The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38 nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4 nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3 nM, whereas that of the 4-C10L17-AZM conjugate was 90 pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to μM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.

将小分子抑制剂乙酰唑酰胺（AZM）与一组设计的多肽缀合，并使用表面等离子体共振对所得缀合物与人碳酸酐酶II（HCA II）的亲和力进行评估。AZM-HCA II复合物的解离常数为38 nM，而AZM共轭多肽（4-C10L17-AZM）与HCA II的解离常数为4 nM，由于多肽的结合，亲和力提高了10倍。对于人碳酸酐酶IX（HCA IX），AZM的解离常数为3 nM，而4-C10L17-AZM共轭物的解离常数为90 pM，亲和力提高了33倍。对于与靶蛋白已经具有高亲和力的小分子配体，由于多肽的缀合而实现了这种显着的亲和力增加。这支持了这样的概念，即由于多肽结合而引起的增强不限于将分子在mM到μM范围内的蛋白质结合的小分子配体，还可以用于nM配体以提供具有pM范围内解离常数的识别元件。对两种不带有蛋白聚糖（PG）结构域的HCA IX构建体的评估未显示AZM与多肽缀合物之间的显着亲和力差异，这提供了证据表明4-C10L17-AZM与HCA IX的结合改善是由于多肽之间的相互作用所致片段和仅在一种碳酸酐酶HCA IX中发现的PG结构域。