Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Synthesis and Application of Constrained Amidoboronic Acids Using Amphoteric Boron-Containing Building Blocks.

Amidoboronic acid-containing peptidomimetics are an important class of scaffolds in chemistry and drug discovery. Despite increasing interest in boron-based enzyme inhibitors, constrained amidoboronic acids have received little attention due to the limited options available for their synthesis. We describe a new methodology to prepare both α- and ß-amidoboronic acids that impose restrictions on backbone angles. Lewis acid-promoted Boyer-Schmidt-Aube lactam ring expansions using an azidoalkylboronate enabled generation of constrained α-amidoboronic acid derivatives, whereas assembly of the homologous ß-amidoboronic acids was achieved through a novel boronic acid-mediated lactamization process stemming from an α-boryl aldehyde. The results of quantum chemical calculations suggest carboxylate-boron coordination to be rate-limiting for small ring sizes, whereas the tetrahedral intermediate formation is rate limiting in the case of larger rings. As part of this study, an application of ß-amidoboronic acid derivatives as novel VIM-2 metallo-ß-lactamase inhibitors has been demonstrated.

TonB-dependent uptake of ß-lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia.

The ß-lactam antibiotic ceftazidime is one of the handful of drugs with proven clinical efficacy against the important opportunistic human pathogen Stenotrophomonas maltophilia. Here, we show that mutations in the energy transducer TonB, encoded by smlt0009 in S. maltophilia, confer ceftazidime resistance and smlt0009 mutants have reduced uptake of ceftazidime. This breaks the dogma that ß-lactams enter Gram-negative bacteria only by passive diffusion through outer membrane porins. We also show that ceftazidime-resistant TonB mutants are cross-resistant to fluoroquinolone antimicrobials and a siderophore-conjugated lactivicin antibiotic designed to target TonB-dependent uptake. This implies that attempts to improve the penetration of antimicrobials into S. maltophilia by conjugating them with TonB substrates will suffer from the fact that ß-lactams and fluoroquinolones coselect resistance to these novel and otherwise promising antimicrobials. Finally, we show that smlt0009 mutants already exist among S. maltophilia clinical isolates and have reduced susceptibility to siderophore-conjugated lactivicin, despite the in vitro growth impairment seen in smlt0009 mutants selected in the laboratory.

Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activates Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia.

Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory-selected mutants and clinical isolates, we show here that damage to the 50S ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.

Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia.

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

Profiling interactions of vaborbactam with metallo-ß-lactamases.

ß-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by ß-lactamases. ß-Lactamases fall into two mechanistic groups: the serine ß-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo ß-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both ß-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-ß-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-ß-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20-100 fold below that by which it inhibits its current clinical targets, the Class A serine ß-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of ß-lactamases and justify further work on the development of boronates as broad-spectrum ß-lactamase inhibitors.

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates.

There are currently no clinically available inhibitors of metallo-ß-lactamases (MBLs), enzymes that hydrolyze ß-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 µM; Ki values of 0.03-1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of ß-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Structural/mechanistic insights into the efficacy of nonclassical ß-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates.

Clavulanic acid and avibactam are clinically deployed serine ß-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo ß-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine ß-lactamase) but not L1 (metallo ß-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like ß-lactams plus nonclassical ß-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.

Disruption of mpl Activates ß-Lactamase Production in Stenotrophomonas maltophilia and Pseudomonas aeruginosa Clinical Isolates.

The hyperproduction of chromosomally encoded ß-lactamases is a key method of acquired resistance to ceftazidime, aztreonam, and, when seen in backgrounds having reduced envelope permeability, carbapenems. Here, we show that the loss of Mpl, a UDP-muramic acid/peptide ligase, is a common and previously overlooked cause of chromosomally encoded ß-lactamase hyperproduction in clinical isolates of Stenotrophomonas maltophilia and Pseudomonas aeruginosa, important pathogens notorious for their ß-lactam-resistant phenotypes.

Sideromimic Modification of Lactivicin Dramatically Increases Potency against Extensively Drug-Resistant Stenotrophomonas maltophilia Clinical Isolates.

Acetamido derivatives of the naturally antibacterial non-ß-lactam lactivicin (LTV) have improved activity against their penicillin binding protein targets and reduced hydrolysis by ß-lactamases, but penetration into Gram-negative bacteria is still relatively poor. Here we report that modification of the LTV lactone with a catechol-type siderophore increases potency 1,000-fold against Stenotrophomonas maltophilia, a species renowned for its insusceptibility to antimicrobials. The MIC90 of modified lactone compound 17 (LTV17) against a global collection of extensively drug-resistant clinical S. maltophilia isolates was 0.063 µg · ml(-1) Sideromimic modification does not reduce the ability of LTVs to induce production of the L1 and L2 ß-lactamases in S. maltophilia and does not reduce the rate at which LTVs are hydrolyzed by L1 or L2. We conclude, therefore, that lactivicin modification with a siderophore known to be preferentially used by S. maltophilia substantially increases penetration via siderophore uptake. LTV17 has the potential to be developed as a novel antimicrobial for treatment of infections by S. maltophilia More generally, our work shows that sideromimic modification in a species-targeted manner might prove useful for the development of narrow-spectrum antimicrobials that have reduced collateral effects.

Studies on the Reactions of Biapenem with VIM Metallo ß-Lactamases and the Serine ß-Lactamase KPC-2.

Carbapenems are important antibacterials and are both substrates and inhibitors of some ß-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-ß-lactamases VIM-1 and VIM-2 and the class A serine-ß-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the ß-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of ß-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Structural Basis of Metallo-ß-lactamase Inhibition by N-Sulfamoylpyrrole-2-carboxylates.

Metallo-ß-lactamases (MBLs) can efficiently catalyze the hydrolysis of all classes of ß-lactam antibiotics except monobactams. While serine-ß-lactamase (SBL) inhibitors (e.g., clavulanic acid, avibactam) are established for clinical use, no such MBL inhibitors are available. We report on the synthesis and mechanism of inhibition of N-sulfamoylpyrrole-2-carboxylates (NSPCs) which are potent inhibitors of clinically relevant B1 subclass MBLs, including NDM-1. Crystallography reveals that the N-sulfamoyl NH2 group displaces the dizinc bridging hydroxide/water of the B1 MBLs. Comparison of crystal structures of an NSPC and taniborbactam (VRNX-5133), presently in Phase III clinical trials, shows similar binding modes for the NSPC and the cyclic boronate ring systems. The presence of an NSPC restores meropenem efficacy in clinically derived E. coli and K. pneumoniae blaNDM-1. The results support the potential of NSPCs and related compounds as efficient MBL inhibitors, though further optimization is required for their clinical development.

Faropenem reacts with serine and metallo-ß-lactamases to give multiple products.

Penems have demonstrated potential as antibacterials and ß-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some ß-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing ß-lactamases, focusing on the class A serine ß-lactamase KPC-2 and the metallo ß-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three ß-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the ß-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with ß-lactamases. They also exemplify how crystal structures of ß-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.

Broad Spectrum ß-Lactamase Inhibition by a Thioether Substituted Bicyclic Boronate.

ß-Lactamases comprise the most widely used mode of resistance to ß-lactam antibiotics. Cyclic boronates have shown promise as a new class of ß-lactamase inhibitor, with pioneering potential to potently inhibit both metallo- and serine-ß-lactamases. We report studies concerning a bicyclic boronate ester with a thioether rather than the more typical ß-lactam antibiotic "C-6/C-7" acylamino type side chain, which is present in the penicillin/cephalosporin antibiotics. The thioether bicyclic boronate ester was tested for activity against representative serine- and metallo-ß-lactamases. The results support the broad inhibition potential of bicyclic boronate based inhibitors with different side chains, including against metallo-ß-lactamases from B1, B2, and B3 subclasses. Combined with previous crystallographic studies, analysis of a crystal structure of the thioether inhibitor with the clinically relevant VIM-2 metallo-ß-lactamase implies that further SAR work will expand the already broad scope of ß-lactamase inhibition by bicyclic boronates.

Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C ß-Lactamase from Escherichia coli.

Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ß­lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ß­lactamases, our data support the proposal that bicyclic boronates are broad-spectrum ß­lactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-ß-Lactamases.

The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of ß-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-ß-lactamases (SBLs) and some clinically important metallo-ß-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the "high-energy-intermediate" analogue approach for broad-spectrum ß-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes.

Studies on the inhibition of AmpC and other ß-lactamases by cyclic boronates.

BACKGROUND: The ß-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of ß-lactamases, which collectively are able to hydrolyse all classes of ß-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) ß-lactamase families. METHODS: Using biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important ß-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem. RESULTS: Crystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of ß-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum ß-lactamase inhibitors. CONCLUSIONS: Together with reported studies on the structural basis of their inhibition of class A, B and D ß-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis. GENERAL SIGNIFICANCE: Bicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.

[Identification of the Mycobacterium tuberculosis Beijing lineage in Ecuador].

INTRODUCTION: Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia. OBJECTIVE: To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported. MATERIALS AND METHODS: We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR. RESULTS: We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported. CONCLUSION: This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.