OVERCOMING INHIBITOR RESISTANCE IN THE SHV ΒETA-LACTAMASE By JODI MICHELLE THOMSON Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Robert A Bonomo Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. For Robert A. Bonomo TABLE OF CONTENTS Table of Contents ............................................................................... 1-2 List of Figures ..................................................................................... 3-8 List of Tables...........................................................................................9 Acknowledgments........................................................................... 10-12 Abbreviations .................................................................................. 13-14 Abstract........................................................................................... 15-16 Chapter 1: Introduction ................................................................... 17-43 Body......................................................................................... 17-31 Figures ..................................................................................... 32-43 Chapter II: SHV Arg244: Clavulanate Resistance.......................... 44-81 Introduction .............................................................................. 44-46 Methods ................................................................................... 47-58 Results ..................................................................................... 59-64 Discussion................................................................................ 65-69 Tables ...................................................................................... 70-72 Figures ..................................................................................... 73-81 Chapter III: Arg 244: Sulbactam Susceptibility ............................. 82-108 Introduction .............................................................................. 82-85 Methods ................................................................................... 86-88 Results ..................................................................................... 89-92 1 Discussion................................................................................ 93-98 Tables .................................................................................... 99-101 Figures ................................................................................. 102-108 Chapter IV: Inhibition of SHV ESBLs by Transition State Analogs ....................................................................... 109-121 Introduction .......................................................................... 109-111 Methods ............................................................................... 112-113 Results ................................................................................. 114-115 Discussion............................................................................ 116-117 Tables ..........................................................................................118 Figures ................................................................................. 119-121 Chapter V: β-Lactamase Inhibition by BLIP ............................... 122-146 Introduction .......................................................................... 122-125 Methods ............................................................................... 126-130 Results ................................................................................. 131-133 Discussion............................................................................ 134-137 Tables .................................................................................. 138-139 Figures ................................................................................. 140-146 Chapter VI: Concluding Remarks............................................... 147-154 Bibliography ................................................................................ 155-171 2 LIST OF FIGURES 1-1 Examples of the 8 generations of β-lactam antibiotics currently on the market exemplify conservation of the lactam backbone and diversity of the side-chains 1-2 The mechanism of attachment of the penicillin binding protein (PBP) to its natural D-Ala D-Ala substrate (top) and a β-lactam antibiotic (bottom) is strikingly similar 1-3 The crystal structure of SHV β-lactamase (Protein Data Bank accession number 1SHV) 1-4 The SHV β-lactamase active site pocket, highlighting important residues for catalysis and substrate specificity 1-5 The numbering scheme for the backbone nucleus of penicillins and cephalosporins 1-6 Structures of the β-lactamase inhibitors currently on the market. The main difference between these and the beta-lactam antibiotics is the presence of a good leaving group at the 1 position 1-7 The pathway of inhibition by clavulanate in the active site of class A β- lactamases is a multi-step process with numerous intermediates 1-8 The proposed contribution of Arg244 to clavulanate inactivation of TEM-1: (A) Direct hydrogen bonding interactions (B) Coordination of a catalytic water molecule essential for inactivation 3 1-9 Structures of cephalosporins and corresponding boronic acid transition state inhibitors (BATSIs): (A) Ceftazidime (B) Ceftazidime BATSI (C) Cefotaxime (D) Cefotaxime BATSI (E) Cephalothin (F) Achiral Cephalothin BATSI (G) Chiral Cephalothin BATSI 1-10 Chemical structure of novel penem inhibitors(1 and 2) developed by Wyeth Pharmaceuticals which display large, bicyclic R1 side chains 1-11 Endo-trig cyclization of Penem 1 in the active site of a β-lactamase results in the formation of a seven-membered ring 1-12 The results of alanine scanning mutagenesis of BLIP indicated fewer “hotspots” of BLIP binding to SHV-1 (left) compared to TEM-1 (right). Residues identified as critical for binding are in red (reduce binding >10 fold when mutated), residues that, when mutated, lead to an increase in affinity are colored cyan, and residues that had a < 10 fold effect on affinity when mutated are colored green. (Zhang and Palzkill, JBC 279-41, 42860-42866) 2-1 Chemical structures of compounds tested in this study. The structures of clavulanic acid and nitrocefin are labeled with the accepted ring numbering system 2-2 The proposed contribution of Arg244 to clavulanic acid inactivation of TEM-1. The guanidinium group of Arg244 is essential for (A) hydrogen bonding interactions with the C3 carboxylate of the inhibitor in the active site, and (B) coordination of a proton donating water molecule essential for the saturation of the double bond of the C2 constituent (Imtiaz et al., 1993) 4 2-3 Synthesis of the chiral cephalothin boronic acid transition state inhibitor (A) (+)-Pinanediol, THF, rt; (B) (dichloromethyl)lithium, THF, –100 °C → 0 °C; (C) lithium bis(trimethylsilyl)amide, THF, –80 °C → rt; (D) 2-thiopheneacetylchloride, 2- thiopheneacetic acid, THF –80 → rt; (E) aqueous HCl 3N, 1h, 100 °C 2-4 Timed inactivation of SHV-1 and Arg244Ser, -Gln, -Leu, and –Glu reveal that Arg244 variants are inactivated more rapidly by clavulanate than SHV-1. Enzymes were incubated with KI concentrations of clavulanate and initial velocities of nitrocefin hydrolysis measured at timepoints 0-600 seconds 2-5 (A) Deconvoluted mass spectra of SHV-1 and Arg244Ser before and after 15- minute incubation with clavulanate show similar intermediates of inactivation. Spectra were obtained on a Q-STAR XL quadrupole-time-of-flight mass spectrometer equipped with a nanospray source. Eight distinct mass shifts were identified with both enzymes. (B) Proposed intermediates in the clavulanate inactivation pathway. The 198 Da and 173 Da adducts are represented by more than one candidate structure (blue). The terminally inactivated 52 Da crosslinked species is highlighted in red 2-6 The time-dependent inhibition of the chiral cephalothin boronic acid transition state inhibitor (compound 2) is more significant with SHV-1 than with SHV Arg244 Ser, -Gln, -Leu, and -Glu. Enzymes were incubated with inhibitor at concentrations equal to the Ki and initial velocities of nitrocefin hydrolysis assessed at 0-3600s. Initial Ki determination was performed after a 5 minute incubation 5 2-7 Arg244 in relation to Val216 in the SHV-1 (1SHV) and TEM-1 (1BTL) in apo- enzyme crystal structure representations. The distance between the backbone carbonyl oxygen of Val216 and the nearest guanidinium nitrogen in SHV is 7.85 Å. In contrast, the distance in TEM-1 is 5.19 Å. Shown in TEM-1 is the bridging water molecule which makes hydrogen bonds with Val216 (2.87 Å) and Arg244 (3.08 Å). The active site Ser70 is shown for reference 2-8 Suggested role of Arg 244 in stabilizing the β-lactam carboxylate of penicillins, cephalosporins,