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

Abstract

Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60% homology exist in human blood with HCAI being present in five- to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K D of 17 nM and HCAI with a K D of 470 nM, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine. Alternatives to antibodies: Polypeptide conjugates from benzenesulfonamide and a set of 42-residue designed polypeptides with no prior relationship to HCAII were screened for affinity towards HCAII. The identified tight binder 4-C37L34-B showed an affinity of 17 nM and discriminated against the enzyme isoform HCAI by a factor of 30 although HCAII and HCAI show 60% homology. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.