Fig. 2: Identification and verification of JLJ648, JLJ600 and JLJ555 as dimerizers with pyroptotic activity. | npj Drug Discovery

Fig. 2: Identification and verification of JLJ648, JLJ600 and JLJ555 as dimerizers with pyroptotic activity.

From: Mechanistic basis for a novel dual-function Gag-Pol dimerizer potentiating CARD8 inflammasome activation and clearance of HIV-infected cells

Fig. 2: Identification and verification of JLJ648, JLJ600 and JLJ555 as dimerizers with pyroptotic activity.

a, b Drug screening. Dox-treated cells were incubated with candidate drugs at 5 µM for 3 h before cell death measurement by the LDH release assay. In a, the clone 14 cells were used to determine drug potency. In b, a control CARD8-KO THP-1 cell line expressing HIV-1 Gag-Pol and auto-processing-deficient CARD8 (CARD8-S297A) was used to measure non-specific cell death. c The Dox-treated clone 14 cells were treated with the indicated compounds at 5 µM for 3 h before cell death measurement by the LDH release assay. Multiple comparisons were made using one-way ANOVA followed by Dunnett's test. ****p < 0.0001 d The table summarizes chemical structures of JLJ555, JLJ600, and JLJ648 and the activity values for antiviral replication, cytotoxicity, pyroptotic cell death, and p66 homodimerization using HTRF. The inhibition of antiviral replication (EC50) and cytotoxicity (CC50) were determined in MT-2 in the presence and absence, respectively, of HIV-1 infection using MTT to measure cell viability. In addition, general cytotoxicity was determined in THP-1 and CD4+ T cells. Pyroptotic cell killing in CD4+ T cells was determined as described in e. Table prepared using Microsoft PowerPoint. The cell killing dose responses. CD4+ T cells were isolated from PBMCs and co-stimulated with anti-CD3 and anti-CD28 antibodies for 3 days. Stimulated cells were infected with VSVG pseudotyped HIV-1 reporter virus NL4-3-ΔEnv-GFP. JLJ555, JLJ600, and JLJ648 in successive three-fold dilutions were used to treat infected cells for 2 days before GFP measurement by flow cytometry. f p66 homodimerization dose response curves. Serial dilutions of JLJ555, JLJ600, and JLJ648 were incubated with 12.5 nM Hp66 and 25 nM HTFp66 for 17 h prior to the addition of A-6HIS-Eu donor and anti-FLAG-M2-XL665 acceptor antibodies. Percent effect is calculated by normalizing the background-subtracted ratios of acceptor:donor fluorescence at each compound concentration to that with the highest acceptor:donor ratio. Data shown is the average of three separate assays performed in triplicate. Panels prepared using GraphPad Prism unless otherwise specified.

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