Different roads to discovery; Prontosil (hence sulfa drugs) and penicillin (hence β-lactams)

The important chemotherapeutic agents, Prontosil and pentenylpenicillin (penicillin F), were investigated initially by two men, Domagk and Fleming, who had been influenced by the horrendous wound infections of World War I. The very different pathways leading to their development and to that of the successor antibacterials (sulfa drugs, further penicillins, semi-synthetic penicillins), including the role played by patents, are discussed.     

Synthesis, docking and in vitro antimalarial evaluation of bifunctional hybrids derived from β-lactams and 7-chloroquinoline using click chemistry

1,2,3-Triazole tethered β-lactam and 7-chloroquinoline bifunctional hybrids were synthesized and evaluated as potential antimalarial agents. Activity against cultured Plasmodium falciparum was dependent on the N-substituent of the β-lactam ring as well as the presence of bis-triazole at the C-3 position. The observed activity profiles were further substantiated by docking studies via inhibition of P. falciparum dihydrofolate reductase (PfDHFR), a potential target for the development of new anti-malarials.     

Thioglycine and L-thiovaline: biologically active H₂S-donors

Thioglycine and l-thiovaline are stable under acidic and basic conditions but in the presence of bicarbonate they liberate the gasotransmitter H(2)S. In cells both thioamino acids were proven to enhance cGMP formation and promote vasorelaxation in mouse aortic rings. Given that H(2)S is known to lower arterial hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H(2)S donors hold promise as novel treatments for cardiovascular diseases.     

Novel non-peptide β-secretase inhibitors derived from structure-based virtual screening and bioassay

This Letter describes an efficient approach by integrating virtual screening with bioassay technology for finding small organic inhibitors targeting β-secretase (BACE-1). Fifteen hits with inhibitory potencies ranging from 2.8 to 118 μM (IC₅₀) against β-secretase were successfully identified. Compound 12 with IC₅₀ of 2.8 μM is the most potent hit against BACE-1. Docking simulation from gold 3.0 suggests putative binding mode of 12 in BACE-1 and potential key pharmacophore groups for further designing of non-peptide compounds as more powerful inhibitors against BACE-1.     

Divergent synthesis of biologically active l-threo-β-hydroxyaspartates from common trans-oxazolidine dicarboxylate

Amino Acids - A divergent synthetic strategy starting from a common trans-oxazolidine dicarboxylate intermediate has been successful to produce several non-proteinogenic...     

Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi

Thirteen thermophilic and thermotolerant fungal cultures isolated from composting soils produced diverse β-glucosidases as indicated by zymograms of PAGE developed using 4-methylumbelliferyl-β-d-glucoside. IEF profiling revealed the presence of 28 β-glucosidases separated on the basis of their pI. Eleven of the β-glucosidases were active under acidic conditions. Two β-glucosidase isoforms, ASCβG-II of Aspergillus caespitosus and HIβG-I of Humicola insolens were resistant to inhibition by glucose and were active in the presence of 300 and 100 (mM) glucose, respectively.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

β-Galactosidase from Ginkgo biloba seeds active against β-galactose-containing N-glycans: purification and characterization

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal’ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-Gal’ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, β-Gal’ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that β-Gal’ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal’ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal’ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.     

Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.     

Propenylamide and propenylsulfonamide cephalosporins as a novel class of anti-MRSA β-lactams

Novel C(3) propenylamide and propenylsulfonamide cephalosporins have been synthesized and tested for their ability to inhibit the penicillin-binding protein 2′ (PBP2′) from Staphylococcus epidermidis and the growth of a panel of clinically relevant bacterial species, including methicillin-resistant Staphylococcus aureus (MRSA). The most potent compounds inhibited the growth of MRSA strains with minimum inhibitory concentrations (MIC) as low as 1 μg/mL. The structure–activity relationship revealed the potential for further optimization of this new cephalosporin class.     

Potential bioisosteres of β-uracilalanines derived from 1H-1,2,3-triazole-C-carboxylic acids

The 1H-1,2,3-triazole-originated derivatives of willardiine were obtained by: (i) construction of the 1H-1,2,3-triazole ring in 1,3-dipolar cycloaddition of the uracil-derived azides and the carboxylate-bearing alkynes or α-acylphosphorus ylide, or (ii) N-alkylation of the uracil derivative with the 1H-1,2,3-triazole-4-carboxylate-derived mesylate. The latter method offered: (i) reproducible results, (ii) a significant reduction of amounts of auxiliary materials, (iii) reduction in wastes and (iv) reduction in a number of manual operations required for obtaining the reaction product. Compound 6a exhibited significant binding affinity to hHS1S2I ligand-binding domain of GluR2 receptor (EC₅₀ = 2.90 µM) and decreased viability of human astrocytoma MOG-G-CCM cells in higher extent than known AMPA antagonist GYKI 52466.     

Inhibitors of Trichoderma reesei β-glucosidase activity derived from autohydrolysis-exploded Eucalyptus regnans

Summary Enzymic saccharification of Eucalyptus regnans pulps pretreated by autohydrolysis-steam explosion resulted in low cellulose conversions into glucose when using trichodermal cellulase preparations. The reduced levels of glucose were attributable to the production of compounds during enzymic hydrolysis which were inhibitory to -d-glucosidase of Trichoderma reesei C-30 and in Meicelase, but not to the cellulases. Aspergillus niger -glucosidase was not inhibited, nor were -d-xylosidase(s) and 1,4--d-xylanase(s). The inhibitory compound(s) could be extracted from the enzymic hydrolyzates with ethyl acetate. The ethyl acetate extractives inhibited -glucosidase in a competitive manner, and inhibitory action was not affected by pH. Addition of the inhibitory compound(s) to trichodermal cellulase digests of cellulose resulted in reduced glucose yields compared to a control. The inhibitory effects could be overcome when cellulase digests were supplemented with A. niger -glucosidase resulting in higher cellulose-to-glucose conversions. The inhibitory compound(s) were localized mainly in the heartwood of E. regnans. An inhibitor compound of this type has not hitherto been reported. The presence of inhibitory compound(s) in the autohydrolysis liquor fraction is also reported.     

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H₂O₂ (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillin N acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillin N) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.     

Production of pharmaceutically important genistein and daidzein from soybean flour extract by using β-glucosidase derived from Penicillium janthinellum NCIM 1171

Genistein and daidzein are isoflavones with well-recognized biological activities. Their glycosidic forms (genistin and daidzin, respectively) are abundant in some plants. In this study, production of β-glucosidase from Penicillium janthinellum NCIM 1171 and its use in the obtaining genistein and daidzein are described. In a response surface methodology (RSM) optimized medium, levels of β-glucosidase under submerged and solid state fermentation conditions were found to be 10.2 ± 0.75 IU/mL and 121 ± 9.3 IU/g, respectively. The supernatants resulting from submerged fermentation were subjected to ion exchange and gel filtration chromatography and the enzyme was purified in a 44.4 fold manner with final recovery of 39.75 %. The β-glucosidase was deduced to be a monomeric protein with a molecular mass of 97.18 kDa. The purified protein showed 46 % sequence coverage matching with β-glucosidase derived from Penicillium sp. ABP88968. The purified enzyme was effective in producing genistein and daidzein from soybean (Glycine max) flour extract with a yield of 92.3 and 95 %, respectively. To the best of our knowledge, this is the first report on the use of a wild type strain of P. janthinellum for the production of genistein and daidzein with high productivity and purity.     

Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

The synthesis of novel sulfamides derived from β-benzylphenethylamines as acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase enzymes inhibitors

In this study, a series of novel β-benzylphenethylamines and their sulfamide derivatives were synthesized starting from (Z)-2,3-diphenylacrylonitriles. Pd-C catalysed hydrogenation of diphenylacrylonitriles, reduction of propanenitriles with LiAlH₄ in the presence of AlCl₃ followed by addition of conc. HCl afforded β-benzylphenethylamine hydrochloride salts. The reactions of these amine hydrochloride salts with chlorosulfonyl isocyanate (CSI) in the presence of tert-BuOH and excess Et₃N gave sulfamoylcarbamates. Removing of Boc group from the synthesized sulfamoylcarbamates with trifluoroacetic acid (TFA) yielded novel sulfamides in good yields. These novel sulfamides derived from β-benzylphenethylamines were effective inhibitors of the cytosolic carbonic anhydrase I and II isoenzymes (hCA I and II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with K_i values in the range of 0.278–2.260 nM for hCA I, 0.187–1.478 nM for hCA II, 0.127–2.452 nM for AChE and 0.494–1.790 nM for BChE. The inhibitory effects of the synthesized novel sulfamides derived from β-benzylphenethylamines were compared to those of acetazolamide and dorzolamide as clinical hCA I and II isoenzymes inhibitors and tacrine as a clinical AChE and BChE enzymes inhibitors. In addition to in vitro tests, molecular modeling approaches are implemented not only for prediction of the binding affinities of the compounds but also to study their inhibition mechanisms in atomic level at the catalytic domains.     

Antiviral effects of β-defensin derived from orange-spotted grouper (Epinephelus coioides)

Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a β-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1β. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.     

Agonists of the γ-aminobutyric acid type B (GABAB) receptor derived from β-hydroxy and β-amino difluoromethyl ketones

β-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA-B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of γ-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of β-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA-B receptor. Moreover, we also report that the synthesis and biological evaluation of β-amino difluoromethyl ketones provided the most potent compound across these two series.