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.
- 1Hubei Key Laboratory of Embryonic Stem Cell Research and Hubei Key Laboratory of Wudang Local Chinese Medicine, Department of Biochemistry and Molecular Biology, Research of Institute of Biomedicine, College of Basic Medicine, Hubei University of Medicine, Shiyan 442000, China.
- 2Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan 442008, China.
Abstract
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.
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BibTeX
@article{wang2026site,
title = {{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.}},
author = {Luyao Wang and Li Ding and Tianxiang Qin and Xinyu Liu and Chenglan Lou and Hongyi Luo and Fang Sun and Chenhu Qin and Xiangdong Ye and Xudong Luo and Zongyun Chen},
journal = {Toxins},
year = {2026},
month = oct,
doi = {10.3390/toxins18100449},
url = {https://europepmc.org/article/MED/42931539},
note = {CC BY},
}
RIS
TY - JOUR
TI - 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.
AU - Luyao Wang
AU - Li Ding
AU - Tianxiang Qin
AU - Xinyu Liu
AU - Chenglan Lou
AU - Hongyi Luo
AU - Fang Sun
AU - Chenhu Qin
AU - Xiangdong Ye
AU - Xudong Luo
AU - Zongyun Chen
PY - 2026
DA - 2026/10/15
JO - Toxins
DO - 10.3390/toxins18100449
UR - https://europepmc.org/article/MED/42931539
AB - 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.
SN - 2072-6651
ER -