Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation

Abstract

Introduction: The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885.

Methods: The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers.

Results: Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies.

Conclusion: Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Keywords: C5 R885 variants; C5 cleavage; C5b6; MAC formation; RBC hemolysis; complement activation; macrocyclic peptide inhibitor; permeability.

Figure 1

Zilucoplan binds human C5 and blocks complement-mediated hemolysis in vitro. (A) SPR assay shows the binding of zilucoplan with human C5 (SPR sensorgram as fluctuating lines and fitting as linear lines at each concentration). (B) Zilucoplan inhibits the lysis of antibody-sensitized sheep red blood cells exposed to 1% NHS. (C) ELISA measurement of C5a and sC5b9 in the supernatant of RBC hemolysis described in panel (B, D) Wieslab assay to measure the production of MAC in CP, LP, and AP activation. (E) Inhibition of ABO incompatibility-based hemolysis by zilucoplan. (F) Graphical representation (generated using PyMOL 2.4.0 based on PDB 5I5K) of the binding site of a peptide analog of zilucoplan (in red, unpublished data) on C5 TED (in yellow), R885 (in orange) as part of eculizumab epitopes on C5b (in gray and including TED), and the cleavage site at R751 (in green) within C5a (in blue). (G) Zilucoplan inhibition of C5 (200 nM) binding to surface C3b measured by SPR, shown as mean ± SD (normalized to the signal at zero zilucoplan, N = 5). (H) Zilucoplan inhibited the deposition of MAC on HUVECs. *, p value <0.05. Fluorescence intensity was shown as mean ± SD (n = 3 wells in each group). Statistical significance was analyzed by ANOVA (Kruskal–Wallis test) using GraphPad software (Version 9.2.0). (I) The activity of hemolysis inhibition of zilucoplan is potent in non-human primates and drastically reduced in guinea peak, rats, and mice. SPR, surface plasmon resonance; NHS, normal human serum; RBC, red blood cell; MAC, membrane attack complex; CP, classical pathway; LP, lectin pathway; AP, alternative pathway; TED, thioester-like domain; HUVECs, human umbilical vein endothelial cells.

Figure 2

Zilucoplan blocks complement activation by C5 variants R885C/H. (A, B) Inhibition of the lysis of EA upon the activation of rhC5 R885C (A) or R885H (B) was tested for zilucoplan (circle) or eculizumab biosimilar (triangle). (C, D) SPR measurement (in a single cycle kinetics mode using Biacore 8K) of zilucoplan or eculizumab biosimilar to rhC5 R885 (C) or R885H (D) at 37°C. Surface densities of C5 proteins for zilucoplan testing: sera C5 wt-5970RU, rhC5 wt-6874RU, rhC5 R885C-6496RU, and rhC5 R885H-6145RU. Zilucoplan concentrations used: 0, 0.31, 0.62, 1.25, and 2.5 nM. Surface densities of C5 proteins for eculizumab testing: sera C5 wt-715RU, rhC5 wt-840RU, rhC5 R885C-731RU, and rhC5 R885H-767RU. Eculizumab biosimilar concentrations used: 0, 0.31, 0.62, 1.25, and 2.5 nM on C5 wt and 0, 11, 33, 100, and 300 nM on C5-R885C/H. Binding kinetics and affinity for zilucoplan and eculizumab biosimilar on C5 wt from human sera or recombinant forms (rhC5 wt, R885C, and R885H) from two independent measurements are also summarized in tabular format. For the clarity of figures, only the SPR sensorgrams for the mutants are shown. SPR, surface plasmon resonance.

Figure 3

Zilucoplan destabilizes C5b6 and inhibits C5b6-mediated hemolysis lysis. (A) Schematic depiction (using PyMOL 2.4.0 based on PDB 4a5w) of the C5b6 complex. C5b is in gray except for the C5 TED in yellow, proposed binding sites for zilucoplan peptidyl portion based on a peptide analog (unpublished data) in pink, and C6 in lime. (B) Native gel analysis of the C5b6 complex in the presence and/or absence of zilucoplan. Lanes 1 and 6: C5b6 incubated at 37°C/h with (Lane 1) or without (Lane 6) 1% DMSO. Lanes 2–6: C5b6 after incubation with various concentrations of zilucoplan (in 1% DMSO) at 37°C for 1 h prior to gel loading. Lanes 7 and 8: C5 and C6 alone. (C) Zilucoplan inhibited the plasmin-mediated lysis of EA. Plasmin without or with increasing concentrations of zilucoplan was added to a mixture of C5 and C6 and incubated for 1 h. The plasmin reaction solution was then added to EA before mixing with purified C7, C8, and C9 to proceed with the lysis. More details are described in the Materials and Methods. Note that no sera were used to exclude any contribution by C3/C5 convertases. (D) A schematic diagram of the dual mechanism of action for zilucoplan: zilucoplan binds C5 (gray/dark blue) to prevent C5 cleavage into C5a (dark blue) and C5b (gray) by C5 convertases (light blue) and remains on C5b to prevent the formation of C5b6 (C6 in green) mediated by nonspecific proteases, both contributing to the blockade of MAC formation (red). TED, thioester-like domain; DMSO, dimethyl sulfoxide.

Figure 4

Analysis of the permeability of zilucoplan and eculizumab biosimilar. In a Matrigel-coated transwell assay, the concentrations of zilucoplan were measured by HPLC-MS to calculate the ratio between the lower and upper chambers. Fluorescence intensities of AF488-labeled eculizumab biosimilar or FITC-dextran (4 or 150 kDa) were measured in each chamber separately to calculate their respective ratios. Error bars represent ranges based on two or more independent plates with duplicates in each plate. Eculizumab biosimilar labeled with fluorophore AF488; dextran-4 kDa and dextran-150 kDa, FITC-dextran 4 kDa, or FITC-dextran 150 kDa. HPLC-MS, high-performance liquid chromatography–mass spectrometry; FITC, fluorescein isothiocyanate.