Using a PCR-Based Method To Analyze and Model Large,Heterogeneous Populations of DNA

The study of populations of large size and high diversity is limited by the capability of collecting data. Moreover, for a pool of individuals, each associated with a unique characteristic feature, as the pool size grows, the possible interactions increase exponentially and quickly go beyond the limit of computation and experimental studies. Herein, the design of DNA libraries with various diversity is reported. By using a facile analytical method based on real-time PCR, the diversity of a pool of DNA can be evaluated to allow extraordinarily high heterogenicity (e.g., >1 trillion). It is demonstrated that these DNA libraries can be used to model heterogeneous populations; these libraries exhibit functions such as self-protection, suitability for biased expansion, and the possibility to evolve into amorphous structures. The method has shown the remarkable power of parallel computing with DNA, since it can resemble an analogue computer and be applied in selection-based biotechnology methods, such as DNA-encoded chemical libraries. As a chemical approach to solve problems traditionally for genetic and statistical analysis, the method provides a quick and cost-efficient evaluation of library diversity for intermediate steps through a selection process.     

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA-Displayed Peptide Libraries

DNA-encoded small-molecule libraries and mRNA displayed peptide libraries both use numerically large pools of oligonucleotide-tagged molecules to identify potential hits for protein targets. They differ dramatically, however, in the ‘drug-likeness’ of the molecules that each can be used to discover. We give here an overview of the two techniques, comparing some advantages and disadvantages of each, and suggest areas where particularly mRNA display can benefit from adopting advances developed with DNA-encoded small molecule libraries. We outline cases where chemical modification of the peptide library has already been used in mRNA display, and survey opportunities to expand this using examples from DNA-encoded small molecule libraries. We also propose potential opportunities for encoding such reactions within the mRNA/cDNA tag of an mRNA-displayed peptide library to allow a more diversity-oriented approach to library modification. Finally, we outline alternate approaches for enriching target-binding hits from a pooled and tagged library, and close by detailing several examples of how an adjusted mRNA-display based approach could be used to discover new ‘drug-like’ modified small peptides.     

Combinatorial strategies for targeting protein families: application to the proteases

Tens of thousands of proteins have been identified as a result of recent large scale genomic and proteomic efforts. With this large influx of new proteins, the formidable task of elucidating their function begins. However, this task becomes more manageable if proteins are divided into families based upon sequence homology, thereby allowing tools for their systematic study to be developed based upon their common structural and mechanistic characteristics. Combinatorial chemistry is ideally suited for the systematic study of protein families because a large amount of diversity can be readily displayed about a common scaffold designed to target a given protein family. Targeted combinatorial libraries have been particularly effective for the study of a ubiquitous family of proteins, the proteases. Substrate-specificity profiles of many proteases have been determined by using combinatorial libraries of appropriately labeled peptides. This specificity information been utilized to identify the physiological protein substrates of these enzymes and has facilitated inhibitor design efforts. Furthermore, combinatorial libraries of small molecules prepared with mechanism-based scaffolds have resulted in the identification of potent, small-molecule inhibitors of numerous proteases. Cell-permeable small-molecule inhibitors identified by these methods have served as powerful chemical tools to study protease function in vitro and in vivo and have served as leads for the development of therapeutic agents.     

Multiple Chemical Features Impact Biological Performance Diversity of a Highly Active Natural Product-Inspired Library

A systematic, diversity-oriented synthesis approach was employed to access a natural product-inspired flavonoid library with diverse chemical features, including chemical properties, scaffold, stereochemistry, and appendages. Using Cell Painting, the effects of these diversity elements were evaluated, and multiple chemical features that predict biological performance diversity were identified. Scaffold identity appears to be the dominant predictor of performance diversity, but stereochemistry and appendages also contribute to a lesser degree. In addition, the diversity of chemical properties contributed to performance diversity, and the driving chemical property was dependent on the scaffold. These results highlight the importance of key chemical features that may inform the creation of small-molecule, performance-diverse libraries to improve the efficiency and success of high-throughput screening campaigns.     

Drug design strategies for targeting G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) form a large protein family that plays an important role in many physiological and pathophysiological processes. Since the sequencing of the human genome has revealed several hundred new members of this receptor family, many new opportunities for developing novel therapeutics have emerged. The increasing knowledge of GPCRs (biological target space) and their ligands (chemical ligand space) enables novel drug design strategies to accelerate the finding and optimization of GPCR leads: The crystal structure of rhodopsin provides the first three-dimensional GPCR information, which now supports homology modeling studies and structure-based drug design approaches within the GPCR target family. On the other hand, the classical ligand-based design approaches (for example, virtual screening, pharmacophore modeling, quantitative structure-activity relationship (QSAR)) are still powerful methods for lead finding and optimization. In addition, the cross-target analysis of GPCR ligands has revealed more and more common structural motifs and three-dimensional pharmacophores. Such GPCR privileged structural motifs have been successfully used by many pharmaceutical companies to design and synthesize combinatorial libraries, which are subsequently tested against novel GPCR targets for lead finding. In the near future structural biology and chemogenomics might allow the mapping of the ligand binding to the receptor. The linking of chemical and biological spaces will aid in generating lead-finding libraries, which are tailor-made for their respective receptor.     

Recent Advances on the Selection Methods of DNA-Encoded Libraries

DNA-encoded libraries (DEL) have come of age and become a major technology platform for ligand discovery in both academia and the pharmaceutical industry. Technological maturation in the past two decades and the recent explosive developments of DEL-compatible chemistries have greatly improved the chemical diversity of DELs and fueled its applications in drug discovery. A relatively less-covered aspect of DELs is the selection method. Typically, DEL selection is considered as a binding assay and the selection is conducted with purified protein targets immobilized on a matrix, and the binders are separated from the non-binding background via physical washes. However, the recent innovations in DEL selection methods have not only expanded the target scope of DELs, but also revealed the potential of the DEL technology as a powerful tool in exploring fundamental biology. In this Review, we first cover the “classic” DEL selection methods with purified proteins on solid phase, and then we discuss the strategies to realize DEL selections in solution phase. Finally, we focus on the emerging approaches for DELs to interrogate complex biological targets.     

New Enantioselective Synthesis of Monofluorinated Pyridines Designed for the Preparation of Chemical Libraries

Chiral pyridines with a fluorine atom in the benzylic position are easily accessible from optically active propargylic fluorides by using the Bohlmann–Rahtz reaction. Such pyridines, possessing four points of molecular diversity, are useful scaffolds for the preparation of chemical libraries.     

Virtual Combinatorial Chemistry and Pharmacological Screening: A Short Guide to Drug Design

Traditionally, drug development involved the individual synthesis and biological evaluation of hundreds to thousands of compounds with the intention of highlighting their biological activity, selectivity, and bioavailability, as well as their low toxicity. On average, this process of new drug development involved, in addition to high economic costs, a period of several years before hopefully finding a drug with suitable characteristics to drive its commercialization. Therefore, the chemical synthesis of new compounds became the limiting step in the process of searching for or optimizing leads for new drug development. This need for large chemical libraries led to the birth of high-throughput synthesis methods and combinatorial chemistry. Virtual combinatorial chemistry is based on the same principle as real chemistry—many different compounds can be generated from a few building blocks at once. The difference lies in its speed, as millions of compounds can be produced in a few seconds. On the other hand, many virtual screening methods, such as QSAR (Quantitative Sturcture-Activity Relationship), pharmacophore models, and molecular docking, have been developed to study these libraries. These models allow for the selection of molecules to be synthesized and tested with a high probability of success. The virtual combinatorial chemistry–virtual screening tandem has become a fundamental tool in the process of searching for and developing a drug, as it allows the process to be accelerated with extraordinary economic savings.     

An Evolutionary Model Encompassing Substrate Specificity and Reactivity of Type I Polyketide Synthase Thioesterases

Bacterial polyketides are a rich source of chemical diversity and pharmaceutical agents. Understanding the biochemical basis for their biosynthesis and the evolutionary driving force leading to this diversity is essential to take advantage of the enzymes as biocatalysts and to access new chemical diversity for drug discovery. Biochemical characterization of the thioesterase (TE) responsible for 6‐deoxyerythronolide macrocyclization shows that a small, evolutionarily accessible change to the substrate can increase the chemical diversity of products, including macrodiolide formation. We propose an evolutionary model in which TEs are by nature non‐selective for the type of chemistry they catalyze, producing a range of metabolites. As one metabolite becomes essential for improving fitness in a particular environment, the TE evolves to enrich for that corresponding reactivity. This hypothesis is supported by our phylogenetic analysis, showing convergent evolution of macrodiolide‐forming TEs.     

Isocyanide Multicomponent Reactions on Solid Phase: State of the Art and Future Application

Drug discovery efforts largely depend on access to structural diversity. Multicomponent reactions allow for time-efficient chemical transformations and provide advanced intermediates with three or four points of diversification for further expansion to a structural variety of organic molecules. This review is aimed at solid-phase syntheses of small molecules involving isocyanide-based multicomponent reactions. The majority of all reported syntheses employ the Ugi four-component reaction. The review also covers the Passerini and Groebke-Blackburn-Bienaymé reactions. To date, the main advantages of the solid-phase approach are the ability to prepare chemical libraries intended for biological screening and elimination of the isocyanide odor. However, the potential of multicomponent reactions has not been fully exploited. The unexplored avenues of these reactions, including chiral frameworks, DNA-encoded libraries, eco-friendly synthesis, and chiral auxiliary reactions, are briefly outlined.     

Mammalian Chemical Genomics towards Identifying Targets and Elucidating Modes-of-Action of Bioactive Compounds

The step of identifying target molecules and elucidating the mode of action of bioactive compounds is a major bottleneck for drug discovery from phenotypic screening. Genetic screening for genes that affect drug sensitivity or phenotypes of mammalian cultured cells is a powerful tool to obtain clues to their modes of action. Chemical genomic screening systems for comprehensively identifying such genes or genetic pathways have been established using shRNA libraries for RNA interference-mediated mRNA knockdown or sgRNA libraries for CRISPR/Cas9-mediated gene knockout. The combination of chemical genomic screening in mammalian cells with other approaches such as biochemical searches for target molecules, phenotypic profiling, and yeast genetics provides a systematic way to elucidate the mode of action by converging various pieces of information regarding target molecules, target pathways, and synthetic lethal pathways.     

Puckering the Planar Landscape of Fragments: Design and Synthesis of a 3D Cyclobutane Fragment Library

Fragment-based drug discovery (FBDD) has a growing need for unique screening libraries. The cyclobutane moiety was identified as an underrepresented yet attractive three-dimensional (3D) scaffold. Synthetic strategies were developed via a key 3-azido-cyclobutanone intermediate, giving potential access to a range of functional groups with accessible growth vectors. A focused set of 33 novel 3D cyclobutane fragments was synthesised, comprising three functionalities: secondary amines, amides, and sulfonamides. This library was designed using Principal Component Analysis (PCA) and an expanded version of the rule of three (RO3), followed by Principal Moment of Inertia (PMI) analysis to achieve both chemical diversity and high 3D character. Cis and trans ring isomers of library members were generated to maximise the shape diversity obtained, while limiting molecular complexity through avoiding enantiomers. Property analyses of the cyclobutane library indicated that it fares favourably against existing synthetic 3D fragment libraries in terms of shape and physicochemical properties.     

Scanning Protein Surfaces with DNA-Encoded Libraries

Understanding the ligandability of a target protein, defined as the capability of a protein to bind drug-like compounds on any site, can give important stimuli to drug-development projects. For instance, inhibition of protein–protein interactions usually depends on the identification of protein surface binders. DNA-encoded chemical libraries (DELs) allow scanning of protein surfaces with large chemical space. Encoded library selection screens uncovered several protein–protein interaction inhibitors and compounds binding to the surface of G protein-coupled receptors (GPCRs) and kinases. The protein surface-binding chemotypes from DELs are predominantly chemically modified and cyclized peptides, and functional small-molecule peptidomimetics. Peptoid libraries and structural peptidomimetics have been less studied in the DEL field, hinting at hitherto less populated chemical space and suggesting alternative library designs. Roughly a third of bioactive molecules evolved from smaller, target-focused libraries. They showcase the potential of encoded libraries to identify more potent molecules from weak, for example, fragment-like, starting points.     

Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering

Genetic selection from libraries expressing proteins with randomized amino acid segments is a powerful approach to identify proteins with novel biological activities. Here, we assessed the utility of deep DNA sequencing to characterize the composition, diversity, size and stability of such randomized libraries. We used 454 pyrosequencing to sequence a retroviral library expressing small proteins with randomized transmembrane domains. Despite the potential for unintended random mutagenesis during its construction, the overall hydrophobic composition and diversity of the proteins encoded by the sequenced library conformed well to its design. In addition, our sequencing results allowed us to calculate a more accurate estimate of the number of different proteins encoded by the library and suggested that the traditional methods for estimating the size of randomized libraries may overestimate their true size. Our results further demonstrated that no significant genetic bottlenecks exist in the methods used to express complex retrovirus libraries in mammalian cells and recover library sequences from these cells. These findings suggest that deep sequencing can be used to determine the quality and content of other libraries with randomized segments and to follow individual sequences during selection.     

The development of chemical engineering in german industry and universities

Chemical engineering is taught at German universities in three different types of curricula: chemical engineering proper, process engineering (“Verfahrenstechnik”), and industrial chemistry (“Technische Chemie”). Independent departments resp. faculties of chemical engineering exist at four universities. At other universities process engineering is offered as a complete curriculum with a smaller amount of chemistry than chemical engineering curricula, mostly by the departments of mechanical engineering. Industrial chemistry is an essential component of chemistry courses at most technical universities and optional subject at several classical universities. The cause of this diversity of approaches to chemical engineering can be traced back to the beginning of the production of high-value organics (dyes, pharmaceuticals) in Germany in the second half of the 19th century.     

New approaches for studying the chemical diversity of natural resources and the bioactivity of their constituents

Natural products (NPs) have historically been an important source of lead molecules in drug discovery. However, the interest that the pharmaceutical industry has had in NPs has declined in part because of the lack of compatibility of traditional natural-product extract libraries with high-throughput screenings and the low hit rate. Furthermore, in contrast to the synthetic libraries, compounds from natural sources are likely to have complex structures which slow down the identification process and contribute to problems related to supply and manufacturing. In this paper, we summarise some of the strategies that are being developed in our research unit to address these issues. On one hand, differential screening strategies were established with the aim of identifying dynamically induced NPs from silent biosynthetic pathways in plants and fungi that had been exposed to different stress situations. On the other hand, high-resolution HPLC techniques were optimised for biological and chemical profiling of crude extracts. This led to an integrated platform for rapid and efficient identification of new drug-leads and biomarkers of interest that were based on miniaturised technological approaches and metabolomics.     

Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica

Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.     

The Catalytic,Formal Homo-Nazarov Cyclization as a Template for Diversity-Oriented Synthesis

Since the formal characterization of diversity-oriented synthesis (DOS), identification of effective examples of DOS remains an important endeavor for synthetic and pharmaceutical chemists requiring new strategies to generate broader skeletal diversity. Over the last decade, the formal homo-Nazarov cyclization has been an increasingly popular strategy for the concise assembly of functionalized six-membered rings due to the advent of catalytic methods to promote the transformation under milder reaction conditions. Moreover, using the formal homo-Nazarov cyclization as a mechanistic template, reactions can be strategically devised as an effort towards diversity-oriented synthesis. The resulting “homo-Nazarov-inspired” reactions are then demonstrated as a means to access broad structural diversity. This review discusses the development of these catalytic approaches and the successful implementation of the formal homo-Nazarov reaction as a viable template for diversity-oriented synthesis.