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Focco Van Den Akker - One of the best experts on this subject based on the ideXlab platform.

  • detecting a quasi stable imine species on the reaction pathway of shv 1 β lactamase and 6β hydroxymethyl Penicillanic Acid sulfone
    Biochemistry, 2015
    Co-Authors: E A Rodkey, Christopher R Bethel, Sivaprakash Shanmugam, Zhe Ding, Marianne Pusztaicarey, Michael D Nottingham, Weirui Chai, John D Buynak, Robert A Bonomo, Focco Van Den Akker
    Abstract:

    For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH2OH group (6β-(hydroxymethyl)Penicillanic Acid). The 6β 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 β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(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 6β-(hydroxymethyl) sulbactam formed longer-lived acyl–enzyme in...

  • design synthesis and crystal structures of 6 alkylidene 2 substituted Penicillanic Acid sulfones as potent inhibitors of acinetobacter baumannii oxa 24 carbapenemase
    Journal of the American Chemical Society, 2010
    Co-Authors: Elena Santillana, Christopher R Bethel, Alejandro Beceiro, Anne M Distler, Sarah M Drawz, Anjaneyulu Sheri, Jared M Sampson, Matthew Kalp, Sundar Ram Reddy Pagadala, Focco Van Den Akker
    Abstract:

    Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted Penicillanic Acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less

Christopher R Bethel - One of the best experts on this subject based on the ideXlab platform.

  • ln 1 255 a Penicillanic Acid sulfone able to inhibit the class d carbapenemase oxa 48
    Journal of Antimicrobial Chemotherapy, 2016
    Co-Authors: Juan A Vallejo, Christopher R Bethel, John D Buynak, Robert A Bonomo, Marta Martinezguitian, Juan C Vazquezucha, Concepcion Gonzalezbello, Margarita Poza, Alejandro Beceiro
    Abstract:

    OBJECTIVES: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted Penicillanic Acid sulfone, to inhibit the activity of the carbapenemase OXA-48. METHODS: The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic Acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. RESULTS: Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ± 1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. CONCLUSIONS: In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.

  • detecting a quasi stable imine species on the reaction pathway of shv 1 β lactamase and 6β hydroxymethyl Penicillanic Acid sulfone
    Biochemistry, 2015
    Co-Authors: E A Rodkey, Christopher R Bethel, Sivaprakash Shanmugam, Zhe Ding, Marianne Pusztaicarey, Michael D Nottingham, Weirui Chai, John D Buynak, Robert A Bonomo, Focco Van Den Akker
    Abstract:

    For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH2OH group (6β-(hydroxymethyl)Penicillanic Acid). The 6β 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 β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(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 6β-(hydroxymethyl) sulbactam formed longer-lived acyl–enzyme in...

  • inactivation of a class a and a class c β lactamase by 6β hydroxymethyl Penicillanic Acid sulfone
    Biochemical Pharmacology, 2012
    Co-Authors: Krisztina M Pappwallace, Christopher R Bethel, Thomas D Gootz, Wenchi Shang, Justin G Stroh, Dale Gordon Mcleod, Loren M Price, Anthony Marfat, Anne M Distler, Sarah M Drawz
    Abstract:

    Abstract β-Lactamase inhibitors (clavulanic Acid, sulbactam, and tazobactam) contribute significantly to the longevity of the β-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6β-(hydroxymethyl)Penicillanic Acid sulfone (6β-HM-sulfone) was tested against isolates expressing the class A TEM-1 β-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6β-HM-sulfone inhibitor to ampicillin was highly effective. 6β-HM-sulfone inhibited TEM-1 with an IC50 of 12 ± 2 nM and PDC-3 with an IC50 of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (kcat/kinact) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6β-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6β-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (H O H). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6β-HM-sulfone is an effective and versatile β-lactamase inhibitor of representative class A and C enzymes.

  • design synthesis and crystal structures of 6 alkylidene 2 substituted Penicillanic Acid sulfones as potent inhibitors of acinetobacter baumannii oxa 24 carbapenemase
    Journal of the American Chemical Society, 2010
    Co-Authors: Elena Santillana, Christopher R Bethel, Alejandro Beceiro, Anne M Distler, Sarah M Drawz, Anjaneyulu Sheri, Jared M Sampson, Matthew Kalp, Sundar Ram Reddy Pagadala, Focco Van Den Akker
    Abstract:

    Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted Penicillanic Acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less

Shahriar Mobashery - One of the best experts on this subject based on the ideXlab platform.

  • regiospecific syntheses of 6α 1r hydroxyoctyl Penicillanic Acid and 6β 1r hydroxyoctyl Penicillanic Acid as mechanistic probes of class d β lactamases
    Organic Letters, 2009
    Co-Authors: Sebastian A Testero, Peter I Odaniel, Dusan Hesek, Akihiro Ishiwata, Bruce C Noll, Shahriar Mobashery
    Abstract:

    The unique hydrophobic surface patches in class D β-lactamases presented an opportunity for designing two compounds, 6α-(1R-hydroxyoctyl)Penicillanic Acid and 6β-(1R-hydroxyoctyl)Penicillanic Acid, as mechanistic probes of these enzymes. In a sequence of three synthetic steps from benzhydryl 6,6-dibromopenicillanate, the targeted compounds were prepared in a stereospecific manner.

  • Quantification of the extent of attenuation of the rate of turnover chemistry of the TEM-1 β-lactamase by the α-1R-hydroxyethyl group in substrates
    Bioorganic & Medicinal Chemistry Letters, 1996
    Co-Authors: Kazuyuki Miyashita, Irina Massova, Shahriar Mobashery
    Abstract:

    Abstract The 6 α -1 R -hydroxyethyl group of imipenem, a clinically used carbapenem antibacterial agent, is believed to displace the hydrolytic water from its optimal position in the active site of class A β-lactamases. This interaction renders the molecule a poor substrate for these bacterial enzymes, hence preserving the antibacterial property of the antibiotic. The extent of the contribution of the 6 α -1 R -hydroxyethyl group in substrates toward stabilization of the antibiotic to the hydrolytic action of class A TEM-1 β-lactamase was studied by the synthesis and evaluation of two Penicillanic Acid derivatives, 6 α -(1 R -hydroxyethyl)Penicillanic Acid ( 2 ) and 6 β -(1 R -hydroxyethyl)Penicillanic Acid ( 3 ). The kinetic evaluation of the enzymic hydrolysis of these two Penicillanic Acid derivatives indicated that the 6 α -1 R -hydroxyethyl group imparts as much as 10 4 -fold to the hydrolytic stability of the β-lactam substrate. The role of the 6α-1 R -hydroxythermal group in imparting hydrolytic stability to β-lactam antibiotics towards class A β-lactamase was investigated.

  • A structure-based analysis of the inhibition of class A beta-lactamases by sulbactam.
    Biochemistry, 1994
    Co-Authors: Uzma Imtiaz, Eric M. Billings, James R. Knox, Shahriar Mobashery
    Abstract:

    : From the crystal structure of the Bacillus licheniformis 749/C beta-lactamase, energy-minimized structures for the precatalytic, the acyl-enzyme intermediate, and the acylated linear inactivating species for sulbactam--a clinically useful mechanism-based inactivator for class A beta-lactamases--were generated. The effect of individual Ser-235-Ala and Arg244-Ser point mutations on the inactivation and turnover processes was consistent with the existence of hydrogen bonds between the side chains of these residues and the sulbactam species. The departure of the sulfinate leaving group from the acyl-enzyme intermediate of sulbactam is believed to be a prerequisite for the inactivation process. In order to explore the influence of the leaving group, Penicillanic Acid (2), Penicillanic Acid alpha-S-oxide (3), and Penicillanic Acid beta-S-oxide (4) were synthesized and studied in kinetic experiments with the TEM-1 beta-lactamase. Penicillanic Acid is only a substrate, but Penicillanic Acid S-oxides were both substrates and inactivators for the enzyme. An argument is presented to rationalize these observations on the basis of the leaving ability of thiolate, sulfenate, and sulfinate from the acyl-enzyme intermediates of Penicillanic Acid (2), the Penicillanic Acid S-oxides (3 and 4), and sulbactam, respectively. The departure of the leaving group does not appear to be rate limiting in the inactivator process, but is an indispensable component of the irreversible inactivation of the enzyme. Molecular dynamics calculations of the putative inactivating species suggest that Lys-73, Lys-234, and Ser-130 are three likely residues that may be modified in the course of the inactivation chemistry. A discussion is presented of the mechanism of formation of the transiently inhibited enzyme species, which comes about as a consequence of the tautomerization of the double bond of the inactivating iminium moiety. In addition, the mechanistic details presented for sulbactam are compared and contrasted with those of clavulanic Acid, another clinically used inactivator for class A beta-lactamases.

Sarah M Drawz - One of the best experts on this subject based on the ideXlab platform.

  • inactivation of a class a and a class c β lactamase by 6β hydroxymethyl Penicillanic Acid sulfone
    Biochemical Pharmacology, 2012
    Co-Authors: Krisztina M Pappwallace, Christopher R Bethel, Thomas D Gootz, Wenchi Shang, Justin G Stroh, Dale Gordon Mcleod, Loren M Price, Anthony Marfat, Anne M Distler, Sarah M Drawz
    Abstract:

    Abstract β-Lactamase inhibitors (clavulanic Acid, sulbactam, and tazobactam) contribute significantly to the longevity of the β-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6β-(hydroxymethyl)Penicillanic Acid sulfone (6β-HM-sulfone) was tested against isolates expressing the class A TEM-1 β-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6β-HM-sulfone inhibitor to ampicillin was highly effective. 6β-HM-sulfone inhibited TEM-1 with an IC50 of 12 ± 2 nM and PDC-3 with an IC50 of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (kcat/kinact) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6β-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6β-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (H O H). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6β-HM-sulfone is an effective and versatile β-lactamase inhibitor of representative class A and C enzymes.

  • design synthesis and crystal structures of 6 alkylidene 2 substituted Penicillanic Acid sulfones as potent inhibitors of acinetobacter baumannii oxa 24 carbapenemase
    Journal of the American Chemical Society, 2010
    Co-Authors: Elena Santillana, Christopher R Bethel, Alejandro Beceiro, Anne M Distler, Sarah M Drawz, Anjaneyulu Sheri, Jared M Sampson, Matthew Kalp, Sundar Ram Reddy Pagadala, Focco Van Den Akker
    Abstract:

    Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted Penicillanic Acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less

Alejandro Beceiro - One of the best experts on this subject based on the ideXlab platform.

  • ln 1 255 a Penicillanic Acid sulfone able to inhibit the class d carbapenemase oxa 48
    Journal of Antimicrobial Chemotherapy, 2016
    Co-Authors: Juan A Vallejo, Christopher R Bethel, John D Buynak, Robert A Bonomo, Marta Martinezguitian, Juan C Vazquezucha, Concepcion Gonzalezbello, Margarita Poza, Alejandro Beceiro
    Abstract:

    OBJECTIVES: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted Penicillanic Acid sulfone, to inhibit the activity of the carbapenemase OXA-48. METHODS: The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic Acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. RESULTS: Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ± 1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. CONCLUSIONS: In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.

  • design synthesis and crystal structures of 6 alkylidene 2 substituted Penicillanic Acid sulfones as potent inhibitors of acinetobacter baumannii oxa 24 carbapenemase
    Journal of the American Chemical Society, 2010
    Co-Authors: Elena Santillana, Christopher R Bethel, Alejandro Beceiro, Anne M Distler, Sarah M Drawz, Anjaneyulu Sheri, Jared M Sampson, Matthew Kalp, Sundar Ram Reddy Pagadala, Focco Van Den Akker
    Abstract:

    Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted Penicillanic Acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanionmore » hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.« less