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

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis

Metabolites of milk intake: a metabolomic approach in UK twins with findings replicated in two European cohorts

Purpose: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies.  Methods: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed.  Results: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10−5) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (β = 0.012, SE = 0.002, P = 2.98 × 10−12) and the nucleotide uridine (β = 0.004, SE = 0.001, P = 9.86 × 10−6) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (β = 0.050, SE = 0.015, P = 7.53 × 10−4) and validated in the urine of 236 UK twins (β = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (β = 0.034, SE = 0.005, P = 9.75 × 10−14) and diacylphosphatidylcholine C28:1 (β = 0.034, SE = 0.004, P = 4.53 × 10−16), were also replicated.  Conclusions: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations

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

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

A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and β-lactamases

Penicillin binding proteins (PBPs) and β-lactamases are involved in interactions with β-lactam antibiotics connected with both antibacterial activity and mediation of bacterial β-lactam resistance. Current methods for identifying inhibitors of PBPs and β-lactamases can be inefficient and are often not suitable for studying weakly and/or reversibly binding compounds. Therefore, improved ligand binding assays for PBPs and β-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and "serine" β-lactamases using a boronic-acid-based, reversibly binding "tracer." The tracer was designed based on a crystal structure of a covalent complex between a boronic acid and PBP1b from Streptococcus pneumoniae. The tracer bound to three different PBPs with modest affinity (K(d)=4-12 μM) and more tightly to the TEM1 serine β-lactamase (K(d)=109 nM). β-Lactams and other boronic acids were able to displace the tracer in competition assays. These results indicate that fluorescent boronic acids are suited to serve as reversibly binding tracers in FP-based assays with PBPs and β-lactamases and potentially with other related enzymes

Modulating carnitine levels by targeting its biosynthesis - selective inhibition of gamma-butyrobetaine hydroxylase

Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart disease. We report the identification of potent, selective and cell active inhibitors of γ-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual 'U-shape' conformation stabilised by inter- and intra-molecular π-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate γ-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies. © 2014 the Partner Organisations

Use of ferrous iron by metallo-β-lactamases.

Metallo--lactamases (MBLs) catalyse the hydrolysis of almost all -lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo--lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo--lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs

Fluorophosphate Nucleotide Analogs 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-n-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)