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CX-397, a novel recombinant hirudin analog having a hybrid sequence of hirudin variants-1 and -3
Y Komatsu1, S Misawa, A Sukesada
1Pharmaceuticals & Biotechnology Laboratory, Nikko Kyodo Co., Ltd., Saitama, Japan.
Insights
Novel hirudin analogs were created to understand thrombin inhibition. CX-397, a hybrid of hirudin variants, demonstrated the strongest inhibitory activity against thrombin, offering insights into structure-activity relationships.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Hirudins are potent thrombin inhibitors.
- Understanding the structure-activity relationship of hirudin variants is crucial for developing novel anticoagulants.
Purpose of the Study:
- To elucidate the differential roles of the N- and C-terminal halves of hirudins in thrombin inhibition.
- To characterize novel recombinant hirudin analogs, CX-397 and CX-397R.
Main Methods:
- Production of recombinant hirudin analogs (CX-397 and CX-397R) with hybrid amino acid sequences.
- Determination of anti-thrombin activity using a fluorogenic enzyme assay.
- Comparison of dissociation constants (Ki) for thrombin inhibition.
Main Results:
- CX-397, composed of the N-terminal half of hirudin variant-1 and the C-terminal half of hirudin variant-3, exhibited the strongest thrombin inhibitory activity.
- The order of dissociation constants (Ki) was CX-397R > rHV-1 > rHV-3 > CX-397.
- CX-397 displayed a dissociation constant of 0.0433 pM, indicating potent inhibition.
Conclusions:
- The N- and C-terminal domains of hirudins play differential roles in thrombin inhibition.
- CX-397 represents a highly effective hirudin analog for thrombin inhibition.
- These findings contribute to the design of optimized anticoagulant therapies.
Abstract:
To elucidate the differential roles of N- and C-terminal halves of hirudins in thrombin inhibition, we produced novel recombinant hirudin analogs, CX-397 and CX-397R, having a hybrid amino acid sequence of hirudin variants-1 (HV-1) and -3 (HV-3). CX-397 is composed of the N-terminal half of HV-1 (HV-11-36) combined with the C-terminal half of HV-3 (HV-337-66). CX-397R is the opposite combination. Their anti-thrombin activity was determined by a fluorogenic enzyme assay and compared with that of recombinant HV-1 (rHV-1) and rHV-3. The order of the magnitude of dissociation constants (Ki) of these four hirudin analogs in thrombin inhibition was as follows: CX-397R (0.294 pM) > rHV-1 (0.148 pM) > rHV-3 (0.0593 pM) > CX-397 (0.0433 pM), indicating that CX-397 is the strongest inhibitor among them.