Generation Of Libraries Research Articles

Expanding Chemical Frontiers: Approaches for Generating Diverse and Bioactive Natural Product-Like Compounds Libraries from Extracts.

The realms of natural products and synthetic compounds exhibit distinct chemical spaces that not only differ but also complement each other. While the convergence of these two domains has been explored through semisynthesis and conventional pharmacomodulation endeavours applied to natural frameworks, a recent and innovative approach has emerged that involves the combinatorial generation of libraries of 'natural product-like compounds' (NPLCs) through the direct synthetic derivatization of natural extracts. This has led to the production of numerous NPLCs that incorporate structural elements from both their natural (multiple saturated rings, oxygen content, chiral centres) and synthetic (aromatic rings, nitrogen and halogen content, drug-like properties) precursors. Through careful selection of extracts and reagents, specific bioactivities have been achieved, and this strategy has been deployed in various ways, showing great promise without reaching its full potential to date. This review seeks to provide an overview of reported examples involving the chemical engineering of extracts, showcasing a spectrum of natural product alterations spanning from simple substitutions to complete scaffold remodelling. It also includes an analysis of the accomplishments, perspectives and technical challenges within this field.

Chemical and Antiplasmodial Investigations on Eremophila-Derived Alkaloids and Semisynthetic Ether Analogues.

Microthecaline A (1), the known antiplasmodial quinoline serrulatane alkaloid from the roots of Eremophila microtheca F. Muell. ex Benth. (Scrophulariaceae), was targeted for isolation and subsequent use in the generation of a semisynthetic ether library. A large-scale extraction and isolation yielded the previously undescribed quinoline serrulatane microthecaline B (2), along with crystalline 1 that enabled the first X-ray crystallographic analysis to be undertaken on this rare alkaloid structure class. The X-ray diffraction analysis of 1 supported the absolute configuration assignment of microthecaline A, which was originally assigned by ECD data analysis. Microthecaline A (1) was converted into 10 new semisynthetic ether derivatives (3-12) using a diverse series of commercially available alkyl halides. Chemical structures of the new serrulatane alkaloid and semisynthetic ether analogues were assigned by spectroscopic and spectrometric analyses. Antiplasmodial evaluations of 1-12 showed that the semisynthetic derivative 5 elicited the most potent activity with an IC50 value of 7.2 μM against Plasmodium falciparum 3D7 (drug-sensitive) strain.

Mapping niche-specific two-component system requirements in uropathogenic Escherichia coli.

Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify-for the first time-a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate-at a systems level-which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

SENSAAS-Flex: a joint optimization approach for aligning 3D shapes and exploring the molecular conformation space.

Popular shape-based alignment methods handle molecular flexibility by utilizing conformational ensembles to select the most fitted conformer. However, the initial conformer library generation step is computationally intensive and limiting to the overall alignment process. In this work, we describe a method to perform flexible alignment of two molecular shapes by optimizing the 3D conformation. SENSAAS-Flex, an add-on to the SENSAAS tool, is able to proceed from a limited set of initial conformers through an iterative process where additional conformational optimizations are made at the substructure level and constrained by the target shape. In self- and cross-alignment experiments, SENSAAS-Flex is able to reproduce the crystal structure geometry of ligands of the AstraZeneca Molecule Overlay Test set and PDBbind refined dataset. Our study shows that the point-based representation of molecular surfaces is appropriate in terms of shape constraint to sample the conformational space and perform flexible molecular alignments. The documentation and source code are available at https://chemoinfo.ipmc.cnrs.fr/Sensaas-flex/sensaas-flex-main.tar.gz.

NeuroClick: software for mimicking click reaction to generate drug-like molecules permeating the blood-brain barrier.

Background: Traditional methods for chemical library generation in virtual screening often impose limitations on the accessible chemical space or produce synthetically irrelevant structures. Incorporating common chemical reactions into generative algorithms could offer significant benefits. Materials & methods: In this study, we developed NeuroClick, a graphical user interface software designed to perform in silico azide-alkyne cycloaddition, a widely utilized synthetic approach in modern medicinal chemistry. Results & conclusion: NeuroClick facilitates the generation and filtering of large combinatorial libraries at a remarkable rate of 10,000 molecules per minute. Moreover, the generated products can be filtered to identify subsets of pharmaceutically relevant compounds based on Lipinski's rule of five and blood-brain barrier permeability prediction. We demonstrate the utility of NeuroClick by generating and filtering several thousand molecules for dopamine D3 receptor ligand screening.

Computational Methods Enable the Prediction of Improved Catalysts for Nickel-Catalyzed Cross-Electrophile Coupling.

Cross-electrophile coupling has emerged as an attractive and efficient method for the synthesis of C(sp2)-C(sp3) bonds. These reactions are most often catalyzed by nickel complexes of nitrogenous ligands, especially 2,2'-bipyridines. Precise prediction, selection, and design of optimal ligands remains challenging, despite significant increases in reaction scope and mechanistic understanding. Molecular parameterization and statistical modeling provide a path to the development of improved bipyridine ligands that will enhance the selectivity of existing reactions and broaden the scope of electrophiles that can be coupled. Herein, we describe the generation of a computational ligand library, correlation of observed reaction outcomes with features of the ligands, and the in silico design of improved bipyridine ligands for Ni-catalyzed cross-electrophile coupling. The new nitrogen-substituted ligands display a 5-fold increase in selectivity for product formation versus homodimerization when compared to the current state of the art. This increase in selectivity and yield was general for several cross-electrophile couplings, including the challenging coupling of an aryl chloride with an N-alkylpyridinium salt.

Changes in DNA methylation associated with a specific mode of delivery: a pilot study.

The mode of delivery represents an epigenetic factor with potential to affect further development of the individual by multiple mechanisms. DNA methylation may be one of them, representing a major epigenetic mechanism involving direct chemical modification of the individual's DNA. This pilot study aims to examine whether a specific mode of delivery induces changes of DNA methylation by comparing the umbilical cord blood and peripheral blood of the newborns. Blood samples from infants born by vaginal delivery and caesarean section were analysed to prepare the Methylseq library according to NEBNext enzymatic Methyl-seq Methylation Library Preparation Kit with further generation of target-enriched DNA libraries using the Twist Human Methylome Panel. DNA methylation status was determined using Illumina next-generation sequencing (NGS). We identified 168 differentially methylated regions in umbilical cord blood samples and 157 regions in peripheral blood samples. These were associated with 59 common biological, metabolic and signalling pathways for umbilical cord and peripheral blood samples. Caesarean section is likely to represent an important epigenetic factor with the potential to induce changes in the genome that could play an important role in development of a broad spectrum of disorders. Our results could contribute to the elucidation of how epigenetic factors, such as a specific mode of delivery, could have adverse impact on health of an individual later in their life.

Osteoblast Transcriptome Changes During Iron Deficiency

Introduction:Chronic kidney disease (CKD) often results in iron deficiency anemia and bone loss. Previously, iron deficiency was found to impede normal osteoblast differentiation. To investigate regulatory mechanisms elicited by iron deficiency we utilized unbiased RNA-sequencing to evaluate transcriptome-level changes over the course of differentiation.
 Methods:Mouse progenitor clone 2 (MPC-2) cells were differentiated in osteogenic media with or without an iron chelator, deferoxamine (DFO; 5 μM). RNA was extracted at 3, 7, and 14 days after initiating differentiation. Library generation and sequence alignment was performed by the Center for Medical Genomics (CMG). Count-based algorithms implemented in R packages (edgeR and limma-voom; default parameters) identified differentially expressed genes between cells in the control and DFO treatment.
 Results:RNA sequencing illustrated transcriptome changes due to iron deficiency including 63 upregulated/81 downregulated genes at day 3, 371 upregulated/780 downregulated genes at day 7, and 342 upregulated/739 downregulated genes at day 14. Importantly, day 14 controls clustered as transcriptional distinct whereas day 14 DFO treated cells clustered with early time points. As expected, genes involved in bone mineralization were suppressed with DFO including Dmp1 (logFC: -5.92, p= 5.7e-10) and Phex (logFC: -4.17, p= 5.5e-16). Interestingly, genes such as Ces2e, Gdf15, and Ano3, not previously found associated with osteoblasts, were significantly upregulated after 3 days with DFO (logFC: 5.14, 2.7, and 2.5 respectively).
 Conclusions:Transcriptomic changes occur even within 3 days of exposure to iron deficiency. Next steps include performing functional enrichment analysis, and validating expression level changes in vitro as well as within in vivo models of CKD-mediated bone loss.
 Scientific Impact and implications:Iron deficiency is intricately linked to impaired osteoblast function. Understanding the underlying molecular mechanisms could elucidate novel avenues for anabolic bone therapies in CKD.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non-Natural Multiply Backbone N-Methylated Peptide Macrocycles in Yeast.

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.

Acquisition and Analysis of DIA-Based Proteomic Data: A Comprehensive Survey in 2023

Data-independent acquisition (DIA) mass spectrometry (MS) has emerged as a powerful technology for high-throughput, accurate, and reproducible quantitative proteomics. This review provides a comprehensive overview of recent advances in both the experimental and computational methods for DIA proteomics, from data acquisition schemes to analysis strategies and software tools. DIA acquisition schemes are categorized based on the design of precursor isolation windows, highlighting wide-window, overlapping-window, narrow-window, scanning quadrupole-based, and parallel accumulation-serial fragmentation–enhanced DIA methods. For DIA data analysis, major strategies are classified into spectrum reconstruction, sequence-based search, library-based search, de novo sequencing, and sequencing-independent approaches. A wide array of software tools implementing these strategies are reviewed, with details on their overall workflows and scoring approaches at different steps. The generation and optimization of spectral libraries, which are critical resources for DIA analysis, are also discussed. Publicly available benchmark datasets covering global proteomics and phosphoproteomics are summarized to facilitate performance evaluation of various software tools and analysis workflows. Continued advances and synergistic developments of versatile components in DIA workflows are expected to further enhance the power of DIA-based proteomics.

Semisynthesis and Cytotoxic Evaluation of an Ether Analogue Library Based on a Polyhalogenated Diphenyl Ether Scaffold Isolated from a Lamellodysidea Sponge.

The known oxygenated polyhalogenated diphenyl ether, 2-(2',4'-dibromophenoxy)-3,5-dibromophenol (1), with previously reported activity in multiple cytotoxicity assays was isolated from the sponge Lamellodysidea sp. and proved to be an amenable scaffold for semisynthetic library generation. The phenol group of 1 was targeted to generate 12 ether analogues in low-to-excellent yields, and the new library was fully characterized by NMR, UV, and MS analyses. The chemical structures for 2, 8, and 9 were additionally determined via single-crystal X-ray diffraction analysis. All natural and semisynthetic compounds were evaluated for their ability to inhibit the growth of DU145, LNCaP, MCF-7, and MDA-MB-231 cancer cell lines. Compound 3 was shown to have near-equivalent activity compared to scaffold 1 in two in vitro assays, and the activity of the compounds with an additional benzyl ring appeared to be reliant on the presence and position of additional halogens.

PolyUniverse: Generation of a Large-scale Polymer Library Using Rule-Based Polymerization Reactions for Polymer Informatics

Recent advancements in machine learning have revolutionized polymer research, leading to the swift integration of diverse computational techniques for de novo molecular design. A crucial aspect of these processes is...

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

Generation of Whole-Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis.

Whole-genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at a single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high-quality libraries amplified with fewer polymerase chain reaction (PCR) cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer clean-up steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with a few PCR cycles to reach the yield needed for sequencing.

Efficient and Fast High-performance Library Generation for Deep Learning Accelerators

Efficient and Fast High-performance Library Generation for Deep Learning Accelerators

Development of multigroup cross section library generation system TPAMS

Kylin-2 is an advanced neutronics lattice code, developed by Nuclear Power Institute of China. High-precision multigroup cross section library is need for KYLIN-2 to carry out simulation of current pressurized water reactor (PWR) and advanced reactor. In this paper a multigroup cross section library generation system named TPAMS was developed, the methods in TPAMS dealing with resonance data such as subgroup parameters, lambda factor, resonance integral were discussed. Moreover, the depletion chain simplification method was studied. TPAMS can produce multigroup library in binary and ASIIC formats, including detailed data contents for resonance, transport and depletion calculations. A multigroup cross section library has been generated for KYLIN-2 based on TPAMS system. The multigroup cross section library was verified through the analysis of various criticality and burnup benchmarks, the values of multiplication factor and isotope density were compared with the experiment data. Numerical results demonstrate the accuracy of the multigroup cross section library and the reliability of the multigroup cross section library generation system TPAMS.

Designing novel inhibitor derivatives targeting SARS-CoV-2 Mpro enzyme: a deep learning and structure biology approach

Graphical abstract showing the overall workflow for the analysis of the ChEMBL dataset: from de novo library generation to MD Simulation.

CARS.jl: Efficient fitting of dual‐pump multi‐species CARS spectra using compressed libraries

AbstractIn this publication, we demonstrate the extension of our loss‐less library approach previously limited to fitting ro‐vibrational N2 CARS spectra for thermometry purposes to accurate and efficient fitting of dual‐pump CARS spectra with multiple species. The fundamental reasoning behind the compression approach is to make use of finite resolution effects arising from the laser linewidths, which are typically larger than the transitional linewidths. For accurate reconstruction at runtime of the fit, in addition to the squared modulus and real component, also the imaginary component as well as intra‐region species cross terms have to be compressed and tabulated for dual‐pump multi‐species spectra. The resulting libraries do not require to tabulate combinations of mole fractions, leading to a near linear growth of the library with respect to the number of species. The accuracy and computational efficiency of the method is benchmarked using a reference implementation provided alongside this publication. Library generation times, mainly depending on the underlying spectral model, are typically in the order of seconds to a few minutes. The simulation of a single dual‐pump spectrum containing three species is sped up by a factor of >5000 without significantly affecting the accuracy of reconstruction. In a simulated experiment with Stokes‐noise imposed data, the time required to it temperature, mole fraction of three resonant species and wavenumber shift on a single spectrum required 90 ms.