Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics

Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architecture and natural product inspired compound library design. Domains and proteins identified as being structurally similar in their ligand-sensing cores are grouped in a protein structure similarity cluster (PSSC). Natural products can be considered as evolutionary pre-validated ligands for multiple proteins and therefore natural products that are known to interact with one of the PSSC member proteins are selected as guiding structures for compound library synthesis. Application of this novel strategy for compound library design provided enhanced hit rates in small compound libraries for structurally similar proteins.     

Microwave-assisted diastereoselective two-step three-component synthesis for rapid access to drug-like libraries of substituted 3-amino-β-lactams

Large, diverse compound libraries are an essential requisite in target-based drug development. In this work, a robust microwave-assisted synthesis for the diastereoselective generation of 3-saccharinyl-trans-β-lactams is reported. The method is optimised for combinatorial library synthesis in which decoration of the scaffold is varied on both the β-lactam and the saccharine moiety. Within the European Lead Factory (ELF) consortium, a library of 263 compounds was efficiently produced using the developed methodology.     

Synthesis of antitrypanosomal 1,2-dioxane derivatives based on a natural product scaffold

A short practical synthesis of a new natural product based scaffold (6), based on antitrypanosomal and antimalarial compounds isolated from different Plakortis species is described. The scaffold contains a peroxide unit that is surprisingly stable to chemical manipulation elsewhere in the molecule, enabling it to be elaborated into a small library of derivatives. It is stable to ozonolysis, reductive work-up with dimethylsulfide and the Wittig reaction with stabilized phosphorus ylides. The scaffold along with its Wittig analogues has displayed low to sub-micro molar (0.2-3.3 μM) antitrypanosomal activity.     

A novel complexity-to-diversity strategy for the diversity-oriented synthesis of structurally diverse and complex macrocycles from quinine

Recent years have witnessed a global decline in the productivity and advancement of the pharmaceutical industry. A major contributing factor to this is the downturn in drug discovery successes. This can be attributed to the lack of structural (particularly scaffold) diversity and structural complexity exhibited by current small molecule screening collections.Macrocycles have been shown to exhibit a diverse range of biological properties, with over 100 natural product-derived examples currently marketed as FDA-approved drugs. Despite this, synthetic macrocycles are widely considered to be a poorly explored structural class within drug discovery, which can be attributed to their synthetic intractability.Herein we describe a novel complexity-to-diversity strategy for the diversity-oriented synthesis of novel, structurally complex and diverse macrocyclic scaffolds from natural product starting materials. This approach exploits the inherent structural (including functional) and stereochemical complexity of natural products in order to rapidly generate diversity and complexity. Readily-accessible natural product-derived intermediates serve as structural templates which can be divergently functionalized with different building blocks to generate a diverse range of acyclic precursors. Subsequent macrocyclisation then furnishes compounds that are each based around a distinct molecular scaffold. Thus, high levels of library scaffold diversity can be rapidly achieved. In this proof-of-concept study, the natural product quinine was used as the foundation for library synthesis, and six novel structurally diverse, highly complex and functionalized macrocycles were generated.     

Reaction modelling and simulation to assess the integrated use of transketolase and ω-transaminase for the synthesis of an aminotriol

The most attractive, as well as challenging, multistep organic syntheses would preferably be carried out in a single reactor, as a one-pot synthesis. For biocatalytic syntheses, multistep reactions in one-pot mode bring a number of advantages, while at the same time raising unique challenges such as the compatibility of different biocatalysts. In this paper, we have developed a transketolase–transaminase (TK-TAm) two-step one-pot aminotriol synthesis reaction model, which integrates reaction kinetic models with process characterization (consisting of component degradation as a function of pH and concentration, aldehyde toxicity towards the enzyme, and ketol donor and acceptor side-reactions with TAm). Based on the analysis of the effect of the TAm/TK activity ratio on product yield, simulations provided guidance for further process and biocatalyst development.     

An alternative method for the synthesis of tailor-made genes

Oligonucleotide synthesis was coupled with amplification by the polymerase chain reaction to generate an exact translational fusion between a plant signal sequence and an animal structural gene. A synthetic 111-mer oligonucleotide representing less than two percent of the reaction products was successfully amplified by using short primers containing restriction sites designed for ease of cloning and providing in-frame fusion. The method overcomes the length-versus-yield dilemma in oligonucleotide synthesis, and is generally adaptable to the construction of a translationally competent coding sequence from any two DNA fragments.     

Parallel synthesis of peptide libraries using microwave irradiation

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-beta-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer alpha- and beta-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).     

Reaction modelling and simulation to assess the integrated use of transketolase and ω-transaminase for the synthesis of an aminotriol

The most attractive, as well as challenging, multistep organic syntheses would preferably be carried out in a single reactor, as a one-pot synthesis. For biocatalytic syntheses, multistep reactions in one-pot mode bring a number of advantages, while at the same time raising unique challenges such as the compatibility of different biocatalysts. In this paper, we have developed a transketolase-transaminase (TK-TAm) two-step one-pot aminotriol synthesis reaction model, which integrates reaction kinetic models with process characterization (consisting of component degradation as a function of pH and concentration, aldehyde toxicity towards the enzyme, and ketol donor and acceptor side-reactions with TAm). Based on the analysis of the effect of the TAm/TK activity ratio on product yield, simulations provided guidance for further process and biocatalyst development.     

Recent Advanced Materials for Electrochemical and Photoelectrochemical Synthesis of Ammonia from Dinitrogen: One Step Closer to a Sustainable Energy Future

Ammonia (NH₃), an important raw material for chemical industry and agriculture, is also considered to be an intriguing energy storage and transportation media for chemical conversion schemes. The world's primary NH₃ supply is based on the natural nitrogen fixation by diazotrophs through an enzymatic nitrogenase process and the industrial nitrogen fixation through a traditional Haber–Bosch process. The natural synthesis of NH₃ can hardly meet the rapidly growing global demand. Meanwhile, the industrial NH₃ production is still dominated by the high‐temperature and high‐pressure reaction between nitrogen and hydrogen (N₂ + 3H₂ → 2NH₃), requiring intensive energy input and generating massive CO₂. Therefore, seeking a breakthrough in the development of catalysts toward efficient ammonia synthesis has become the frontier of energy and chemical conversion schemes. This review summarizes and discusses the recent progress on developing new strategies to optimize the efficiency of NH₃ production coupled with renewable energy sources, with a specific focus on electrocatalytic and photoelectrocatalytic conversion of N₂ to NH₃. The most recent advances in the development of catalytic materials, the design of the reaction systems, and the computational insights for electrochemical and photoelectrochemical ammonia synthesis are covered.     

Design and evaluation of 3-aminopyrazolopyridinone kinase inhibitors inspired by the natural product indirubin

A lead-like kinase inhibitor screening library containing new 3-aminopyrazolopyridinones and closely related compounds was designed that contained hydrogen-bond donor-acceptor motifs and substitution vectors inspired by the natural product kinase inhibitor indirubin. The solubility of the 3-aminopyrazolopyridinone scaffold was more than 1000-fold greater than that of indirubin itself, and solubility was enhanced by reduction of the proportion of lipophilic aryl substituents or the introduction of basic groups. Several components of the library showed kinase inhibitory activity. A subset of diaryl-substituted analogues preferentially inhibited tyrosine kinases with low micromolar activity and good ligand efficiency, and showed cellular antiproliferative activity. The evaluation of the library shows that new, non-natural compounds with relevant biological activity and improved physicochemical properties can be generated from the natural product indirubin, providing compounds that may be useful for kinase inhibitor drug discovery.     

Novel protein science enabled by total chemical synthesis

Chemical synthesis is a well‐established method for the preparation in the research laboratory of multiple‐tens‐of‐milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d ‐protein and l ‐protein enantiomers facilitate crystallization and determination of protein structures by X‐ray diffraction. d ‐Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d ‐protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear‐loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time‐resolved FTIR for the elucidation of protein structure–activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein‐inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d ‐protein forms of polymerase enzymes.     

γ-Pyrone natural products—A privileged compound class provided by nature

In “Biology Oriented Synthesis” (BIOS), the inherent biological relevance of natural products is employed for the design and synthesis of compound libraries. Towards this end, library generation in BIOS is focused on compound classes from biologically relevant space such as the natural product space or also the drug space and only scaffolds of these areas of proven relevance are employed for synthesis of small focused libraries with limited diversity. We here present a short overview of γ-pyrone natural products, highlighting their biological properties and their potential applicability in a BIOS of a compound library.     

Identification of Francisella tularensis in natural water samples by PCR

Abstract Francisella tularensis , the causative agent of the epizootic disease tularemia in mammals, can be isolated from mud and water. To study the spread and persistence of Francisella tularensis in water, different strategies for pre-treatment of natural water samples prior to identification of the bacterium by polymerase chain reaction (PCR) were evaluated. A method for handling of samples taken from natural waters was developed. Applied on natural water samples amended with F. tularensis , the method rendered identification by PCR reproducible and it resulted in an amplified Francisella -specific product in all samples from natural waters tested. In addition, by employing primers targeting conserved regions of the 16S rDNA the presence of bacteria was demonstrated in all samples investigated. The results presented will, in combination with other techniques that allow identification, improve studies on the epizootiology and epidemiology of the genus Francisella .     

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)₃-GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)₃-GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki–Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)₃-GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.     

Solid-phase synthesis and anti-infective activity of a combinatorial library based on the natural product anisomycin

The solid-phase synthesis of a library based on the natural product anisomycin is described. The resulting library was tested against a panel of bacterial and fungal targets, and active compounds were identified in a Staphylococcus aureus whole-cell assay and an efflux-deficient fungal whole-cell assay.     

Gel shift selection of translation enhancer sequences using messenger RNA display

We designed a new approach for selection of translation enhancer sequences that enables efficient protein synthesis in cell-free systems. The selection is based on a gel shift assay of a messenger RNA (mRNA)–protein fusion product that is synthesized in a cell-free translation system using an mRNA display method. A library of randomized 20-nt-long sequences, with all possible combinations of the four nucleotides, upstream of a coding region was screened by successive rounds of screening in which the translation time of the succeeding round was reduced compared with the previous round. An efficient translation enhancer sequence capable of more rapid initiation of cell-free protein synthesis, with a minimal translation time of 5 min, than a natural longer enhancer sequence (Xenopus β-globin 5′UTR) was selected using rabbit reticulocyte extract as a model cell-free translation system. Furthermore, a successful screening of cap-independent translation enhancer sequence and a significant sequence similarity of the selected candidates validated the efficiency of the combined mRNA display and gel shift assay method for the rapid development of advanced cell-free translation systems.     

Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design of focused libraries that might provide protein ligands with enhanced quality and probability. The combined application of natural product derived scaffolds with a new approach that clusters proteins according to structural similarity of their ligand sensing cores provides a new principle for the design and synthesis of such libraries. This article discusses recent advances in the synthesis of natural product inspired compound collections and the application of protein structure similarity clustering for the development of such libraries.     

Novel approach for optimization of a ‘difficult’ peptide synthesis by utilizing quantitative reaction monitoring assays

Three different synthetic strategies were tested to synthesize EGFRvIII peptide (1), which is a functional variant of the epidermal growth factor receptor, a protein that has been well validated as a target for cancer therapy. The initial synthesis was performed with Applied Biosystem (Carlsbad, CA, USA) 433A peptide synthesizer and it indicated that the last three amino acids coupling and Fmoc removal rates were lower than the rest of the sequence. Purity of the crude peptide was 54.5%. The second synthesis was performed manually utilizing C S Bio (Menlo Park, CA, USA) synthesizer and the Kaiser test for reaction monitoring. Because of non‐optimized reaction conditions and an unexpected by‐product, lower purity crude peptide (40.5%) was obtained. Quantitative assays to monitor reactions were developed and demonstrated in gram scale synthesis with C S Bio synthesizer. The optimized synthetic conditions improved the peptide purity to 68.1%. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.