Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M ß-Lactamases: Biochemical and Structural Analyses.

Design of novel ß-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two α-amido-ß-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum ß-lactamase [ESBL]) ß-lactamases were evaluated. The 50% inhibitory concentrations (IC50s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k2/K for CTX-M-96 (24,000 M-1 s-1) was twice the reported value for KPC-2 (12,000 M-1 s-1); for MB_076, the k2/K values ranged from 1,200 M-1 s-1 (KPC-2) to 3,900 M-1 s-1 (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-Å resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to ß-lactamase inhibitor design.

Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C ß-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

Straightforward synthesis of chiral non-racemic α-boryl isocyanides.

A straightforward concise synthesis of chiral non-racemic aliphatic α-boryl isocyanides, relay intermediates for boron-based bioactive molecules in multicomponent reactions, is presented. The short synthetic sequence comprises as key steps copper-catalysed asymmetric borylation of imines, simultaneous nitrogen formylation/boron-protecting group interconversion and the final formamide dehydration reaction.

Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog.

Extended-spectrum class C ß-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by ß-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki , 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.

Small-molecule inhibitors prevent the genotoxic and protumoural effects induced by colibactin-producing bacteria.

OBJECTIVE: Colorectal cancers (CRCs) are frequently colonised by colibactin toxin-producing Escherichia coli bacteria that induce DNA damage in host cells and exhibit protumoural activities. Our objective was to identify small molecules inhibiting the toxic effects induced by these colibactin-producing bacteria. DESIGN: A structural approach was adopted for the identification of a putative ligand for the ClbP enzyme involved in the synthesis of colibactin. Intestinal epithelial cells and a CRC mouse model were used to assess the activity of the selected compounds in vitro and in vivo. RESULTS: Docking experiments identified two boron-based compounds with computed ligand efficiency values (-0.8 and -0.9 kcal/mol/atom) consistent with data expected for medicinal chemistry leads. The crystalline structure of ClbP in complex with the compounds confirmed that the compounds were binding to the active site of ClbP. The two compounds (2 mM) suppressed the genotoxic activity of colibactin-producing E coli both in vitro and in vivo. The mean degree of suppression of DNA damage for the most efficient compound was 98±2% (95% CI). This compound also prevented cell proliferation and colibactin-producing E coli-induced tumourigenesis in mice. In a CRC murine model colonised by colibactin-producing E coli, the number of tumours decreased by 3.5-fold in animals receiving the compound in drinking water (p<0.01). CONCLUSIONS: These results demonstrate that targeting colibactin production controls the genotoxic and protumoural effects induced by this toxin.

Crystal Structures of KPC-2 and SHV-1 ß-Lactamases in Complex with the Boronic Acid Transition State Analog S02030.

Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to ß-lactamases emerges in part via the expression of KPC-2 and SHV-1 ß-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this ß-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism-based inactivators (e.g., clavulanate). In order to develop new ß-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54- and 1.87-Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 ß-lactamases, respectively, as well as a comparative analysis of the S02030 binding modes, including a previously determined S02030 class C ADC-7 ß-lactamase complex. S02030 is able to inhibit vastly different serine ß-lactamases by interacting with the conserved features of these active sites, which includes (i) forming the bond with catalytic serine via the boron atom, (ii) positioning one of the boronic acid oxygens in the oxyanion hole, and (iii) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, S02030 is able to overcome more distantly located structural differences between the ß-lactamases. This unique feature is achieved by repositioning the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring, allowing it to potentially form a unique C-H…O 2.9-Å hydrogen bond with S130 in KPC-2.

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A ß-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design.

Boronic acid transition state inhibitors (BATSIs) are competitive, reversible ß-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A ß-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of ß-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 ß-lactamases, nine possessed 50% inhibitory concentrations (IC50s) of ≤ 600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k2/K) of 1.2 ± 0.2 × 10(4) M(-1) s(-1) for KPC-2 and 4.7 ± 0.6 × 10(3) M(-1) s(-1) for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or blaKPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A ß-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.

Structure-based approach for identification of novel phenylboronic acids as serine-ß-lactamase inhibitors.

ß-Lactamases are bacterial enzymes conferring resistance to ß-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of ~1400 boronic acids as potential AmpC ß-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant ß-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography.

Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three ultrahigh resolution X-ray crystal structures of CTX-M ß-lactamase, directly visualizing protonation state changes along the enzymatic pathway: apo protein at 0.79 Å, precovalent complex with nonelectrophilic ligand at 0.89 Å, and acylation transition state (TS) analogue at 0.84 Å. Binding of the noncovalent ligand induces a proton transfer from the catalytic Ser70 to the negatively charged Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73, with a length of 2.53 Å and the shared hydrogen equidistant from the heteroatoms. QM/MM reaction path calculations determined the proton transfer barrier to be 1.53 kcal/mol. The LBHB is absent in the other two structures although Glu166 remains neutral in the covalent complex. Our data represents the first X-ray crystallographic example of a hydrogen engaged in an enzymatic LBHB, and demonstrates that desolvation of the active site by ligand binding can provide a protein microenvironment conducive to LBHB formation. It also suggests that LBHBs may contribute to stabilization of the TS in general acid/base catalysis together with other preorganized features of enzyme active sites. These structures reconcile previous experimental results suggesting alternatively Glu166 or Lys73 as the general base for acylation, and underline the importance of considering residue protonation state change when modeling protein-ligand interactions. Additionally, the observation of another LBHB (2.47 Å) between two conserved residues, Asp233 and Asp246, suggests that LBHBs may potentially play a special structural role in proteins.

Synthesis of 1,2,3-triazol-1-yl-methaneboronic acids via click chemistry: an easy access to a new potential scaffold for protease inhibitors.

Stereoselective synthesis of previously unreported 1,2,3-triazol-1-yl-methaneboronic acids has been achieved from azidomethaneboronates by Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC). The proximity of the cycloaddition reaction center to the boronic group is not detrimental for the stability of the sp3-carbon-boron bond nor to the stereoisomeric composition, further expanding the field of application of click chemistry to new boronate substrates and offering a new potential scaffold for protease inhibitors.

Fragment-guided design of subnanomolar ß-lactamase inhibitors active in vivo.

Fragment-based design was used to guide derivatization of a lead series of ß-lactamase inhibitors that had heretofore resisted optimization for in vivo activity. X-ray structures of fragments overlaid with the lead suggested new, unanticipated functionality and points of attachment. Synthesis of three derivatives improved affinity over 20-fold and improved efficacy in cell culture. Crystal structures were consistent with the fragment-based design, enabling further optimization to a K(i) of 50 pM, a 500-fold improvement that required the synthesis of only six derivatives. One of these, compound 5, was tested in mice. Whereas cefotaxime alone failed to cure mice infected with ß-lactamase-expressing Escherichia coli, 65% were cleared of infection when treated with a cefotaxime:5 combination. Fragment complexes offer a path around design hurdles, even for advanced molecules; the series described here may provide leads to overcome ß-lactamase-based resistance, a key clinical challenge.

Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii.

ß-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C ß-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, ß-lactamase inhibitors are structurally similar to ß-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from ß-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in ß-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-ß-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel ß-lactamase inhibitors against a key resistance target.

Interactions of oximino-substituted boronic acids and ß-lactams with the CMY-2-derived extended-spectrum cephalosporinases CMY-30 and CMY-42.

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the Ω-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-ß-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the Ω-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (Ω-loop-Q120 loop-R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

Defining the minimum inhibitory concentration of 22 rifamycins in iron limited, physiologic medium against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates.

Recently, we reported rifabutin hyper-activity against Acinetobacter baumannii. We sought to characterize if any additional rifamycins (n = 22) would also display hyper-activity when tested in iron-limited media against A. baumannii, K. pneumoniae, and E. coli. MICs were determined against representative clinical isolates using the iron-limited media RPMI-1640. Only rifabutin was hyperactive against A. baumannii.

Sulfonamidoboronic Acids as "Cross-Class" Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to ß-lactams. One of the most important mechanisms is the production of ß-lactamase enzymes capable of hydrolyzing ß-lactam antibiotics. Co-expression of multiple classes of ß-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical ß-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C ß-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other ß-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum ß-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

Synthesis of a Novel Boronic Acid Transition State Inhibitor, MB076: A Heterocyclic Triazole Effectively Inhibits Acinetobacter-Derived Cephalosporinase Variants with an Expanded-Substrate Spectrum.

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC ß-lactamase variants with Ki values <1 µM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Optically active α-phenylethylamine as efficient organocatalyst in the solvent-free reactions between 2,3-butanedione and conjugated nitroolefins.

(R)-(+) and (S)-(-)-1-phenylethylamine have been shown to promote highly diastereoselective and complementary enantioselective formal [3 + 2]carbocyclization reactions between 2,3-butanedione and conjugated nitroalkenes with formation of enantiomerically rich 2-hydroxy-3-nitrocyclopentanone derivatives. The reactions were carried out both in solvent and under solvent-free conditions. The absolute configurations of the products were assigned by X-ray and circular dichroism spectra analyses.

Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin-binding protein.

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cß bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

Novel insights into the mode of inhibition of class A SHV-1 beta-lactamases revealed by boronic acid transition state inhibitors.

Boronic acid transition state inhibitors (BATSIs) are potent class A and C ß-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 ß-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 ß-lactamase with micromolar affinity that is considerably weaker than their inhibition of other ß-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other ß-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

Insights Into the Inhibition of MOX-1 ß-Lactamase by S02030, a Boronic Acid Transition State Inhibitor.

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C ß-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 ß-lactamase, a plasmid-borne, expanded-spectrum AmpC ß-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the ß-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 µg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 µg/ml when S02030 is added at a concentration of 4 µg/ml. The IC50 and K i of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 µM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC ß-lactamases.