Previously we demonstrated that human astrovirus CP binds C1q (Bonaparte et al., 2008
) and MBL (Hair et al., 2010
), inhibiting classical and lectin pathway activation, respectively. A critical 30 amino acid region of the CP molecule was subsequently identified that bound C1q and inhibited classical pathway activation. A derivative of this construct named E23A was found to have enhanced ability to inhibit classical complement activation. In order to test the ability of this compound to inhibit an important clinical disease process, we utilized an in vitro
model of ABO incompatibility using mismatched human erythrocytes and serum. ABO incompatibility, which can occur due to inadvertent incompatible transfusions or passive transplacental transfer of incompatible maternal antibody to the fetus, results in life threatening rapid intravascular complement-mediated hemolysis of critical importance in neonatal and transfusion medicine (Jantapour et al., 2008
). E23A and AcPA potently inhibited human ABO incompatibility ( and ) demonstrating the potential therapeutic value of CPPs in clinical disease. We have also performed proof-of-concept in vivo
testing of E23A and AcPA ( and ) showing that injection into rats inhibited serum lysis of antibody-sensitized erythrocytes. Thus, these compounds appear to demonstrate similar functionality in vivo
as has been demonstrated in vitro
, such that future determination of pharmacodynamics and pharmacokinetics in this rat model is feasible and warranted.
A fifteen amino acid derivative of the 30 residue coat protein peptide, Δ8-22, retained functional activity and was chosen for further manipulation. Residues were rearranged to yield a construct with the proline in the middle of the sequence and the majority of the charged and polar residues at the carboxy terminus, termed polar assortant (PA) (). As predicted, aqueous solubility of the compound was significantly improved. Interestingly, the ability of this construct to inhibit classical complement activation was increased over Δ8-22 (). Deletion analysis of the PA revealed that removal of one residue at the N or C terminus resulted in loss of inhibitory activity in the hemolytic assay () suggesting that 15 residues is the minimal length of the CPP necessary to inhibit complement activation. Altering the sequence appeared to alter function as shown by the polar assortant (PA). Altering the composition also appeared to affect function as seen with the alanine scan and truncation peptides. We speculate that this improved functionality could be due to having increased the polarity of the molecule, or having a proline-induced angular ‘kink’ in the molecule, or both.
Our previous work has shown that CP can displace the cognate serine proteases C1s-C1r-C1r-C1s from C1q CLR and that substitution of a lysine critical for MASP-2 binding to MBL abolished CP binding to MBL (Hair et al., 2010
). In order to disrupt the interaction of the pattern recognition molecules, C1q and MBL, with their cognate serine proteases, CPPs would be predicted to have similar binding affinities. It has been previously shown by SPR that immobilized C1q binds to its cognate serine proteases (C1s-C1r-C1r-C1s) with a KD
of 2.72 nM (Phillips et al., 2009
). The binding of E23A, Δ8-22, and PA to C1q with similar affinity for C1s-C1r-C1r-C1s (i.e., nM range) ( and ) is consistent with our hypothesis that these peptides may function by competitively displacing, at least partially, C1s-C1r-C1r-C1s from the CLR of C1q. Binding of PA to the CLR of C1q was also verified in a binding assay (). It is of interest to note that while PA displays superior inhibitory activity compared to all the other CPPs, its binding affinity to C1q as assessed by SPR is lower than that of E23A and Δ8-22 (33.3 nM versus 6.08 and 5.43, respectively). It had been previously demonstrated by ELISA that E23A and Δ8-22 bound C1q with lower affinity to other CPPs even though they showed increased inhibitory activity against classical pathway activation (Gronemus et al., 2010
). Thus, tighter binding does not necessarily correlate with inhibitory activity. That is, the overall strength of binding does not inform one about the orientation of the peptide and it may be the case that a lower affinity orientation has a greater effect on preventing the conformational changes necessary to elicit C1s-C1r-C1r-C1s activation. In addition to these SPR results, CPP E23A inhibits lectin pathway activation () suggesting that as with the parental CP molecule, CPPs can also bind MBL to inhibit lectin pathway activation. Studies to analyze the precise molecular details as to how the CPPs bind C1q and MBL to inhibit classical and lectin pathway activation are currently underway.
One of the ultimate goals of this research is to develop the CPP into a therapeutic molecule for use in disease processes induced by dysregulated classical and lectin pathway activation. To our knowledge there has been only one other report of a peptidic inhibitor of classical pathway activation that functions by binding to C1q. This 15 residue cyclical peptide termed 2J (CEGPFGPRHDLTFCW), initially identified through panning a phage-display library against C1q binding, bound to the globular head domain of C1q and inhibited classical pathway activation in serum isolated from humans, non-human primates, rats and mice in vitro
(Roos et al., 2001
). While promising, no further work on this molecule has been reported including testing of its efficacy in vivo
. In contrast to peptide 2J, CPPs are postulated to bind to the CLRs of C1q and MBL (Hair et al., 2010
and ) inhibiting both the classical and lectin pathways respectively and retaining their activity in vivo
. Another very well characterized peptidic complement inhibitor is Compstatin, a 13 residue molecule that binds C3 preventing its activation. Compstatin is currently in clinical trials for age-related macular degeneration and is likely to have other indications as well (reviewed in Ricklin and Lambris, 2008
). The success of Compstatin clearly demonstrates that peptidic inhibitors of specific complement factors have genuine potential as therapeutics for complement-mediated disease processes.
In conclusion, CPPs show proof-of-concept that these peptidic molecules are reasonable candidates as potential therapeutic agents for complement-mediated diseases such as ABO incompatibility and that these compounds appear to be functional in vivo. The polar assortant (PA) construct appears to be superior to prior compounds in terms of aqueous solubility and potency for inhibiting classical complement pathway activation. These studies pave the way for in vivo testing to determine the pharmacological profile of PA as a prerequisite for testing this compound in animal models of disease caused by dysregulated activation of the classical and lectin pathways.