P2′ Site Scanning of Snake Kunitz-Type Toxin Analogue BF9-N17H with Unique P1 Site Histidine Highlights Novel XIa Inhibitory Peptides Without Plasmin Inhibitory Activity

Kunitz-type peptides from venomous and blood-sucking animals represent an important natural molecular resource for anticoagulant development towards XIa, a new target for antithrombotic drug discovery. However, the plasmin-inhibitory activity of these Kunitz-type XIa inhibitory anticoagulants limits their application. Here, using the snake Kunitz-type toxin analogue BF9-N17H, an XIa inhibitor with a unique P1-site histidine, as a template, P2′ scanning was performed to enhance its selectivity towards XIa. Ten BF9-N17H analogues were designed using different amino acid classification replacement at the P2′ site, including non-polar amino acids A and P, acidic amino acids D and E, polar amino acids S, N, and Y, and basic amino acids K, R, and H. Coagulation factor inhibitory experiments showed that three BF9-N17H analogues, BF9N17HP2, BF9N17HK8 and BF9N17HR9, lost their XIa inhibitory activity, but seven other analogues, BF9N17HA1, BF9N17HD2, BF9N17HS4, BF9N17HN5, BF9N17HY6, BF9N17HE7 and BF9N17HH10, effectively maintained their inhibitory activity towards XIa, with IC50 values from 61.19 ± 7.04 nM to 4.54 ± 3.65 nM. One analogue, BF9N17HD2, inhibited XIa with an IC50 of 6.19 ± 1.38 nM and had a selectivity index of 142.79 towards plasmin. It only had weak inhibitory activity toward plasmin, with an IC50 of 883.88 ± 148.35 nM. Excitingly, five analogues, BF9N17HS4, BF9N17HN5, BF9N17HY6, BF9N17HE7 and BF9N17HH10, almost lost their plasmin-inhibitory activity completely, indicating their high selectivity towards XIa. To the best of our knowledge, this is the first report of Kunitz-type XIa inhibitors without plasmin-inhibitory activity, and it highlights that the Kunitz-type peptide scaffold might be used as a potential candidate for the development of potent and selective XIa-inhibitory antithrombotic agents.