人類第五型磷酸二酯酶與其抑制藥物阿伐那非之複合體結構揭示其亞型選擇性機制以及針對α螺旋主鍊氧原子之鹵素鍵設計方針

Structure of Human Phosphodiesterase 5A1 Complexed with Avanafil Reveals Molecular Basis of Isoform Selectivity and Guidelines for Targeting α-Helix Backbone Oxygen by Halogen Bonding

謝詔名(Chao-Ming Hsieh)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2020年)

https://doi.org/10.6342/NTU202004042

摘要

3',5'-環核苷酸磷酸二酯酶酵素家族包含11種亞型，其成員負責調控細胞內cAMP及cGMP兩種3',5'-環核苷酸的降解。cAMP及cGMP為細胞內非常重要的第二傳訊者，可活化多種訊息傳遞途徑，進而影響細胞增值、分化、存活、細胞凋亡等生理作用，可知3',5'-環核苷酸磷酸二酯酶的活性對於細胞的運作非常重要，也因此3',5'-環核苷酸磷酸二酯酶是很好的藥物設計標的。藉著抑制其催化活性，可以調節細胞的功能進而達到緩解相關疾病的目的。在已開發完成的各類亞型抑制藥物中，第五型磷酸二酯酶的專一性抑制藥物是最成功的案例之一。目前已於臨床上使用的西地那非 (sildenafil)、伐地那非 (vardenafil)、他達拉非 (tadalafil)及阿伐那非 (avanafil)皆已廣泛的用來治療肺動脈高壓，心血管疾病，前列腺增生和男性性功能障礙等疾病。在這些抑制劑中，阿伐那非為唯一的第二代藥物。與其他第一代藥物相比，阿伐那非具有更好的亞型選擇性，且產生副作用的機率較低。然而目前尚無第五型磷酸二酯酶與阿伐那非的複合體結構，所以尚無法從結構的角度上得知阿伐那非相較於其他的抑制劑有更好亞型選擇性的原因。為了得知阿伐那非作用的結構機轉，並獲得可提供下一代藥物設計的結構資訊，我們將第五型磷酸二酯酶與阿伐那非進行共結晶，並成功獲得解析度達1.92 Å的複合體晶體結構。基於所解出的結構資訊，我們得知了阿伐那非的結合模式，並搭配磷酸二酯酶亞型間的序列比對結果，推論不同亞型之間數個位置的胺基酸序列差異是決定阿伐那非有較好亞型選擇性的關鍵。除此之外，藥物結構中氯原子與酵素活性中心之α螺旋主鏈氧原子形成的鹵素鍵，亦提供了一個新的藥物設計概念，我們認為鹵素鍵的形成除了可增強藥物的結合力，並可提高藥物的選擇性。而這些資訊，應有助於設計出具有更高選擇性以及更低副作用新一代抑制劑。

關鍵字

人類第五型磷酸二酯酶 ； 阿伐那非 ； 鹵素鍵 ； 主鏈氧原子 ； α螺旋

並列摘要

Cyclic nucleotide phosphodiesterases (PDEs) constitute a large enzyme superfamily that, depending on sequence variation and differences in substrate specificity, is further classified into 11 subfamilies. PDEs are responsible for the breakdown of two important secondary messengers - cAMP and cGMP. Because these two cyclic nucleotides relay the extracellular signals and directly control the activities of various signal transduction pathways to induce physiological changes, such as cell proliferation, differentiation, anti-apoptosis and apoptosis, thus their cellular concentrations are tightly regulated. And the catalytic activities of PDEs play an important role in controlling cAMP and cGMP cellular concentrations. For this reason, PDEs are valid targets for drug development, whose inhibition may alleviate several symptoms by increasing cAMP or cGMP levels. Among numerous PDEs inhibitors, the development of PDE5-specific inhibitors represent one of the most successful cases in medicinal chemistry. The sildenafil, vardenafil, tadalafil, and avanafil are four PDE5 inhibitors in wide clinical use for treating pulmonary arterial hypertension, cardiovascular disease, benign prostatic hyperplasia, and erectile dysfunction. Among them, avanafil is the only FDA-approved second generation inhibitor which exhibits improved PDE isoform selectivity and reduced adverse effects compared to the first generation drugs. However, the molecular basis underlying avanafil’s better isoform selectivity has remained elusive due to the lack of PDE5-avanafil complex structure. To this end, we co-crystallized human PDE5 and avanafil and successfully determined the complex structure at 1.92 Å. Analysis of protein-drug interactions revealed structural basis of avanafil’s superior isoform selectivity. Moreover, a halogen bonding was observed between avanafil and a backbone carbonyl oxygen of an adjacent α-helix, whose contribution to inhibitory potency illustrates the feasibility of exploiting α-helix backbone in structure-based drug design.

並列關鍵字

human Phosphodiesterase 5A1 ； avanafil ； halogen bonding ； backbone carbonyl oxygen ； α-helix

參考文獻

1. S. H. Francis, M. A. Blount, J. D. Corbin, Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 91, 651-690 (2011).

2. M. F. Azevedo, F. R. Faucz, E. Bimpaki, A. Horvath, I. Levy, R. B. de Alexandre, F. Ahmad, V. Manganiello, C. A. Stratakis, Clinical and molecular genetics of the phosphodiesterases (PDEs). Endocr Rev 35, 195-233 (2014).

3. M. Conti, J. Beavo, Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76, 481-511 (2007).

4. D. H. Maurice, H. Ke, F. Ahmad, Y. Wang, J. Chung, V. C. Manganiello, Advances in targeting cyclic nucleotide phosphodiesterases. Nat Rev Drug Discov 13, 290-314 (2014).

5. G. S. Baillie, G. S. Tejeda, M. P. Kelly, Therapeutic targeting of 3',5'-cyclic nucleotide phosphodiesterases: inhibition and beyond. Nat Rev Drug Discov 18, 770-796 (2019).