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Published on: February 28, 2025
Pyrazole-Based Transthyretin Kinetic Stabilizers Identified Using a Covalent Fluorescent Probe Assay for Selectivity
Kyung Ah Kim1, Chaeeun Lee1, Nayoung Lim1
1Department of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Republic of Korea.
Abstract:
Transthyretin (TTR) amyloidosis arises from the extracellular aggregation of misfolded TTR monomers into β-sheet-rich fibrils, leading to progressive tissue damage. To inhibit this process, we designed and synthesized pyrazole-based kinetic stabilizers targeting the thyroxine-binding sites of TTR. Structure-activity relationship studies revealed that derivatives with hydrophobic trans-alkene linkers and 3,5-substituted pyrazole rings showed enhanced stabilizing potency, particularly those bearing carboxylic acid, amide, or sulfonamide groups. A covalent fluorescent probe derived from trans-styrylpyrazole was developed to selectively react with Lys15, enabling fluorescence probe exclusion and native PAGE assays to evaluate stabilizer selectivity in human serum. Among these, 3,5-dichloropyrazole derivatives exhibited efficacy comparable to that of tafamidis and acoramidis. X-ray crystallography of the TTR-17 complex confirmed hydrogen bonding with Ser117/117' and electrostatic interactions with Lys15. Pharmacokinetic studies of compounds 16 and 17 demonstrated favorable exposure, bioavailability, and metabolic stability, supporting their preclinical development for hereditary- and wild-type TTR amyloidosis.
Insights
Researchers developed novel pyrazole-based compounds to stabilize transthyretin (TTR), preventing amyloidosis. These stabilizers show promise for treating hereditary and wild-type TTR amyloidosis, with some matching existing therapies.
Area of Science:
- Medicinal Chemistry
- Biochemistry
- Drug Discovery
Background:
- Transthyretin (TTR) amyloidosis is caused by misfolded TTR protein forming amyloid fibrils.
- These fibrils deposit in tissues, leading to progressive organ damage.
Purpose of the Study:
- To design and synthesize novel pyrazole-based kinetic stabilizers for TTR.
- To evaluate the efficacy and selectivity of these stabilizers in inhibiting TTR aggregation.
Main Methods:
- Structure-activity relationship (SAR) studies of pyrazole derivatives.
- Development of a covalent fluorescent probe for selectivity assays.
- Evaluation of stabilizer efficacy using fluorescence probe exclusion and native PAGE.
- X-ray crystallography to elucidate binding interactions.
- Pharmacokinetic studies to assess drug-like properties.
Main Results:
- Pyrazole derivatives with hydrophobic trans-alkene linkers and specific substituents showed potent TTR stabilization.
- 3,5-dichloropyrazole derivatives demonstrated efficacy comparable to tafamidis and acoramidis.
- X-ray crystallography confirmed specific hydrogen bonding and electrostatic interactions.
- Compounds 16 and 17 exhibited favorable pharmacokinetic profiles, including good bioavailability and metabolic stability.
Conclusions:
- Novel pyrazole-based compounds effectively stabilize TTR and inhibit amyloid formation.
- These compounds represent promising preclinical candidates for treating TTR amyloidosis.
- The developed covalent probe is valuable for assessing stabilizer selectivity in complex biological matrices.
