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Summary: Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of β-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization. [Copyright &y& Elsevier]

The biological significance of protein and lipid glycosylation is well established. For example, cells respond to environmental stimuli by altering glycan structures on their surfaces, and cancer cells evade normal growth regulation in part by remodeling their surface glycans. In general, glycan chemical properties differ significantly from those of proteins, lipids, nucleic acids, and small molecule metabolites. Thus, advances in glycomics, a comprehensive study to identify all glycans in an organism, rely on the development of specialized analytical methods. Mass spectrometry (MS) is emerging as an enabling technology in the field of glycomics. This review summarizes recent developments in mass spectrometric analysis methods for protein-based glycomics and glycoproteomics workflows. [Copyright &y& Elsevier]

Summary: We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model. [Copyright &y& Elsevier]

Summary: Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase—R2 variant—was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur. [Copyright &y& Elsevier]

Summary: Conversion of cellular prion protein (PrPC) into the pathological conformer (PrPSc) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrPPalm) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrPSc conversion. [Copyright &y& Elsevier]

Summary: The highly substrate-specific strictosidine synthase (EC 4.3.3.2) catalyzes the biological Pictet-Spengler condensation between tryptamine and secologanin, leading to the synthesis of about 2000 monoterpenoid indole alkaloids in higher plants. The crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine has been elucidated here, allowing the rational site-directed mutation of the active center of STR1 and resulting in modulation of its substrate acceptance. Here, we report on the rational redesign of STR1 by generation of a Val208Ala mutant, further describing the influence on substrate acceptance and the enzyme-catalyzed synthesis of 10-methyl- and 10-methoxystrictosidines. Based on the addition of strictosidine to a crude strictosidine glucosidase preparation from Catharanthus cells, a combined chemoenzymatic approach to generating large alkaloid libraries for future pharmacological screenings is presented. [Copyright &y& Elsevier]

Summary: Osteoporosis is a major age-related source of morbidity and mortality. Increased bone resorption mediated by osteoclasts is central to its pathogenesis. Cytokines, particularly RANKL and TNFα, are often increased under pathologic conditions, leading to enhanced osteoclastogenesis. Black cohosh (Actaea/Cimicifuga racemosa L), a popular herbal supplement for the treatment of menopausal symptoms, was recently shown to have the beneficial effect of preventing bone loss. Here, we demonstrate that 25-acetylcimigenol xylopyranoside (ACCX), a triterpenoid glycoside isolated from black cohosh, potently blocks in vitro osteoclastogenesis induced by either RANKL or TNFα. This blockage of osteoclastogenesis elicited by ACCX results from abrogation of the NF-κB and ERK pathways induced by either RANKL or TNFα, respectively. Importantly, this compound attenuates TNFα-induced bone loss in vivo. Therefore, ACCX represents a potential lead for the development of a new class of antiosteoporosis agents. [Copyright &y& Elsevier]

Summary: A series of contrast agents for magnetic resonance imaging (MRI) aimed at noninvasively determining the hormone receptor status of cancer in vitro was developed. These MRI contrast agents were prepared by conjugating progesterone to clinically used Gd(III) chelates. These agents exhibited higher progesterone receptor binding affinities in the nanomolar range and intracellular accumulation. High logP values of the modified compounds suggested that the lipophilicity of the steroid conjugates may have contributed to membrane permeability. Synchrotron radiation X-ray fluorescence microscopy and magnetic resonance images revealed that the synthesized conjugates showed the greatest cellular accumulation and significant increase in relaxivity in vitro compared to the previously developed steroid-modified agent. Transcriptional assays using the progesterone response element linked to luciferase indicated that the contrast agents entered the cell, interacted with the biological target, and drove specific progesterone-mediated transcription. [Copyright &y& Elsevier]

Summary: Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewisx trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core. [Copyright &y& Elsevier]

Summary: Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg2+-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria. [Copyright &y& Elsevier]

Summary: Histone deacetylases (HDACs), enzymes involved in chromatin remodeling, are promising targets for anticancer drug development. Several HDAC inhibitors (HDACi) are in clinical trials. One limitation of present HDACi is their nonspecificity, affecting many HDACs with similar effectiveness. We have identified a small molecule, the sesquiterpene lactone parthenolide (PN), which specifically depletes HDAC1 protein without affecting other class I/II HDACs. HDAC1 depletion occurred through proteasomal degradation and resulted in transcriptional consequences comparable to those observed with pan-HDACi. Surprisingly, HDAC1 depletion did not occur through the inflammation mediator IKK2, a known PN target and regulator of HDAC1. Rather, PN promoted HDAC1 depletion and cell death through the DNA-damage-transducer ataxia telangiectasia mutated. Our study suggests that modulating cellular HDAC protein levels with small molecules provides an alternative approach to specific HDAC inhibition and effective cancer treatment. [Copyright &y& Elsevier]

Summary: Several polyketide secondary metabolites are predicted to undergo isoprenoid-like β-alkylations during biosynthesis. One such secondary metabolite is myxovirescin A1, produced by Myxococcus xanthus. Myxovirescin is of special interest in that it appears to undergo two distinct β-alkylations. Additionally, the myxovirescin biosynthetic gene cluster lacks tandem thiolation domains required in the synthesis of other β-branched secondary metabolites. To probe the origins of the β-branches in myxovirescin, we heterologously overexpressed the proteins predicted to be responsible for myxovirescin β-alkylation and reconstituted their activities in vitro on model substrates. Our results confirm that myxovirescin undergoes two isoprenoid-like β-alkylations during its biosynthesis, including an unprecedented β-ethylation. The study of its biosynthesis should shed light on the scope and requirements for isoprenoid-like biosynthetic logic in a polyketide context. [Copyright &y& Elsevier]

Summary: Aptamers targeting reverse transcriptase (RT) from HIV-1 inhibit viral replication in vitro, presumably by competing with binding of the primer/template complex. This site is not targeted by the currently available small-molecule anti-HIV-1 RT inhibitors. We have identified SY-3E4, a small-molecule inhibitor of HIV-1 RT, by applying a screening assay that utilizes a reporter-ribozyme regulated by the anti-HIV-1 RT aptamer. SY-3E4 displaces the aptamer from the protein, selectively inhibits DNA-dependent, but not RNA-dependent, polymerase activity, and inhibits the replication of both the wild-type virus and a multidrug-resistant strain. Analysis of available structural data of HIV-1 and HIV-2 RTs rationalizes many of the observed characteristics of the inhibitory profiles of SY-3E4 and the aptamer and suggests a previously not considered region in these RTs as a target for antiviral therapy. Our study reveals unexplored ways for rapidly identifying alternative small-molecule target sites in proteins and illustrates strategies for overcoming resistance-conferring mutations with small molecules. [Copyright &y& Elsevier]

Summary: Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the β-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems. [Copyright &y& Elsevier]

Summary: Acyl carrier protein (ACP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS II ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed. [Copyright &y& Elsevier]

Summary: Flavonoids and stilbenes have attracted much attention as potential targets for nutraceuticals, cosmetics, and pharmaceuticals. We have developed a system for producing “unnatural” flavonoids and stilbenes in Escherichia coli. The artificial biosynthetic pathway included three steps. These included a substrate synthesis step for CoA esters synthesis from carboxylic acids by 4-coumarate:CoA ligase, a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthase and chalcone isomerase, and into stilbenes by stilbene synthase, and a modification step for modification of the flavanones by flavone synthase, flavanone 3β-hydroxylase and flavonol synthase. Incubation of the recombinant E. coli with exogenously supplied carboxylic acids led to production of 87 polyketides, including 36 unnatural flavonoids and stilbenes. This system is promising for construction of a larger library by employing other polyketide synthases and modification enzymes. [Copyright &y& Elsevier]

Summary: Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A λ-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a “dehydroproline acrylamide” moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin. [Copyright &y& Elsevier]

Summary: The last stages of assembly of the aminocoumarin antibiotics, clorobiocin and coumermycin A1, which target the GyrB subunits of bacterial DNA gyrase, involve enzymatic transfer of the pyrrolyl-2-carbonyl acyl group from a carrier protein (CloN1/CouN1) to the 3′-OH of the noviosyl moiety of the antibiotic scaffold. The enzyme, CouN7, will catalyze both the forward and back reaction on both arms of the coumermycin scaffold. This occurs via an O-acyl-Ser101-CouN7 intermediate, as shown by transient labeling of the enzyme with [14C]acetyl-S-CouN1 as donor and by inactivating mutation of the active site, Ser101, to Ala. The intermediacy of the pyrrolyl-2-carbonyl-O-CouN7 allows net pyrrole transfer between distinct aminocoumarin scaffolds, for example, between the descarbamoylnovobiocin scaffold and coumermycin A1 and vice versa. CouN7 also allows shuttling of surrogate acyl groups between noviosyl-aminocoumarin scaffolds to generate new antibiotic variants. [Copyright &y& Elsevier]

Summary: Cyclic peptides are of considerable interest as potential protein ligands. It has been postulated that cyclic molecules might be more cell permeable than their linear counterparts due to their reduced conformational flexibility. We report a study that tests this hypothesis by using a quantitative, reporter gene-based assay that measures the relative cell permeability of steroid conjugates of molecules of interest. We demonstrate that cyclic peptides are, in fact, not generally more permeable than their linear counterparts. [Copyright &y& Elsevier]

Summary: Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comparison of the nigericin biosynthetic genes with their counterparts in the biosynthetic clusters for other polyketides has prompted a significant revision of the proposed common pathway for polyether biosynthesis. In particular, we present evidence that in nigericin, nanchangmycin, and monensin, an unusual ketosynthase-like protein, KSX, transfers the initially formed linear polyketide chain to a discrete acyl carrier protein, ACPX, for oxidative cyclization. Consistent with this, deletion of either monACPX or monKSX from the monensin gene cluster effectively abolished monensin A biosynthesis. [Copyright &y& Elsevier]

Summary: To increase the chemical diversity of bioactive molecules by incorporating unusual elements, we have examined the replacement of a C=C double bond with the isoelectronic, isostructural B-N bond in the context of nonsteroidal estrogen receptor (ER) ligands. While the B-N bond was hydrolytically labile in the unhindered cyclofenil system, the more hindered anilino dimesitylboranes, analogs of triarylethylene estrogens, were easily prepared, hydrolytically stable, and demonstrated substantial affinity for ERs. X-ray analysis of one ERα-ligand complex revealed steric clashes with the para methyl groups distorting the receptor; removal of these groups resulted in an increase in affinity, potency, and transcriptional efficacy. These studies define the structural determinants of stability and cellular bioactivity of a B-N for C=C substitution in nonsteroidal estrogens and provide a framework for further exploration of “elemental isomerism” for diversification of drug-like molecules. [Copyright &y& Elsevier]

Summary: Bis-indolylquinones represent a class of fungal natural products that display antiretroviral, antidiabetes, or cytotoxic bioactivities. Recent advances in Aspergillus genomic mining efforts have led to the discovery of the tdiA-E-gene cluster, which is the first genetic locus dedicated to bis-indolylquinone biosynthesis. We have now genetically and biochemically characterized the enzymes TdiA (bis-indolylquinone synthetase) and TdiD (L-tryptophan:phenylpyruvate aminotransferase), which, together, confer biosynthetic abilities for didemethylasterriquinone D to Aspergillus nidulans. This compound is the universal intermediate for all bis-indolylquinones. In this biochemical study of a bis-indolylquinone synthetase and a fungal natural product transaminase, we present a one-pot chemoenzymatic protocol to generate didemethylasterriquinone D in vitro. As TdiA resembles a nonribosomal peptide synthetase, yet catalyzes carbon-carbon-bond formation, we discuss the implications for peptide synthetase chemistry. [Copyright &y& Elsevier]

Summary: The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug''s potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved α-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA''s potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases. [Copyright &y& Elsevier]

Summary: Reversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS, and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT-specific small-molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells, and consequently inhibit the multiplication of HIV. [Copyright &y& Elsevier]

Summary: Group II introns are mobile genetic elements that have been implicated as agents of genetic diversity, and serve as important model systems for investigating RNA catalysis and pre-mRNA splicing. In the absence of an atomic-resolution structure of the intron, detailed understanding of its catalytic mechanism has remained elusive. Previous identification of a divalent metal ion stabilizing the leaving group in both splicing steps suggested that the group II intron may employ a “two-metal ion” mechanism, a catalytic strategy used by a number of protein phosphoester transfer enzymes. Using metal rescue experiments, we now reveal the presence of a second metal ion required for nucleophile activation in the exon-ligation step of group II intron splicing. Coupled with biochemical and structural evidence of at least two metal ions at the group I intron reaction center, these results suggest a mechanistic paradigm for describing catalysis by large ribozymes. [Copyright &y& Elsevier]

Summary: The first step of the reaction catalyzed by the homodimeric FabH from a dissociated fatty acid synthase is acyl transfer from acyl-CoA to an active site cysteine. We report that C1 to C10 alkyl-CoA disulfides irreversibly inhibit Escherichia coli FabH (ecFabH) and Mycobacterium tuberculosis FabH with relative efficiencies that reflect these enzymes'' differential acyl-group specificity. Crystallographic and kinetic studies with MeSSCoA show rapid inhibition of one monomer of ecFabH through formation of a methyl disulfide conjugate with this cysteine. Reaction of the second subunit with either MeSSCoA or acetyl-CoA is much slower. In the presence of malonyl-ACP, the acylation rate of the second subunit is restored to that of the native ecFabH. These observations suggest a catalytic model in which a structurally disordered apo-ecFabH dimer orders on binding either the first substrate, acetyl-CoA, or the inhibitor MeSSCoA, and is restored to a disordered state on binding of malonyl-ACP. [Copyright &y& Elsevier]

Summary: We systematically compare X-ray structures of inhibitor complexes of four well-known enzymes and correlate two- and three-dimensional (2D and 3D) similarity of inhibitors with their potency. The analysis reveals the presence of unexpected systematic relationships between molecular similarity and potency. These findings explain why apparently inconsistent structure-activity relationships (SARs) can coexist in different targets, and they have general implications for compound screening and optimization. The results suggest that (1) even for active sites with significant binding constraints, there is a high probability that structurally diverse ligands with similar activity can be identified, (2) different types of SARs are not mutually exclusive, and (3) the chemical nature of ligands is of comparable importance for SARs as the features of active sites. These insights aid in the understanding of target-specific SARs and their intrinsic degree of variability. [Copyright &y& Elsevier]

Summary: The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1′ site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC. [Copyright &y& Elsevier]

Summary: Chitinases hydrolyse the β(1,4)-glycosidic bonds of chitin, an essential fungal cell wall component. Genetic data on a subclass of fungal family 18 chitinases have suggested a role in cell wall morphology. Specific inhibitors of these enzymes would be useful as tools to study their role in cell wall morphogenesis and could possess antifungal properties. Here, we describe the crystallographic structure of a fungal “plant-type” family 18 chitinase, that of Saccharomyces cerevisiae CTS1. The enzyme is active against 4-methylumbelliferyl chitooligosaccharides and displays an unusually low pH optimum for activity. A library screen against ScCTS1 yielded hits with Ki''s as low as 3.2 μM. Crystal structures of ScCTS1 in complex with inhibitors from three series reveal striking mimicry of carbohydrate substrate by small aromatic moieties and a pocket that could be further exploited in optimization of these inhibitors. [Copyright &y& Elsevier]

Summary: The circumsporozoite protein (CSP) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. We describe here the design of a conformationally constrained synthetic peptide, designated UK-39, which has structural and antigenic similarity to the NPNA-repeat region of native CSP. NMR studies on the antigen support the presence of helical turn-like structures within consecutive NPNA motifs in aqueous solution. Intramuscular delivery of UK-39 to mice and rabbits on the surface of reconstituted influenza virosomes elicited high titers of sporozoite crossreactive antibodies. Influenza virus proteins were crucially important for the immunostimulatory activity of the virosome-based antigen delivery system, as a liposomal formulation of UK-39 was not immunogenic. IgG antibodies elicited by UK-39 inhibited invasion of hepatocytes by P. falciparum sporozoites, but not by antigenically distinct P. yoelii sporozoites. Our approach to optimized virosome-formulated synthetic peptide vaccines should be generally applicable for other infectious and noninfectious diseases. [Copyright &y& Elsevier]

Summary: The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo. [Copyright &y& Elsevier]

Summary: Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-(N-amidino-Δ3-pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aerA-aerI as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety. [Copyright &y& Elsevier]

Summary: Bioreductive alkylating agents are an important class of clinical antitumor antibiotics that crosslink and monoalkylate DNA. Here, we use a synthetic, photochemically activated derivative of FR400482 to investigate the molecular mechanism of this class of drugs in a biologically relevant context. We find that the organization of DNA into nucleosomes effectively protects it against drug-mediated crosslinking, while permitting monoalkylation. This modification has the potential to lead to the formation of covalent crosslinks between chromatin and nuclear proteins. Using in vitro approaches, we found that interstrand crosslinking of free DNA results in a significant decrease in basal and activated transcription. Finally, crosslinked plasmid DNA is inefficiently assembled into chromatin. Our studies suggest pathways for the clinical effectiveness of this class of reagents. [Copyright &y& Elsevier]

Summary: Rv0098 is part of an operon, Rv0096–Rv0101, from Mycobacterium tuberculosis (Mtb) that is essential for Mtb''s survival in mouse macrophages. This operon also contains an acyl carrier protein and one of the only two nonribosomal peptide synthases in Mtb. Rv0098 is annotated in the genome as a hypothetical protein and was proposed to be an acyl-coenzyme A (CoA) dehydratase. The structure of Rv0098, together with subsequent biochemical analysis, indicated that Rv0098 is a long-chain fatty acyl-CoA thioesterase (FcoT). However, FcoT lacks a general base or a nucleophile that is always found in the catalytic site of type II and type I thioesterases, respectively. The active site of Mtb FcoT reveals the structural basis for its substrate specificity for long-chain acyl-CoA and allows us to propose a catalytic mechanism for the enzyme. The characterization of Mtb FcoT provides a putative function of this operon that is crucial for Mtb pathogenicity. [Copyright &y& Elsevier]

Summary: Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N3-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids. [Copyright &y& Elsevier]

Summary: Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network. [Copyright &y& Elsevier]

Summary: High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (Kd ∼150 μM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties. [Copyright &y& Elsevier]

Summary: Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles. [Copyright &y& Elsevier]

Summary: The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired α,β-unsaturated δ-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity. [Copyright &y& Elsevier]

Summary: The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. [Copyright &y& Elsevier]

Summary: Nanomedicine directed at diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. Here, we targeted a spherical, icosahedral viral nanoplatform to a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding mediated through specific protein-ligand interactions exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto cells. The fluorescence-imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections. [Copyright &y& Elsevier]

Summary: Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a γ-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective γ-glutamyl group is then cleaved by BtrG via an uncommon γ-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis. [Copyright &y& Elsevier]

Summary: Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I. [Copyright &y& Elsevier]

Summary: The mammalian target of rapamycin (mTOR) signaling network is central to the regulation of cell growth in response to both growth factors and nutrients. We developed a high-throughput, cell-based assay to identify small-molecule modulators of the mTOR signaling network. One such compound, which we name quinostatin, potently inhibits this network by directly targeting the lipid-kinase activity of the catalytic subunits of class Ia PI3Ks. This study illustrates the power of unbiased, phenotypic screening as a means for illuminating cell circuitry, and resulted in the identification of a chemotype for selective inhibition of the class Ia PI3Ks. [Copyright &y& Elsevier]

Summary: The crystal structures of a wild-type and a mutant PCS, a novel plant type III polyketide synthase from a medicinal plant, Aloe arborescens, were solved at 1.6 Å resolution. The crystal structures revealed that the pentaketide-producing wild-type and the octaketide-producing M207G mutant shared almost the same overall folding, and that the large-to-small substitution dramatically increases the volume of the polyketide-elongation tunnel by opening a gate to two hidden pockets behind the active site of the enzyme. The chemically inert active site residue 207 thus controls the number of condensations of malonyl-CoA, solely depending on the steric bulk of the side chain. These findings not only provided insight into the polyketide formation reaction, but they also suggested strategies for the engineered biosynthesis of polyketides. [Copyright &y& Elsevier]

Summary: There is an urgent, global need for the development of new antibacterial agents. We have applied the small-molecule macroarray approach to the synthesis and screening of antibacterial compounds active against the Gram-positive pathogen Staphylococcus aureus. Several macroarrays of 1,3-diphenyl-2-propen-1-ones (chalcones), cyanopyridines, and pyrimidines were synthesized on a planar cellulose support system on the order of days. This support system was found to be highly compatible with antibacterial assay formats, including disk-diffusion and agar-overlay visualization methods. Further, sufficient compound was isolated from each spot of the macroarray for both compound characterization and minimum inhibitory concentration (MIC) estimation. Analysis of the small-molecule macroarrays in these assays uncovered a set of antibacterial agents with in vitro MIC values against methicillin-resistant S. aureus comparable to certain antibacterial drugs in use today. [Copyright &y& Elsevier]

Summary: Tyrosine kinases and phosphatases establish the crucial balance of tyrosine phosphorylation in cellular signaling, but creating specific inhibitors of protein Tyr phosphatases (PTPs) remains a challenge. Here, we report the development of a potent, selective inhibitor of Mycobacterium tuberculosis PtpB, a bacterial PTP that is secreted into host cells where it disrupts unidentified signaling pathways. The inhibitor, (oxalylamino-methylene)-thiophene sulfonamide (OMTS), showed an IC50 of 440 ± 50 nM and >60-fold specificity for PtpB over six human PTPs. The 2 Å resolution crystal structure of PtpB in complex with OMTS revealed a large rearrangement of the enzyme, with some residues shifting >27 Å relative to the PtpB:PO4 complex. Extensive contacts with the catalytic loop provide a potential basis for inhibitor selectivity. Two OMTS molecules bound adjacent to each other, raising the possibility of a second substrate phosphotyrosine binding site in PtpB. The PtpB:OMTS structure provides an unanticipated framework to guide inhibitor improvement. [Copyright &y& Elsevier]

Summary: Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different β-lactam antibiotics. We have constructed P. chrysogenum strains expressing cefD1, cefD2, cefEF, and cefG genes cloned from Acremonium chrysogenum. Northern analysis revealed that the four genes were expressed in P. chrysogenum. The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth. [Copyright &y& Elsevier]

Summary: The gene cluster responsible for pimaricin biosynthesis in Streptomyces natalensis contains a cholesterol oxidase-encoding gene (pimE) surrounded by genes involved in pimaricin production. Gene-inactivation and -complementation experiments revealed that pimE encodes a functional cholesterol oxidase and, surprisingly, that it is also involved in pimaricin biosynthesis. This extracellular enzyme was purified from S. natalensis culture broths to homogeneity, and it was shown to restore pimaricin production when added to the mutant culture broths. Other cholesterol oxidases also triggered pimaricin production, suggesting that these enzymes could act as signaling proteins for polyene biosynthesis. This finding constitutes the description of a cholesterol oxidase gene with an involvement in antibiotic biosynthesis, and it broadens the scope of the biological functions for this type of oxidase. [Copyright &y& Elsevier]

Summary: Wortmannin (Wm), a steroid-like molecule of 428.4 Da, appears to be unstable in biological fluids (apparent chemical instability), yet it exhibits an antiproliferative activity in assays employing a 48 hr incubation period (prolonged bioactivity), a situation we refer to as the “wortmannin paradox.” Under physiological conditions, Wm covalently reacts with nucleophiles such as the side chains of cysteine, N-methyl hexanoic acid, lysine, or proline at the C20 position on the furan ring. Like Wm, WmC20 amino acid derivatives had significant antiproliferative activities. Three Wm derivatives, WmC20-proline, WmC20-cysteine, and a WmC20-N-methyl hexanoic acid, generated Wm that then reacted with lysine in an exchange-type reaction. This unusual, reversible, covalent reaction of Wm with nucleophiles under physiological conditions provides an explanation for the wortmannin paradox. [Copyright &y& Elsevier]