Báo cáo khoa học: Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives ) a screening based on affinity for the UMP-CMP binding site

Nucleotide Binding To Human UMP-CMP Kinase Using Fluorescent Derivatives – A Screening Based On Affinity For The UMP-CMP Binding Site

Tác giả: Dimitri Topalis, Hiroki Kumamoto, Maria-Fernanda Amaya Velasco, Laurence Dugué, Ahmed Haouz, Julie Anne C. Alexandre, Sarah Gallois-Montbrun, Pedro Maria Alzari, Sylvie Pochet, Luigi André Agrofoglio and Dominique Deville-Bonne

Lĩnh vực: Hóa sinh, Dược lý

Nội dung tài liệu: Nghiên cứu này trình bày một phương pháp sàng lọc mới dựa trên ái lực đối với vị trí liên kết UMP-CMP của enzyme UMP-CMP kinase ở người. Phương pháp này sử dụng các dẫn xuất huỳnh quang của nucleotide để đánh giá ái lực liên kết của các hợp chất dị vòng phosphonate. Các kết quả cho thấy enzyme này có ái lực cao với một số dẫn xuất uracil vinylphosphonate, cao hơn so với cidofovir. Các hợp chất này hoạt động như các chất ức chế thay vì cơ chất. Nghiên cứu cũng cung cấp thông tin về cấu trúc của enzyme và cách nó tương tác với các phối tử khác nhau, làm sáng tỏ cơ chế hoạt động và tiềm năng phát triển các tác nhân kháng virus và ung thư mới.

Mục lục chi tiết:

• Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives – a screening based on affinity for the UMP-CMP binding site
• Keywords
• Correspondence
• *These authors contributed equally to this work
• *Present address
• (Received 12 February 2007, revised 25 May 2007, accepted 29 May 2007)
• doi:10.1111/j.1742-4658.2007.05902.x
• Human UMP-CMP kinase (UCK) plays a key role in the ribonucleoside and deoxyribonucleoside salvage pathway and in the anabolic phosphorylation of nucleoside analogs used as antiviral and anticancer agents. The phosphorylation of nucleoside analogs requires three steps involving the action of deoxyribonucleoside kinase
• Abbreviations
• UMP-CMP kinase and acyclic phosphonate analogs
• Results and Discussion
• Competitive fluorescence experiments to determine the binding of natural substrates to the donor and the acceptor sites of human UCK
• Fig. 1. Fluorescence competition assays with Mant-ATP bound to the human UCK donor-binding site.
• Dictyostelium UCK has been reported to specifically bind MABA-CDP at the CMP-binding site [16].
• Fig. 2. (A) Formula of MABA-CDP used in the fluorescent competitive titration assay to determine dissociation constants of acyclic nucleotides. (B) Formula of C5-substituted vinyl phosphonates (Y = H, Cl, Br, phenyl, fluorophenyl, phenyl-S).
• Binding of acyclic phosphonate nucleosides to the acceptor site of human UCK using fluorescent MABA-CDP
• Fig. 3. Fluorescence assays with MABA-CDP bound to the human UCK acceptor site.
• Table 1. Equilibrium dissociation constants and kinetic parameters of human UCK for natural NMP and cidofovir.
• Scheme 1. Synthesis of novel acyclic phosphononucleoside analogs.
• Table 2. Binding affinities of human UCK for new uracil acyclic phosphonates determined in the MABA-CDP fluorescent assay and inhibitory constants in activity assays.
• Fig. 4. Inhibition of human UCK activity by 5-chloro-UVP (compound 6c).
• affinity of the enzyme for this compound.
• Structural analysis of human UCK
• Fig. 5. (A) Superpositions of the polypeptide backbone of human UCK (green) and pig adenylate kinase (orange; Protein Data Bank code 3ADK), Dictyostelium discoideum UCK (blue; 2UKD), human adenylate kinases 1 (cyan; 1Z83) and 2 (light gray; 2C9Y), yeast uridylate kinase (yellow; 1UKY) and yeast adenylate kinase (pink; 1DVR). (B) Crystallographic dimer of human UCK, with each monomer shown in a different color.
• Fig. 6. Model of the acceptor-binding site in the closed form of human UCK.
• Conclusion
• Experimental procedures
• Materials
• Synthesis of UVPs
• Protein expression and purification
• Fluorescence measurements
• Enzymatic activity measurements
• Crystallographic studies
• Structural models
• Acknowledgements
• References