Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d

Complement
activation plays a major role in many acute and chronic
inflammatory conditions. C3d, a terminal product of complement activation,
remains covalently attached to cells and is an excellent biomarker
of complement-mediated inflammation. We employed a virtual high-throughput
screening protocol to identify molecules with predicted binding to
complement C3d and with intrinsic fluorescence properties to enable
detection. Pharmacophore models were developed based on known C3d–ligand
interactions and information from computational analysis of structural
and molecular dynamics data. Iterative pharmacophore-based virtual
screening was performed to identify druglike molecules with physicochemical
similarity to the natural C3d ligand CR2. Hits from the pharmacophore
screens were docked to C3d and ranked based on predicted binding free
energies. Top-ranked molecules were selected for experimental validation
of binding affinity to C3d, using microscale thermophoresis, and for
their suitability to become molecular imaging agents, using fluorescence
spectroscopy. This work serves as a foundation for identifying additional
fluorescent molecules with high-affinity for C3d that will subsequently
be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal
monitoring of disease progression, and for targeting therapeutics
to sites of inflammation.