Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

Genomic Approaches to Antifungal Drug Target Identification and Validation.

The last several decades have witnessed a surge in drug-resistant fungal infections that pose a serious threat to human health. While there is a limited arsenal of drugs that can be used to treat systemic infections, scientific advances have provided renewed optimism for the discovery of novel antifungals. The development of chemical-genomic assays using Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of molecules in a living cell. Advances in molecular biology techniques have enabled complementary assays to be developed in fungal pathogens, including Candida albicans and Cryptococcus neoformans. These approaches enable the identification of target genes for drug candidates, as well as genes involved in buffering drug target pathways. Here, we examine yeast chemical-genomic assays and highlight how such resources can be utilized to predict the mechanisms of action of compounds, to study virulence attributes of diverse fungal pathogens, and to bolster the antifungal pipeline.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

Seasonal changes in NRF2 antioxidant pathway regulates winter depression-like behavior.

Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

Comparative chemical genomic profiling across plant-based hydrolysate toxins reveals widespread antagonism in fitness contributions.

The budding yeast Saccharomyces cerevisiae has been used extensively in fermentative industrial processes, including biofuel production from sustainable plant-based hydrolysates. Myriad toxins and stressors found in hydrolysates inhibit microbial metabolism and product formation. Overcoming these stresses requires mitigation strategies that include strain engineering. To identify shared and divergent mechanisms of toxicity and to implicate gene targets for genetic engineering, we used a chemical genomic approach to study fitness effects across a library of S. cerevisiae deletion mutants cultured anaerobically in dozens of individual compounds found in different types of hydrolysates. Relationships in chemical genomic profiles identified classes of toxins that provoked similar cellular responses, spanning inhibitor relationships that were not expected from chemical classification. Our results also revealed widespread antagonistic effects across inhibitors, such that the same gene deletions were beneficial for surviving some toxins but detrimental for others. This work presents a rich dataset relating gene function to chemical compounds, which both expands our understanding of plant-based hydrolysates and provides a useful resource to identify engineering targets.

The impact of the genetic background on gene deletion phenotypes in Saccharomyces cerevisiae.

Loss-of-function (LoF) mutations associated with disease do not manifest equally in different individuals. The impact of the genetic background on the consequences of LoF mutations remains poorly characterized. Here, we systematically assessed the changes in gene deletion phenotypes for 3,786 gene knockouts in four Saccharomyces cerevisiae strains and 38 conditions. We observed 18.5% of deletion phenotypes changing between pairs of strains on average with a small fraction conserved in all four strains. Conditions causing higher wild-type growth differences and the deletion of pleiotropic genes showed above-average changes in phenotypes. In addition, we performed a genome-wide association study (GWAS) for growth under the same conditions for a panel of 925 yeast isolates. Gene-condition associations derived from GWAS were not enriched for genes with deletion phenotypes under the same conditions. However, cases where the results were congruent indicate the most likely mechanism underlying the GWAS signal. Overall, these results show a high degree of genetic background dependencies for LoF phenotypes.

Strategies for Design of Molecular Structures with a Desired Pharmacophore Using Deep Reinforcement Learning.

The goal of drug design is to discover molecular structures that have suitable pharmacological properties in vast chemical space. In recent years, the use of deep generative models (DGMs) is getting a lot of attention as an effective method of generating new molecules with desired properties. However, most of the properties do not have three-dimensional (3D) information, such as shape and pharmacophore. In drug discovery, pharmacophores are valuable clues in finding active compounds. In this study, we propose a computational strategy based on deep reinforcement learning for generating molecular structures with a desired pharmacophore. In addition, to extract selective molecules against a target protein, chemical genomics-based virtual screening (CGBVS) is used as post-processing method of deep reinforcement learning. As an example study, we have employed this strategy to generate molecular structures of selective TIE2 inhibitors. This strategy can be adopted into general use for generating selective molecules with a desired pharmacophore.

Inducible Cell Fusion Permits Use of Competitive Fitness Profiling in the Human Pathogenic Fungus Aspergillus fumigatus.

Antifungal agents directed against novel therapeutic targets are required for treating invasive, chronic, and allergic Aspergillus infections. Competitive fitness profiling technologies have been used in a number of bacterial and yeast systems to identify druggable targets; however, the development of similar systems in filamentous fungi is complicated by the fact that they undergo cell fusion and heterokaryosis. Here, we demonstrate that cell fusion in Aspergillus fumigatus under standard culture conditions is not predominately constitutive, as with most ascomycetes, but can be induced by a range of extracellular stressors. Using this knowledge, we have developed a barcode-free genetic profiling system that permits high-throughput parallel determination of strain fitness in a collection of diploid A. fumigatus mutants. We show that heterozygous cyp51A and arf2 null mutants have reduced fitness in the presence of itraconazole and brefeldin A, respectively, and a heterozygous atp17 null mutant is resistant to brefeldin A.

Integrating yeast chemical genomics and mammalian cell pathway analysis.

Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/ß-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.

Synthetic genome readers target clustered binding sites across diverse chromatin states.

Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to "read" specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

BACKGROUND: Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. RESULTS: Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. CONCLUSIONS: We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana.

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.

Plant-derived antifungal agent poacic acid targets ß-1,3-glucan.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited ß-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding ß-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.

BACKGROUND: The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features. RESULTS: Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics. CONCLUSIONS: Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

Merging roads: chemical tools and cell biology to study unconventional protein secretion.

The endomembrane trafficking network is highly complex and dynamic, with both conventional and so-called unconventional routes which are in essence recently discovered pathways that are poorly understood in plants. One approach to dissecting endomembrane pathways that we have pioneered is the use of chemical biology. Classical genetic manipulations often deal with indirect pleiotropic phenotypes resulting from the perturbation of key players of the trafficking routes. Many of these difficulties can be circumvented using small molecules to modify or disrupt the function or localization of key proteins regulating these pathways. In this review, we summarize how small molecules have been used as probes to define these pathways, and how they could be used to increase current knowledge of unconventional protein secretion pathways.

Genomic tools to profile antibiotic mode of action.

The increasing emergence of antimicrobial multiresistant bacteria is of great concern to public health. While these bacteria are becoming an ever more prominent cause of nosocomial and community-acquired infections worldwide, the antibiotic discovery pipeline has been stalled in the last few years with very few efforts in the research and development of novel antibacterial therapies. Some of the root causes that have hampered current antibiotic drug development are the lack of understanding of the mode of action (MOA) of novel antibiotic molecules and the poor characterization of the bacterial physiological response to antibiotics that ultimately causes resistance. Here, we review how bacterial genetic tools can be applied at the genomic level with the goal of profiling resistance to antibiotics and elucidating antibiotic MOAs. Specifically, we highlight how chemical genomic detection of the MOA of novel antibiotic molecules and antibiotic profiling by next-generation sequencing are leveraging basic antibiotic research to unprecedented levels with great opportunities for knowledge translation.

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

Development of quantitative high-throughput screening assays to identify, validate, and optimize small-molecule stabilizers of misfolded ß-glucocerebrosidase with therapeutic potential for Gaucher disease and Parkinson's disease.

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small pro-luminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and non-inhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: the fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 provided direct visualization of GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy, by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically-relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of new chemical matter targeting GCase, ultimately leading to a viable therapeutic for two protein-misfolding diseases.

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.