Sundar Ram Reddy Pagadala

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.

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.

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.

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

ABSTRACT Among Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an “extended-spectrum” β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced Ki values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

Penam Sulfones and β-Lactamase Inhibition: SA2-13 and the Importance of the C2 Side Chain Length and Composition

β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-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 β-lactamases that possess the K234R substitution.

The Importance of the trans‐Enamine Intermediate as a β‐Lactamase Inhibition Strategy Probed in Inhibitor‐Resistant SHV β‐Lactamase Variants

The ability of bacteria to express inhibitor‐resistant (IR) β‐lactamases is stimulating the development of novel inhibitors of these enzymes. The 2′β‐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) β‐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.