigatus, and A. flavus. Cytotoxicity of smHDPMs on 3T3 and HepG2 cell lines. CC_50; concentration of drug that reduces 50% cell viability.

Figure 2

smHDPMs activate human mast cells via MRGPRX2. (A) LAD2 cells pretreated with or without pertussis toxin (PTx, 100 ng/mL, 16h) were exposed to vehicle (Control) or smHDPMs compounds (Cpd) 1, 2, and 3 (3 µM each) for 30 min and percentage of β-hexosaminidase release was measured. (B) Western blotting was performed to determine the expression level of MRGPRX2 in Control and MRGPRX2 knockdown LAD2 cells. (C) Control and knockdown cells were stimulated with smHDPMs Cpd 1, 2, and 3 (3 µM each) and percentage of β-hexosaminidase release was determined. All the points are expressed as a mean ± SEM of three experiments in triplicate. Statistical significance was determined by two-tailed unpaired t-Test. *** indicates p value <0.001,** indicates p value <0.01 and * indicates p value <0.05.

Figure 3

smHDPMs induce degranulation in RBL-2H3 cells expressing MRGPRX2. (A) Untransfected RBL-2H3 cells (RBL-2H3) and cells stably expressing MRGPRX2 (RBL-2H3-MRGPRX2) were exposed to vehicle (Control) or smHDPMs, (Cpd 1, 2, and 3, 3 µM) for 30 min and percentage of β-hexosaminidase release was determined. Concentration–response curves for (B) Cpd 1, (C) Cpd 2, and (D) Cpd 3 were determined using RBL-2H3-MRGPRX2 cells. Data are presented as a mean ± SEM and are representative of three independent experiments in triplicate. Statistical significance was determined by two-tailed unpaired t-Test. *** indicates p value <0.001.

Figure 4

Structure and antibacterial activity of three smHDPMs compounds. (A) Structure of compound (Cpd) 2 and two of its derivatives, Cpd 4 and Cpd 5. (B) Minimum inhibitory concentration (MIC) values (μg/mL) of smHDPMs against three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia) and two Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis). Cytotoxicity of smHDPMs on 3T3 and HepG2 cell lines. CC₅₀ concentration of drug that reduces 50% cell growth.

Figure 5

Compound 4 and Compound 5 induce degranulation in RBL-2H3 cells expressing MRGPRX2. (A) Untransfected RBL-2H3 cells (RBL-2H3) and cells stably expressing MRGPRX2 (RBL-2H3-MRGPRX2) were exposed to vehicle (Control), smHDPMs compound (Cpd) 4, and Cpd 5 (3 µM each) for 30 min and percentage of β-hexosaminidase release was determined. Concentration–response curves for (B) Cpd 4 and (C) Cpd 5 were performed using RBL-2H3-MRGPRX2 cells. Data are presented as mean ± SEM and are representative of three independent experiments in triplicate. Statistical significance was determined by two-tailed unpaired t-Test. *** indicates p value <0.001.

Figure 6

smHDPMs induce degranulation in PMCs via Mrgprb2. (A) Genotyping of wild-type (WT) and Mrgprb2⁻/⁻ by PCR using Mrgprb2-specific primers. (B) PMCs isolated from WT and Mrgprb2⁻/⁻ mice were exposed to IgE (1 µg/mL, 16 h) and then stimulated with antigen (DNP-BSA, 100 ng/mL, 30 min) and β-hexosaminidase release was determined. (C) PMCs were stimulated with compound 48/80 (10 µg/mL) or smHDPMs (Cps 1–5, 10 µM) for 30 min and β-hexosaminidase release was determined. (D) BMMCs were exposed to IgE (1 µg/mL, 16 h) and stimulated with antigen (DNP-BSA, 100 ng/mL), 48/80 (10 µg/mL), smHDPMs (10 µM) for 30 min and β-hexosaminidase release was determined. All the points expressed as a mean ± SEM of three experiments in triplicate. Statistical significance was determined by two-way and one-way ANOVA. **** indicates p value <0.0001, *** indicates p value <0.001 and ** indicates p value <0.01.

Figure 7

Missense MRGPRX2 variants do not respond to smHDPMs for degranulation. (A) RBL-2H3 cells were transiently transfected with cDNA encoding MRGPRX2 or its missense variants D184H and G165E. Receptor expression was determined by flow cytometry using anti-MRGPRX2-PE antibody. (B) RBL-2H3 expressing MRGPRX2 variants (D184H and G165E) were stimulated with smHDPMs (Cpds 1–5, 3.0 µM) for 30 min and β-hexosaminidase release was determined. All the points are expressed as a mean ± SEM of three experiments in triplicate. Statistical significance was determined by one-way ANOVA. **** indicates p value <0.0001,*** indicates p value <0.001 and ** indicates p value <0.01.