药物设计学：核酸化学及以核酸为靶点的药物设计

1、核酸化学及以核酸为靶点的药物设计 RNA Is AscendingScience, 2001, 294, 2443RNA-building enzyme revealed1、基于核酸代谢合成途径的药物设计2、反义寡核苷酸药物研究3、cADPR模拟物的设计、合成及性质研究de novo synthesis of purinesPRPP激酶PRPP氨基转移酶嘌呤合成的分之点之一对核苷酸反馈抑制敏感Asp5-氨基咪唑-4-羧酸核苷酸IMP-亚甲基四氢叶酸(AICAR)salvage pathway嘌呤磷酸核糖转移酶在某些器官、组织中，如脑、骨髓等，只能通过补救合成途径合成嘌呤核苷；在某些肿瘤细胞或病

2、毒感染细胞中，核苷酸合成十分旺盛而补救合成是主要途径。反馈调节de novo synthesis of pyrimidinesUMPUDP dUDPUTP dUMPCTP dTMP还原酶 CN CC C N123456氨基甲酰磷酸天冬氨酸嘧啶碱合成的元素来源嘧啶 + PRPP 一磷酸嘧啶核苷 + PPi嘧啶磷酸核苷转移酶嘧啶核苷酸的补救途径尿嘧啶核苷 + ATP UMP + ADP尿苷激酶salvage pathway核酸的合成途径中涉及许多关键性的酶 （如次黄嘌呤鸟嘌呤磷酸核糖转移酶，PRPP酰胺转移酶, etc.），多种底物（如IMP，AMP，GMP, etc.）以及原料(如一碳单位、氨基

3、酸, etc.)，从这些入手都可以进行战略性的药物设计。利用生物电子等排原理等方法将代谢物的结构作细微的合理改变就可以得到很多有效的代谢拮抗物。药物设计途径叶酸类似物叶酸（二氢叶酸、四氢叶酸）为嘌呤核苷酸合成提供重要原料一碳单位，设计叶酸类似物可以竞争性地抑制二氢叶酸还原酶，使叶酸不能还原成二氢叶酸和四氢叶酸。从而抑制了嘌呤核苷酸的合成。RR叶酸OHH氨基蝶呤NH2H氨甲蝶呤NH2CH3MTX与二氢叶酸还原酶的亲和力比二氢叶酸强1000倍，同时也能抑制胸苷酸合成酶。氨基酸类似物氨基酸类似物主要是核苷酸合成过程中重要原料谷氨酰胺的结构类似物，干扰谷氨酰胺在核苷酸合成中的作用。谷氨酰胺氮杂丝氨酸（

4、重氮乙酰丝氨酸）6-重氮-5-氧正亮氨酸嘌呤类似物6-巯基嘌呤（6MP）磺巯嘌呤钠（Sulfomercaprine Sodium） 后者是前者的前药，6MP与次黄嘌呤结构的结构相似，在体内经酶促转变为活性的6硫代次黄嘌呤核苷酸，抑制腺酰琥珀酸合成酶，阻止IMP转变为腺苷酸，抑制DNA和RNA的合成。还能竞争性抑制次黄嘌呤鸟嘌呤磷酸核糖转移酶以及反馈抑制PRPP酰胺转移酶。尿嘧啶类似物尿嘧啶进入肿瘤组织的速度比其它嘧啶快，根据电子等排原理以卤原子替代氢原子。5-氟尿嘧啶（Fluorouracil）由于氟原子的半径和氢原子半径相近，氟化物的体积与原化合物几乎相等，加之CF键特别稳定，在代谢过程中不

5、易分解，能在分子水平代替正常代谢物，因而是胸腺嘧啶合成酶（TS）抑制剂，使TMP的合成受到阻断。5-FU还能在体内转变成FUTP，FUTP可以以FUMP的形式掺入RNA分子，异常核苷酸破坏了RNA的结构和功能。尿嘧啶类似物氟尿嘧啶由于存在严重的消化道反应和骨髓抑制等副作用，因而研制了大量的5氟尿嘧啶的衍生物。替加氟（Tegafur）双呋氟尿嘧啶（Difuradin）去氧氟尿苷（Doxifluridine）卡莫氟（Carmofur）核酸分解代谢中的抗代谢物嘌呤核苷酸嘌呤碱尿酸（uric acid）别嘌呤醇与次黄嘌呤结构类似，抑制黄嘌呤氧化酶，与酶紧密结合使之失活，抑制尿酸的生成。并可生成别嘌呤核

6、苷酸抑制嘌呤核苷酸的合成。临床上用于治疗痛风症（gout）。黄嘌呤氧化酶HDMS or BSAThe presence of HMDS can inactivate the catalystN7-isomer is formed if SnCl4 is used.TMSOTf and suitable protecting groups can eliminate N7-product formation.A-Form B-Form Z-Form可逆相互作用纺锤霉素（a）和咪唑-纺锤霉素（b）分别与A:T和G:C碱基对富集序列结合示意图。空心箭头表示从小分子给体到DNA受体之间的氢键，实心箭头

7、表示从DNA给体到小分子受体之间的氢键，虚线表示范德华非键相互作用 Key features to be met by antisense oligonucleotides:High affinity and high specificity of an oligonucleotide to its mRNA target.Rapid crossing of cellular and nuclear membranes (bioavailability),Increased stability of oligonucleotide analogues in a cellular environ

8、ment (biostability),Minimization of toxicity, immunogenicity and non-antisense activities arising from unspecific binding to elements of the cellular matix,Feasibility of mass production at a realistic price.SYNTHESIS OF ISONUCLEOSIDESi) Acetone, H+; ii) TsCl, Pyridine; iv) 1% TFA, MeOH; v) K2CO3, M

9、eOH; vi) KOBu-t, DMF; vii) 0.3% TFA, NaBH4SYNTHESIS OF ISONUCLEOSIDESi) Acetone, H+; ii) Na2CO3; iii) TsCl, Pyridine; iv) 1% TFA, MeOH; v) K2CO3, MeOH; vi) KOBu-t, DMF; vii) 0.3% TFA, NaBH4SYNTHESIS OF ISONUCLEOSIDESi) NaNO2, H+; ii) NaBH4; iii) BzCl, Pyridine;iv) PPh3; v) Base, DBU; vi) NaOCH3, CH3

10、OHSYNTHESIS OF ISONUCLEOSIDESi) Acetone, H+; ii) TsCl, Pyridine; iii) 1% TFA, MeOH;iv) K2CO3, MeOH; v) KOBu-t, DMF; vi) a. 0.3% TFA; b. NaBH4The root mean square position derivatives for all heavy atoms from the average structure as a function of time dA14:dT14dA14:AdA14:BGlobal twist along the sequ

11、ence for the MD-averaged structures of the duplexes(the first and the last base pairs are not considered) End and side views of (from left to right) canonical B DNA; average structure of dA14:dT14; average structure of dA14:A; average structure of dA14:BPeptide Conjugated Oligonucleotide肽缀合寡核苷酸的合成方法

12、肽缀合寡核苷酸的合成方法Bioconjugate Chem. 2003, 14, 153-157.ESR signal strength of spin labeled conjugates of peptide and PNA R15T10 (1), R10T10(2), R6T10 (3) and spin labeled PNA T10 (4) incubated with erythrocyte. HPLC chromatogram of arginine-rich peptide and oligonucleotide conjugation for 2 h (a) and 8 h

13、(b).Delivery of FITC-oligonucleotide (a) and (L-Arg)9Cys-conjugated FITC-labeled oligonucleotide (b) incubated with HepG-2 cells by confocal laser scanning microscopy.cAMPcGMPcADPRPharmacol. Therap., 2000, 87, 199-226.cADPRcADPR对细胞内钙库的调控作用DirectlycADPR与Ryanodine受体作用的可能途径Stability of cADPRJ. Med. Che

14、m. 2002, 45, 5340-5352.J. Cell Sci. 2004, 117, 2141-2149.Effect of the cADPR mimic cIDPRE on Ca2+ signaling in T cellsJ. Med. Chem. 2004, 47, 5674-5682.Effect of the cADPR mimic cIDPRE on Ca2+ signaling in T cellsEffect of the cIDPRE and 8-substituted cIDPRE analogues on Ca2+ signaling in T cellsSup

15、erimposition of minimized conformations of cADPR (yellow) and cIDP-DE (pink). (a) superimposed based on nucleobases; (b) superimposed based on diphosphates; (c) superimposed based on whole molecules. (a)(b)(c)Reagents and condition: (a) BzCl，Py；（b）ClCH2OCH2CH2OAC , BSA，TMSOTf，DCE；（c）NH3/CH3OH；（d）NaN

16、O2，AcOH; (e) TBDMSCl, Imidazole, DMF, r.t.Reagents and condition: (a) DBU, ClCH2OCH2CH2OAC, CH2Cl2, rt; (b) TBAF, THF, rt; (c) (PhNH)2POCl, tetrazole, Py, rt; (d) CH3ONa, CH3OH, rt; (e) PSS, TPSCl, tetrazole, Py, rt; (f) isoamyl nitrite, Py:ACOH:AC2O(2:1:1), H3PO2, Et3N, Py; (g) I2, 3MS, Py, rt. em

17、= 355 nmExcitation spectrum of cIDP-DEEmission spectrum of cIDP-DE excited at 278 nm= 1 mMCa2+extra = 0 mM Ca2+i nM time secIMIMcIDP-DECa2+i was determined in Jurkat T cells in the absence of extracellular Ca2+. Intracellular Ca2+ pools were completely depleted by addition of ionomycin; a second add

18、ition of ionomycin did not release any further Ca2+. Under these conditions cIDP-DE did not change the fura2 ratio Ca2+ nMtime secCa2+extra = 0 mM= 1 mMcIDPDEcIDPDE induced a transient Ca2+ release after a short delay when intracellular Ca2+ pools were filled, and Ca2+ store depletion by cIDP-DE act

19、ivated Ca2+ entry upon re-addition of extracellular Ca2+ nucleotide uMCa2+ nMcIDPDE released Ca2+ from intracellular stores in saponin-permeabilized Jurkat T cells, However, cIDP-DE was a much weaker agonist as compared to cADPR at least at high concentrations. 100 seccIDPDE 500 uM cIDPDE 1 mM anti-

20、CD3 OKT3 Ca2+ nM Effect of cIDP-DE on Ca2+ signaling in intact Jurkat T cells. Both 500 uM and 1 mM cIDPDE activated biphasic Ca2+ signaling consisting of a dose-dependent initial peak and a less dose-dependent sustained elevation of Ca2+i cIDPDE, added at 1 mM extracellular concentration, induced C

21、a2+ signaling comparable to the anti-CD3 mAb OKT3 nucleotide uMCa2+ nMEffect of cIDPDE on Ca2+ signaling in intact Jurkat T cells. Both 500 uM and 1 mM cIDPDE activated biphasic Ca2+ signaling consisting of a dose-dependent initial peak.2222Ca2+i nM46.415 96.793 102.812 105.054 sec45.984 96.826 102.

22、846 105.078 seccontrolcIDPDE (500 uM)Ca2+ mobilizing activity of cIDP-DE observed in Ca2+ imaging experiments. J. Biol. Chem. 2005, 280, 15952-15959 time sec Ca2+i nM cIDPDE control Ca2+ mobilizing activity of cIDPDE observed in Ca2+ imaging experiments. 药物发现之路开发成本：1962, $4 million/drug 1996, $350 m

23、illion/drug 2000, $500 million/drug 2003, $800 million/drug开发周期：1960-1980，almost quadrupled 1980-, 12-15 years 1962, tragedy of thalidomide led to the passing of the Harris-Kefauver Amendments 成功几率： 1950, 7000 compds/drug 1979, 10000 compds/drug 2004, 20000 compds/drug“I have not failed. Ive just fo

24、und 10,000 ways that wont work.”结语Medicinal chemistry is the sciences that deals with the discovery and design of new therapeutic chemicals and their development into useful medicines.Medicines are substances used to treat diseases.Drugs are molecules used as medicines or as components in medicines

25、to diagnose, cure, mitigate, treat, or prevent disease.Medicinal chemistry may involve isolation of compounds from nature or the synthesis of new molecules, investigations of the relationships between the structure of natural and/or synthetic compounds and their biological activitiesINTRODUCTIONNatu

26、re is still an excellent source of new drugs or, more commonly, of precursors to drugs.1999, 9 of the 20 leading drugs were derived from natural products;1983-1994, almost 40% of the 520 new drugs were natural products or derived from natural products;More than 60% of the anticancer and anti-infective agents are of natural product origin or deri