Conjugation to albumin-binding molecule tags as a strategy to improve both efficacy and pharmacokinetic properties of the complement inhibitor compstatin

Abstract

The compstatin family of complement inhibitors has shown promise in various immuno-inflammatory disorders. Although recent analogues show beneficial pharmacokinetics, further extension of the plasma half-life is expected to benefit systemic application of these peptidic inhibitors. We therefore synthesized conjugates of compstatin analogues and albumin-binding molecules (ABM) to increase circulatory residence. Equilibrium dialysis in complement-depleted serum showed a marked increase in plasma protein binding from <8 % to >99 % for a resulting chimera (ABM2-Cp20). Further analysis confirmed interaction with albumin from different species, primarily via site II. Importantly, ABM2-Cp20 bound 20-fold stronger to its target protein C3b (KD =150 pM) than the parent peptide. Kinetic and in silico analysis suggested that ABM2 occupies a secondary site on C3b and improves the dissociation rate via additional contacts. Addition of an ABM modifier thereby not only improved plasma protein binding but also produced the most potent compstatin analogue to date with potential implications for the treatment of systemic complement-related diseases.

Keywords: albumin binding; complement inhibitor; compstatin; conjugation; peptide drugs.

Figure 1

Structure and proposed binding model of ABM2-Cp20. a) Structure of ABM2-Cp20 with the ABM2 tag shown in red. b) Docking of ABM2-Cp20 (yellow spheres) into the compstatin binding site of C3c (green cartoon/surface representation; PDB code: 2QKI); the primary compstatin binding site and the proposed extended contact site for ABM2 are marked with blue and red arrows, respectively. c) Close-up of ABM2-Cp20 (stick representation) docked to C3c (green surface; positive and negative surface charges are shown in red and blue, respectively). The hydrogen bond between ABM2-Cp20 and lysine residue 386 of C3c (K386) predicted from the computational analysis is highlighted by a white circle.

Scheme 1

Synthesis of conjugates between compstatin analogues Cp20 and Cp40 with albumin-binding molecules. Reagents and conditions: a) coupling of RCOOH: DIPEA, HATU, DMF (R=ABM1), or DIPEA, PyBOP, NMP, CH₂Cl₂ (R=ABM2);^{[17, 18]} b) resin cleavage with 90% TFA, 5% thioanisole, 3% EDT, 2% anisole; c) cyclization with H₂O₂. (Detailed procedures are given in the Supporting Information.)