Powerful Protein Binders from Designed Polypeptides and Small Organic Molecules—A General Concept for Protein Recognition

Abstract

High‐affinity binders for the C‐reactive protein (CRP), with dissociation constants in the pM to nM range and selectivities in human serum comparable to those of antibodies, were obtained by conjugation of 16 designed polypeptides to phosphocholine, a small molecule that binds CRP with a KD value of 5 μM (see picture). The polypeptides were not designed specifically to recognize CRP and bind by an adapted fit mechanism.

A synthetic polypeptide conjugate from a 42‐residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity

“…Reagents were used as obtained from commercial sources unless otherwise stated. Concentrations were performed at reduced pressure and a temperature below 40 C. Measurement of pH was carried out using pH paper (pH range [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Flash chromatography was performed using silica gel 60 (0.040-0.063 mm) as the stationary phase.…”

“…The binder concept has been described in detail previously [9][10][11]. In short, it is based on the idea that if a small molecule that is known to bind to a protein, albeit with poor affinity and selectivity, is conjugated to a polypeptide with hydrophobic as well as charged residues, a dramatic increase in affinity and selectivity is achieved although the polypeptide itself binds to the protein with poor affinity.…”

“…The concept is unlike that of binder molecules prepared by molecular biology technologies that form pre-organized structures that are shape complimentary to the target protein. The set of 16 polypeptide sequences was described in detail previously (Figure 2) [9][10][11]. The sequences were designed to have some propensity for forming helix-loop-helix motifs and were varied with regard to the number and position of charged residues and also with regard to the site of incorporation of the small molecules.…”

“…Recently, we proposed a general binder concept that uses conjugates from small organic molecules and 42-residue polypeptides to form hybrid molecules that bind proteins with affinities that are enhanced by three to four orders of magnitude in comparison with those of the small molecules and selectivities that are enhanced by one to two orders of magnitude [9][10][11]. The concept is robust and can be used to prepare selective, high-affinity binders for, in principle, any protein in short time by organic synthesis, provided that a small molecule or peptide is available, which binds the protein with at least modest affinity and selectivity.…”

Polypeptide Conjugate Binders that Discriminate between Two Isoforms of Human Carbonic Anhydrase in Human Blood

“…functionalized benzene sulfonamide ligand to each member of a set of 16 designed polypeptides, as previously described for the C-reactive protein (CRP). [12] The extracted proteins were identified by mass spectrometry and SDS-PAGE, whereas the biomolecular interactions were evaluated by surface plasmon resonance (SPR) biosensor analysis.…”