BACKGROUND: Numerous studies have shown that high C-reactive protein (CRP) levels predict cardiovascular disease and augur a poor prognosis in patients with acute coronary syndromes. Much in vitro and in vivo data support of a role for CRP in atherogenesis. There is an urgent need to develop inhibitors that specifically block the biological effects of CRP in vivo. The one-bead-one-compound (OBOC) combinatorial library method has been used to discover ligands against several biological targets. In this study, we use a novel fluorescence-based screening method to screen an OBOC combinatorial library for the discovery of peptides against human CRP. METHODS: Human CRP was labeled with fluorescein isothiocyanate (FITC) and human serum albumin (HuSA) was labeled with phycoerythrin (PE) and used for screening. The OBOC library LWH-01 was synthesized on TentaGel resin beads using a standard solid-phase "split/mix" approach. RESULTS: By subtraction screening, eight peptides that bind specifically to CRP and not to HuSA were identified. In human aortic endothelial cells (HAECs) incubated with CRP, inhibitors CRPi-2, CRPi-3, and CRPi-6 significantly inhibited CRP-induced superoxide, cytokine release, and nuclear factor-κB (NFκB) activity. Molecular docking studies demonstrate that CRPi-2 interacts with the two Ca(2+) ions in the single subunit of CRP. The binding of CRPi-2 is reminiscent of choline binding. CONCLUSIONS: Future studies will examine the utility of this inhibitor in animal models and clinical trials.
BACKGROUND: Numerous studies have shown that high C-reactive protein (CRP) levels predict cardiovascular disease and augur a poor prognosis in patients with acute coronary syndromes. Much in vitro and in vivo data support of a role for CRP in atherogenesis. There is an urgent need to develop inhibitors that specifically block the biological effects of CRP in vivo. The one-bead-one-compound (OBOC) combinatorial library method has been used to discover ligands against several biological targets. In this study, we use a novel fluorescence-based screening method to screen an OBOC combinatorial library for the discovery of peptides against humanCRP. METHODS:HumanCRP was labeled with fluorescein isothiocyanate (FITC) and humanserum albumin (HuSA) was labeled with phycoerythrin (PE) and used for screening. The OBOC library LWH-01 was synthesized on TentaGel resin beads using a standard solid-phase "split/mix" approach. RESULTS: By subtraction screening, eight peptides that bind specifically to CRP and not to HuSA were identified. In human aortic endothelial cells (HAECs) incubated with CRP, inhibitors CRPi-2, CRPi-3, and CRPi-6 significantly inhibited CRP-induced superoxide, cytokine release, and nuclear factor-κB (NFκB) activity. Molecular docking studies demonstrate that CRPi-2 interacts with the two Ca(2+) ions in the single subunit of CRP. The binding of CRPi-2 is reminiscent of choline binding. CONCLUSIONS: Future studies will examine the utility of this inhibitor in animal models and clinical trials.
Authors: Mark B Pepys; Gideon M Hirschfield; Glenys A Tennent; J Ruth Gallimore; Melvyn C Kahan; Vittorio Bellotti; Philip N Hawkins; Rebecca M Myers; Martin D Smith; Alessandra Polara; Alexander J A Cobb; Steven V Ley; J Andrew Aquilina; Carol V Robinson; Isam Sharif; Gillian A Gray; Caroline A Sabin; Michelle C Jenvey; Simon E Kolstoe; Darren Thompson; Stephen P Wood Journal: Nature Date: 2006-04-27 Impact factor: 49.962
Authors: Rebecca A Roof; Katarzyna Sobczyk-Kojiro; Anjanette J Turbiak; David L Roman; Irina D Pogozheva; Levi L Blazer; Richard R Neubig; Henry I Mosberg Journal: Chem Biol Drug Des Date: 2008-07-15 Impact factor: 2.817