Guangzhou, China – Researchers from the South China University of Technology identified a novel approach to effectively silence the expression of transthyretin (TTR), with potential implications for innovative therapeutic approaches in TTR amyloidosis, including transthyretin-mediated amyloid cardiomyopathy (ATTR-CM).
The proposed strategy involves stabilizing the G-quadruplex structure—a fundamental genomic structural feature—and reducing the production of associated proteins.
“Herein, we propose a novel strategy to silence TTR mRNA expression by stabilizing the G-quadruplex in the 5′UTR region of the TTR gene. Further in vivo study to verify this strategy in treating ATTR is on progress in our lab,” the researchers wrote in Bioorganic & Medicinal Chemistry Letters.
Using advanced analysis tools, such as the software program QGRS mapper, the researchers demonstrated that small molecule ligands—TMPyP4, Braco-19, NMM, and TO—could induce and stabilize specific G-quadruplex motifs in the TTR gene. Among them, TmPyP4 and Braco-19 exhibited superior induction.
Further investigation identified 4 putative G2-quadruplex sequences forming stable antiparallel, parallel, and hybrid G2-quadruplex structures. Among them, Ttrg 3 (5′-GGAAGGAAGGGAGGGAGGG-3′) exhibited the highest stability in forming G-quadruplex.
Subsequent experiments revealed that TmPyP4, Braco-19, NMM, and TO effectively stabilized the parallel topology of Ttrg 3. After a 48-hour incubation period, quantitative real-time polymerase chain reaction experiments showed a significant reduction in the transcription of TTR mRNA in HepG2 cells treated with 20 μM TmPyP4 and Braco-19. This occurred without significant apoptosis induction, suggesting that the ligands have a high level of biocompatibility with low cytotoxicity.
Moreover, ultraviolet-vis spectroscopy experiments showed the binding behavior of ligands to the G-quadruplex structure, revealing a red shift in absorption and increased π-π stacking interactions. Fluorescence titration experiments further confirmed the strong binding of ligands, such as TmPyP4, TO, NMM, and CX-5461, to the G-quadruplex structure.
The thermal stability of Ttrg 3 was enhanced by ligands, with TmPyP4 and NMM showing the most pronounced effect, increasing the melting point by over 20 °C.
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