High-affinity recognition of the human C-reactive protein independent of phosphocholine

• Published in: Organic & biomolecular chemistry Vol. 15; no. 21; pp. 4644 - 4654
• Main Authors: Yang, Jie, Gustavsson, Anna-Lena, Haraldsson, Martin, Karlsson, Goran, Norberg, Thomas, Baltzer, Lars
• Format: Journal Article
• Language: English
• Published: CAMBRIDGE Royal Soc Chemistry 2017
• Subjects: Amino Acid Sequence; binding; C-Reactive Protein - chemistry; C-Reactive Protein - metabolism; Chemistry; Chemistry, Organic; Drug Design; Humans; Medicin och hälsovetenskap; Models, Molecular; Organic Chemistry; Organisk kemi; Phosphorylcholine - metabolism; Physical Sciences; Protein Conformation; Science & Technology; BINDING
• ISSN: 1477-0520; 1477-0539; 1477-0539
• DOI: 10.1039/c7ob00684e

A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 mu M. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the mu M to nM range for which no small molecule ligands are known.