苯醌还原酶2与抗癌前药CB1954的复合晶体结构
摘要
目的
肿瘤治疗是现代医学的重要课题。传统的抗肿瘤药物选择性不高,既杀伤肿瘤细胞,也损害体内繁殖旺盛的细胞或某些特定类型的正常细胞,常出现较明显的毒性反应。自杀基因/前药疗法可使前药在表达自杀基因的肿瘤内,转为高效毒性物质,通过直接细胞毒性或旁观者效应发挥抗肿瘤作用,这不仅使正常组织免受损伤,而且可清除无法用手术切除的癌灶或转移灶。CB1954(5-(aziridin-1-yl)-2,4-dinitrobenzamide)是一种抗癌前药(prodrug),人们最初发现它对大鼠Walker 256肿瘤具有高选择性活性并能治愈该肿瘤。目前用于抗体导向的酶-前药治疗(antibody directed enzyme-prodrug therapy,ADEPT)和基因导向的酶-前药治疗(gene-directed enzyme prodrug therapy,GDEPT,在英国已进入前列腺癌治疗的一期临床试验。作为前药,CB1954对造血系统具有很低的毒性。由于它并不是典型的苯醌衍生物,它不能被NADPH:细胞色素P450还原酶或黄嘌呤氧化酶等单电子转移酶还原,因为这些酶不能还原硝基基团。大鼠QR1(quinone reductase 1)能还原CB1954产生5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide,这是一种具有很高毒性的化合物,能诱导DNA链间交联(interstrand cross-linking),进而杀死癌细胞。然而人QR1对CB1954的催化能力很弱。最近发现人QR2在体外对CB1954的还原能力是QR1的3000多倍。CB1954和QR2电子供体同时给药的协同抗体介导的抗癌前药治疗(co-substrate-mediated antitumor prodrug therapy)由此得以发展,此方法将CB1954的IC_(50)由几百μM降至小于1μM。由此可见,QR2极有可能是在细胞内负责活化CB1954的酶。
由于QR2在某些癌症(如前列腺癌)中高度表达,且CB1954在癌症治疗中有重要作用,研究QR2活化CB1954的机制及特异性将对理解CB1954作用机理及设计针对QR2或QR1的特异性更高的抗癌前药有重要作用。
ObjectiveTumor therapy is an important topic in medical research nowadays. Traditional Cancer chemotherapy is often hampered by an insufficient therapeutic index , lacking of specificity, and the emergence of drug — resistant cell subpopu-lations. Enzyme - Prodrug Therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic products, is an approach aimed at enhancing the selectivity of cancer chemotherapy for solid tumors. CB1954(5 -(aziridin -1 - yl) -2,4-dinitrobenzamide)is a cancer prodrug,which exhibits dramatic and highly selective activity against the Rat Walker tumor and could actually cure this tumor. It has been of intense interest as a prodrug in the treatment of cancer in antibody - directed enzyme prodrug therapy ( ADEPT) and gene — directed enzyme prodrug therapy ( GDEPT) , and it is currently in phase I clinical trials for prostate cancer treatment in Britain. As a prodrug, CB1954 exhibits minimal toxic effects on the hematopoietic system. It is not a quinone derivative and therefore, it cannot be reduced by typical quinone reduetases, such as NADPH: cytochrome P450 reductase or xanthine oxidase. However, rat QR1 can serve as a nitroreductase in reducing CB1954, and the reduction product, 5 - ( aziridin -1 -yl) -4- hydroxylamino -2 - nitrobenzamide, is a highly toxic compound that reacts bifunctionally in cells to induce DNA - DNA inter-strand cross -linking, which consequently kills the cancer cells. However, the reduction of CB1954 by human QR1 is very inefficient. Recently it was discovered that QR2 is a much more efficient enzyme in the reductions of CB1954. It is 3000 times more efficient than human QR1 in the reduction of CB1954 when assayed in vitro. This led to the development of a novel co - substrate mediated
anti -tumor prodrug therapy, in which CB1954 id given simultaneously. This in .turn decreases IC50of CB1954 from a concentration in the range of hundreds of micro molar to less than 1 micro molar, and suggests that QR2 may be the cellular enzyme responsible for the activation of CB1954.Because QR2 is highly expressed in certain kinds of cancer ( such as prostate cancer) , and CB1954 plays an important role in cancer therapy, it is necessary to investigate the mechanism and specificity of the activation of CB1954 by QR2, which will aid the design of more specific cancer prodrugs towards QR2 orQRl.Material and Methods1. Chemicals and reagentsCB1954, nicotinamide, 1 - (2 - hydroxyethylnicotinamide) and menadione were purchased from Sigma. All other chemicals were of highest purity commercially available. 1 - ( carbamoylmethyl) -3 - carbamoylpyridinium iodide was prepared by stirring and heating a mixture of nicotinamide and 2 - iodoactamide in DMF at 60 for 3 hours as reported. Subsequent reduction to produce the di-hydronicotinamide derivatives ( SUB10R (1 - ( 2 - hydroxyethyldihydronicoti-namide) was carried out as reported.2. Protein MethodsTo produce recombinant QR2, the coding region of QR2 cDNA fragments were amplified by PCR and cloned into pET23d vector ( Novagen) between Ncol and Xhol. The insert was verified by DNA sequencing, and then transformed in to B834 E. coli strain. The recombinant protein was purified by passing through DEAE Sepharose, Superdex 75 (Pharmacia) and Mono -Q column (Pharmacia) sequentially to obtain the homogeneous protein sample. Protein concentration was determined by BioRad protein assay kit. The mutant QR2 ( Asnl61His) was generated by site -directed mutagenesis method. The mutant protein was expressed and purified similarly to the wild -type QR2.3. CrystallizationThe crystal of QR2 - CB1954 complex was obtained by co - crystallization
in the presence of 1 mM CB1954 with ammonium sulfate as precipitants.Crystallographic AnalysisAll diffraction data were collected at 100 ( K using a CCD detector at beam line 19BM of Advanced Photon Source (APS) , Argonne National Laboratories. Raw data were processed using the HKL2000 software. Crystal of the native QR2, QR2 - CB1954 complex all belong to F2l2l2lspace group. The cell dimensions of all the crystals are nearly identical. And consequently the complex structures were directly refined with the native structure ( 1 qr2, Protein Data Bank) with CNS and the density of CB1954 was clearly shown after Fourier -difference transformation. Figures were prepared using PDB viewer and rendered with POV - ray software packages. The atomic coordinates of QR2 - CB1954 complex have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University with access code 1X12.4. Enzymatic assaysQR2 QR2N161H activities were determined spectrophotometrically with menadione or CB1954 as substrate, and SUB10R as co - substrate. The catalysis was monitored at wavelength of 360 and 370 nm with a Unicam UV - Vis spectrophotometer.Results1. The QR2 - CB1954structure indicates that CB1954 is bound to the active site of QR2. The CB1954 molecule occupies the entire cavity, whereas resvera-trol occupies the entire cavity. The benzene ring of CB1954 sits near parallel to the plane of the isoalloxazine ring, stacked on top of ring C of the isoalloxazine moiety of FAD. Three phenylalanine side -chains (F131, F178 and F126) sitting on top of the benzene ring of C1954. Those hydrophobic interactions hold the benzene ring of CB1954 parallel to the plane of isoalloxazine ring of co - factor FAD. The orientation of CB1954 molecule is unmistakable, with the aziri-dine functional group and the amide group pointing towards the solvent. One of the two oxygens in the 2 - nitro group forms one hydrogen bond with the amide group of N161, while the other oxygen forms a hydrogen bond through a water
肿瘤治疗是现代医学的重要课题。传统的抗肿瘤药物选择性不高,既杀伤肿瘤细胞,也损害体内繁殖旺盛的细胞或某些特定类型的正常细胞,常出现较明显的毒性反应。自杀基因/前药疗法可使前药在表达自杀基因的肿瘤内,转为高效毒性物质,通过直接细胞毒性或旁观者效应发挥抗肿瘤作用,这不仅使正常组织免受损伤,而且可清除无法用手术切除的癌灶或转移灶。CB1954(5-(aziridin-1-yl)-2,4-dinitrobenzamide)是一种抗癌前药(prodrug),人们最初发现它对大鼠Walker 256肿瘤具有高选择性活性并能治愈该肿瘤。目前用于抗体导向的酶-前药治疗(antibody directed enzyme-prodrug therapy,ADEPT)和基因导向的酶-前药治疗(gene-directed enzyme prodrug therapy,GDEPT,在英国已进入前列腺癌治疗的一期临床试验。作为前药,CB1954对造血系统具有很低的毒性。由于它并不是典型的苯醌衍生物,它不能被NADPH:细胞色素P450还原酶或黄嘌呤氧化酶等单电子转移酶还原,因为这些酶不能还原硝基基团。大鼠QR1(quinone reductase 1)能还原CB1954产生5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide,这是一种具有很高毒性的化合物,能诱导DNA链间交联(interstrand cross-linking),进而杀死癌细胞。然而人QR1对CB1954的催化能力很弱。最近发现人QR2在体外对CB1954的还原能力是QR1的3000多倍。CB1954和QR2电子供体同时给药的协同抗体介导的抗癌前药治疗(co-substrate-mediated antitumor prodrug therapy)由此得以发展,此方法将CB1954的IC_(50)由几百μM降至小于1μM。由此可见,QR2极有可能是在细胞内负责活化CB1954的酶。
由于QR2在某些癌症(如前列腺癌)中高度表达,且CB1954在癌症治疗中有重要作用,研究QR2活化CB1954的机制及特异性将对理解CB1954作用机理及设计针对QR2或QR1的特异性更高的抗癌前药有重要作用。
ObjectiveTumor therapy is an important topic in medical research nowadays. Traditional Cancer chemotherapy is often hampered by an insufficient therapeutic index , lacking of specificity, and the emergence of drug — resistant cell subpopu-lations. Enzyme - Prodrug Therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic products, is an approach aimed at enhancing the selectivity of cancer chemotherapy for solid tumors. CB1954(5 -(aziridin -1 - yl) -2,4-dinitrobenzamide)is a cancer prodrug,which exhibits dramatic and highly selective activity against the Rat Walker tumor and could actually cure this tumor. It has been of intense interest as a prodrug in the treatment of cancer in antibody - directed enzyme prodrug therapy ( ADEPT) and gene — directed enzyme prodrug therapy ( GDEPT) , and it is currently in phase I clinical trials for prostate cancer treatment in Britain. As a prodrug, CB1954 exhibits minimal toxic effects on the hematopoietic system. It is not a quinone derivative and therefore, it cannot be reduced by typical quinone reduetases, such as NADPH: cytochrome P450 reductase or xanthine oxidase. However, rat QR1 can serve as a nitroreductase in reducing CB1954, and the reduction product, 5 - ( aziridin -1 -yl) -4- hydroxylamino -2 - nitrobenzamide, is a highly toxic compound that reacts bifunctionally in cells to induce DNA - DNA inter-strand cross -linking, which consequently kills the cancer cells. However, the reduction of CB1954 by human QR1 is very inefficient. Recently it was discovered that QR2 is a much more efficient enzyme in the reductions of CB1954. It is 3000 times more efficient than human QR1 in the reduction of CB1954 when assayed in vitro. This led to the development of a novel co - substrate mediated
anti -tumor prodrug therapy, in which CB1954 id given simultaneously. This in .turn decreases IC50of CB1954 from a concentration in the range of hundreds of micro molar to less than 1 micro molar, and suggests that QR2 may be the cellular enzyme responsible for the activation of CB1954.Because QR2 is highly expressed in certain kinds of cancer ( such as prostate cancer) , and CB1954 plays an important role in cancer therapy, it is necessary to investigate the mechanism and specificity of the activation of CB1954 by QR2, which will aid the design of more specific cancer prodrugs towards QR2 orQRl.Material and Methods1. Chemicals and reagentsCB1954, nicotinamide, 1 - (2 - hydroxyethylnicotinamide) and menadione were purchased from Sigma. All other chemicals were of highest purity commercially available. 1 - ( carbamoylmethyl) -3 - carbamoylpyridinium iodide was prepared by stirring and heating a mixture of nicotinamide and 2 - iodoactamide in DMF at 60 for 3 hours as reported. Subsequent reduction to produce the di-hydronicotinamide derivatives ( SUB10R (1 - ( 2 - hydroxyethyldihydronicoti-namide) was carried out as reported.2. Protein MethodsTo produce recombinant QR2, the coding region of QR2 cDNA fragments were amplified by PCR and cloned into pET23d vector ( Novagen) between Ncol and Xhol. The insert was verified by DNA sequencing, and then transformed in to B834 E. coli strain. The recombinant protein was purified by passing through DEAE Sepharose, Superdex 75 (Pharmacia) and Mono -Q column (Pharmacia) sequentially to obtain the homogeneous protein sample. Protein concentration was determined by BioRad protein assay kit. The mutant QR2 ( Asnl61His) was generated by site -directed mutagenesis method. The mutant protein was expressed and purified similarly to the wild -type QR2.3. CrystallizationThe crystal of QR2 - CB1954 complex was obtained by co - crystallization
in the presence of 1 mM CB1954 with ammonium sulfate as precipitants.Crystallographic AnalysisAll diffraction data were collected at 100 ( K using a CCD detector at beam line 19BM of Advanced Photon Source (APS) , Argonne National Laboratories. Raw data were processed using the HKL2000 software. Crystal of the native QR2, QR2 - CB1954 complex all belong to F2l2l2lspace group. The cell dimensions of all the crystals are nearly identical. And consequently the complex structures were directly refined with the native structure ( 1 qr2, Protein Data Bank) with CNS and the density of CB1954 was clearly shown after Fourier -difference transformation. Figures were prepared using PDB viewer and rendered with POV - ray software packages. The atomic coordinates of QR2 - CB1954 complex have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University with access code 1X12.4. Enzymatic assaysQR2 QR2N161H activities were determined spectrophotometrically with menadione or CB1954 as substrate, and SUB10R as co - substrate. The catalysis was monitored at wavelength of 360 and 370 nm with a Unicam UV - Vis spectrophotometer.Results1. The QR2 - CB1954structure indicates that CB1954 is bound to the active site of QR2. The CB1954 molecule occupies the entire cavity, whereas resvera-trol occupies the entire cavity. The benzene ring of CB1954 sits near parallel to the plane of the isoalloxazine ring, stacked on top of ring C of the isoalloxazine moiety of FAD. Three phenylalanine side -chains (F131, F178 and F126) sitting on top of the benzene ring of C1954. Those hydrophobic interactions hold the benzene ring of CB1954 parallel to the plane of isoalloxazine ring of co - factor FAD. The orientation of CB1954 molecule is unmistakable, with the aziri-dine functional group and the amide group pointing towards the solvent. One of the two oxygens in the 2 - nitro group forms one hydrogen bond with the amide group of N161, while the other oxygen forms a hydrogen bond through a water
引文
1. Knox RJ, Friedlos F, Boland MP. The bioactivation of CB 1954 and its use as a prodrug in antibody - directed enzyme prodrug therapy ( ADEPT). Cancer Metastasis Rev, 1993, 12 (2) : 195 -212.
2. Denny WA. Nitroreductase - based GDEPT. Curr Pharm Des, 2002, 8 (15): 1349-61.
3. Chung -Faye G, Palmer D, Anderson D, Clark J, Downes M, Baddeley J, Hussain S, Murray PI, Searle P, Seymour L, Harris PA, Ferry D, Kerr DJ. Virus - directed, enzyme prodrug therapy with nitroimidazole reductase; a phase I and pharmacokinetic study of its prodrug, CB1954. Clin Cancer Res, 2001, 7 (9) : 2662 -8.
4. Knox RJ, Boland MP, Friedlos F, Coles B, Southan C, Roberts JJ. The nitroreductase enzyme in Walker cells that activates 5 - ( aziridin -1 - yl) 2,4 - dinitrobenzamide ( CB 1954) to 5 - ( aziridin -1 -yl) -4- hydroxylamino — 2 - nitrobenzamide is a form of NAD ( P) H dehydrogenase ( quinone) (EC 1.6.99.2). Biochem Pharmacol, 1988, 37 (24) : 4671 -7.
5. Knox RJ, Jenkins TC, Hobbs SM, Chen S, Melton RG, Burke PJ. Bioactivation of 5 - ( aziridin -1 -yl) -2,4- dinitrobenzamide ( CB 1954 ) by human NAD( P) H quinone oxidoreductase 2: a novel co - substrate - mediated antitumor prodrug therapy. Cancer Res,2000, 60 (15) : 4179 -86.
6. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse - Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography & NMR system; A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr, 1998, 54 ( Pt 5) : 905 -21.
7. Guex N, Peitsch MC. SWISS - MODEL and the Swiss - PdbViewer; an environment for comparative protein modeling. Electrophoresis, 1997, 18 (15): 2714-23.
8. Harris M, Jones TA. Molray-----a web interface between O and the POV Ray ray tracer. Acta Crystallogr D Biol Crystallogr, 2001, 57 ( Pt 8) : 1201-3.
9. Knox RJ, Friedlos F, Marchbank T, Roberts JJ. Bioactivation of CB 1954: reaction of the active 4 - hydroxylamino derivative with thioesters to form the ultimate DNA - DNA interstrand crosslinking species. Biochem Pharmacol, 1991, 42 (9): 1691-7.
10. Wu K, Knox R, Sun XZ, Joseph P, Jaiswal AK, Zhang D, Deng PS, Chen S. Catalytic properties of NAD ( P) H: quinone oxidoreductase - 2 (NQO2) , a dihydronicotinamide riboside dependent oxidoreductase. Arch Biochem Biophys, 1997, 347 (2) : 221 -8.
11. Li R, Bianchet MA, Talalay P, Amzel LM. The three -dimensional structure of NAD ( P) H: quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two - electron reduction. Proc Natl Acad Sci U S A, 1995, 92 (19): 8846 -50.
12. Buryanovskyy L, Fu Y, Boyd M, Ma Y, Hsieh TC, Wu JM, Zhang Z. Crystal structure of quinone reductase 2 in complex with resveratrol. Biochemistry, 2004, 43 (36): 11417 -26.
13. Chen S, Wu K, Knox R. Structure - function studies of DT - diaphorase ( NQO1) and NRH: quinone oxidoreductase ( NQO2 ). Free Radic Biol Med, 2000, 29 (3 -4): 276 -84.
14.Liao S, Williams - Ashman HG. Enzymatic oxidation of some non - phosphorylated derivatives of dihydronicotinamide. Biochem Biophys Res Commun, 1961, 4: 208-13.
15. Foster CE, Bianchet MA, Talalay P, Faig M, Amzel LM. Structures of mammalian cytosolic quinone reductases. Free Radic Biol Med, 2000, 29 (3-4): 241-5.
16. Harada S, Fujii C, Hayashi A, Ohkoshi N. An association between idiopathic Parkinsons disease and polymorphisms of phase II detoxification enzymes : glutathione S - transferase Ml and quinone oxidoreductase 1 and 2. Biochem Biophys Res Commun, 2001, 288 (4) : 887 -92.
17. Harada S, Tachikawa H, Kawanishi Y. A possible association between an insertion/deletion polymorphism of the NQO2 gene and schizophrenia. Psychiatr Genet, 2003, 13 (4) : 205 -9.
2. Denny WA. Nitroreductase - based GDEPT. Curr Pharm Des, 2002, 8 (15): 1349-61.
3. Chung -Faye G, Palmer D, Anderson D, Clark J, Downes M, Baddeley J, Hussain S, Murray PI, Searle P, Seymour L, Harris PA, Ferry D, Kerr DJ. Virus - directed, enzyme prodrug therapy with nitroimidazole reductase; a phase I and pharmacokinetic study of its prodrug, CB1954. Clin Cancer Res, 2001, 7 (9) : 2662 -8.
4. Knox RJ, Boland MP, Friedlos F, Coles B, Southan C, Roberts JJ. The nitroreductase enzyme in Walker cells that activates 5 - ( aziridin -1 - yl) 2,4 - dinitrobenzamide ( CB 1954) to 5 - ( aziridin -1 -yl) -4- hydroxylamino — 2 - nitrobenzamide is a form of NAD ( P) H dehydrogenase ( quinone) (EC 1.6.99.2). Biochem Pharmacol, 1988, 37 (24) : 4671 -7.
5. Knox RJ, Jenkins TC, Hobbs SM, Chen S, Melton RG, Burke PJ. Bioactivation of 5 - ( aziridin -1 -yl) -2,4- dinitrobenzamide ( CB 1954 ) by human NAD( P) H quinone oxidoreductase 2: a novel co - substrate - mediated antitumor prodrug therapy. Cancer Res,2000, 60 (15) : 4179 -86.
6. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse - Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography & NMR system; A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr, 1998, 54 ( Pt 5) : 905 -21.
7. Guex N, Peitsch MC. SWISS - MODEL and the Swiss - PdbViewer; an environment for comparative protein modeling. Electrophoresis, 1997, 18 (15): 2714-23.
8. Harris M, Jones TA. Molray-----a web interface between O and the POV Ray ray tracer. Acta Crystallogr D Biol Crystallogr, 2001, 57 ( Pt 8) : 1201-3.
9. Knox RJ, Friedlos F, Marchbank T, Roberts JJ. Bioactivation of CB 1954: reaction of the active 4 - hydroxylamino derivative with thioesters to form the ultimate DNA - DNA interstrand crosslinking species. Biochem Pharmacol, 1991, 42 (9): 1691-7.
10. Wu K, Knox R, Sun XZ, Joseph P, Jaiswal AK, Zhang D, Deng PS, Chen S. Catalytic properties of NAD ( P) H: quinone oxidoreductase - 2 (NQO2) , a dihydronicotinamide riboside dependent oxidoreductase. Arch Biochem Biophys, 1997, 347 (2) : 221 -8.
11. Li R, Bianchet MA, Talalay P, Amzel LM. The three -dimensional structure of NAD ( P) H: quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two - electron reduction. Proc Natl Acad Sci U S A, 1995, 92 (19): 8846 -50.
12. Buryanovskyy L, Fu Y, Boyd M, Ma Y, Hsieh TC, Wu JM, Zhang Z. Crystal structure of quinone reductase 2 in complex with resveratrol. Biochemistry, 2004, 43 (36): 11417 -26.
13. Chen S, Wu K, Knox R. Structure - function studies of DT - diaphorase ( NQO1) and NRH: quinone oxidoreductase ( NQO2 ). Free Radic Biol Med, 2000, 29 (3 -4): 276 -84.
14.Liao S, Williams - Ashman HG. Enzymatic oxidation of some non - phosphorylated derivatives of dihydronicotinamide. Biochem Biophys Res Commun, 1961, 4: 208-13.
15. Foster CE, Bianchet MA, Talalay P, Faig M, Amzel LM. Structures of mammalian cytosolic quinone reductases. Free Radic Biol Med, 2000, 29 (3-4): 241-5.
16. Harada S, Fujii C, Hayashi A, Ohkoshi N. An association between idiopathic Parkinsons disease and polymorphisms of phase II detoxification enzymes : glutathione S - transferase Ml and quinone oxidoreductase 1 and 2. Biochem Biophys Res Commun, 2001, 288 (4) : 887 -92.
17. Harada S, Tachikawa H, Kawanishi Y. A possible association between an insertion/deletion polymorphism of the NQO2 gene and schizophrenia. Psychiatr Genet, 2003, 13 (4) : 205 -9.