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

Mechanistic and inhibition studies on gamma-butyrobetaine hydroxylase

Carnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or 19F NMR, respectively. Furthermore, the use of 19F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols.</p

Mechanistic and inhibition studies on gamma-butyrobetaine hydroxylase

Carnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or 19F NMR, respectively. Furthermore, the use of 19F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols.This thesis is not currently available in ORA

Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme

Adenosine 5'-phosphoramidate (NH2-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH2-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH2-pA. The enzyme functions as a homodimer (2 × 15 800 Da). At the optimum pH of 7.0, the Km for NH2-pA was 0.5 µM and kcat 0.8 s-1 (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms

The Impact of Four High-Altitude Training Camps on the Aerobic Capacity of a Short Track PyeongChang 2018 Olympian: A Case Study

This study characterizes high-altitude training camps and their effect on the aerobic capacity of a Polish national team member (M.W.), who was a participant in the PyeongChang 2018 Winter Olympic Games (body weight: 59.6 kg, body height: 161.0 cm, fat mass: 10.9 kg and 18.3% of fat tissue, fat-free mass: 48.7 kg, muscle mass: 46.3 kg, and BMI = 23.0 kg/m2). The tests were conducted in the periods from April 2018 to September 2018 and April 2019 to September 2019 (period of general and special preparation). The study evaluated aerobic and anaerobic capacity determined by laboratory tests, a cardiopulmonary graded exercise test to exhaustion performed on a cycle ergometer (CPET), and the Wingate anaerobic test. Based on the research, training in hypobaric conditions translated into significant improvements in the skater&rsquo;s exercise capacity recorded after participating in the Olympic Winter Games in Korea (February 2018). In the analyzed period (2018&ndash;2019), there was a significant increase in key parameters of aerobic fitness such as anaerobic threshold power output (AT-PO) [W]&mdash;223; power output POmax [W]&mdash;299 and AT-PO [W/kg]&mdash;3.50; (POmax) [W/kg]&mdash;4.69; and AT-VO2 [mL/kg/min]&mdash;51.3; VO2max [mL/kg/min]&mdash;61.0. The athlete showed high-exercise-induced adaptations and improvements in the aerobic metabolic potential after two seasons, in which four training camps were held in altitude conditions

Selected Indices of Anaerobic Capacity and Their Changes during Special Judo Fitness Tests at Different Ambient Temperatures Performed among Judo Athletes

Background: Thermoregulatory processes play an important role during athletic competition. When athletes compete in an elevated ambient temperature, metabolic processes in their bodies become intensified. The main objective of the study was to determine changes in anaerobic total work (TW) and relative peak power (RPP) during a special judo fitness test at different ambient temperatures performed among judo athletes. Methods: The study included 15 judo athletes aged 20.7 &plusmn; 2.0 years, with a body height of 178 &plusmn; 6.3 cm, body mass totalling 76.3 &plusmn; 12.6 kg, VO2max at 43.2 &plusmn; 7.8 mL&middot;kg&minus;1, and peak power of 12.1 W&middot;kg&minus;1. A complete set of results was obtained for 10 athletes. In the main part of the examinations, judo athletes performed five sequences (7.20 min each), alternating efforts on a leg cycle and arm cycle ergometer in a thermal chamber at 21 &plusmn; 0.5 &deg;C and 31 &plusmn; 0.5 &deg;C. The efforts differed from typical interval exercise by alternating upper- and lower-limb efforts, as well as with regard to the duration of those efforts. Each sequence was followed by a 15 min interval for rest. In each sequence, subjects performed four anaerobic tests with the upper and lower limbs. Results: In the first of five series of efforts performed with the lower limbs (LL) at an ambient temperature of 21 &deg;C, statistically significant differences (p &lt; 0.001) were found between the mean RPP values recorded during the first and third and fourth repetitions, and between the second versus third and fourth repetitions. Statistically significant differences were also observed between the first and fourth efforts performed by the LL at 31 &deg;C (p &lt; 0.001) and between the second and third performed using the upper limbs (UL) at an ambient temperature of 21 &deg;C Conclusions: Varying ambient thermal conditions do not affect the size of generated relative peak power or the volume of work performed in pulsating anaerobic exercise