Insight into the Effect of Inhibitor Resistant S130G Mutant on Physico-Chemical Properties of SHV Type Beta-Lactamase: A Molecular Dynamics Study

Bacterial resistance is a serious threat to human health. The production of β-lactamase, which inactivates β-lactams is most common cause of resistance to the β-lactam antibiotics. The Class A enzymes are most frequently encountered among the four β-lactamases in the clinic isolates. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. SHV and TEM type are known to be most common class A β-lactamases. In the present study, we have analyzed the effect of inhibitor resistant S130G point mutation of SHV type Class-A β-lactamase using molecular dynamics and other in silico approaches. Our study involved the use of different in silico methods to investigate the affect of S130G point mutation on the major physico-chemical properties of SHV type class A β-lactamase. We have used molecular dynamics approach to compare the dynamic behaviour of native and S130G mutant form of SHV β-lactamase by analyzing different properties like root mean square deviation (RMSD), H-bond, Radius of gyration (Rg) and RMS fluctuation of mutation. The results clearly suggest notable loss in the stability of S130G mutant that may further lead to decrease in substrate specificity of SHV. Molecular docking further indicates that S130G mutation decreases the binding affinity of all the three inhibitors in clinical practice.     

Raman crystallographic studies of the intermediates formed by Ser130Gly SHV, a beta-lactamase that confers resistance to clinical inhibitors

Antibiotic resistance to beta-lactam compounds in Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae is often mediated by beta-lactamase enzymes like TEM and SHV. Previously, a limited number of inhibitors have shown efficacy in combating such bacterial drug resistance. However, many Gram-negative pathogens have evolved inhibitor resistant forms of these hydrolytic enzymes. A single point mutation of the active site residue Ser130 to a Gly in either TEM or SHV results in resistance to amoxicillin and clavulanic acid, an important clinical beta-lactam-beta-lactamase inhibitor combination antibiotic. Previous structural and modeling studies of the S130G mutants of TEM and SHV have shown differences in how these two distinct but closely related enzymes compensate for the loss of the Ser130 residue. In the case of S130G SHV, a structure of tazobactam in the active site has suggested that the inhibitor preferentially assumes a cis-enamine intermediate form when the Ser130 hydroxyl is absent. Raman crystallographic studies of S130G SHV inhibited with tazobactam, sulbactam, clavulanic acid, and 2'-glutaroxy penem sulfone (SA2-13) were performed with the aim of identifying the type and amount of intermediate formed with each drug to understand the role of the S130G mutation in formation of the important enamine intermediates. It is demonstrated that with the exception of sulbactam, each compound forms observable trans-enamine intermediates. For S130G reacted with tazobactam, identical steady state levels of enamine are achieved when compared to those of wild-type (WT) or even deacylation deficient forms of the enzyme. With clavulanic acid, slightly smaller amounts of enamine are observed within the first 30 min of the reaction but are not significantly different than those for tazobactam. Thus, the resistance mutation does not substantially affect the amount of trans-enamine formed with clavulanic acid during the critical early time period of inhibition. This finding has important implications in the design of beta-lactamase inhibitors for drug resistant variants like S130G SHV.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Peroxisome proliferator-activated receptor β/δ cross talks with E2F and attenuates mitosis in HRAS-expressing cells

The role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in Harvey sarcoma ras (Hras)-expressing cells was examined. Ligand activation of PPARβ/δ caused a negative selection with respect to cells expressing higher levels of the Hras oncogene by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing Pparβ/δ-null keratinocytes compared to HRAS-expressing wild-type keratinocytes. Ligand-activated PPARβ/δ repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation and increasing promoter recruitment of p130/p107. These results demonstrate a novel mechanism of PPARβ/δ cross talk with E2F signaling. Since cotreatment with a PPARβ/δ ligand and various mitosis inhibitors increases the efficacy of increasing G₂/M arrest, targeting PPARβ/δ in conjunction with mitosis inhibitors could become a suitable option for development of new multitarget strategies for inhibiting RAS-dependent tumorigenesis.     

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC₅₀-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics.     

A��42-to-A��40- and Angiotensin-converting Activities in Different Domains of Angiotensin-converting Enzyme

Amyloid β-protein 1–42 (Aβ42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Aβ. In contrast, Aβ40 is the predominant secreted form of Aβ and recent studies have suggested that Aβ40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Aβ42 to Aβ40, and its inhibition enhances brain Aβ42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628–8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Aβ42 to Aβ40. Interestingly, Aβ42-to-Aβ40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Aβ42-to-Aβ40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Aβ42-to-Aβ40-converting activity of ACE or increasing neurotoxic Aβ42.     

Exploring sequence requirements for C₃/C₄ carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C₃/C₄ carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C₃/C₄ carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C₃/C₄ β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C₃/C₄ β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C₃/C₄ carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.     

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host''s neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting ‘finger-loop’ interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA.     

Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion — a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO₂-HCO₃ ^- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.     

Dynamic diagnosis of familial prion diseases supports the β2-α2 loop as a universal interference target

Background

Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrP^C and on the properties of PrP^Sc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.

Methodology/Principal Findings

Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.

Conclusions/Significance

Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.     

Discovery of Novel New Delhi Metallo-β-Lactamases-1 Inhibitors by Multistep Virtual Screening

The emergence of NDM-1 containing multi-antibiotic resistant "Superbugs" necessitates the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor library containing 298 compounds of which 44 chemical compounds were purchased and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1 inhibitors with micromolar IC₅₀ values were validated. The most potent inhibitor, VNI-41, inhibited NDM-1 with an IC₅₀ of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results demonstrate the feasibility of applying virtual screening methodologies in identifying novel inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a functional scaffold.     

Crystal structure of H2O2-dependent cytochrome P450SPalpha with its bound fatty acid substrate: insight into the regioselective hydroxylation of fatty acids at the alpha position

Cytochrome P450_SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H₂O₂-dependent P450. P450_SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450_SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the C_α of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450_SPα. Mutations at the active site and the F–G loop of P450_SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450_BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450_SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450_BSβ.     

Crystallization and preliminary crystal structure of the complex of 17beta-hydroxysteroid dehydrogenase with a dual-site inhibitor

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyzes the synthesis of 17β-estradiol (E₂) from estrone, in the ovary and peripheral tissues. While the structures of 17β-HSD1 alone and in complex with E₂ have been determined (D. Ghosh, V. Pletnev, D.-W. Zhu, Z. Wawrzak, W.-L. Duax, W. Pangborn, F. Labrie, S.-X. Lin, Structure of human 17β-hydroxysteroid dehydrogenase at 2.20 Å resolution, Structure 3 (1995) 503–513; A. Azzi, P.H. Rhese, D.-W. Zhu, R.L. Campbell, F. Labrie, S.-X. Lin, Crystal structure of human estrogenic 17β-hydroxysteroid dehydrogenase complexed with 17_β-estradiol, Nature Struct. Biol. 3 (1996) 665–668, no structures of inhibitor/enzyme complex, either modeled or from crystallography, have been reported before the submission of the present paper. The best available inhibitors are among the ‘dual-site inhibitors’, blocking estrogenic 17β-HSD and the estrogen receptor. These compounds belong to a family of estradiol analogues having an halogen atom at the 16 position and an extended alkyl-amide chain at the 7 position (C. Labrie, G. Martel, J.M. Dufour, G. Levesque, Y. Merand, F. Labrie, Novel compounds inhibit estrogen formation and action, Cancer Res. 52 (1992) 610–615). We now report the crystallization of this enzyme/inhibitor complex. The complex of the best available dual-site inhibitor, EM-139, with 17β-HSD1 has been crystallized using both cocrystallization and soaking methods. Crystals are isomorphous to the native crystals grown in the presence of 0.06% β-octyl-glucoside and polyethyleneglycol 4000, with a monoclinic space group C2. Data at 1.8 Å have been collected from a synchrotron source. Even though the size of the inhibitor is greater than that of the substrate, our preliminary X-ray-diffraction study shows that EM-139 fits into the active site in a position similar to that of estrogen. The availability of such structural data will help design more potent inhibitors of estrogenic 17β-HSD.     

G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION*

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G_o protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G_o proteins by active α_2A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1''s central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

Cyclin D3 regulates the G1/S transition and is frequently overexpressed in several cancer types including breast cancer, where it promotes tumor progression. Here we show that a cytoskeletal protein keratin 19 (K19) physically interacts with a serine/threonine kinase GSK3β and prevents GSK3β-dependent degradation of cyclin D3. The absence of K19 allowed active GSK3β to accumulate in the nucleus and degrade cyclin D3. Specifically, the head (H) domain of K19 was required to sustain inhibitory phosphorylation of GSK3β Ser9, prevent nuclear accumulation of GSK3β, and maintain cyclin D3 levels and cell proliferation. K19 was found to interact with GSK3β and K19–GSK3β interaction was mapped out to require Ser10 and Ser35 residues on the H domain of K19. Unlike wildtype K19, S10A and S35A mutants failed to maintain total and nuclear cyclin D3 levels and induce cell proliferation. Finally, we show that the K19–GSK3β-cyclin D3 pathway affected sensitivity of cells toward inhibitors to cyclin-dependent kinase 4 and 6 (CDK4/6). Overall, these findings establish a role for K19 in the regulation of GSK3β-cyclin D3 pathway and demonstrate a potential strategy for overcoming resistance to CDK4/6 inhibitors.     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.     

Virtual screening, identification and in vitro testing of novel inhibitors of O-acetyl-L-serine sulfhydrylase of Entamoeba histolytica

The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite Entamoeba histolytica is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of E. histolytica. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. In vitro screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between in silico prediction and in vitro inhibition studies. This compound is thus a good starting point for further development of strong inhibitors.     

Replication Initiation at a Distance: DETERMINATION OF THE CIS- AND TRANS-ACTING ELEMENTS OF REPLICATION ORIGIN �� OF PLASMID R6K*

Plasmid R6K, which contains 3 replication origins called α, γ, and β, is a favorable system to investigate the molecular mechanism(s) of action at a distance, i.e. replication initiation at a considerable distance from the primary initiator protein binding sites (iterons). The centrally located γ origin contains 7 iterons that bind to the plasmid-encoded initiator protein, π. Ori α, located at a distance of ∼4 kb from γ, contains a single iteron that does not directly bind to π but is believed to access the protein by π-mediated α-γ iteron-iteron interaction that loops out the intervening ∼3.7 kb of DNA. Although the cis-acting components and the trans-acting proteins required for ori γ function have been analyzed in detail, such information was lacking for ori α. Here, we have identified both the sequence elements located at α and those at γ, that together promoted α activity. The data support the conclusion that besides the single iteron, a neighboring DNA primase recognition element called G site is essential for α-directed plasmid maintenance. Sequences preceding the iteron and immediately following the G site, although not absolutely necessary, appear to play a role in efficient plasmid maintenance. In addition, while both dnaA1 and dnaA2 boxes that bind to DnaA protein and are located at γ were essential for α activity, only dnaA2 was required for initiation at γ. Mutations in the AT-rich region of γ also abolished α function. These results are consistent with the interpretation that a protein-DNA complex consisting of π and DnaA forms at γ and activates α at a distance by DNA looping.