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1 Printed in the Republic of Korea Beta-lactamase 저해제이성체의결합양상과활성관계 Á Á Á½ * y w w w ( ) Relationship Between the Binding Mode and the Activity of Stereoisomers of Beta-Lactamase Inhibitors Sung Hee Mok, Inhee Choi, Insun Park, and Choonmi Kim* College of Pharmacy, Ewha Womans University, Seoul , Korea (Received September 8, 2005). ful docking v FlexiDock w sulbactam allyl ey q k- Bacteroides fragilis k-lactamase docking w š w 3-phenyl-clavams 6-methyl 3-phenyl clavams Enterobacter cloacae P99 k-lactamase dockingw k-lactamase ligand w ³ w. k-lactamase wy w x w y w w, y û ù w w w ww ù x w x w w w y ùkü. docking w w w y kw w w flexible docking zw ƒ k-lactamase w w w. : FlexiDock, k-lactamase, w,, w ABSTRACT. Using the computer docking program, FlexiDock, two sets of α and β isomers of novel allyl substituted derivatives of sulbactam were docked into β-lactamase of Bacteroides fragilis and four diastereomers of 3-phenyl-clavams and four diastereomers of 6-methyl 3-phenyl clavams were docked into β-lactamase of Enterobacter cloacae P99 to determine the binding modes of β-lactamase-ligand complex. Isomers with high activities were docked stably inside the active sites with five hydrogen bonds whereas the others with low or no activities formed one to three hydrogen bonds or no bond at all, demonstrating the positive correlation between the binding modes and the activities. These results indicate that the biologically active stereoisomers can be selected by the binding mode differences through docking study. Thus, flexible docking used in this work could be a useful and effective method in the development of therapeutically effective stereoisomers of β-lactamase inhibitors. Keywords: FlexiDock, β-lactamase, Inhibitor, Stereoisomers, Binding Modes Enterobacter species penicillin cephalosporin β-lactam w w ü ùkù z vancomycin w ü»» w l w w ü w. ü 1,2 l w β- lactamaseƒ w β-lactam ring amide bond ƒ ww w z w

2 462 Á Á Á½ w» w w wì β- lactamase w n wš z β-lactamase w w ƒ š. β-lactamase» j serine z metal z ù. class A, C D z ƒ sw z class B z ƒ. Class A z w w x š sulbactam tazobactam ù w class Bù C z w, p class B w metalloβ-lactamase w ƒ. z w y ù 3,4 kü Ÿ w w w ƒ š. ƒ β-lactamase w sulbactam tazobactam w β-lactamase active site w C-6 e w j» group w w œ x š, Sandanayaka penam sulfones C-6 e y» w allyl substituted penam sulfones w w (Table 1). 3,3-dimethyl-4,4,7-trioxo-6-(4-oxo-pent- 2enyl)-4lamda*6*-thia-1-aza-bicyclo[3.2.0]heptane-2- carboxylic acid(opca) 6-(3-methoxycarbonyl-allyl)3,3- dimethyl-4,4,7-trioxo-4lamda*6*-thia-1-aza-bicyclo [3.2.0]heptane-2-carboxylic acid(mcca) α β- Bacteroides fragilis β-lactamase w y d β- y α- sulbactam ù tazobactam j ƒw C-6 e yw y g. 2 w β-lactamase w w clavams w clavulanic acid O-acyl class A z w w w ù ký w chiral vinyl ether [2+2] cycloaddition mw 3-phenyl clavams(3-pc) 6-methyl-3-phenyl clavams(6-mp) w š, w y class C w Enterobacter cloacae P99 β-lactamase w d. 5 C-3 C-5 eƒ R configuration y ƒ j ùkû. computer aided drug design(cadd) y ù x» y w š. CADD w molecular docking X-ray crystallographyù NMR ƒ x w ligandƒ y ww w y ü j» w. p y p p y y w y» w w š y w» w computer aided molecular docking z k., ligand ƒƒ binding mode w» w Tripos Co. SYBYL package sw docking module FlexiDock w ww. 6 FlexiDock genetic algorithm w, van der Waals overlap ù» ƒ w ligand binding pocket protein ligand flexible w w induced-fit docking ww program. 7 X-ray crystallography x class B C w w Bacteroides fragilis β-lactamase Enterobacter cloacae P99 β-lactamase protein data bank(pdb) kw, FlexiDock program X- protein-ligand y ù yw xw w z, w dockingw w k w w β- lactamase y w x y w wš w. Silicon Graphics Octane Workstation SYBYL package(version 7.0) flexible docking v FlexiDock ww. Selection of β-lactamase complex PDB Bacteroides fragilis β-lactamase tricyclic inhibitor 7,8-dihydroxy-1-methoxy-3-methyl- 10-oxo-4,10-dihydro-1H,3H-pyrano[4,3-B]chromene-9- carboxylic acid(sb236050) complex x wš x

3 1KR3 kw (resolution=2.5å). z 464» ƒ š š sodium zincƒ sw homodimer. 9 Enterobacter cloacae P99 β-lactamase phosphonate monoester inhibitor(ipp) complex x wš 1BLS kw (resolution=2.3å). complex chain 722» ligand (P-Iodophenylacetylamino)methyl phosphonic acid ƒ š. 1 Preperation of β-lactamase and ligands for docking PDB kw β-lactamase w kw wš ƒw z X- ligand wš Kollman-all-atom charge w. w ligand y w w w š š wù 9,10 ƒwš Tripos force field Gasteiger-Hückel charge w. w ligand bond order wš ƒw ƒ Tripos force field Gasteiger-Hückel charge w. ƒƒ charge, ligand w β-lactamase-ligand w w š Powell method w gradient 0.1 Kcal/mol yw. e SYBYL default w w. Docking w SYBYL biopolymer module w z conjugate gradient method gradientƒ Kcal/mol ¾ Tripos force field Gasteiger-Hückel charge w y w. Docking the ligands into the β-lactamase X- ligand w β-lactamase w docking ww. Binding pocket active site» l 4Å ü» sw g w š, β-lactamase active site» ligands flexiblew w, hydrogen donor acceptor w. x w y ú» w preposition jš simulation ww, β-lactamase w w 463 ƒ ûš orientation w kw. Docking w» w X- ligand w z ligand dockingw w w X- w reproducibility test ww, X- ligand w ligand root mean square deviation(rmsd) w. docking active site» w, y w» p w q w w ³ w. š Reproducibility X- ligand w z û β- lactamase w ligand dockingw w X- y, mw FlexiDock docking protocol w ƒ ù yw x w. Bacteroides fragilis β-lactamase - SB complex: SB û³ q Chaetomium funicola tricyclic heterocycle ƒ metallo-β-lactamase w. w 8 dockingw w X- ligand RMSD 0.28Å orientation conformation, x ùkü (Fig. 1a). His162, Asn193, Lys184, š w w w y w y w. Enterobacter cloacae P99 β-lactamase - IPP complex: Phosphonate monoester β-lactamase active site serine phosphorylationw w w. 10 w dockingw ligand RMSD 0.43Å orientation conformation w (Fig. 1b). ligandƒ Ser64 œ w y e z viewer 4.13Å. œ w ñš ligand w z û w ligand docking w w ƒ 3.53Å ùkù, w y ú w q. w Gln120,

residues, Black dotted lines: hydrogen bonds. Table 1.

Chemical name Ligand code IC 50 (µm) Structure 3,3-Dimethyl-4,4,7-trioxo-6-(4-oxo-pent- 2enyl)-4lamda*6*-thia-1-aza-bicyclo[3.

4 464 Á Á Á½ Fig. 1. Docking of SB into Bacteroides fragilis β-lactamase(a) and IPP into Enterobacter cloacae β-lactamase(b) for reproducibility. Dark grey stick: the ligand in X-ray structure, Grey stick: the ligand in the docked structure, Black thin line: active site residues, Black dotted lines: hydrogen bonds. Table 1. Inhibitory effects of substituted penam sulfone derivatives on β-lactamase of Bacteroides fragilis with their structures Chemical name Ligand code IC 50 (µm) Structure 3,3-Dimethyl-4,4,7-trioxo-6-(4-oxo-pent- 2enyl)-4lamda*6*-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid OPCA-α 71.0 OPCA-β (3-methoxycarbonyl-allyl)3,3-dimethyl- 4,4,7-trioxo-4lamda*6*-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid MCCA-α >100 MCCA-β 1.4 Sulbactam >100 Tazobactam >100 Lys315, Tyr150 Asn152 w w ùkù x. Docking of OPCA-α, β and MCCA-α, β isomers Sandanayaka w w allyl substituted penam sufones metallo-β-lactamase w y d (Table 1) y w w ùkü š w. 2 in vitro d y e docking w w y

5 β-lactamase w w 465 Fig. 2. Representation of isomers, OPCA-α(a), OPCA-β(b), MCCA-α(c), and MCCA-β(d) in the active site of Bacteroides fragilis β-lactamase. Dark grey stick: isomers, Black thin lines: active site residues, Black dotted lines: hydrogen bonds. y ³ w ƒ. OPCA-α β-, š MCCA-α β- β-lactamase w w β- α- w y e w ùkü. y û OPCA-α (Fig. 2a) active site» Asn193wš w w š docking w e C-6 e ey 6-(4-oxo-pent-2enyl)»ƒ active site ƒ wš Á ew. IC 50 OPCA-β(Fig. 2b) active site» His162, Lys184, Asn193 w x w active site ¾ docking. His162 ligand w w w» š, Lys184 S. maltophilia L1 w metallo-β-lactamase sequence» β-lactam w w w w carboxylate salt bridge x w active site» ww. Asn Chryseobacterium strains BlaB IND-1 w metallo-β-lactamase w» ù» y w» y yw w w. β- 11 C-6 e ey» active site ü w w, α- ww ligand active site» ww y w. C-6 ey» e ƒ y C-6 e yw y w j q. w amide ring C-O» Zn2 β- 3.47Å, α- 4.77Å { ƒ À ew. Zn w w w w Zn2 ƒ ƒ¾ Zn mw ù» w v w w q. MCCA (Fig. 2c), α- Asn193

6 466 Á Á Á½ His223 w w ù ey» C-6 e Á Ê active site ƒ w. ù y MCCA-β(Fig. 2d) Asn193 His162 w w š C-6 e y» active site w w. Amide ring carbonyl» Zn2 β- ƒ 4.71Å, α- ƒ 5.87Å β- ƒ { ƒ¾. OPCA ƒ MCCA C-6 ey» e Zn2 ƒ y w ƒ q. w IC 50 ƒ x š. IC 50 w» ƒ¾ š w y C-6 ey» active site œ ƒ w docking y w. Docking of isomers of 3-phenyl-clavams ey 4 3-phenyl clavams docking w Cierpucha w d y (Table 2) w, 5 C-3 e phenyl»ƒ ey clavams ƒ z ƒ d RR-PC(Fig. 3a) active site Ser64, Gln120 Asn152 w w. β-lactam Table 2. Inhibitory effects of phenyl clavams on β-lactamase of Enterobacter cloacae P99 cephalosporinase with their structures Chemical name Ligand code IC 50(M) structure (3R,5R) 3-Phenyl-clavam RR-PC 0.88*10-2 (3R,5S) 3-Phenyl-clavam RS-PC 0.80*10-1 (3S,5R) 3-Phenyl-clavam SR-PC - (3S,5S) 3-Phenyl-clavam SS-PC - (3R,5R,6S) 6-Methyl-3-phenyl-clavam RRS-MP 0.95*10-2 (3S,5S,6R) 6-Methyl-3-phenyl-clavam SSR-MP 0.26*10-1 (3S,5R,6S) 6-Methyl-3-phenyl-clavam SRS-MP 0.69*10-1 (3R,5S,6R) 6-Methyl-3-phenyl-clavam RSR-MP 0.76*10-1 -: lack of β-lactamase inhibition

7 β-lactamase w w 467 Fig. 3. Representation of isomers, RR-PC(a), RS-PC(b), SR-PC(c), and SS-PC(d) in the active site of the Enterobacter cloacae β-lactamase. Dark grey stick: isomers, Black thin lines: active site residues, Black dotted lines: hydrogen bonds. ring N atom carbonyl» O atom Ser64 w x w Asn152 w x w. Ser64 nucleophilic serine class C z» w w w». lignad Ser318 main chain N atom 3.95Å Ser318 Ser64 oxyanion pocket x w ligand binding» w w w. 10 IC 50 RR-PC û ùkù RS-PC(Fig. 3b) RR-PCƒ w x w» w x w ù RR-PC carbonyl group O atom Ser64 w w š ù» ƒƒ w w x w. w Ser318 main chain N atom 4.14Å RR-PC w ew. RS-PC RR-PC docking w y w. wr β-lactamase wy ùkù SR-PC(Fig. 3c) SS- PC(Fig. 3d) active site ü x w x w w. Ser64 ƒ ƒƒ 3.37Å 3.73Å w w» š, Ser 318 N atom ƒƒ 5.44Å 6.65Å Ser64 oxyanion pocket x w» ew. phenyl»ƒ w C-3 configuration S R steric hindrance ƒ f active site ƒ w

8 468 Á Á Á½ Fig. 4. Representation of isomers, RRS-MP(a), SSR-MP(b), SRS-MP(c), and RSR-MP(d) in the active site of the Enterobacter cloacae β-lactamase. Dark grey stick: isomers, Black thin lines: active site residues, Black dotted lines: hydrogen bonds. ƒ. Docking ùkù w w wy ƒ RR-PC Ser64, š 5 w x w active site w š y SR- PC SS-PC β-lactamase x w x w w w y w. Docking of isomers of 6-methyl-3-phenyl-clavams C-3 e phenyl»ƒ, C-6 e methyl»ƒ ey 4 dockingw IC 50 ùkü. ƒ wy RRS-MP(Fig. 4a) w r β-lactam N atom carbonyl» O atom ƒƒ Ser64 w w š, active site» Lys67, Gln120, Asn152 w w w docking. ù carbonyl group O atom Ser318 main chain N atom 5.78Å RR-PC. IC 50 ùkü SSR-MP(Fig. 4b) β-lactam ring N atom O atom Ser64 w w š RRS-MP w w carbonyl group O atom Ser64 O atom 4.10Å w x w w. Ser Å RRS-MP w. wy ùkù SRS-MP (Fig. 4c) β-lactam ring N atom Ser64 w w x w š, carbonyl» O atom 4.94Å SSR-MP ew. w e ƒ w y ƒ š w w y wy w w w š w w. ƒ wy û RSR- MP(Fig. 4d) active site ü» w x w Ser Å

9 β-lactamase w w 469 w w» š, Ser Å oxyanion pocket x w». IC 50 ƒ RRS- MP Enterobacter cloacae P99 β-lactamase active site» 5 w x w š, p» ƒ w š ƒ Ser64 w ƒà ew w w x w y w. w docking w ù kù w IC 50 w w y k yw ƒ š ƒ. w β-lactamase w metallo-β-lactamase Bacteroides fragilis β-lactamase (class B) serine dependent β-lactamase Enterobacter cloacae P99 β-lactamase(class C) dockingw w ³ wš y w. w v y w» w reproducibility test ww š v X- yw xw y w. Ligands dockingw y w ƒ IC 50 docking k w y y w. β-lactamase y w œw y w z y. Computer-aided molecular docking ligand w ³ mw t w w w š w ù z kw» z w w. y z k w flexible docking program z ƒ. y w w w w. x 1. Lobkovsky, E.; Moews, P. C.; Liu, H.; Zhao, H.; Frere, J. M.; Knox, J. R. Proc. Natl. Acad. Sci. U S A. 1993, 90, Sandanayaka, V. P.; Feigelson, G. B.; Prashad, A. S.; Yang, Y.; Petersen, P. J. Bioorg. Med. Chem. Lett. 2001, 11, Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. Rev. 2005, 105, Antony, J.; Gresh, N.; Olsen, L.; Hemmingsen, L.; Schofield, C. J.; Bauer, R. J. Comput. Chem. 2002, 23, Cierpucha, M.; Solecka, J.; Frelek, J.; Szczukiewicz, P.; Chmielewski, M. Bioorg. Med. Chem. 2004, 12, Tripos associates. Sybyl Molecular Modeling Software, Sybyl theory manual., 6.6ed.; Tripos Associates: St. Louis, Kim, S. H.; Katzenellenbogen, J. A. Bioorg. Med. Chem. 2000, 8, Ahn, M. H.; Choi, I. H.; Kim, C. M. Yakhak Hoeji. 2002, 46, Payne, D. J.; Hueso-Rodriguez, J. A.; Boyd, H.; Concha, N. O.; Janson, C. A.; Gilpin, M.; Bateson, J. H.; Cheever, C.; Niconovich, N. L.; Pearson, S.; Rittenhouse, S.; Tew, D.; Diez, E.; Perez, P.; De La Fuente, J.; Rees, M.; Rivera-Sagredo, A. Antimicrob. Agents. Chemother. 2002, 46, Lobkovsky, E.; Billings, E. M.; Moews, P. C.; Rahil, J.; Pratt, R. F.; Knox, J. R. Biochemistry. 1994, 33, Yanchak, M. P.; Taylor, R. A.; Crowder, M. W. Biochemistry. 2000, 39,

10(3)-09.fm

10(3)-09.fm w y wz 10«3y 253~258 (2010.12.) Journal of Korean Society of Urban Environment ³ w Á» Á Á y w y œw (2010 11 22, 2010 12 9 k) Study on Determine of Detention Pond in Small Developed Area In-Soo Chang ½