Identification of fragments targeting SMYD3 using highly sensitive kinetic and multiplexed biosensor-based screening.

A 1056-membered fragment library has been screened against SMYD3 using a novel multiplexed experimental design implemented in a grating coupled interferometry (GCI)-based biosensor. SMYD3 is a prospective target for anticancer drugs and the focus has initially been on discovery of inhibitors of its lysine methyl transferase activity. However, it has multiple protein interaction partners and several potential roles in carcinogenesis. It therefore remains unclear what mode of action ligands targeting the protein should have. Our goal was therefore to identify new ligands and discriminate hits that interact with the active site and those that interact with other sites. In addition, we were interested in selecting hits based on kinetic features rather than affinity. Screening was done in parallel against SMYD3 alone or SMYD3 with the active site blocked by a tight binding inhibitor. Hit selection was primarily based on dissociation rates. In total, 20 fragments were selected as hits, of which half apparently targeted the active site and half targeted other sites. Twelve of the hits were selected for structural analysis using X-ray crystallography in order to identify binding sites and modes of binding. Four of the hits were successfully identified in crystal structures with SMYD3; the others did not show any electron densities for ligands in the crystals. Although it might be possible to optimize the crystallography approach for a better success rate, it was clear that the sensitivity and time resolution of the biosensor assay was exceptional and enabled kinetic rate constants to be estimated for fragments. Fragments are typically considered to interact too rapidly for such quantification to be possible. This approach consequently represents a paradigm shift. In addition, the multiplexed approach allows ligands targeting different sites to be rationally selected already in the fragment library screening stage.

Accurate Computation of Thermodynamic Activation Parameters in the Chorismate Mutase Reaction from Empirical Valence Bond Simulations.

Chorismate mutase (CM) enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes' prominence in the literature, the extent of the roles that activation enthalpy and entropy play in catalyzing the conversion of chorismate to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismate mutases. Within this study, we utilize EVB/MD free energy perturbation calculations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of activation free energies of the reaction catalyzed by a monofunctional Bacillus subtilis CM and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed CM reactions are enthalpically driven. Furthermore, we observe that the monofunctional CM from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically driven nature of the reactions and also the difference in efficiencies between the two enzymes.

The Ambivalence of Connexin43 Gap Peptides in Cardioprotection of the Isolated Heart against Ischemic Injury.

The present study investigates infarct-reducing effects of blocking ischemia-induced opening of connexin43 hemichannels using peptides Gap19, Gap26 or Gap27. Cardioprotection by ischemic preconditioning (IPC) and Gap peptides was compared, and combined treatment was tested in isolated, perfused male rat hearts using function and infarct size after global ischemia, high-resolution respirometry of isolated mitochondrial and peptide binding kinetics as endpoints. The Gap peptides reduced infarct size significantly when given prior to ischemia plus at reperfusion (Gap19 76.2 ± 2.7, Gap26 72.9 ± 5.8 and Gap27 71.9 ± 5.8% of untreated control infarcts, mean ± SEM). Cardioprotection was lost when Gap26, but not Gap27 or Gap19, was combined with triggering IPC (IPC 73.4 ± 5.5, Gap19-IPC 60.9 ± 5.1, Gap26-IPC 109.6 ± 7.8, Gap27-IPC 56.3 ± 8.0% of untreated control infarct). Binding stability of peptide Gap26 to its specific extracellular loop sequence (EL2) of connexin43 was stronger than Gap27 to its corresponding loop EL1 (dissociation rate constant Kd 0.061 ± 0.004 vs. 0.0043 ± 0.0001 s-1, mean ± SD). Mitochondria from IPC hearts showed slightly but significantly reduced respiratory control ratio (RCR). In vitro addition of Gap peptides did not significantly alter respiration. If transient hemichannel activity is part of the IPC triggering event, inhibition of IPC triggering stimuli might limit the use of cardioprotective Gap peptides.

ThermoSlope: A Software for Determining Thermodynamic Parameters from Single Steady-State Experiments.

The determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three α-amylases from different species and habitats. The experiments with the α-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.

Biochemical and biophysical characterization of the OXA-48-like carbapenemase OXA-436.

The crystal structure of the class D ß-lactamase OXA-436 was solved to a resolution of 1.80 Å. Higher catalytic rates were found at higher temperatures for the clinically important antibiotic imipenem, indicating better adaptation of OXA-436 to its mesophilic host than OXA-48, which is believed to originate from an environmental source. Furthermore, based on the most populated conformations during 100 ns molecular-dynamics simulations, it is postulated that the modulation of activity involves conformational shifts of the α3-α4 and ß5-ß6 loops. While these changes overall do not cause clinically significant shifts in the resistance profile, they show that antibiotic-resistance enzymes exist in a continuum. It is believed that these seemingly neutral differences in the sequence exist on a path leading to significant changes in substrate selectivity.

The biological assembly of OXA-48 reveals a dimer interface with high charge complementarity and very high affinity.

Many class D ß-lactamases form dimers in solution. The functional basis of the dimerization of OXA-48-like class D ß-lactamases is not known, but in order to understand the structural requirements for dimerization of OXA-48, we have characterized the dimer interface. Size exclusion chromatography, small angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) were used to confirm the oligomeric state of OXA-48 in solution. X-ray crystallographic structures were used to elucidate the key interactions of dimerization. In silico residue scanning combined with site-directed mutagenesis was used to probe hot spots of dimerization. The affinity of dimerization was quantified using microscale thermophoresis, and the overall thermostability was investigated using differential scanning calorimetry. OXA-48 was consistently found to be a dimer in solution regardless of the method used, and the biological assembly found from the SAXS envelope is consistent with the dimer identified from the crystal structures. The buried chloride that interacts with Arg206 and Arg206' at the dimer interface was found to enhance the thermal stability by > 4 °C and crystal structures and mutations (R189A, R189A/R206A) identified several additional important ionic interactions. The affinity for OXA-48 R206A dimerization was in the picomolar range, thus revealing very high dimer affinity. In summary, OXA-48 has a very stable dimer interface, facilitated by noncovalent and predominantly charged interactions, which is stronger than the dimer interfaces previously described for other class D ß-lactamases. PDB CODES: The oxacillinase-48 (OXA-48) R206A structure has PDB ID: 5OFT and OXA-48 R189A has PDB ID: 6GOA.

A focused fragment library targeting the antibiotic resistance enzyme - Oxacillinase-48: Synthesis, structural evaluation and inhibitor design.

ß-Lactam antibiotics are of utmost importance when treating bacterial infections in the medical community. However, currently their utility is threatened by the emergence and spread of ß-lactam resistance. The most prevalent resistance mechanism to ß-lactam antibiotics is expression of ß-lactamase enzymes. One way to overcome resistance caused by ß-lactamases, is the development of ß-lactamase inhibitors and today several ß-lactamase inhibitors e.g. avibactam, are approved in the clinic. Our focus is the oxacillinase-48 (OXA-48), an enzyme reported to spread rapidly across the world and commonly identified in Escherichia coli and Klebsiella pneumoniae. To guide inhibitor design, we used diversely substituted 3-aryl and 3-heteroaryl benzoic acids to probe the active site of OXA-48 for useful enzyme-inhibitor interactions. In the presented study, a focused fragment library containing 49 3-substituted benzoic acid derivatives were synthesised and biochemically characterized. Based on crystallographic data from 33 fragment-enzyme complexes, the fragments could be classified into R1 or R2 binders by their overall binding conformation in relation to the binding of the R1 and R2 side groups of imipenem. Moreover, binding interactions attractive for future inhibitor design were found and their usefulness explored by the rational design and evaluation of merged inhibitors from orthogonally binding fragments. The best inhibitors among the resulting 3,5-disubstituted benzoic acids showed inhibitory potential in the low micromolar range (IC50 = 2.9 µM). For these inhibitors, the complex X-ray structures revealed non-covalent binding to Arg250, Arg214 and Tyr211 in the active site and the interactions observed with the mono-substituted fragments were also identified in the merged structures.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D ß-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D ß-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Structure, activity and thermostability investigations of OXA-163, OXA-181 and OXA-245 using biochemical analysis, crystal structures and differential scanning calorimetry analysis.

The first crystal structures of the class D ß-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20 Šresolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07 Šresolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8°C), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4°C) and OXA-181 (Tm = 52.6°C). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.

Screening and Design of Inhibitor Scaffolds for the Antibiotic Resistance Oxacillinase-48 (OXA-48) through Surface Plasmon Resonance Screening.

The spread of antibiotic resistant bacteria is a global threat that shakes the foundations of modern healthcare. ß-Lactamases are enzymes that confer resistance to ß-lactam antibiotics in bacteria, and there is a critical need for new inhibitors of these enzymes for combination therapy together with an antibiotic. With this in mind, we have screened a library of 490 fragments to identify starting points for the development of new inhibitors of the class D ß-lactamase oxacillinase-48 (OXA-48) through surface plasmon resonance (SPR), dose-rate inhibition assays, and X-ray crystallography. Furthermore, we have uncovered structure-activity relationships and used alternate conformations from a crystallographic structure to grow a fragment into a more potent compound with a KD of 50 µM and an IC50 of 18 µM.

A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement.

BACKGROUND: In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. RESULTS: In our case study, three homologous ß-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene. CONCLUSION: We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.