Sideromimic Modification of Lactivicin Dramatically Increases Potency against Extensively Drug-Resistant <i>Stenotrophomonas maltophilia </i>Clinical Isolates

Acetamido derivatives of the naturally antibacterial non-β-lactam lactivicin (LTV) have improved activity against their penicillin binding protein targets and reduced hydrolysis by β-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 β-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

Comparison of verona integron-borne metallo-beta-lactamase (VIM) variants reveals differences in stability and inhibition profiles

DUZGUN, AZER OZAD/0000-0002-6301-611X; Abboud, Martine I./0000-0003-2141-5988; Brem, Jurgen/0000-0002-0137-3226; McDonough, Michael A/0000-0003-4664-6942; Rydzik, Anna/0000-0003-3158-0493; DUZGUN, AZER OZAD/0000-0002-6301-611X; McDonough, Michael/0000-0003-4664-6942; Schofield, Christopher/0000-0002-0290-6565; SANDALLI, Cemal/0000-0002-1298-3687WOS: 000376490800025PubMed: 26666919Metallo-beta-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. the Verona integron-borne metallo-beta-lactamase (VIM) enzymes are among the most widely distributed MBLs, with > 40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of beta-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. the results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.Rhodes Trust; Scientific and Technology Council of Turkey; Recep Tayyip Erdogan Universitesi Research FundRecep Tayyip Erdogan University [BAP-2013.102.03.13]; Medical Research CouncilMedical Research Council UK (MRC) [MR/L007665/1]; Medical Research Council/Canadian Grant [G1100135]; Biochemical Society Krebs Memorial Award; Medical Research CouncilMedical Research Council UK (MRC) [G1100135, MR/N002679/1] Funding Source: researchfishThe Rhodes Trust provided funding to Anne Makena. Scientific and Technology Council of Turkey provided funding to Cemal Sandalli. Recep Tayyip Erdogan Universitesi Research Fund provided funding to Aysegul Saral, Aysegul C. Cicek, and Cemal Sandalli under grant number BAP-2013.102.03.13. Medical Research Council provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number MR/L007665/1. Medical Research Council/Canadian Grant provided funding to Jurgen Brem, Michael A. McDonough, Anna M. Rydzik, and Christopher J. Schofield under grant number G1100135. Biochemical Society Krebs Memorial Award provided funding to Martine I. Abboud

¹⁹F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-β-Lactamase

The role of metallo-β-lactamases (MBLs) in β-lactam antibiotic resistance is a growing problem. We describe the use of protein-observe 19F-NMR (PrOF NMR) to study the dynamics of the São Paolo MBL (SPM-1) from β-lactam resistant Pseudomonas aeruginosa. Cysteinyl-variants on the α3 and L3 regions, which flank the di-Zn(II) active site, were selectively 19F-labeled using 3-bromo-1,1,1,-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in both inhibitor and hydrolyzed β-lactam product binding to SPM-1. They have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-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 beta-lactamase stable beta-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.Peer reviewe

Assay platform for clinically relevant metallo-beta-lactamases

Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors

Structure-Guided Design of Cell Wall Biosynthesis Inhibitors That Overcome beta-Lactam Resistance in Staphylococcus aureus (MRSA).

beta-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering beta-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low beta-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing beta-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.Eur-Intafa

Synthesis of Fluorophosphate Nucleotide Analogues and Their Characterization as Tools for ¹⁹F NMR Studies

To broaden the scope of existing methods based on ¹⁹F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)­nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)­phosphate moiety and evaluated them as tools for ¹⁹F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-<i>n</i>-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m⁷G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5′-phosphorylated oligos we also synthesized oligonucleotide 5′-(2-fluorodiphosphates), which are potentially useful as ¹⁹F NMR hybridization probes. The compounds were characterized by ¹⁹F NMR and evaluated as ¹⁹F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E)

Studying the active-site loop movement of the São Paolo metallo-β-lactamase-1

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics. We report biophysical and kinetic studies on the São Paulo MBL (SPM-1), which reveal its Zn(ii) ion usage and mechanism as characteristic of the clinically important di-Zn(ii) dependent B1 MBL subfamily. Biophysical analyses employing crystallography, dynamic (19)F NMR and ion mobility mass spectrometry, however, reveal that SPM-1 possesses loop and mobile element regions characteristic of the B2 MBLs. These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity. SPM-1 thus appears to be a hybrid B1/B2 MBL. The results have implications for MBL evolution and inhibitor design