Detecting a quasi-stable imine species on the reaction pathway of SHV-1 ß-lactamase and 6ß-(hydroxymethyl)penicillanic acid sulfone.

For the class A ß-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6ß position by a CH2OH group (6ß-(hydroxymethyl)penicillanic acid). The 6ß substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a ß-aminoacrylate) in 1 h for sulbactam and in 4 h for 6ß-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6ß-(hydroxymethyl) sulbactam formed longer-lived acyl-enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6ß-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6ß-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.

Penam sulfones and ß-lactamase inhibition: SA2-13 and the importance of the C2 side chain length and composition.

ß-Lactamases are the major reason ß-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, ß-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k(react) than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k(react) compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV ß-lactamases that possess the K234R substitution.

ß-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme.

The inhibition of the class A SHV-1 ß-lactamase by 7-(tert-butoxycarbonyl)methylidenecephalosporin sulfone was examined kinetically, spectroscopically, and crystallographically. An 1.14 Å X-ray crystal structure shows that the stable acyl-enzyme, which incorporates an eight-membered ring, is a covalent derivative of Ser70 linked to the 7-carboxy group of 2-H-5,8-dihydro-1,1-dioxo-1,5-thiazocine-4,7-dicarboxylic acid. A cephalosporin-derived enzyme complex of this type is unprecedented, and the rearrangement leading to its formation may offer new possibilities for inhibitor design. The observed acyl-enzyme derives its stability from the resonance stabilization conveyed by the ß-aminoacrylate (i.e., vinylogous urethane) functionality as there is relatively little interaction of the eight-membered ring with active site residues. Two mechanistic schemes are proposed, differing in whether, subsequent to acylation of the active site serine and opening of the ß-lactam, the resultant dihydrothiazine fragments on its own or is assisted by an adjacent nucleophilic atom, in the form of the carbonyl oxygen of the C7 tert-butyloxycarbonyl group. This compound was also found to be a submicromolar inhibitor of the class C ADC-7 and PDC-3 ß-lactamases.

Design and exploration of novel boronic acid inhibitors reveals important interactions with a clavulanic acid-resistant sulfhydryl-variable (SHV) ß-lactamase.

Inhibitor resistant (IR) class A ß-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV ß-lactamase, from Klebsiella pneumoniae , results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting ß-lactamases revealed that only -Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM K(i) for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR ß-lactamases.

Crystal structure of a preacylation complex of the ß-lactamase inhibitor sulbactam bound to a sulfenamide bond-containing thiol-ß-lactamase.

The rise of inhibitor-resistant and other ß-lactamase variants is generating an interest in developing new ß-lactamase inhibitors to complement currently available antibiotics. To gain insight into the chemistry of inhibitor recognition, we determined the crystal structure of the inhibitor preacylation complex of sulbactam, a clinical ß-lactamase inhibitor, bound in the active site of the S70C variant of SHV-1 ß-lactamase, a resistance enzyme that is normally present in Klebsiella pneumoniae. The S70C mutation was designed to affect the reactivity of that catalytic residue to allow for capture of the preacylation complex. Unexpectedly, the 1.45 Å resolution inhibitor complex structure revealed that residue C70 is involved in a sulfenamide bond with K73. Such a covalent bond is not present in the wild-type SHV-1 or in an apo S70C structure also determined in this study. This bond likely contributed significantly to obtaining the preacylation complex with sulbactam due to further decreased reactivity toward substrates. The intact sulbactam is positioned in the active site such that its carboxyl moiety interacts with R244, S130, and T235 and its carbonyl moiety is situated in the oxyanion hole. To our knowledge, in addition to being the first preacylation inhibitor ß-lactamase complex, this is also the first observation of a sulfenamide bond between a cysteine and lysine in an active site. Not only could our results aid, therefore, structure-based inhibitor design efforts in class A ß-lactamases, but the sulfenamide-bond forming approach to yield preacylation complexes could also be applied to other classes of ß-lactamases and penicillin-binding proteins with the SXXK motif.

The importance of the trans-enamine intermediate as a ß-lactamase inhibition strategy probed in inhibitor-resistant SHV ß-lactamase variants.

The ability of bacteria to express inhibitor-resistant (IR) ß-lactamases is stimulating the development of novel inhibitors of these enzymes. The 2'ß-glutaroxypenicillinate sulfone, SA2-13, was previously designed to enhance the stabilization of the deacylation-refractory, trans-enamine inhibitory intermediate. To test whether this mode of inhibition can overcome different IR mutations, we determined the binding mode of SA2-13 through X-ray crystallography, obtaining co-crystals of the inhibitor-protein complex by soaking crystals of the IR sulfhydryl variable (SHV) ß-lactamase variants S130G and M69V with the inhibitor. The 1.45 Å crystal structure of the S130G SHV:SA2-13 complex reveals that SA2-13 is still able to form the stable trans-enamine intermediate similar to the wild-type complex structure, yet with its carboxyl linker shifted deeper into the active site in the space vacated by the S130G mutation. In contrast, data from crystals of the M69V SHV:SA2-13 complex at 1.3 Å did not reveal clear inhibitor density indicating that this IR variant disfavors the trans-enamine conformation, likely due to a subtle shift in A237.

Racial/ethnic differences in survival among elderly patients with a primary glioblastoma.

BACKGROUND: Few studies have assessed racial/ethnic differences in survival after primary glioblastoma diagnosis. We investigate these differences, incorporating information on White, Hispanics and Asians, as well as White, non-Hispanics and Blacks, among elderly individuals with a primary glioblastoma utilizing the population-based Surveillance, Epidemiology and End Results (SEER) Program-Medicare linked database. METHODS: A total of 1,530 individuals diagnosed > = 66 years of age from 6/1/91 to 12/31/99 in the SEER data were linked with Medicare information from 1/1/91 to 12/31/01. All individuals had Medicare Parts A and B and were non-HMO for 6 months before and 12 months after diagnosis to gather pre-diagnosis co-morbidities and post-diagnosis first course of treatment. Survival differences by race/ethnicity and by race/ethnicity stratified by treatment type and/or median household income were examined using Kaplan-Meier and multivariable Cox proportional hazards models. RESULTS: Significant racial/ethnic differences existed between White, non-Hispanics and Blacks in marital status, income and SEER registry region for the entire US. In analysis limited to the West region, significant racial/ethnic differences existed for income only. Overall there were no differences in survival between White, non-Hispanics and Blacks, however, in analysis limited to the West region, Asians had a lower risk of death compared to White, non-Hispanics [HR = 0.67, 95% CI (0.43, 1.03)]. Asians who had multiple treatments also had a lower risk of death compared to White, non-Hispanics [HR = 0.65, 95% CI (0.41, 1.01)]. CONCLUSIONS: Racial/ethnic differences in survival after primary glioblastoma diagnosis exist and may be partially explained by racial/ethnic differences in treatment and income.