John D. Buynak

Strategic Design of an Effective β-Lactamase Inhibitor LN-1-255, A 6-ALKYLIDENE-2′-SUBSTITUTED PENICILLIN SULFONE

In an effort to devise strategies for overcoming bacterial β-lactamases, we studied LN-1-255, a 6-alkylidene-2′-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among β-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing blaSHV extended-spectrum and inhibitor-resistant β-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 β-lactamases with nm affinity (KI = 110 ± 10 and 100 ± 10 nm, respectively). When LN-1-255 inactivated SHV β-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55Å resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the “tail” of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2′-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel β-lactamase inhibitor design.

The synthesis and evaluation of 3-substituted-7-(alkylidene)cephalosporin sulfones as β-lactamase inhibitors

A series of 3-substituted-7-(alkylidene)cephaloporin sulfones were prepared and evaluated as inhibitors of representative class A and class C serine beta-lactamase. Appropriate substituents resulted in a 1000-fold improvement in the inhibition of the class A enzymes and a simultaneous 20-fold improvement in the inhibition of class C. These new compounds have achieved the goal of creating broad scale inhibitors in the cephalosporin series.

Synthesis and reactivity of sulfur and silyl substituted α-alkylidene-β-lactams

Abstract Synthetically valuable α-alkylidene-β-lactams are produced from the addition of chlorosulfonyl isocyanate to allenyl sulfides.

Structure-Activity Relationships of Different β-Lactam Antibiotics against a Soluble Form of Enterococcus faecium PBP5, a Type II Bacterial Transpeptidase

ABSTRACT Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. β-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel β-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different β-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Δ1-36 rPBP5). The soluble protein, Δ1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH2-terminal protein sequence was verified by amino acid sequencing. Using β-lactams with different R1 side chains, we show that azlocillin has greater affinity for Δ1-36 rPBP5 than piperacillin and ampicillin (apparent Ki = 7 ± 0.3 μM, compared to 36 ± 3 and 51 ± 10 μM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k2 = 15 ± 4 M−1 s−1). Meropenem demonstrates an affinity for Δ1-36 rPBP5 comparable to that of ampicillin (apparent Ki = 51 ± 15 μM) but is slower at acylating (apparent k2 = 0.14 ± 0.02 M−1 s−1). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a β-lactam, and suggests that the specific side chain interactions of β-lactams could be modified to improve inactivation of resistant PBPs.

Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

ABSTRACT Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a Km value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a Km value of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

Penicillin Sulfone Inhibitors of Class D β-Lactamases

ABSTRACT OXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low Ki values for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant Ki improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low Kis for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [tn] = 0) and tns ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.

A convenient method for the production of 6-oxopenicillinates and 7-oxocephalosporinates

Abstract A convenient and economical method for the preparation of 6-oxopenicillinates and 7-oxocephalosporinates from the corresponding amines is presented. These ketones arekey intermediates in the synthesis of inhibitors of β-lactamase elastase.

Approaches to the simultaneous inactivation of metallo-and serine-β-lactamases

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-beta-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-beta-lactamase. With respect to interactions with the classes A, C, and D serine beta-lactamases, as judged by their correspondingly low K(m) values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine beta-lactamases, the OC-NR-OH functionality can function as a structural isostere of the OC-NR-H group, with the N-O-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k(cat) values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine beta-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.

The synthesis and lactamase inhibitory activity of 6-(carboxymethylene)penicillins and 7-(carboxymethylene)cephalosporins

Abstract A series of 6-(carboxymethylene)penicillinates and 7-(carboxymethylene)cephalosporanates were synthesized and evaluated as inhibitors of one type A and two type C β-lactamases. Disodium 6-(carboxymethylene)penicillinate sulfone ( 15 ) showed broad spectrum activity. A kinetic analysis demonstrated that 15 was a potent, partially irreversible inhibitor of the β-lactamase derived from Enterobacter cloacae P99.

Cephalosporin-derived inhibitors of β-lactamase. Part 4: The C3 substituent

New C3-substituted β-lactamase inhibitors were prepared and evaluated against representative class A and class C enzymes. It was possible to improve simultaneous inhibitory activity of both classes of serine hydrolase. Other inhibitors showed high selectivity for either the class C cephalosporinases or the class A penicillinases. This represents the first time that cephalosporin-derived inhibitors have demonstrated selectivity for the class A β-lactamases.