揭示人類降鈣素雙位點突變變異體其抑制聚集機制
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2025
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胜肽藥物面臨的挑戰之一是其不可逆的聚集特性，從而降低治療效果和生物利用度。例如，人類降鈣素 (human calcitonin, hCT)，這個賀爾蒙多肽含有32個胺基酸，由甲狀腺濾泡旁細胞 (C-cells) 所分泌在維持骨骼結構和調節血液中的鈣含量起到重要的作用，這些特性使其可用於治療骨質疏鬆症和佩吉特氏症等骨骼相關疾病，但因聚集特性嚴重限制了其作為藥物的潛力。我們實驗室過去的研究發現序列中的 Tyr12 和 Asn17 在hCT聚集形成類澱粉蛋白纖維 (amyloid fibril) 的過程中發揮關鍵作用。這些位置的雙突變點 (Y12LN17H hCT, DM hCT) 抑制了多肽聚集的能力，進而提升其治療潛力，然而，這些突變點為何可抵擋多肽的聚集，目前尚不明確。在本研究中，為了闡明 DM hCT 抗聚集特性的潛在機制及其在防止 hCT 聚集方面的抑制作用。分析並比較了 DM hCT 與另外三個雙變異體 (Y12LN17A、Y12LN17D、Y12LN17DAB) 各方面的性質，這些變異體皆針對 Asn-17 位點進行改變，並各自具有不同的胺基酸特性。利用硫磺素-T (thioflavin T, ThT) 螢光動力學實驗評估它們的聚集程度及使用圓二色光譜 (circular dichroism spectra, CD spectra) 了解它們形成α-螺旋 (α-helix) 構象的傾向，試圖討論構象穩定性與聚集之間的關係，並增進我們對該胜肽DM hCT相較於hCT較不容易聚集的原因，除此之外，我們還探討了這些序列取代對生物活性的影響。希望這些發現將有助於優化 DM hCT 並推進臨床應用胜肽藥物的開發。
One major challenge for peptide drugs is their tendency for irreversible aggregation, significantly reducing therapeutic efficacy and bioavailability. For example, human calcitonin (hCT) is a hormone polypeptide composed of 32 amino acids, secreted by thyroid parafollicular cells (C-cells). It plays a essential role in maintaining bone structure and regulating blood calcium levels. These properties make hCT suitable for treating bone-related diseases such as osteoporosis and Paget's disease. However, its therapeutic potential is severely limited by its strong tendency to aggregate. Previous research in our laboratory revealed that Tyr12 and Asn17 play key roles in the amyloid fibril formation of hCT during its aggregation process. Double mutations at these positions (Y12LN17H hCT, DM hCT) have been shown to inhibit peptide aggregation, thereby enhancing its therapeutic potential. Nevertheless, the exact mechanism by which these mutations prevent peptide aggregation remains unclear.In this study, we aimed to elucidate the mechanism underlying the anti-aggregation properties of DM hCT and its inhibitory effect in preventing hCT aggregation. We analyzed and compared the properties of DM hCT with three other double mutants (Y12LN17A, Y12LN17D, Y12LN17DAB), all of which were designed with substitutions at the Asn17 residue, each incorporating different amino acid replacements. The extent of aggregation was evaluated using thioflavin T (ThT) fluorescence kinetics assays, while circular dichroism (CD) spectroscopy was employed to assess their propensity to form α-helical conformations. These experiments aimed to explore the relationship between conformational stability and aggregation, and to further elucidate the underlying reasons why DM hCT exhibits reduced aggregation compared to wild-type hCT. In addition, we examined the impact of these sequence modifications on their biological activity were evaluated using cAMP assays. These results may help optimize DM hCT and facilitate the development of peptide-based therapeutics for clinical applications.
One major challenge for peptide drugs is their tendency for irreversible aggregation, significantly reducing therapeutic efficacy and bioavailability. For example, human calcitonin (hCT) is a hormone polypeptide composed of 32 amino acids, secreted by thyroid parafollicular cells (C-cells). It plays a essential role in maintaining bone structure and regulating blood calcium levels. These properties make hCT suitable for treating bone-related diseases such as osteoporosis and Paget's disease. However, its therapeutic potential is severely limited by its strong tendency to aggregate. Previous research in our laboratory revealed that Tyr12 and Asn17 play key roles in the amyloid fibril formation of hCT during its aggregation process. Double mutations at these positions (Y12LN17H hCT, DM hCT) have been shown to inhibit peptide aggregation, thereby enhancing its therapeutic potential. Nevertheless, the exact mechanism by which these mutations prevent peptide aggregation remains unclear.In this study, we aimed to elucidate the mechanism underlying the anti-aggregation properties of DM hCT and its inhibitory effect in preventing hCT aggregation. We analyzed and compared the properties of DM hCT with three other double mutants (Y12LN17A, Y12LN17D, Y12LN17DAB), all of which were designed with substitutions at the Asn17 residue, each incorporating different amino acid replacements. The extent of aggregation was evaluated using thioflavin T (ThT) fluorescence kinetics assays, while circular dichroism (CD) spectroscopy was employed to assess their propensity to form α-helical conformations. These experiments aimed to explore the relationship between conformational stability and aggregation, and to further elucidate the underlying reasons why DM hCT exhibits reduced aggregation compared to wild-type hCT. In addition, we examined the impact of these sequence modifications on their biological activity were evaluated using cAMP assays. These results may help optimize DM hCT and facilitate the development of peptide-based therapeutics for clinical applications.
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Keywords
人類降鈣素, 胺基酸取代, 分子對接, Human calcitonin, Amino acid substitution, Molecular docking