Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins

Abstract

The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional “hot-spot” on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins.

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics

“…We used an alternative cheminformatics approach to screen for small molecules that are chemically similar to a region of S. aureus SCIN-B that has previously been shown to be critical for C3b-binding and complement inhibition (25, 38). A novel chemical information software tool, ChemVassa (39), was used to calculate a numeric fingerprint for residues originating from the second α-helix of SCIN-B.…”

“…The ability of each compound to inhibit complement activation by the alternative pathway (AP) or classical pathway (CP) was performed as previously described (25, 41) with the following modifications. Each experiment began with overnight coating of ELISA plates (Costar EIA/RIA, Fisher Scientific) with either 25 μg ml −1 Salmonella typhosa lipopolysaccharides (LPS) for the AP (Sigma Aldrich) or 3 μg ml −1 human IgM (ImmunoReagents) for the CP in a buffer containing 100 mM Na 2 CO 3 /NaHCO 3 pH (9.6) followed by blocking with a buffer of PBS, 1% (w/v) bovine serum albumin, and 0.05% (v/v) Tween-20 for 1 h at 37°C.…”

“…Compounds were diluted from a 10 mM stock in neat DMSO to yield a 250 μM final compound concentration and 2.5% (v/v) DMSO concentration into serum containing AP or CP buffer. The serum/compound mixture was transferred to the washed ELISA plate and incubated at 37°C for 1 h. After washing, deposited complement components were detected using monoclonal antibodies (anti-C3b: WM1, Sigma; anti-C4b: C4-1, Cell Sciences; anti-MAC/C5b-9: aE11, Santa Cruz Biotechnology) at 1/300 dilutions and detected as previously described (25). All measurements were normalized by treating the mean value of vehicle control as 100% signal.…”

“…For all LE-SPR experiments a C3b biosensor was created in a manner which uniformly captures C3b in a physiologically relevant orientation and has been described in detail elsewhere (25, 38, 42). Briefly, C3b was site-specifically biotinylated using a CVF-based C3 convertase and EZ-link Maleimide-PEG 2 -biotin (Thermo Scientific) through a protocol identical to that described by Garcia et al (38).…”

“…While compstatin is of synthetic origin, the feasibility of lower molecular weight complement inhibitors has also been provided by nature itself. The human bacterial pathogen, Staphylococcus aureus , secretes several small C3b-binding proteins which act as potent inhibitors of complement (24) and peptidic mimics (< 1.5 kDa) of one family of these immune evasion molecules, staphylococcal complement inhibitor (SCINs), have been reported (25). …”