Search

Francisella tularensis , the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity. [ABSTRACT FROM AUTHOR]

Abstract: The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping–Pong Bi–Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution. [Copyright &y& Elsevier]

Dihydroorotase (EC 3.5.2.3) catalyzes the reversible cyclization of N-carbamoyl- l-aspartate to l-dihydroorotate in the third step of the pyrimidine-biosynthesis pathway in Bacillus anthracis. A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure. These differences demonstrate the utility of exploring multiple structures of a molecular target as expressed from different organisms and how these differences can be exploited for structure-based drug discovery. [ABSTRACT FROM AUTHOR]

Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD+ has been solved to a resolution of 2.1 Å. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code ) which is bound to only NAD+ reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD+ cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors. [ABSTRACT FROM AUTHOR]

We have solved the crystal structure of a segment of nonerythroid α-spectrin (αII) consisting of the first 147 residues to a resolution of 2.3 Å. We find that the structure of this segment is generally similar to a corresponding segment from erythroid α-spectrin (αI) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C'); (ii) a helical conformation in the junction region connecting Helix C' with the first structural domain (D1); (iii) a long A1B1 loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in αII, and the lack of such hydrogen bond networks in αI leads to flexibility in αI. We have previously shown the junction region connecting Helix C' to D1 to be unstructured in a! (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) 1. Biol. Chem. 278, 218372 1844) and now find it to be helical in all, an important difference for α-spectrin association with β-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in α-spectrin will affect Helix C' structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions. [ABSTRACT FROM AUTHOR]

Glutamate racemase (RacE) is a bacterial enzyme that converts L-glutamate to n-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with n-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the n-Glu to L-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure. [ABSTRACT FROM AUTHOR]

Abstract: Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-γ-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 Å and 2.0 Å. Both enzymes are dimers with monomers arranged in a “tail-to-tail” orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases. [Copyright &y& Elsevier]

We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of a and C-terminal end of β) of erythroid spectrin. The values of the inverse line width parameter (ΔH0-1) from a family of SpαI-l -368Δ peptides scanning residues 21-30 exhibited a periodicity of ∼-3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C', a helix with a heptad pattern sequence. The residues with low AH0-1 values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the a spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the ΔH0-1 values from a family of spin-labeled SpβI-1898-2083Δ peptides also exhibited a periodicity of ∼3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high ΔH0-1 values and a hydrophobic side with low ΔH0-1 values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in αβ spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies. [ABSTRACT FROM AUTHOR]

We used Sp&alpah;I-1--156 peptide, a well-characterized model peptide of the αN-terminal region of erythrocyte spectrin, and SpαII-1--149, an αII brain spectrin model peptide similar in sequence to SpαI-1--156, to study their association affinities with a βI-spectrin peptide, SpβI-1898--2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with β-spectrin. However, our studies show that the affinity of αII-1--149 with SpβI-1898--2083 is much higher than that of SpαI-1--156. Our SAXS findings also indicate a significantly more extended conformation for SpαII-1--149 than for SpαI-1--156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 Å for SpαI-1--156 and of about 30 Å for SpαII-1--149, despite the fact that SpαI-1--156 has seven amino acid residues more than SpαII-1--149. For SpαI-1--156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpαII-1--149 support the hypothesis that this junction region is rigid (and probably helical) for αII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in αI to another extreme as a rigid segment in αII, determines the orientation of helix C' relative to the first structural domain. [ABSTRACT FROM AUTHOR]

Abstract: Many hereditary hemolytic anemias are due to spectrin mutations at the C -terminal region of β-spectrin (the βC region) that destabilize spectrin tetramer formation. However, little is known about the βC region of spectrin. We have prepared four recombinant β-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C -terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N -terminal region α-peptide (Spα1–156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the α-peptide. Circular dichroism results showed that the unassociated helices in both α- and β-peptides became associated, presumably to form a helical bundle, for those β-peptides that formed an αβ complex, but not for those β-peptides that did not form an αβ complex. In addition, upon association, an increase in the α-helical content was observed. These results showed that the β-peptides ending prior to residue 2072 (Thr) would not associate with α-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C -terminal segment of β-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C -terminal region ending with residue 2072 is important for its association with α-spectrin in forming tetramers. [ABSTRACT FROM AUTHOR]

Examines the association between human erythrocytes alpha and beta spectrin at tetramerization site. Involvement of interactions between helices in the alpha-beta spectrin association; Formation of the alphabeta complex; Use of circular dichroism techniques to monitor the secondary structures of the individual peptides and complex.

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GBneck2, PM3, and the mbondi2 radii set, generate the closest agreement with experimental values ( r2 = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called 'multiple independent sampling'), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GBHCT and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values ( r2 = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive 'multiple independent sampling method' may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

1. FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties, including metabolic stability, are unknown. 2. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. 3. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of four model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to nine newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. 4. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and non-linear regression models based on the selected molecular descriptors were developed and validated. 5. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior. [ABSTRACT FROM AUTHOR]

Acyl carrier proteins (ACPs) are important components in fatty‐acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9 Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target. [ABSTRACT FROM AUTHOR]

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis ( Ft FabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward Ft FabI. The IC 50 of benzimidazole (−)- 1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (−)/(+)- 1 and (−)/(+)- 2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (−)-rotation for both compounds 1 and 2 , while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with Ft FabI show much tighter binding MM-GBSA free energies for S - 1 and S - 2 than for their enantiomers, R - 1 and R - 2 , consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (−) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (−) to ( S )- and (+) to ( R )- for compounds 1 and 2 , and to further evaluate structural changes to improve efficacy. [ABSTRACT FROM AUTHOR]

Enoyl‐acyl carrier protein (ACP) reductase II (FabK) is a critical rate‐limiting enzyme in the bacterial type II fatty‐acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty‐acid synthesis represent viable drug targets for Gram‐negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram‐negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS‐II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow‐spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping‐pong mechanism. Here, the structure of the PgFabK enzyme as determined using X‐ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM‐barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure‐based drug‐discovery efforts towards the prevention and treatment of P. gingivalis infection. [ABSTRACT FROM AUTHOR]

S. aureus and A. baumannii are among the ESKAPE pathogens that are increasingly difficult to treat due to the rise in the number of drug resistant strains. Novel therapeutics targeting these pathogens are much needed. The bacterial enoyl reductase (FabI) is as potentially significant drug target for developing pathogen-specific antibiotics due to the presence of alternate FabI isoforms in many other bacterial species. We report the identification and development of a novel N -carboxy pyrrolidine scaffold targeting FabI in S. aureus and A. baumannii , two pathogens for which FabI essentiality has been established. This scaffold is unrelated to other known antibiotic families, and FabI is not targeted by any currently approved antibiotic. Our data shows that this scaffold displays promising enzyme inhibitory activity against FabI from both S. aureus and A. baumannii , as well as encouraging antibacterial activity in S. aureus . Compounds also display excellent synergy when combined with colistin and tested against A. baumannii . In this combination the MIC of colistin is reduced by 10-fold. Our first generation compound displays promising enzyme inhibition, targets FabI in S. aureus with a favorable selectivity index (ratio of cytotoxicity to MIC), and has excellent synergy with colistin against A. baumannii , including a multidrug resistant strain. [ABSTRACT FROM AUTHOR]

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC = 1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography. [ABSTRACT FROM AUTHOR]

We present an approach that accelerates protein solid-state NMR 5–20-fold using paramagnetic doping to condense data-collection time (to ∼0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic 1H T1 longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional 13C-13C and 13C-15N solid-state NMR spectra for several to tens of nanomoles of β-amyloid fibrils and ubiquitin in 1–2 d. [ABSTRACT FROM AUTHOR]

The true identity of the protein found in the crystals reported by Bondoc et al.[(2019), Acta Cryst. F75, 646–651] is given. [ABSTRACT FROM AUTHOR]