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.

Intrinsic binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI

“…Several CA isoforms have been purified from the human body . CA I and CA II were purified from human erythrocytes and their inhibition was studied .…”

An efficient strategy to enhance binding affinity and specificity of a known isozyme inhibitor

“…Since these two isozymes have nearly identical molecular weights, they are inseparable by SDS-PAGE. 15 Therefore, bindings between the conjugates and the target enzymes were investigated separately as shown in Fig. 3a.…”

“…14 Selective inhibition through binding to enzyme active sites is also challenging due to great structural homology among the isozymes. 15 The lack of selectivity of conventional inhibitors against isozyme family demonstrates a clear demand for developing efficient strategies to enhance specificity of such inhibitors for diagnostic purposes as well as pharmaceutical application.…”

“…18 It was reported that conjugation of a synthetic polypeptide to this benzenesulfonamide cooperatively enhanced binding affinity as well as specificity. 15,19 The enhanced specificity might be attributed to selective binding of the polypeptides in a slightly varied fashion in the proximity to the active site of each isozyme. These findings suggest that the more selective inhibitors can be developed by conjugating a known small molecule inhibitor to second ligand that binds to proximal sites with compositional and/or structural variations in a cooperative fashion.…”

“…Other approaches, for example, phage display, microarray and mechanism-based designation, are also reported to develop such conjugate ligands. 15,20 …”