Life Sciences Discovery and Technology Highlights

High-Throughput Chemistry and Drug Discovery

Fragment-Based Screening with Natural Products for Novel Antiparasitic Disease Drug Discovery

Introduction: Fragment-based drug discovery (FBDD) can identify relatively simple compounds with low binding affinity due to fewer binding interactions with protein targets. FBDD reduces the library size and provides simpler starting points for subsequent chemical optimization of initial hits. A much greater proportion of chemical space can be sampled in fragment-based screening compared to larger molecules with typical molecular weights (MWs) of 250–500 g mol⁻¹ used in high-throughput screening (HTS) libraries.

Areas covered: The authors cover the role of natural products in FBDD against parasitic disease targets. They review the approaches to develop fragment-based libraries using either natural products or natural product–like compounds. The authors present approaches to FBDD against parasitic diseases and compare these libraries with the three-dimensional (3D) attributes of natural products.

Expert opinion: To effectively use the 3D properties and the chemical diversity of natural products in FBDD against parasitic diseases, there needs to be a mind shift. Library design, in the medicinal chemistry area, has acknowledged that escaping flat-land is very important to increase the chances of clinical success. Attempts to increase sp³ richness in fragment libraries are acknowledged. Sufficient low-molecular-weight natural products are known to create true natural product fragment libraries (Liu, M., and Quinn, R. J., Expert Opin Drug Discov. 2019, doi:10.1080/17460441.2019.1653849).

Tailored Therapeutics Based on 1,2,3-1H-Triazoles: A Mini Review

Contemporary drug discovery approaches rely on library synthesis coupled with combinatorial methods and high-throughput screening to identify leads. Due to the multitude of components involved, however, a majority of optimization techniques face persistent challenges related to the efficiency of synthetic processes and the purity of compound libraries. These methods have recently found an upgradation as fragment-based approaches for target-guided synthesis of lead molecules with active involvement of their biological target. The click chemistry approach serves as a promising tool for tailoring the therapeutically relevant biomolecules of interest, improving their bioavailability and bioactivity and redirecting them as efficacious drugs. The 1,2,3-1H-triazole nucleus, being a planar and biologically acceptable scaffold, plays a crucial role in the design of biomolecular mimetics and tailor-made molecules with therapeutic relevance. This versatile scaffold also forms an integral part of the current fragment-based approaches for drug design, kinetic target-guided synthesis, and bioorthogonal methodologies (Prasher, P., and Sharma, M., Medchemcomm. 2019, 10(8), 1302–1328).

Automated “Cells-to-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes

Mass spectrometry (MS)-based quantitative proteomic analysis has proven valuable for clinical and biotechnology-related research and development. Improvements in sensitivity, resolution, and robustness of mass analyzers have also added value. Manual sample preparation protocols are often, however, a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications in which thousands of samples per month must be analyzed. To alleviate these issues, Chen et al. developed a “cells-to-peptides” automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes 2 h to process 96 samples from cell pellets to the initiation of the tryptic digestion step, and it can process 384 samples in parallel. The authors measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography–mass spectrometry (LC-MS) systems. The automated workflow was tested by preparing 96 Escherichia coli samples and quantifying more than 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly multiplexed LC–multiple reaction monitoring (MRM)–MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications (Chen Y. et al., J. Proteome Res. 2019, doi:10.1021/acs.jproteome.9b00455).

In-Air Production of 3D Co-Culture Tumor Spheroid Hydrogels for Expedited Drug Screening

Three-dimensional (3D) in vitro tumor spheroids are becoming popular as preclinical platforms for testing the performance of existing drugs or for discovery of innovative anticancer therapeutics. This focus is correlated with in vitro 3D tumor models’ ability to mimic the multicellular compact structure and spatial architecture of human solid tumors. These microphysiological systems, however, generally lack the preexistence of tumor–extracellular matrix (ECM), a critical aspect that can affect the overall therapeutic performance and the decision to advance candidate drugs to later stages of the pipeline. Aiming to face this drawback and mimic tumor–ECM, herein the authors rapidly fabricated in-air hyaluronan–methacrylate (HA-MA) and gelatin–methacrylate (GelMA) photocrosslinkable 3D spheroid microgels by using superhydrophobic surfaces. These platforms were used for establishing heterotypic 3D co-culture models of prostate cancer cells (PC3 cells) and human osteoblasts (hOBs) to mimic prostate cancer–to-bone metastasis cellular heterogeneity and the tumor–ECM microenvironment. The 3D microgel microtumors’ morphology, size, and cell number were easily controlled via digital droplet generation on polystyrene superhydrophobic surfaces and under solvent-free conditions when compared to microfluidics or electrospray. Co-culture 3D microgels formed by 2.5% HA-MA–5% GelMA and 5% HA-MA–5% GelMA ratios showed the highest calcium deposition after 14 days of culture, evidencing osteoblasts’ viability and the establishment of functional mineralization in the 3D hydrogel matrix. Cisplatin cytotoxicity evaluation showed that 3D microgels are more resistant to platin chemotherapeutics than their single or co-cultured 3D multicellular spheroid counterparts. Overall, our findings indicate that solvent-free, in-air-produced 3D microgel microenvironments are cost-effective and robust tumor-mimicking platforms for in vitro high-throughput screening of therapeutics targeted to prostate-to-bone metastasis microenvironments.

Statement of significance: The generation of robust microphysiological systems that recapitulate the complexity of the metastatic prostate cancer–to-bone tumor microenvironment is crucial for preclinical evaluation of new therapeutics that can eradicate these secondary tumors. In this study, Antunes et al. used superhydrophobic (SH) surfaces to rapidly fabricate photocrosslinkable HA-MA and GelMA 3D spheroid microgels for PC3 cell and hOB co-culture models that simultaneously mimic the cellular and ECM tumor components. The use of SH platforms overcomes the issues of standard in-liquid microgel production technologies by providing a robust control over 3D microgels’ size, morphology, and cell–cell co-encapsulation numbers, while avoiding the use of oil-based microgel droplet generation. Overall, SH surfaces allowed a solvent-free, cost-effective, reproducible, and adaptable fabrication of heterotypic 3D spherical microgels for high-throughput drug screening (Antunes, J. et al., Acta Biomater. 2019, 94, 392–409).

A High-Throughput Apoptosis Assay Using 3D Cultured Cells

A high-throughput apoptosis assay using three-dimensional (3D) cultured cells was developed with a micropillar–microwell chip platform. Live cell apoptosis assays based on fluorescence detection have been useful in high-content screening. To check the autofluorescence of drugs, controls (no caspase-3/7 reagent in the assay) for the drugs are necessary, which require twice the test space. Thus, a high-throughput capability and highly miniaturized format for reducing reagent usage are necessary in live cell apoptosis assays. Especially, the expensive caspase-3/7 reagent should be reduced in a high-throughput screening system. To solve this issue, Lee et al. developed a miniaturized apoptosis assay using micropillar–microwell chips for which the authors tested 70 drugs (six replicates) per chip and reduced the assay volume to 1 µL. This reduced assay volume can decrease the assay costs compared to the 10–40 µL assay volumes used in 384-well plates. In our experiments, among the 70 drugs, four drugs (cediranib, cabozatinib, panobinostat, and carfilzomib) induced cell death by apoptosis. Those results were confirmed with Western blot assays and proved that the chip platform could be used to identify high-potency apoptosis-inducing drugs in 3D cultured cells with alginate (Lee, S. Y. et al., Molecules. 2019, 24, doi:10.3390/molecules24183362).

Open-Source Software Tools, Databases, and Resources for Single-Cell and Single-Cell-Type Metabolomics

In this age of -omics data–guided big data revolution, metabolomics has received significant attention as compared to genomics, transcriptomics, and proteomics for its proximity to the phenotype, the promises it makes, and the challenges it throws. Although metabolomes of entire organisms, organs, biofluids, and tissues are of immense interest, a cell-specific resolution is deemed critical for biomedical applications in which a granular understanding of cellular metabolism at cell-type and subcellular resolution is desirable. Mass spectrometry (MS) is a versatile technique that is used to analyze a broad range of compounds from different species and cell types, with high accuracy, resolution, sensitivity, and selectivity and fast data acquisition speeds. With recent advances in MS and spectroscopy-based platforms, the research community is able to generate high-throughput data sets from single cells. It is challenging, however, to handle, store, process, analyze, and interpret data in a routine manner. In this treatise, the author presents a workflow of metabolomics data generation from single cells and single-cell types to their analysis, visualization, and interpretation for obtaining biological insights (Misra, B. B., Methods Mol Biol. 2020, 2064, 191–217).

Microfluidics in Biology

Large-Scale Antitumor Screening Based on Heterotypic 3D Tumors Using an Integrated Microfluidic Platform

Chemotherapy screening plays a crucial role in cancer drug discovery and clinical medicine. While conventional methods have contributed greatly to macromanipulation of cell populations, a profounder insight related to the tumor microenvironment requires approaches for completing integrated cell–three-dimensional (3D) tumor micromanipulation, massive tumor simulation and production, and dynamic and high-throughput tumor analysis. In this study, Liu et al. introduced an integrated microfluidic platform with multiparallel components for heterotypic 3D tumor reconstruction and antitumor screening. Sequential microfluidic manipulations, including sample loading, precise localization, 3D tumor formation, chemical stimulation, on-chip analysis, and tumor recovery for off-chip assessment, were permitted and experimentally confirmed in the device based on facile and efficient pneumatic control. Heterotypic 3D tumors with tissue-biomimetic phenotypes can be produced in massive and size-uniform manners. Notably, the authors accomplished screening-like chemotherapy assessment involving different heterotypic 3D tumors and antitumor drugs, and demonstrated the versatility of the platform in large-scale tumor manipulation and analysis. This advancement in microfluidics has potential applications in the fields of oncology, pharmacology, and tissue engineering, and provides insight into the construction of high-performance microsystems for drug development and cancer research (Liu, W. et al., Anal Chem. 2019, doi:10.1021/acs.analchem.9b02768).

Cell-Based Assays on Microfluidics for Drug Screening

Microfluidics is an appealing platform for drug screening and discovery. Compared with the conventional drug-screening methods based on Petri dishes and experimental animals, microfluidic devices have many advantages, including miniaturized size, ease of use, high sensitivity, and high throughput. More importantly, bioassays on microfluidics can avoid ethical issues, which can be a big obstacle hindering the performance of experiments on animals or human beings. Furthermore, three-dimensional (3D) microchips can recapitulate various biochemical and biophysical conditions in vivo and mimic the natural microenvironment of the tissues and organs, providing versatile in vitro models for biomedical applications. In this Perspective article, Liu et al. focuses on cell-based microfluidic assays for drug screening. Meanwhile, the authors also propose potential solutions for the difficulties in this field and discuss the prospects of microfluidics-based technologies for drug screening (Liu, X. et al., ACS Sens. 2019, 4(6), 1465–1475).

Microfluidics for Personalized Drug Screening of Cancer

Resistance to targeted therapies is a major clinical challenge in cancer treatment. Despite technological advances, robust biomarkers or platforms predictive of treatment response are lacking owing to the inherent nature of the complex genomic landscape of carcinoma. Nevertheless, recent efforts centered on performing direct drug screening on patient-derived cells through their ex vivo expansion and maintenance have enabled personalized stratification of treatment modalities. Microfluidics is one such technology that allows high-throughput drug screening through parallelization and automation using small-volume samples. In this review, Menon et al. present recent microfluidic platforms that have been successfully applied for the maintenance and expansion of patient-derived tumor cells spanning diverse cancer types and sources [solid tumors or liquid biopsies (circulating tumor cells)] for personalized drug-screening applications (Menon, V. N. et al., Curr. Opin. Pharmacol. 2019, 48, 155–161).

Advances in Microbial Genomics and Biology

Comparison of Long-Read Sequencing Technologies in the Hybrid Assembly of Complex Bacterial Genomes

Illumina sequencing allows rapid, cheap, and accurate whole-genome bacterial analyses, but short reads [<300 base pairs (bp)] do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). It is not clear, however, how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process that avoids multiple bespoke filtering and data manipulation steps is also crucial to facilitating high-throughput complete bacterial genome reconstruction. In this study, De Maio et al. compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. The authors chose isolates from the family Enterobacteriaceae because these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. The authors assembled genomes de novo using the hybrid assembler Unicycler and compared different read-processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower cost of consumables per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly (De Maio, N. et al., Microb Genom. 2019, doi:10.1099/mgen.0.000294).

Microbiome as an Immunological Modifier

Humans are living ecosystems composed of human cells and microbes. The microbiome is the collection of microbes (microbiota) and their genes. Recent breakthroughs in high-throughput sequencing technologies have made it possible for us to understand the composition of the human microbiome. Launched by the US National Institutes of Health, the Human Microbiome Project indicated that our bodies harbor a wide array of microbes, specific to each body site with interpersonal and intrapersonal variabilities. Numerous studies have indicated that several factors influence the development of the microbiome, including genetics, diet, use of antibiotics, and lifestyle. The microbiome and its mediators are in a continuous crosstalk with the host immune system; hence, any imbalance on one side is reflected on the other. Dysbiosis (microbiota imbalance) was shown in many diseases and pathological conditions, such as inflammatory bowel disease, celiac disease, multiple sclerosis, rheumatoid arthritis, asthma, diabetes, and cancer. The microbial composition mirrors inflammation variations in certain disease conditions, within various stages of the same disease; hence, it has the potential to be used as a biomarker (Kumar, M. et al., Methods Mol. Biol. 2020, 2055, 595–638).

Host–Microbe–Drug–Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. Pryor et al. set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium Escherichia coli and the nematode Caenorhabditis elegans, the authors developed a high-throughput four-way screen to define the underlying host–microbe–drug–nutrient interactions. The authors show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug–nutrient–microbiome interactions to improve host health and longevity through targeted microbiome therapies (Pryor, R. et al., Cell. 2019, 178(6), 1299–1312).

Microbe–Host Interplay in Atopic Dermatitis and Psoriasis

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here, the authors perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analyzed by 16S amplicon or whole-genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. The authors find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers’ microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and it is associated with a disease-relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism, and immune activation. In contrast, psoriasis is characterized by cooccurring communities of microbes with weak associations with disease-related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis (Fyhrquist, N. et al., Nat. Commun. 2019, 10(1), 4703).

Microbial Functional Diversity: From Concepts to Applications

Functional diversity is increasingly recognized by microbial ecologists as the essential link between biodiversity patterns and ecosystem functioning, determining the trophic relationships and interactions between microorganisms, their participation in biogeochemical cycles, and their responses to environmental changes. Consequently, its definition and quantification have practical and theoretical implications. In this opinion article, we present a synthesis on the concept of microbial functional diversity from its definition to its application. Initially, Escalas et al. revisit the original definition of functional diversity, highlighting two fundamental aspects, the ecological unit under study and the functional traits used to characterize it. Then, the authors discuss how the particularities of the microbial world disallow the direct application of the concepts and tools developed for macroorganisms. Next, the authors provide a synthesis of the literature on the types of ecological units and functional traits available in microbial functional ecology. The authors also provide a list of more than 400 traits covering a wide array of environmentally relevant functions. Lastly, the authors provide examples of the use of functional diversity in microbial systems based on the different units and traits discussed herein. The authors hope that this article will stimulate discussions and help the growing field of microbial functional ecology to realize a potential that thus far has been attained only in macrobial ecology (Escalas, A. et al., Ecol. Evol. 2019, 9(20), 12000–12016).