Showing papers in "ChemBioChem in 2010"
TL;DR: The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
Abstract: There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
654 citations
TL;DR: The potential of azides to undergo metal‐free reactions with strained unsaturated systems has inspired the development of an increasing number of reactive probes, which are comprehensively summarized here.
Abstract: Covalently bound azide on a (small) organic molecule or a (large) biomolecular structure has proven an important handle for bioconjugation. Azides are readily introduced, small, and stable, yet undergo smooth ligation with a range of reactive probes under mild conditions. In particular, the potential of azides to undergo metal-free reactions with strained unsaturated systems has inspired the development of an increasing number of reactive probes, which are comprehensively summarized here. For each individual probe, the synthetic preparation is described, together with reaction kinetics and the full range of applications, from materials science to glycoprofiling. Finally, a qualitative and quantitative comparison of azido-reactive probes is provided.
206 citations
TL;DR: An overview on the recent developments in BVMO‐mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective bioc atalytic methodologies to apply Baeyer–Villiger monooxygenases for the synthesis of high added value compounds are provided.
Abstract: Baeyer-Villiger monooxygenases (BVMOs) represent a specific class of monooxygenases that are capable of catalyzing a variety of oxidation reactions, including Baeyer-Villiger oxidations. The recently elucidated BVMO crystal structures have provided a more detailed insight into the complex mechanism of these flavin-containing enzymes. Biocatalytic studies on a number of newly discovered BVMOs have shown that they are very potent oxidative biocatalysts. In addition to catalyzing the regio- and enantioselective Baeyer-Villiger oxidations of a wide range of carbonylic compounds, epoxidations, and enantioselective sulfoxidations have also been shown to be part of their catalytic repertoire. This review provides an overview on the recent developments in BVMO-mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective biocatalytic methodologies to apply BVMOs for the synthesis of high added value compounds.
185 citations
TL;DR: It is shown for the first time that gua-nine, adenine, and hypoxanthine can be produced from forma-mide in a single model prebiotic reaction at lower tempera-tures than previously reported, if formamide is subjected to UVirradiation during heating.
Abstract: Here, we show for the first time that gua-nine, adenine, and hypoxanthine can be produced from forma-mide in a single model prebiotic reaction at lower tempera-tures than previously reported, if formamide is subjected to UVirradiation during heating; this observation relaxes the require-ments for prebiotic purine nucleobase formation. The yieldand diversity of purines produced in heated/UV-irradiated for-mamide are further enhanced by the presence of inorganiccatalysts, as solids or as dissolved ions. We also analyzed theproducts of formamide solutions to which specific hydrogencyanide (HCN) condensation products
177 citations
TL;DR: It is not the end for aminoglycosides, but rather, the challenges faced by researchers have led to ingenuity and a change in how this class of compounds is viewed, a renaissance.
Abstract: Although aminoglycosides have been used as antibacterials for decades, their use has been hindered by their inherent toxicity and the resistance that has emerged to these compounds. It seems that such issues have relegated a formerly front-line class of antimicrobials to the proverbial back shelf. However, recent advances have demonstrated that novel aminoglycosides have a potential to overcome resistance as well as to be used to treat HIV-1 and even human genetic disorders, with abrogated toxicity. It is not the end for aminoglycosides, but rather, the challenges faced by researchers have led to ingenuity and a change in how we view this class of compounds, a renaissance.
174 citations
TL;DR: The crystal structure of human MAGL is reported, and Cys201 is identified as the crucial residue in MAGL inhibition by N‐arachidonylmaleimide, a sulfhydryl‐reactive compound.
Abstract: 2-Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2-arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2-arachidonoylglycerol transmission.
160 citations
TL;DR: The 3DM method was used to generate a comprehensive database of the a/s-hydrolase fold enzyme superfamily, which facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made.
Abstract: Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/s-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition high-quality libraries for protein engineering can be easily designed.
137 citations
TL;DR: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts and all three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold.
Abstract: Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells.
136 citations
TL;DR: The purified recombinant SPS showed activity for the formation of (−)‐solanapyrone A from achiral prosolanAPyrone II, establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels–Alder enzyme.
Abstract: The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.
133 citations
TL;DR: Evidence is presented that membrane closure can accumulate a remarkable number of solutes inside some compartments, and the possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed.
Abstract: One question in the origin of life is the time at which membrane compartments came into the picture as hosts for the first forms of metabolism. If we assume the proteins and nucleic acids came first, then it is difficult to conceive how all the macromolecular components could have been entrapped at a later time in a single compartment. On the other hand, the hypothesis that metabolism originated from inside the compartment means that we would then have to conceive semipermeable, sophisticated membranes in prebiotic times, which does not appear plausible. With this study, we believe that we can offer a partial solution to this riddle, at the same time opening a new vista on the principles of the entrapment of solute in vesicles. We used cryo-TEM to study the entrapment of the protein ferritin in liposomes. The novel, surprising principle that appears is that when lipid surfaces close up in a proteincontaining solution to form vesicles, the entrapment frequency does not follow the expected Poisson distribution, but tends to assume a power-law behaviour, characterized by many “empty” vesicles (no or very little entrapped solute), and a long decreasing tail with extremely crowded vesicles. CryoTEM analysis shows indeed some extremely crowded liposomes adjacent to empty ones. The conclusion is that membrane closure can accumulate a remarkable number of solutes inside some compartments. The possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed. The spontaneous formation of lipid vesicles (liposomes) in an aqueous phase containing one or more solutes produces a heterogeneous population of liposomes in terms of solute content. Such entrapments have generally been studied by averaging techniques, such as batch absorbance or fluorescence, whereas little attention has been devoted to studying individual encapsulation. This is partly due to the technical difficulty of directly counting molecules inside liposomes. The encapsulation of biomacromolecules inside liposomes, on the other hand, is an important issue in origins of life research (protocell models) as well as in recent studies on synthetic cells. A series of recent experiments within our project on the construction of minimal living cells revealed possible deviations from the number of macromolecules expected to be entrapped inside liposomes of diameter d<200 nm. In particular, with the aim of producing green fluorescent protein (GFP) inside liposomes, we prepared liposomes in the presence of the transcription–translation macromolecular machinery, namely E. coli extracts as well as PURESYSTEM (a cell-free protein synthesis kit containing 36 purified components, t-RNAs, ribosomes, for a total of about 80 different macromolecules). We showed that GFP was synthesised inside liposomes, despite of the fact that the Poisson probability of liposome co-entrapment of about 80 different macromolecules (each at a concentration of 0.1–1 mm) is vanishingly small (~10!26). In order to explain the observed positive results, it was suggested that the local (inside vesicles) concentrations of the proteins and nucleic acids required for GFP expression exceeded the expected value by a factor 20 or more. The hypothesis of the spontaneous emergence of a small, yet measurable, fraction of synthetic cells with exceptionally high solute content implies that, in origins-of-life scenarios, functional solutes like RNAs and peptides even if present in dilute solutions could have become entrapped inside vesicles to give rise to functional protocells. How would it be possible to entrap such a large number of molecules inside one single vesicle against the expected Poisson statistics? Here we present evidence that shows that the entrapment frequency does not follow the expected Poisson behaviour. As shown in our previous reports, it is possible to directly visualize and count ferritin molecules inside liposomes by cryoTEM. Being an iron-storage protein, ferritin consists of a nucleus of about 4500 iron ions, spherically enclosed by 24 protein subunits; it is endowed with a large scattering power so that it is classically used as a probe for electron microscopy. Ferritin-containing liposomes are therefore suitable for direct investigation of macromolecular entrapment in cell-like compartments. When liposomes are prepared in a ferritin solution, ferritin is entrapped spontaneously. It is expected that the average number (N0) of ferritin molecules per vesicle is proportional to the ferritin concentration in the bulk (C0) and to the vesicle volume V (i.e. , N0=C0V). At low N0 values, the occupancy frequency of water-soluble molecules in liposomes is classically described by a Poisson distribution, which also accounts for deviations around the average value, as shown in Figure S1. We explored the pattern of ferritin entrapment inside liposomes under different conditions by using ferritin solutions at concentrations of 4, 8, 16 and 32 mm. The liposome composition and concentration were first optimised in order to obtain [a] Prof. P. L. Luisi, M. Allegretti, Dr. P. Stano Dipartimento di Biologia, Universit! degli Studi RomaTre Viale G. Marconi 446, 00146 Rome (Italy) Fax: (+39)0657336321 E-mail : luisi@mat.ethz.ch [b] Dr. T. Pereira de Souza, Prof. Dr. A. Fahr Institut f"r Pharmazie, Friedrich Schiller Universit#t Jena Lessingstrasse 8, 07743 Jena (Germany) [c] Dr. F. Steiniger Elektronenmikroskopisches Zentrum, Friedrich Schiller Universit#t Jena Ziegelm"hlenweg 1, 07743 Jena (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000381.
128 citations
TL;DR: An innovative strategy for ligation at Xaa-Leu sites by using b-mercaptoleucine combined with desulfurization is reported and its application in the total chemical synthesis of HIV-1 Tat protein is reported.
Abstract: Total chemical synthesis of proteins offers exceptional opportunities for preparing targets with exquisite control over the covalent structure, high purity and large quantities for functional and structural analysis. In this regard, native chemical ligation (NCL) continues to be the method of choice for joining two unprotected peptides through thiol capture followed by S–N acyl transfer to form the amide bond at the ligation site. The desulfurization reaction introduced by Yan and Dawson as a post-NCL step greatly expanded the scope of ligation chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Ala sites accessible in the synthesis of functional proteins. This seminal work has prompted several groups to extend this strategy to other band g-mercapto amino acid derivatives (e.g. , Phe and Val) as part of the ongoing efforts to extend NCL to essentially any ligation junction. In addition, by positioning a thiol handle at the Lys side chain and at the 2-acetamido group of the glycan moiety in a glycopeptide allowed NCL to be used for peptide ubiquitylation and glycopeptide ligation respectively, and after a desulfurization step, the native structures could be achieved. The above-described examples testify to the power of desulfurization when combined with NCL to assist the synthesis of naturally occurring proteins. In addition, recent reports on the mild and highly versatile free-radical Cys reduction protocol, 8] as well as the compatibility of these methods in the presence of other thiol functionalities permit the use of this two-step approach in the synthesis of a variety of protein targets. Despite the introduction of mercaptoPhe and mercaptoVal to assist efficient peptide ligation, their utility in protein synthesis has not yet been demonstrated. The preparation of proteins by using ligation methods is often challenging because decreases in rate and chemoselectivity only come to light in more complex systems. To date, only ligation at XaaCys followed by desulfurization has been demonstrated in the synthesis of full-length proteins, this diminishes the generality of the desulfurization approach in the total chemical synthesis of proteins. Here we report an innovative strategy for ligation at Xaa-Leu sites by using b-mercaptoleucine combined with desulfurization (Scheme 1) and its application in the total chemical synthesis of HIV-1 Tat protein. To implement the approach outlined in Scheme 1, we first had to design a synthetic strategy for b-mercaptoleucine, the key residue in this strategy. We envisioned two routes to achieve the synthesis of this building block by starting from the commercially available threo-b-hydroxy-l-leucine. In the first route, the b-hydroxy group could be converted to a good leaving group followed by nucleophilic substitution with a thiol nucleophile, whereas in the second one an aziridine intermediate could be employed to introduce the thiol functionality through a regioselective ring opening. Applying the first strategy by activation of the b-hydroxy group as a mesylate or tosylate derivative, then substitution with PMB-SH or thioacetic acid as a source of the thiolate functionality led to formation of the b-elimination product in a substantial amount (>60 %). Additionally, it is very likely that under these conditions the thiol-substituted product could also be formed through a Michael addition, which would lead to a racemization at the acarbon, rendering this method less desirable for us. Inspired by the recent work on the racemization-free synthesis of b-methylcysteine by starting from Thr, we decided to follow a similar path to prepare the desired building block (Scheme 2). Thus, b-hydroxy-l-leucine was converted to the methyl ester derivative followed by treatment with p-nitrophenylsulfonyl chloride (NsCl) to give the p-nitrosulfonamide 2 in 70 % yield (two steps). Ring closure of 2 under Mitsunobu conditions afforded the aziridine 3 in 96 % yield. Subsequently, BF3·OEt2mediated ring opening with PMB-SH furnished the two regioisomers 4 and 5 in a quantitative yield in a 6:4 ratio respectively. Apparently, the additional steric hindrance in our case compared to Thr (isopropyl vs. methyl) decreased the desired regioselectivity. Notwithstanding the unsatisfactory regioselectivity of the aziridine opening, the regioisomers 4 and 5 were Scheme 1. General strategy for NCL at Leu.
TL;DR: The number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased, and a significant number of hits with improved rates and enantioselectivities were identified in these “smart” libraries.
Abstract: Simultaneous multiple site-saturation mutagenesis was performed at four active-site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3-phenylbutyric acid esters (3-PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of alpha/beta-hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased. Whereas the wild-type esterase showed only marginal activity and poor enantioselectivity (E(true)=3.2) towards 3-PBA-ethyl ester, a significant number of hits with improved rates (up to 240-fold) and enantioselectivities (up to E(true)=80) were identified in these "smart" libraries.
TL;DR: The creation, characterization, and application of the 8–17 RNA‐cleaving DNAzyme is explored, which has been regarded with special importance because it can catalyze sequence‐specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields.
Abstract: Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA-based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as "8-17", has enhanced our understanding of DNAzyme-mediated catalysis. Moreover, the function of 8-17 has been regarded with special importance because it can catalyze sequence-specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8-17 RNA-cleaving DNAzyme.
TL;DR: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphal morphology in the opportunistic fungus Candida albicans, and could be relevant in shaping multispecies Candida bacteria biofilms in the human body.
Abstract: In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer-2 (AI-2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell-free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC-MS and proved to be trans-2-decenoic acid. We show that trans-2-decenoic acid does not inhibit AI-2-specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans-2-decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal-specific signature gene of C. albicans, is abolished by trans-2-decenoic acid. trans-2-Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom-signaling molecules and is the first member of this family from a Gram-positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.
TL;DR: A protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]‐labeled proteins, based on a suite of complementary three‐dimensional solid‐state NMR spectroscopy experiments, directed towards the application to proteins with more than about 100 amino acid residues is presented.
Abstract: The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential C-13 and N-15 resonance assignment of uniformly [N-15,C-13]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The, assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well-dispersed C-beta resonances. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N-terminal globular domain of the HET-s prion, a 227-residue protein.
TL;DR: An overview of potential strategies for tackling protein aggregation and its associated toxicity, focusing on protein aggregation in human disease is given.
Abstract: Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation processes and the intertwined events account for the fact that no effective disease-modifying treatments for these disorders are currently available. Nevertheless, in-depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation-mediated cell toxicity, offering new targets for drug development. In addition, recent findings in the field have identified similar features, revealing the possibility of shared mechanisms and hence potential common approaches for intervention. This review aims to give an overview of potential strategies for tackling protein aggregation and its associated toxicity, focusing on protein aggregation in human disease.
TL;DR: The crystal structure of a thermophilic “ene” reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39 reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes.
Abstract: We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.
TL;DR: The results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.
Abstract: The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide-functionalized Qbeta capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qbeta particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin-mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP-labeled clathrin light chains. By testing Qbeta particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.
TL;DR: This review aims to survey the mechanisms by which macromolecular protease inhibitors function, divided into categories based on their mechanism in order to illustrate that a relatively small number of design principles can be combined to develop new and effective prote enzyme inhibitors.
Abstract: Proteolytic enzymes are ubiquitous in all organisms and constitute 2–4% of the encoded gene products. They are critical for diverse biological processes such as digestion, blood clotting, host defense, pathogenic infection, viral replication, wound healing, and disease progression, to name a few. Because proteases trigger an irreversible event - the cleavage of a protein - their activity must be tightly controlled. Dysregulated proteolytic activity causes a disruption in the homeostatic balance of a biological system and can result in any number of poor biological outcomes. As a result, nature has developed a number of strategies to control proteolysis, including spatial and temporal regulation, zymogen activation and protease degradation, and through the inhibition of proteases by macromolecular inhibitors. Somewhat surprisingly, relatively few design principles underlie the mechanisms of inhibition of a myriad range of macromolecular protease inhibitors. Significant engineering efforts have gone into modifying and improving inhibitor potency and specificity, and to a large extent, the same design principles that work well for naturally occurring protease inhibitors have proved valuable for inhibitors developed in the laboratory.
This review aims to survey the mechanisms by which macromolecular protease inhibitors function. To do this, inhibitors have been divided into categories based on their mechanism in order to illustrate that a relatively small number of design principles can be combined to develop new and effective protease inhibitors. These divisions are not strict, and many inhibitors could be grouped in a number of classes. The list of mechanisms presented here is not exhaustive in its treatment of all inhibitors, but aims to be illustrative of the many ways proteases can be inhibited. For more information on genome-wide protease mining,[1] protease mechanism,[2] pre-clinical inhibition,[3] and drug discovery efforts,[4] the reader is directed to excellent reviews that have been written in recent years. Figure 1 provides an overview of basic substrate and protease nomenclature that will be used in this review.
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Figure 1
(A) Diagram of a protease active site. A protease cleaves a peptide at the scissile bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminal side of the scissile bond and their prime-side residues C-terminal to the scissile bond. The catalytic residues determine the class of protease. Serine, cysteine, and threonine proteases hydrolyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, glutamic and metalloproteases activate a water molecule to hydrolyze the peptide bond in a non-covalent manner. (B) A serine protease (matriptase/MT-SP1, 1EAX.pdb) with the catalytic triad in yellow and the surface loops that surround the active site colored in blue. While the catalytic architecture of proteases is remarkably conserved, the surface loops are areas of high sequential and structural diversity.
TL;DR: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates, and selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was made possible.
Abstract: Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_(BM3) variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates.
TL;DR: These new apratoxins are illustrative of a mechanism (the modification of an NRPS adenylation domain specificity pocket) for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.
Abstract: A collection of Lyngbya bouillonii from Palmyra Atoll in the Central Pacific, a site several thousand kilometers distant from all previous collections of this chemically prolific species of cyanobacterium, was found to contain two new cancer cell cytotoxins of the apratoxin family. The structures of the new compounds, apratoxins F and G, were determined by 1-D and 2-D NMR techniques in combination with mass spectrometric methods. Stereochemistry was explored using chromatographic analyses of the hydrolytically released fragments in combination with NMR and optical rotation comparisons with known members of the apratoxin family. Apratoxins F and G add fresh insights into the SAR of this family because they incorporate an N-methyl alanine residue at a position where all prior apratoxins have possessed a proline unit, yet they retain high potency as cytotoxins to H-460 cancer cells with IC50 values of 2 and 14 nM, respectively. Additional assays using zone inhibition of cancer cells and clonogenic cells give a comparison of the activities of apratoxin F to apratoxin A. Additionally, the clonogenic studies in combination with MTD studies provided insights as to dosing schedules that should be used for in vivo studies, and preliminary in vivo evaluation validated the predicted in vivo efficacy for apratoxin A. These new apratoxins are illustrative of a mechanism, the modification of an NRPS adenylation domain specificity pocket, for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.
TL;DR: This work has used another approach to investigate the biosynthesis of biologically active compounds in fungi that of heterologous gene expression, which involves transfer of a gene of interest from a donor strain to a suitable host.
Abstract: Fungal natural products include antibiotics such as the penicillins, antirejection drugs such as cyclosporins and cholesterollowering drugs such as the statins. High productivity of these pharmaceutically important compounds is desirable and has regularly been pursued by strain improvement and metabolic engineering. Recently partial and full genome sequencing has revealed clusters of genes encoding the biosynthesis of these compounds in fungi. Surprisingly it has been found that while rich in bioactive compounds, fungi are even richer in biosynthetic gene clusters. For example, in the well-characterised species Aspergillus nidulans, there are at least 54 secondary metabolite gene clusters, only half of which are characterised at a chemical level. Fungal secondary metabolite gene clusters can be readily identified from key genes within the clusters, such as nonribosomal peptide synthetases (NRPS), terpene cyclases or polyketide synthases (PKS). Although it is relatively easy to identify such gene clusters, it is currently almost impossible to predict the chemical product synthesised, as the programming of the synthase cannot be predicted from sequence information alone. This is further compounded by the presence of genes for many tailoring enzymes, which usually act after the core synthase has completed synthesis of the skeleton, further modifying the resulting compound, making it extremely difficult to elucidate the likely metabolite from gene sequence data alone. A number of different strategies have been employed to decipher fungal gene clusters. These include: chemical profiling of cultures produced under a range of different conditions; over-expression of pathway regulatory genes (where present) ; manipulation of transcriptional activators (e.g. , LaeA) ; manipulation of the pH regulatory system (e.g. , PacC) ; use of chromatin modifying (i.e. , epigenetic) chemicals ; and cofermentation of bacteria and fungi. Unfortunately all of these techniques are restricted in their general utility. Many of the biosynthetic clusters do not include specific transcriptional regulators, many pathways do not respond to LaeA or PacC regulation and chromatin modifiers only activate a small subset of the gene clusters within a genome. In addition, many fungi are difficult to cultivate on a large scale and are often recalcitrant to molecular techniques, so these approaches cannot be applied. We and others have used another approach to investigate the biosynthesis of biologically active compounds in fungi that of heterologous gene expression. This method involves transfer of a gene of interest from a donor strain to a suitable host. In principle a bacterial host such as E. coli could be used, but several problems are usually encountered. First, bacterial hosts are unable to process eukaryotic introns and so these must be removed. Second, expression of eukaryotic genes in bacteria can be problematic, especially if there is a significant codon bias. Third, bacteria can experience difficulty in correctly folding fungal polypeptides. Fourth, proteins such as PKS and NRPS require selective post-translational phosphopantetheinylation for them to be active in vivo. Finally, bacteria may not supply specific metabolites for biosynthesis. While each of these problems can be overcome in isolation, cumulative effects can make the use of bacteria as expression hosts for fungal genes troublesome and inefficient. This is illustrated by the recent expression of the beauvericin NRPS (bbBeas) from
TL;DR: Although all the radical‐AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.
Abstract: Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B(12), AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.
TL;DR: The stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses, and it was suggested that CorB is a trans‐acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains.
Abstract: Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS-derived and the other NRPS/PKS-derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans-AT-type mixed PKS/NRPS gene cluster, containing a beta-branching cassette. Striking features of this gene cluster are a NRPS-like adenylation domain that is part of a PKS-type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans-acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses.
TL;DR: It is proposed that oxidation‐stable mutants might well be a “prerequisite” for thermostabilization, because laboratory‐evolved thermostability in CHMO might be masked by a high degree of oxidation instability.
Abstract: Baeyer–Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo-, regio-, and enantioselectivity in many cases. Large-scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface-exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H2O2 (0.2 m), whereas the wild-type enzyme’s activity was completely abolished at concentrations as low as 5 mm H2O2. We propose that oxidation-stable mutants might well be a “prerequisite” for thermostabilization, because laboratoryevolved thermostability in CHMO might be masked by a high degree of oxidation instability.
TL;DR: The evaluated multimers showed very high avidities—increasing with the number of cRGD moieties—in in vitro studies on immobilized ανβ3 integrin and U87MG cells, of up to 131‐ and 124‐fold, respectively, relative to the underivatized monomer.
Abstract: Integrin α(ν)β(3) is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin-binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of α(ν)β(3) integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD-multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin α(ν)β(3.) The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA-based chelator developed for the derivatization of sterically demanding structures and successfully labeled with (68)Ga for a potential in vivo application. The evaluated multimers showed very high avidities-increasing with the number of cRGD moieties-in in vitro studies on immobilized α(ν)β(3) integrin and U87MG cells, of up to 131- and 124-fold, respectively, relative to the underivatized monomer.
TL;DR: A new strategy for the preparation of stabilized α-helices, termed hydrogen bond surrogate (HBS) helices, which involves replacement of one of the main chain hydrogen bonds with a covalent linkage to constrain very short peptides into highly stable α-helical conformation.
Abstract: Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors.[1–8] Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity.[9–10] The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. We recently introduced a new strategy for the preparation of stabilized α-helices, termed hydrogen bond surrogate (HBS) helices, which involves replacement of one of the main chain hydrogen bonds with a covalent linkage (Figure 1A).[11] The salient feature of the HBS approach is its ability to constrain very short peptides into highly stable α-helical conformation without blocking any molecular recognition surfaces. We have extensively analyzed the conformation adopted by HBS α-helices with 2D NMR, X-ray, and circular dichroism spectroscopies.[12–14] In addition, HBS helices have been shown to target their expected protein partners with high affinity in cell-free and cell culture assays.[15–17]
TL;DR: A more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on the b-peptoid–peptide hybrid backbone architecture were performed to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity.
Abstract: While natural host-defense antimicrobial peptides (AMPs) and analogues thereof have been investigated intensely in the last two decades with the purpose of combating the still increasing threat from emerging multiresistant pathogenic microbes, 2] compounds with peptidomimetic backbones have received considerable attention due to their superior stability against proteolytic enzymes. 4] Typically, studies of peptidomimetic AMP analogues have involved a brief microbiological evaluation of an array of oligomers, and only occasionally has testing been performed across a broader range of microorganisms or involved systematic structure–activity relationship (SAR) studies. Such investigations have proven fruitful for a-peptidic AMPs, 18] and might reveal unexpected lead structures and selectivity profiles when applied to peptidomimetics as well. Therefore, we have performed a more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on our b-peptoid–peptide hybrid backbone architecture. 20] Antimicrobial activities were determined alongside the archetypal cationic AMP magainin-2 and its clinically tested derivative pexiganan against a series of five important pathogens belonging to different classes. The obtained SAR data were subsequently correlated with various measurements of toxicity towards mammalian cells. Thereby we were able to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity. Three subclasses 1 a–3 d (Scheme 1) were originally designed to address the general effects of length, type of cationic side chains, and presence of a-chirality in the b-peptoid residues. These series had previously been confirmed to possess membrane activity, as indicated by testing for hemolytic and prehemolytic effects, as well as by calcein release experiments with model liposomes, albeit the interaction of these compounds with intracellular targets cannot be ruled out based on our data. The all-aliphatic compound 4 and the mixed aromatic–aliphatic chimera 5 were included to address the importance of lipophilicity and type of cationic residue. Finally, we included three 5/6-carboxyfluorescein-labeled oligomers (6–8) to assess the influence of the presence of this widely used fluorophore on the antimicrobial activity, which might have important implications for confocal fluorescence microscopy studies of the interaction of labeled compounds with live bacteria. The chimeras 4–8 were of dodecamer length to minimize undesired mammalian cell toxicity that might be observed with increasing length. This compound collection was tested against a variety of clinically relevant pathogens and human red blood cells (Table 1). Scheme 1. Chemical structures of the examined hybrid oligomers. The abbreviations used for the b-peptoid units were adapted from the abbreviations commonly used for peptoids (i.e. , N-alkylglycines), 7] by adding the b-prefix. bNspe = N-(S)-1-phenylethyl-balanine, bNphe =b-N-phenylalanine, bNsce = N-(S)-1-cyclohexylethyl-b-alanine, hArg = homoarginine, CF = 5/6-carboxyfluoresceinoyl.
TL;DR: Biochemical characterization now shows that SPHKAP can be considered as the first mammalian AKAP that preferentially binds to PKA‐RIα, and is a very promising model for studying the function of the less explored cytosolic PKA•RI signaling nodes.
Abstract: The compartmentalization of kinases and phosphatases plays an important role in the specificity of second-messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase-anchoring proteins (AKAPs). Most AKAPs bind avidly to PKA-RII, while some have dual specificity for both PKA-RI and PKA-RII; however, no mammalian PKA-RI-specific AKAPs have thus far been assigned. This has mainly been attributed to the observation that PKA-RI is more cytosolic than the more heavily compartmentalized PKA-RII. Chemical proteomics screens of the cAMP interactome in mammalian heart tissue recently identified sphingosine kinase type 1-interacting protein (SKIP, SPHKAP) as a putative novel AKAP. Biochemical characterization now shows that SPHKAP can be considered as the first mammalian AKAP that preferentially binds to PKA-RIalpha. Recombinant human SPHKAP functions as an RI-specific AKAP that utilizes the characteristic AKAP amphipathic helix for interaction. Further chemical proteomic screening utilizing differential binding characteristics of specific cAMP resins confirms SPHKAPs endogenous specificity for PKA-RI directly in mammalian heart and spleen tissue. Immunolocalization studies revealed that recombinant SPHKAP is expressed in the cytoplasm, where PKA-RIalpha also mainly resides. Alignment of SPHKAPs' amphipathic helix with peptide models of PKA-RI- or PKA-RII-specific anchoring domains shows that it has largely only PKA-RIalpha characteristics. Being the first mammalian PKA-RI-specific AKAP with cytosolic localization, SPHKAP is a very promising model for studying the function of the less explored cytosolic PKA-RI signaling nodes.
TL;DR: A new concept for CYP106A2‐catalyzed steroid hydroxylations is developed by which the productivity of progesterone and testosterone conversion could be increased up to 18‐fold to yield an absolute productivity up to 5.5 g L−1 d−1.
Abstract: Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.