Crystal Structure Of Quinone Reductase 2 In Complex With Cancer Prodrug CB1954

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 mediatedanti -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 - crystallizationin 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...