Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors.

With the addition of the compstatin-based complement C3 inhibitor pegcetacoplan, another class of complement targeted therapeutics have recently been approved. Moreover, compstatin derivatives with enhanced pharmacodynamic and pharmacokinetic profiles are in clinical development (e.g., Cp40/AMY-101). Despite this progress, the target binding and inhibitory modes of the compstatin family remain incompletely described. Here, we present the crystal structure of Cp40 complexed with its target C3b at 2.0-Å resolution. Structure-activity-relationship studies rationalize the picomolar affinity and long target residence achieved by lead optimization, and reveal a role for structural water in inhibitor binding. We provide explanations for the narrow species specificity of this drug class and demonstrate distinct target selection modes between clinical compstatin derivatives. Functional studies provide further insight into physiological complement activation and corroborate the mechanism of its compstatin-mediated inhibition. Our study may thereby guide the application of existing and development of next-generation compstatin analogs.

Carbohydrate-Lectin Interactions: An Unexpected Contribution to Affinity.

Uropathogenic E. coli exploit PapG-II adhesin for infecting host cells of the kidney; the expression of PapG-II at the tip of bacterial pili correlates with the onset of pyelonephritis in humans, a potentially life-threatening condition. It was envisaged that blocking PapG-II (and thus bacterial adhesion) would provide a viable therapeutic alternative to conventional antibiotic treatment. In our search for potent PapG-II antagonists, we observed an increase in affinity when tetrasaccharide 1, the natural ligand of PapG-II in human kidneys, was elongated to hexasaccharide 2, even though the additional Siaα(2-3)Gal extension is not in direct contact with the lectin. ITC studies suggest that the increased affinity results from partial desolvation of nonbinding regions of the hexasaccharide; this is ultimately responsible for perturbation of the outer hydration layers. Our results are in agreement with previous observations and suggest a general mechanism for modulating carbohydrate-protein interactions based on nonbinding regions of the ligand.

Epitope mapping of sialyl Lewis(x) bound to E-selectin using saturation transfer difference NMR experiments.

A complex between sialyl Lewisx (alpha-D-Neu5Ac-[2-->3]- beta-D-Gal-[1-->4]-[alpha-L-Fuc-(1-->3)]-beta-D-GlcNAc-O-[CH2]8 COOMe) and E-selectin was studied using saturation transfer difference (STD) nuclear magnetic resonance (NMR) experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semiquantitative analysis of STD total correlation spectroscopy spectra provides clear evidence that the galactose residue receives the largest saturation transfer. The protons H4 and H6 of the galactose residue are in especially close contact to the amino acids of the E-selectin binding pocket. The fucose residue also receives a significant saturation transfer. The GlcNAc and Neu5Ac residues, with the exception of H3 and H3' of Neu5Ac, were found to interact weakly with the protein surface. These findings are in excellent agreement with a recently published X-ray structure and with the earlier findings from syntheses and activity assays. To further characterize the binding pocket of E-selectin, an inhibitory peptide, Ac-TWDQLWDLMK-CONH2, was synthesized and the binding to E-selectin studied utilizing transfer nuclear Overhauser effect spectroscopy (trNOESY) experiments. Finally, competitive trNOESY experiments were performed, showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.