Identification of Dual-Target Compounds with Antifungal and Anti-NLRP3 Inflammasome Activity

Abstract

Invasive and superficial infections caused by the Candida species result in significant global morbidity and mortality. As the pathogenicity of these organisms is intimately intertwined with host immune response, therapies to target both the fungus and host inflammation may be warranted. Structural similarities exist between established inhibitors of the NLRP3 inflammasome and those of fungal acetohydroxyacid synthase (AHAS). Therefore, we leveraged this information to conduct an in silico molecular docking screen to find novel polypharmacologic inhibitors of these targets that resulted in the identification of 12 candidate molecules. Of these, compound 10 significantly attenuated activation of the NLPR3 inflammasome by LPS + ATP, while also demonstrating growth inhibitory activity against C. albicans that was alleviated in the presence of exogenous branched chain amino acids, consistent with targeting of fungal AHAS. SAR studies delineated an essential molecular scaffold required for dual activity. Ultimately, 10 and its analog 10a resulted in IC₅₀ (IL-1β release) and MIC₅₀ (fungal growth) values with low μM potency against several Candida species. Collectively, this work demonstrates promising potential of dual-target approaches for improved management of fungal infections.

Keywords: AHAS; Candida; NLRP3; antifungal; dual-target; inflammasome.

Figure 1.

Approach used to identify dual-target inhibitors. (A) Established inhibitors of the NLRP3 inflammasome include sulfonylureas MCC950 and hypoglycemic agents such as glyburide. Inhibitors of yeast acetohydroxyacid synthase (AHAS) include a series of herbicidal sulfonylurea agents including chlorimuron ethyl and sulfometuron. Identical chemical features in both sets of compounds are highlighted in green and similar chemical features are highlighted in blue. The predicted NLRP3 and known AHAS binding sites are indicated by red circles. (B) The Maybridge screening collection of over 53000 compounds was used to perform in silico molecular docking for both NLRP3 and AHAS. Compounds scoring in the top 1% for each target were cross-referenced and common hits were ordered for experimental validation. Generally, compounds were first tested for their capacity to inhibit IL-1β release in THP1 cells stimulated with the inflammasome inducers LPS and ATP. Compounds showing good efficacy were tested for their capacity to inhibit C. albicans growth. Compounds possessing both anti-inflammatory and antifungal properties at a dose of 50 μM were further characterized by establishing inhibitory concentration 50 (IC₅₀) and modified minimal inhibitory concentration (MIC) values.

Figure 2.

AHAS and NLRP3 Binding Sites. (A) MCC950 and key binding residues in proposed NLRP3 binding site. (B) 2D ligand interaction diagram of MCC950 in NLRP3 binding site. (C) Sulfometuron methyl and key binding residues in Sc-AHAS active site. FAD cofactor visible in background. (D) 2D ligand interaction diagram of sulfometuron methyl in AHAS active site.

Figure 3.

Compound 10 identified by molecular docking possesses both anti-inflammasome and antifungal activity. (A) THP1 cells were treated with 50 μM of each lead compound or vehicle alone (0.5% DMSO) for 1 h, followed by 20 ng of LPS for 3.5 h, and then 5 mM ATP for 30 min where indicated. IL-1β was measured by ELISA. (B) THP1 cells were used as described in panel A and treated with vehicle, 50 μM compound 10, or 0.1 μM MCC950. Processed Caspase-1 was measured by bioluminescence assay and values expressed as relative light units (RLU). (C) ASC-Speck GFP reporter cells were used and treated as described in panel B. The number of GFP+ Specks were enumerated in 10 random fields and calculated as percentage of the vehicle control. Cell culture experiments were conducted in technical quadruplicate (or duplicate for imaging) and results reported as the mean plus SD from independent experiments (n = 3). * indicates p < 0.05 using one-way ANOVA and Dunnet’s post-test. (D) A growth assay was performed by growing 2.5 × 10³ C. albicans cells in YNB without amino acids for 24 h supplemented with 50 μM compound 10 or vehicle only. Images were captured on a digital scanner and are representative of 3 independent experiments. (E) Growth curves were conducted and OD600 nm monitored kinetically at 30 °C for 24 h in YNB media without (closed shapes) and with (open shapes) 10 mM each isoleucine and valine in the presence (red) or absence (black) of 50 μM compound 10. Experiments were conducted in biological triplicate and reported as mean plus SD * indicates p < 0.05 using multiple t test and Holm-Sidak post-test. (F) WT (SC5314) or genetically altered strains with respect to C. albicans AHAS expression (Δilv2/ILV2, Δ/Δilv2+PrTEF1-ILV2, Δ/Δilv2+PrACT1-ILV2) were grown in vehicle or 50 μM compound 10. Percent growth inhibition was calculated for each strain and normalized to WT values (black bars, left y-axis). Expression levels of ILV2 were assessed for each strain at the same time point using qRT-PCR (gray bars, right y-axis). Values were calculated using the ΔΔC_T method by comparing to the housekeeping gene ACT1 and strain SC5314. Experiments were conducted in biological triplicate and reported as mean plus SD *, # p < 0.05, **, ## p < 0.01 using one-way ANOVA and Dunnett’s post-test.

Figure 4.

Structure activity relationship of compound 10 analogs with respect to IL-1β inhibition. (A) A series of compound 10 analogs were ordered containing substitutions in key groups deemed important for anti-inflammatory or antifungal activity. (B) THP1 cells were treated with 50 μM of each analog or vehicle alone (0.5% DMSO) for 1 h, followed by 20 ng of LPS for 3.5 h, and then 5 mM ATP for 30 min where indicated. Experiments were conducted in technical quadruplicate and results reported as the mean ± SD from independent experiments (n = 3). * indicates p < 0.05 using one-way ANOVA and Dunnett’s post-test. (C) C. albicans was grown in YNB medium at 30 °C for 24 h and in the presence of vehicle (0.5% DMSO) or each analog (50 μM). After incubation, wells were resuspended by pipetting, OD600 nm measured, and data expressed as percentage of vehicle treated control. Experiments were repeated in biological triplicate and are reported as mean + SD * indicates p < 0.05 using one-way ANOVA and Dunnet’s post-test.

Figure 5.

SAR analysis of the thiobenzoate scaffold. The highlighted portion of the thiobenzoate scaffold is required for both anti-NLRP3 and antifungal activity.

Figure 6.

Binding Site Interactions of compound 10. (A) Compound 10 docking pose in AHAS active site. (B) 2D ligand interaction diagram of compound 10 docked into AHAS active site. (C) compound 10 docking pose in NLRP3 binding site. (D) 2D ligand interaction diagram of compound 10 in NLRP3 binding site.

Figure 7.

Identified dual-target inhibitors demonstrate antifungal activity that is rescued by branched chain amino acid supplementation. C. albicans cells were diluted to 2.5³ cells/mL in YNB without amino acids with (open shapes) or without (closed shapes) 10 mM each isoleucine and valine. Cultures were treated with 0.5% vehicle alone (diamonds), 6.4 μM compound 10 (squares), 5.3 μM compound 10a (circles), or 0.5 μM compound 13 (triangles). Microtiter plates were incubated with orbital shaking at 200 rpm and optical density values at 600 nm collected over 24 h. * indicates p < 0.05 for all compounds when comparing amino acid supplemented and unsupplemented readings at each time point using multiple t tests and Holm-Sidak post-test.