Identification of complement inhibitory activities of two chemotherapeutic agents using a high-throughput cell imaging-based screening assay

Abstract

Excessive complement activation contributes significantly to the pathogeneses of various diseases. Currently, significant developmental research efforts aim to identify complement inhibitors with therapeutic uses have led to the approval of one inhibitor for clinical use. However, most existing complement inhibitors are based on monoclonal antibodies, which have many drawbacks such as high costs and limited administration options. With this report, we establish an inexpensive, cell imaging-based high-throughput assay for the large-scale screening of potential small molecule complement inhibitors. Using this assay, we screened a library containing 3115 bioactive chemical compounds and identified cisplatin and pyridostatin as two new complement inhibitors in addition to nafamostat mesylate, a compound with known complement inhibitory activity. We further demonstrated that cisplatin and pyridostatin inhibit C5 convertases in the classical pathway of complement activation but have no effects on the alternative pathway of complement activation. In summary, this work has established a simple, large-scale, high-throughput assay for screening novel complement inhibitors and discovered previously unknown complement activation inhibitory activities for cisplatin and pyridostatin.

Keywords: Complement; complement inhibitor; high-throughput; screening; small compound.

Figure 1.

Evaluation of complement activity using automated cell imaging and image analysis. A-D, Representative images of wells with E^shA in the presence of absence of complement. E^shA were incubated in wells of a 384 well plate the presence (A) or absence (B) of complement, then the numbers of intact red blood cells were analyzed by the Operetta image system. A, B, Digital phase-contrast images. C, D, Automated image analysis. Intact E^shA are outlined in green. E, Complement inhibitory effect of EDTA as determined by imaging and image analysis. F, Correlation between the inhibition values obtained from two independent experiments with a set of 353 small molecules. EXP, experiment.

Figure 2.

Screening of 3115 individual small molecules and identification of several potential complement inhibitors. A, The complement inhibitory activities of 3115 compounds are plotted relative to the negative control. Compound IDs are listed on the X-axis, while complement inhibition (%) is shown along the Y-axis. Compound names are listed for the small molecules with the strongest inhibitory activities. B-G, representative digital phase-contrast images of control wells (B-C) and wells containing treated red blood cells (D-G). NHS, normal human serum.

Figure 3.

Further analysis of the hit compounds and others in a concentration-dependent manner. A, Nafamostat mesylate, a known complement inhibitor, was independently identified using our screening method. B-D, Our screening method identified three new complement activation inhibitors—cisplatin, pyridostatin, and cDPCP. All three compounds inhibit complement activation in a concentration-dependent manner. E-F, Cisplatin and pyridostatin inhibit the classical pathway (CP) of complement activation in a sensitized sheep erythrocyte (E^shA)-based hemolysis assay (upper panels), but not in the alternative pathway (AP) in a rabbit erythrocyte (E^rabb)-based hemolysis assay (lower panels). G-H. Two other platinum-containing agents, oxaliplatin and carboplatin, do not inhibit complement activation. All experiments were performed in duplicate, and the results are shown as mean ± standard deviations from one representative experiment. NHS, normal human serum.

Figure 4.

Classical pathway C3 and C5 convertase inhibition assay The inhibitory activities of cisplatin and pyridostatin against the C3 and C5 convertases in the classical pathway of complement activation were evaluated in an antibody-sensitized sheep erythrocyte (E^shA) hemolysis assay. A, Effects of cisplatin on C3 convertase. B, Effects of pyridostatin on C3 convertase. C, Effects of cisplatin on C5 convertase. D, Effects of pyridostatin on C5 convertase. Equivalent volumes of PBS were used as controls (PBS). *p <0.05.