Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen–Manganese Transporter MntC

MntC is a metal-binding protein component of the Mn^2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn^2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn^2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn^2 +.     

Structure–activity relationships of diverse xanthones against multidrug resistant human tumor cells

Thirteen xanthones were isolated naturally from the stem of Securidaca inappendiculata Hassk, and structure-activity relationships (SARs) of these compounds were comparatively predicted for their cytotoxic activity against three human multidrug resistant (MDR) cell lines MCF-7/ADR, SMMC-7721/Taxol, and A549/Taxol cells. The results showed that the selected xanthones exhibited different potent cytotoxic activity against the growth of different human tumor cell lines, and most of the xanthones exhibited selective cytotoxicity against SMMC-7721/Taxol cells. Furthermore, some tested xanthones showed stronger cytotoxicity than Cisplatin, which has been used in clinical application extensively. The SARs analysis revealed that the cytotoxic activities of diverse xanthones were affected mostly by the number and position of methoxyl and hydroxyl groups. Xanthones with more free hydroxyl and methoxyl groups increased the cytotoxic activity significantly, especially for those with the presence of C-3 hydroxyl and C-4 methoxyl groups.     

Ion-pairing liquid chromatography–tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway

Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-l-Ala, UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ala-d-Glu-l-Lys, and UDP-MurNAc-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5 pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2 nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3 mg dry weight analyzed) treated with fosfomycin, d-boroAla, d-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway.     

Discovery and initial structure–activity relationships of N-benzyl tricyclic indolines as antibacterials for methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent drug resistant bacteria. In 2012, over 11,000 fatalities in the United States were directly attributable to MRSA. In an effort to develop novel structural and mechanistic classes of antibacterial agents to fight against MRSA, we have optimized a hit compound, Of4, previously discovered in a screening campaign of a bio-inspired polycyclic indoline library previously developed in our lab. We took advantage of our concise and versatile synthetic strategy to conduct initial structure–activity relationship studies of Of4, and we now report the discovery of compound 4k as a more potent antibacterial agent against S. aureus. Compound 4k also displayed equivalent activity in four MRSA and a methicillin-susceptible strains while demonstrating an improved mammalian cytotoxicity profile compared to Of4. Interestingly, 4k shares the same tricyclic indoline core as Of1, a β-lactam-selective resistance-modifying agent, but harbors a distinct modification pattern conferring unique bioactivity. This phenomenon is reminiscent of many bioactive natural products.     

Design,synthesis, and structure–activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2–8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2–8. Compounds 2 and 6 had two-digit nanomolar potency (IC₅₀) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.     

Structure-activity relationships in a series of C2-substituted gluco-configured tetrahydroimidazopyridines as β-glucosidase inhibitors

Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent β-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K(i)=0.64 nM) and 5 (K(i)=0.58 nM) showed stronger inhibitory potency against β-glucosidase. Maestro 9.1 was used to study the structure-activity relationships by docking the compounds into the β-glucosidase active sites.     

A hemicellulosic β-glucan from the hypocotyls of Phaseolus aureus

A glucan of $\text{DP}$_nca 80 has been isolated from the hypocotyls of mung bean plants (Phaseolus aureus). Methylation analysis and periodate oxidation studies showed that the glucan has (1 → 3) and (1 → 4) linked d-glucopyranosyl residues in the molar ratio 1·0:1·7. Oligosaccharides containing both β(1 → 3) and β(1 → 4) linked residues were isolated from partial hydrolysates.     

Structure–activity relationships of 2-aminothiazoles effective against Mycobacterium tuberculosis

A series of 2-aminothiazoles was synthesized based on a HTS scaffold from a whole-cell screen against Mycobacterium tuberculosis (Mtb). The SAR shows the central thiazole moiety and the 2-pyridyl moiety at C-4 of the thiazole are intolerant to modification. However, the N-2 position of the aminothiazole exhibits high flexibility and we successfully improved the antitubercular activity of the initial hit by more than 128-fold through introduction of substituted benzoyl groups at this position. N-(3-Chlorobenzoyl)-4-(2-pyridinyl)-1,3-thiazol-2-amine (55) emerged as one of the most promising analogues with a MIC of 0.024 μM or 0.008 μg/mL in 7H9 media and therapeutic index of nearly ～300. However, 55 is rapidly metabolized by human liver microsomes (t_1/2 = 28 min) with metabolism occurring at the invariant aminothiazole moiety and Mtb develops spontaneous low-level resistance with a frequency of ～10^?5.     

Design and synthesis of cell selective α/β-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or β-amino acids. Sequence A-12 was protease susceptible whereas its α/β-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2–32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/β-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.     

Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.     

A structure–activity relationship study of flavonoids as inhibitors of E. coli by membrane interaction effect

Flavonoids exhibit a broad range of biological activities including antibacterial activity. However, the mechanism of their antibacterial activity has not been fully investigated. The antibacterial activity and membrane interaction of 11 flavonoids (including 2 polymethoxyflavones and 4 isoflavonoids) against Escherichia coli were examined in this study. The antibacterial capacity was determined as flavonoids > polymethoxyflavones > isoflavonoids. Using fluorescence, it was observed that the 5 flavonoids rigidified the liposomal membrane, while the polymethoxyflavones and isoflavonoids increased membrane fluidity. There was a significant positive correlation between antibacterial capacity and membrane rigidification effect of the flavonoids. A quantitative structure–activity relationship (QSAR) study demonstrated that the activity of the flavonoid compounds can be related to molecular hydrophobicity (CLogP) and charges on C atom at position3 (C3). The QSAR model could be used to predict the antibacterial activity of flavonoids which could lead to natural compounds having important use in food and medical industry.     

Structure–activity relationships of new 4-hydroxy bis-coumarins as radical scavengers and chain-breaking antioxidants

The main antioxidant properties of five new 4-hydroxy-bis-coumarins during bulk lipid autoxidation at 80 °C and 0.1 mM and 1.0 mM concentrations were studied and compared with 4-hydroxy-2H-chromen-2-one (1). These compounds are: 3,3′-((3,4-dihydroxy-phenyl) methylene) bis (4-hydroxy-2H-chromen-2-one) (2), 3,3′-((3,4-dimethoxyphenyl) methylene) bis (4-hydroxy-2H-chromen-2-one) (3), 3,3′-((4-hydroxy-3,5-dimethoxy-phenyl) methylene) bis(4-hydroxy-2H-chromen-2-one) (4) 3,3′-((3,4,5- trimethoxyphenyl) methylene) bis (4-hydroxy-2H-chromen-2-one) (5) 3,3′-((4-hydroxy-3-methoxy-5-nitrophenyl) methylene) bis (4-hydroxy-2H-chromen-2-one) (6), It was found that compound 2 with a catecholic structure in the aromatic nucleus showed the strongest antioxidant activity. Compound 4 showed a moderate antioxidant activity, and all the other compounds didn't show any capacity as chain-breaking antioxidants. Both 4-hydroxy-bis-coumarins (2 and 4) demonstrated also stronger radical scavenging activity towards DPPH radical by using TLC DPPH rapid test, than compound 1. The other compounds (3, 5, 6) didn't show any capacity as radical scavengers. The structure–activity relationship was discussed on the base of comparable kinetic analysis of studied 4-hydroxy-bis-coumarins with the known and standard antioxidants as α-tocopherol (TOH), caffeic acid (CA), sinapic acid (SA), ferulic acid (FA), and p-coumaric acid (p-CumA). In order to study the possible synergism between two phenolic antioxidants, the antioxidant efficiency and reactivity of two equimolar binary mixtures of coumarins and TOH (2 + TOH and 4 + TOH) and of corresponding cinnamic acid with TOH (CA + TOH and SA + TOH) were also tested and compared. The oxidation stability of the lipid substrate in presence of binary mixtures CA + TOH, SA + TOH and 2 + TOH appeared to be higher than that of the individual antioxidants. However, no synergism was obtained for all tested binary mixtures.     

Structure–activity relationships of diverse annonaceous acetogenins against human tumor cells

Twelve annonaceous acetogenins (ACGs) with different stereochemical structures and configuration were selected to test for their inhibitions on the growth of Hela, SMMC-7541, SGC-7901, MCF-7 and A-5408 tumor cell lines using MTT method. This was the first to simultaneously investigate effects of structural factors of stereochemical structures and configuration on cytotoxicities with structure–activity relationship. The present study showed that cytotoxic selectivities of ACGs with threo/trans/threo/trans/erythro stereochemical arrangement were gently more active than those with threo/trans/threo/trans/threo stereochemical arrangement, and ACGs with cis THF ring partly produced notable cytotoxic selectivities. Furthermore, ACGs with S configuration at C-24 exhibited gently more cytotoxic selectivities potency than those with R configuration at C-24.     

Multiple forms of Vicia faba α-galactosidases and their relationships

Three highly purified α-galactosidases, I, II¹ and II² have been isolated from resting Vicia faba seeds. Form I (MW 160 000) is a tetrame     

Structure–activity relationships of fraxamoside as an unusual xanthine oxidase inhibitor

Fraxamoside, a macrocyclic secoiridoid glucoside featuring a hydroxytyrosol group, was recently identified as a xanthine oxidase inhibitor (XOI) comparable in potency in vitro to the standard antigout drug allopurinol. However, this activity and its considerably higher value than its derivatives oleuropein, oleoside 11-methyl ester, and hydroxytyrosol are not explained by structure–activity relationships (SARs) of known XOIs. To exclude allosteric mechanisms, we first determined the inhibition kinetic of fraxamoside. The resulting competitive mechanism prompted a computational SAR characterization, combining molecular docking and dynamics, which fully explained the behavior of fraxamoside and its derivatives, attributed the higher activity of the former to conformational properties of its macrocycle, and showed a substantial contribution of the glycosidic moiety to binding, in striking contrast with glycoside derivatives of most other XOIs. Overall, fraxamoside emerged as a lead compound for a new class of XOIs potentially characterized by reduced interference with purine metabolism.     

Synthesis and structure–activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase

A series of per-O-benzoylated 5-β-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(β-d-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated β-d-glucopyranosyl cyanide gave the corresponding 5-β-d-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-β-d-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K_i = 8.8 and 11.6 μM, respectively). A detailed analysis of the structure–activity relationships is presented.     

Characterization of the ‘reserve cellulose’ of the endosperm of Carum carvi as a β(1–4)-mannan

The reserve polysaccharide of the endosperm of Carum carvi consists of more than 90% mannose and was characterized as a β(1–4)-mannan. Total or partial acid hydrolysis, enzymatic breakdown or acetolysis of either palm or Carum carvi mannan yielded the same mono- and oligosaccharides, indicating a similar chemical structure of the two reserve polysaccharides. However, Carum carvi contains only traces of the alkali soluble mannan A dominant in the palm endosperm polysaccharide.