Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics
Figure Lengend Snippet: (A) Dose-dependent inhibition of complement by cmp-5 was assessed by a CP ELISA assay where the deposition of C3b, C4b, and MAC in 1% (v/v) serum were monitored. While cmp-5 had no effect on C3b or C4b deposition at cmp-5 concentrations up to 625 μM, an IC50 of 530 μM for cmp-5 was measured when MAC deposition was detected. Detection of (B) C3b or (C) C4b deposition using a CP ELISA in the presence of 250 μM cmp-5 analogues. (D) MAC deposition was measured in the presence of a fixed concentration of cmp-5 (500 μM) or 2.5% (v/v) DMSO vehicle control under varying serum concentrations (0–4% (v/v)). An EC50 of 0.88% (v/v) serum (DMSO control) vs. 1.43% (v/v) serum (cmp-5) was calculated (MAC deposition). (E) A C5a capture ELISA was used to determine the relative amount of C5a produced in the corresponding CP ELISA reactions presented in panel B. An EC50 of 1.55% (v/v) serum (DMSO control) vs. 2.04% (v/v) serum (cmp-5) was observed, indicating a correlation of lower MAC deposition with less C5 cleavage. Data are represented as the mean ± S.D. of three or four independent experiments. All fits were obtained by 4-parameter variable slope nonlinear regression analysis using GraphPad Prism v5.04. (F) An LE-SPR-based competition assay was used to assess whether compstatin or cmp-5 compounds can interfere with C5/C3b binding (see Fig. S3D–F for corresponding assay validation experiments and representative epigrams). A fixed concentration of B23-C5 (250 nM) was co-injected with either unlabeled C5 (250 nM) (i.e. “cold” C5), TRX-4W9A (12 μM), or compstatin peptide (65 μM) in a running buffer of HBS-T. Each of these analytes reduces the B23-C5 specific LE-SPR signal. The presence of 500 μM cmp-5, cmp5-2, and cmp5-14 did not affect the B23-C5-associated LE-SPR signal relative to DMSO vehicle control. Each competition experiment was performed between two and four times (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001). Injections of 250 nM B23-C5 were treated as the control signal for “cold” C5, TRX-4W9A, and compstatin. In contrast, each of the small molecule compounds has been normalized to injections of 250 nM B23-C5 in the presence of vehicle only control (5% (v/v) DMSO).
Article Snippet: As an additional positive control for competition, we used the original version of the compstatin peptide obtained commercially from Tocris.
Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Concentration Assay, Produced, Competitive Binding Assay, Binding Assay, Injection