核磁共振波谱在药物发现中的应用

摘要/Abstract

摘要：

核磁共振波谱通过检测组成有机化合物分子的原子核跃迁而得到反映核性质的参数以及周围化学环境对这些参数的影响规律. 这些参数的相关内容包含了极其详尽的有机化合物分子结构和分子间相互作用的信息，并构成了核磁共振结构解析和生物靶分子-配体相互作用研究的理论基础. 在生物医药研发领域内，科研院所和公司企业的研发工作者们一直在努力探索利用核磁共振波谱监测生物靶分子-配体相互作用作为药物发现工具的潜能. 本文旨在针对核磁共振波谱在药物发现过程中活性化合物筛选的最新研究进展进行综述.

关键词: 核磁共振(NMR), 药物发现, DOSY, G-四链体, 筛选方法

Abstract:

Nuclear magnetic resonance (NMR) spectroscopy is often valued for its ability to shed light on molecular structure, but its greatest potential in drug discovery probably lies in the information it can reveal about molecular interactions at atomic level. The NMR parameters of the atoms in a compound, such as chemical shift, diffusion coefficient and relaxation time, are highly sensitive to the chemical environment surrounding them. Measuring these parameters therefore can provide information on whether a small molecule binds to a target protein or nucleic acid, and what are the interacting parts of the small molecule and the macromolecular target. The NMR approaches can be used to validate ligand binding and/or to identify potential ligands in the mixtures of compounds. In the last decade, the ability of NMR spectroscopy as a tool to monitor intermolecular interactions in drug discovery has been increasingly appreciated in both academia and industry. In this perspective, we highlight some major applications of NMR in drug discovery in this review article, with focus on hit screening.

Key words: NMR, drug discovery, DOSY, G-quadruplex, screening methods

中图分类号:

O482.53

周秋菊, 向俊锋, 唐亚林. 核磁共振波谱在药物发现中的应用[J]. 波谱学杂志, 2010, 27(1): 68-79.

ZHOU Qiu-Ju, XIANG Jun-Feng, TANG Ya-Lin. Applications of Nuclear Magnetic Resonance Spectroscopy in Drug Discovery[J]. Chinese Journal of Magnetic Resonance, 2010, 27(1): 68-79.

参考文献

[1] Fadeev E A, Sam M D, Clubb R T. NMR structure of the amino-terminal domain of the lambda integrase protein in complex with DNA: immobilization of a flexible tail facilitates beta-sheet recognition of the major groove[J]. J Mol Biol, 2009, 388(4): 682-690.

[2] Christen B, Hornemann S, Damberger F F, et al. Prion protein NMR structure from tammar wallaby (Macropus eugenii) shows that the beta2-alpha2 loop is modulated by long-range sequence effects[J]. J Mol Biol, 2009, 389(5): 833-845.

[3] Pellecchia M, Sem D S, Wuthrich K. NMR in drug discovery[J]. Nat Rev Drug Discov, 2002, 1(3): 211-219.

[4] Pellecchia M, Bertini I, Cowburn D, et al. Perspectives on NMR in drug discovery: a technique comes of age
[J]. Nat Rev Drug Discov, 2008, 7(9): 738-745.

[5] Sun H, Tang Y, Xiang J, et al. Spectroscopic studies of the interaction between quercetin and G-quadruplex DNA
[J]. Bioorg Med Chem Lett, 2006, 16(13): 3 586-3 589.

[6] Sun H, Xiang J, Tang Y, et al. Regulation and recognization of the extended G-quadruplex by rutin[J]. Biochem Biophys Res Commun, 2007, 352(4): 942-946.

[7] Li Q, Xiang J, Li X, et al. Stabilizing parallel G-quadruplex DNA by a new class of ligands: Two non-planar alkaloids through interaction in lateral grooves[J]. Biochimie, 2009, 91(7): 811-819.

[8] Yang Q, Xiang J, Yang S, et al. Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly: Ⅱ. The binding characterization with specific intramolecular G-quadruplex and the recognizing mechanism[J]. Nucleic Acids Res, 2009, doi:10.1093/nar/gkp1045.

[9] Weidauer S E, Schmieder P, Beerbaum M, et al. NMR structure of the Wnt modulator protein Sclerostin[J]. Biochem Biophys Res Commun, 2009, 380(1): 160-165.

[10] Laurents D V, Bruix M, Jimenez M A, et al. The ¹H, ¹³C, ¹⁵N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy[J]. Biopolymers, 2009, 91(12): 1 018-1 028.

[11] Eletsky A, Sukumaran D K, Xiao R, et al. NMR structure of protein YvyC from Bacillus subtilis reveals unexpected structural similarity between two PFAM families[J]. Proteins, 2009, 76(4): 1 037-1 041.

[12] Nanga R P, Brender J R, Xu J, et al. Three-dimensional structure and orientation of rat islet amyloid polypeptide protein in a membrane environment by solution NMR spectroscopy[J]. J Am Chem Soc, 2009, 131(23): 8 252-8 261.

[13] Macek P, Chmelik J, Krizova I, et al. NMR structure of the N-terminal domain of capsid protein from the mason-pfizer monkey virus[J]. J Mol Biol, 2009, 392(1): 100-114.

[14] Ryabov Y, Suh J Y, Grishaev A, et al. Using the experimentally determined components of the overall rotational diffusion tensor to restrain molecular shape and size in NMR structure determination of globular proteins and protein-protein complexes[J]. J Am Chem Soc, 2009, 131(27): 9 522-9 531.

[15] Theisgen S, Scheidt H A, Magalhaes A, et al. A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2[J]. Biochim Biophys Acta, 2010, 1798(2): 266-274. 

[16] Saio T, Yokochi M, Inagaki F. The NMR structure of the p62 PB1 domain, a key protein in autophagy and NFkappaB signaling pathway[J]. J Biomol NMR, 2009, 45(3): 335-341.

[17] Wang Y, Patel D J. Solution structure of a parallel-stranded G-quadruplex DNA[J]. J Mol Biol, 1993, 234(4): 1 171-1 183.

[18] Kettani A, Bouaziz S, Wang W, et al. Bombyx mori single repeat telomeric DNA sequence forms a G-quadruplex capped by base triads[J]. Nat Struct Biol, 1997, 4(5): 382-389.

[19] Zhang N, Phan A T, Patel D J. (3 + 1) Assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex[J]. J Am Chem Soc, 2005, 127(49): 17 277-17 285.

[20] Phan A T, Kuryavyi V, Gaw H Y, et al. Smallmolecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter[J]. Nat Chem Biol, 2005, 1(3): 167-173.

[21] Luu K N, Phan A T, Kuryavyi V, et al. Structure of the human telomere in K⁺ solution: an intramolecular (3+1) G-quadruplex scaffold[J]. J Am Chem Soc, 2006, 128(30): 9 963-9 970.

[22] Phan A T, Kuryavyi V, Burge S, et al. Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter[J]. J Am Chem Soc, 2007, 129(14): 4 386-4 392.

[23] Lim K W, Amrane S, Bouaziz S, et al. Structure of the human telomere in K⁺solution: a stable basket-type G-quadruplex with only two G-tetrad layers[J]. J Am Chem Soc, 2009, 131(12): 4 301-4 309.

[24] Chataigner I I, Gennari C, Piarulli U, et al. Discovery of a new efficient chiral ligand for copper-catalyzed enantioselective michael additions by high-throughput screening of a parallel library[J]. Angew Chem Int Ed Engl, 2000, 39(5): 916-918.

[25] Seneci P, Miertus S. Combinatorial chemistry and high-throughput screening in drug discovery: different strategies and formats[J]. Mol Divers, 2000, 5(2): 75-89.

[26] Potyrailo R A, Chisholm B J, Olson D R, et al. Development of combinatorial chemistry methods for coatings: high-throughput screening of abrasion resistance of coatings libraries[J]. Anal Chem, 2002, 74(19): 5 105-5 111.

[27] Fonseca M H, List B. Combinatorial chemistry and high-throughput screening for the discovery of organocatalysts[J]. Curr Opin Chem Biol, 2004, 8(3): 319-326.

[28] Diller D J. The synergy between combinatorial chemistry and high-throughput screening[J]. Curr Opin Drug Discov Devel, 2008, 11(3): 346-355.

[29] Zhou Q, Li L, Xiang J, et al. Screening potential antitumor agents from natural plant extracts by G-quadruplex recognition and NMR methods[J]. Angew Chem Int Ed Engl, 2008, 47(30): 5 590-5 592.

[30] Zhou Q, Li L, Xiang J, et al. Fast screening and structural elucidation of G-quadruplex ligands from a mixture via G-quadruplex recognition and NMR methods[J]. Biochimie, 2009, 91(2): 304-308.

[31] Hajduk P J, Olejniczak E T, Fesik S W. One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules[J]. J Am Chem Soc, 1997, 119(50): 12 257-12 261.

[32] Price W S. Pulsed-field gradient nuclear magnetic resonance as a tool for studying translational diffusion .1. Basic theory[J]. Concepts Magn Reson, 1997, 9(5): 299-336.

[33] Price W S. Pulsed-field gradient nuclear magnetic resonance as a tool for studying translational diffusion: Part Ⅱ. Experimental aspects[J]. Concepts Magn Reson, 1998, 10(4): 197-237.

[34] Dehner A, Kessler H. Diffusion NMR spectroscopy: Folding and aggregation of domains in p53[J]. Chembiochem, 2005, 6(9): 1 550-1 565.

[35] Stebbins J L, Jung D, Leone M, et al. A structure-based approach to retinoid X receptor-alpha inhibition[J]. J Biol Chem, 2006, 281(24): 16 643-16 648.

[36] Fejzo J, Lepre C A, Peng J W, et al. The SHAPES strategy: an NMR-based approach for lead generation in drug discovery[J]. Chem Biol, 1999, 6(10): 755-769.

[37] Johnson E C, Feher V A, Peng J W, et al. Application of NMR SHAPES screening to an RNA target[J]. J Am Chem Soc, 2003, 125(51): 15 724-15 725.

[38] Lepre C A, Peng J, Fejzo J, et al. Applications of SHAPES screening in drug discovery[J]. Comb Chem High Throughput Screen, 2002, 5(8): 583-590.

[39] Leone M, Freeze H H, Chan C S, et al. The Nuclear Overhauser Effect in the lead identification process[J]. Curr Drug Discov Technol, 2006, 3(2): 91-100.

[40] Meyer B, Klein J, Mayer M, et al. Saturation transfer difference NMR spectroscopy for identifying ligand epitopes and binding specificities[J]. Ernst Schering Res Found Workshop, 2004, 44: 149-167.

[41] Hajduk P J, Greer J. A decade of fragment-based drug design: strategic advances and lessons learned[J]. Nat Rev Drug Discov, 2007, 6(3): 211-219.

[42] Shuker S B, Hajduk P J, Meadows R P, et al. Discovering high-affinity ligands for proteins: SAR by NMR[J]. Science, 1996, 274(5292): 1 531-1 534.

[43] Hajduk P J, Sheppard G, Nettesheim D G, et al. Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR[J]. J Am Chem Soc, 1997, 119(25): 5 818-5 827.

[44] MacPherson L J, Bayburt E K, Capparelli M P, et al. Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits[J]. J Med Chem, 1997, 40(16): 2 525-2 532.

[45] Petros A M, Dinges J, Augeri D J, et al. Discovery of a potent inhibitor of the antiapoptotic protein Bcl-xL from NMR and parallel synthesis[J]. J Med Chem, 2006, 49(2): 656-663.

[46] Mayer M, Meyer B. Characterization of ligand binding by saturation transfer difference NMR spectroscopy[J]. Angew Chem Int Ed Engl, 1999, 38(12): 1 784-1 788.

[47] Mayer M, Meyer B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor[J]. J Am Chem Soc, 2001, 123(25): 6 108-6 117.

[48] Mayer M, James T L. NMR-based characterization of phenothiazines as a RNA binding scaffold[J]. J Am Chem Soc, 2004, 126(13): 4 453-4 460.

[49] Salvatella X, Martinell M, Gairi M, et al. A tetraguanidinium ligand binds to the surface of the tetramerization domain of protein P53[J]. Angew Chem Int Ed Engl, 2004, 43(2): 196-198.

[50] Mayer M, Lang P T, Gerber S, et al. Synthesis and testing of a focused phenothiazine library for binding to HIV-1 TAR RNA[J]. Chem Biol, 2006, 13(9): 993-1 000.

[51] Mayer M, James T L. Detecting ligand binding to a small RNA target via saturation transfer difference NMR experiments in D(2)O and H(2)O[J]. J Am Chem Soc, 2002, 124(45): 13 376-13 377.

[52] Yan J, Kline A D, Mo H, et al. The effect of relaxation on the epitope mapping by saturation transfer difference NMR[J]. J Magn Reson, 2003, 163(2): 270-276.

[53] Ramelot T A, Raman S, Kuzin A P, et al. Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study[J]. Proteins, 2009, 75(1): 147-167.

[54] Williamson M P, Craven C J. Automated protein structure calculation from NMR data[J]. J Biomol NMR, 2009, 43(3): 131-143.

[55] Sharma S, Zheng H, Huang Y J, et al. Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry[J]. Proteins, 2009, 76(4): 882-894.

[56] Berjanskii M, Tang P, Liang J, et al. GeNMR: a web server for rapid NMR-based protein structure determination[J]. Nucleic Acids Res, 2009, 37(Web Server issue): W670-677.

[57] Salmon L, Bouvignies G, Markwick P, et al. Protein conformational flexibility from structure-free analysis of NMR dipolar couplings: quantitative and absolute determination of backbone motion in ubiquitin[J]. Angew Chem Int Ed Engl, 2009, 48(23): 4 154-4 157.

[58] Raman S, Huang Y J, Mao B, et al. Accurate automated protein NMR structure determination using unassigned NOESY data[J]. J Am Chem Soc, 2010, 132(1): 202-207. 

[59] Columbus L, Lipfert J, Jambunathan K, et al. Mixing and matching detergents for membrane protein NMR structure determination[J]. J Am Chem Soc, 2009, 131(21): 7 320-7 326.

[60] Sakakibara D, Sasaki A, Ikeya T, et al. Protein structure determination in living cells by in-cell NMR spectroscopy[J]. Nature, 2009, 458(7234): 102-105.

[61] Pellecchia M, Meininger D, Dong Q, et al. NMR-based structural characterization of large protein-ligand interactions[J]. J Biomol NMR, 2002, 22(2): 165-173.

[62] Mergny J L, Helene C. G-quadruplex DNA: a target for drug design[J]. Nat Med, 1998, 4(12): 1 366-1 367.

[63] Hurley L H. DNA and its associated processes as targets for cancer therapy[J]. Nat Rev Cancer, 2002, 2(3): 188-200.

[64] Neidle S, Parkinson G. Telomere maintenance as a target for anticancer drug discovery[J]. Nat Rev Drug Discov, 2002, 1(5): 383-393.

[65] Hurley L H, Wheelhouse R T, Sun D, et al. G-quadruplexes as targets for drug design[J]. Pharmacol Ther, 2000, 85(3): 141-158.

[66] Hurley L H. Secondary DNA structures as molecular targets for cancer therapeutics[J]. Biochem Soc Trans, 2001, 29(Pt 6): 692-696.

[67] Neidle S, Read M A. G-quadruplexes as therapeutic targets[J]. Biopolymers, 2000, 56(3): 195-208.

[68] Koeppel F, Riou J F, Laoui A, et al. Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes[J]. Nucleic Acids Res, 2001, 29(5): 1 087-1 096.

[69] Ren J, Chaires J B. Preferential Binding of 3,3′-Diethyloxadicarbocyanine to Triplex DNA[J]. J Am Chem Soc, 2000, 122(2): 424-425.

[70] Borman S. Ascent of quadruplexes[J]. Chem Eng News, 2007, 85(22): 12-14.

[71] Newman D J. Natural products as leads to potential drugs: an old process or the new hope for drug discovery?
[J]. J Med Chem, 2008, 51(9): 2 589-2 599.

[72] Kintzios S E. Terrestrial plant-derived anticancer agents and plant species used in anticancer research[J]. Crit Rev Plant Sci, 2006, 25(2): 79-113.

[73] Balandrin M F, Klocke J A, Wurtele E S, et al. Natural plant chemicals: sources of industrial and medicinal materials[J]. Science, 1985, 228(4704): 1 154-1 160.

[74] Pezzuto J M. Plant-derived anticancer agents[J]. Biochem Pharmacol, 1997, 53(2): 121-133.

[75] Koeppel F, Riou J F, Laoui A, et al. Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes[J]. Nucleic Acids Res, 2001, 29(5): 1 087-1 096.

[76] Gomez D, Mergny J L, Riou J F. Detection of telomerase inhibitors based on G-quadruplex ligands by a modified telomeric repeat amplification protocol assay[J]. Cancer Res, 2002, 62(12): 3 365-3 368.

[77] Rosu F, De Pauw E, Guittat L, et al. Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis[J]. Biochemistry, 2003, 42(35): 10 361-10 371.

[78] Ladame S, Whitney A M, Balasubramanian S. Targeting nucleic acid secondary structures with polyamides using an optimized dynamic combinatorial approach[J]. Angew Chem Int Ed Engl, 2005, 44(35): 5 736-5 739.

[79] Meiboom S, Gill D. Modified Spin-Echo Method for Measuring Nuclear Relaxation Times[J]. Rev Sci Instrum, 1958, 29(8): 688-691.

[80] Stockman B J, Dalvit C. NMR screening techniques in drug discovery and drug design[J]. Prog Nucl Magn Reson Spectrosc, 2002, 41(3-4): 187-231. 

[81] Zhou Q, Bi Y, Xiang J, et al. Investigation on a potential targeting drug delivery system consisting of folate, mitoxantrone and human serum albumin[J]. Chinese J Chem, 2008, 26(8): 1 385-1 389.

[82] Zartler E R, Yan J, Mo H, et al. 1D NMR Methods in ligand-receptor interactions[J]. Curr Top Med Chem, 2003, 3(1): 25-37.

[83] Xu Q, Sachs J R, Wang T C, et al. Quantification and identification of components in solution mixtures from 1D proton NMR spectra using singular value decomposition[J]. Anal Chem, 2006, 78(20): 7 175-7 185.

[84] Lepre C A, Moore J M, Peng J W. Theory and applications of NMR-based screening in pharmaceutical research[J]. Chem Rev, 2004, 104(8): 3 641-3 676.