Identification of novel small-molecule inhibitors of SARS-CoV-2 by chemical genetics.

There are only eight approved small molecule antiviral drugs for treating COVID-19. Among them, four are nucleotide analogues (remdesivir, JT001, molnupiravir, and azvudine), while the other four are protease inhibitors (nirmatrelvir, ensitrelvir, leritrelvir, and simnotrelvir-ritonavir). Antiviral resistance, unfavourable drug‒drug interaction, and toxicity have been reported in previous studies. Thus there is a dearth of new treatment options for SARS-CoV-2. In this work, a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity. One compound, designated 172, demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern. Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease (3CLpro) by binding to an allosteric site and reduces 3CLpro dimerization. A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro. In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice. Overall, this study identified an alternative druggable site on the SARS-CoV-2 3CLpro, proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

Promising animal models for amyotrophic lateral sclerosis drug discovery: a comprehensive update.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Several animal models have been generated to understand ALS pathogenesis. They have provided valuable insight into disease mechanisms and the development of therapeutic strategies. AREAS COVERED: In this review, the authors provide a concise overview of simple genetic model organisms, including C. elegans, Drosophila, zebrafish, and mouse genetic models that have been generated to study ALS. They emphasize the benefits of each model and their application in translational research for discovering new chemicals, gene therapy approaches, and antibody-based strategies for treating ALS. EXPERT OPINION: Significant progress is being made in identifying new therapeutic targets for ALS. This progress is being enabled by promising animal models of the disease using increasingly effective genetic and pharmacological strategies. There are still challenges to be overcome in order to achieve improved success rates for translating drugs from animal models to clinics for treating ALS. Several promising future directions include the establishment of novel preclinical protocol standards, as well as the combination of animal models with human induced pluripotent stem cells (iPSCs).

A novel chemical genetic approach reveals paralog-specific role of ERK1/2 in mouse embryonic stem cell fate control.

Introduction: Mouse embryonic stem cell (ESC) self-renewal can be maintained through dual inhibition of GSK3 and MEK kinases. MEK has two highly homologous downstream kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2). However, the exact roles of ERK1/2 in mouse ESC self-renewal and differentiation remain unclear. Methods: We selectively deleted or inhibited ERK1, ERK2, or both using genetic and chemical genetic approaches combined with small molecule inhibitors. The effects of ERK paralog-specific inhibition on mouse ESC self-renewal and differentiation were then assessed. Results: ERK1/2 were found to be dispensable for mouse ESC survival and self-renewal. The inhibition of both ERK paralogs, in conjunction with GSK3 inhibition, was sufficient to maintain mouse ESC self-renewal. In contrast, selective deletion or inhibition of only one ERK paralog did not mimic the effect of MEK inhibition in promoting mouse ESC self-renewal. Regarding ESC differentiation, inhibition of ERK1/2 prevented mesendoderm differentiation. Additionally, selective inhibition of ERK1, but not ERK2, promoted mesendoderm differentiation. Discussion: These findings suggest that ERK1 and ERK2 have both overlapping and distinct roles in regulating ESC self-renewal and differentiation. This study provides new insights into the molecular mechanisms of ERK1/2 in governing ESC maintenance and lineage commitment, potentially informing future strategies for controlling stem cell fate in research and therapeutic applications.

Chemical Driving the Subtype Selectivity of Phytohormone Receptors Is Beneficial for Crop Productivity.

Chemicals that modulate phytohormones serve as a research tool in plant science and as products to improve crop productivity. Subtype selectivity refers to a ligand to selectively bind to specific subtypes of a receptor rather than binding to all possible subtypes indiscriminately. It allows for precise and specific control of cellular functions and is widely used in medicine. However, subtype selectivity is rarely mentioned in the realm of plant science, and it requires integrated knowledge from chemistry and biology, including structural features of small molecules as ligands, the redundancy of target proteins, and the response of signaling factors. Here, we present a comprehensive review and evaluation of phytohormone receptor subtype selectivity, leveraging the chemical characteristics of phytohormones and their analogues as clues. This work endeavors to provide a valuable research strategy that integrates knowledge from chemistry and biology to advance research efforts geared toward enhancing crop productivity.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid, and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, together with pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, resulted in adventitious root formation, which is a desirable trait in both agriculture and horticulture.

Chemical genomic analysis reveals the interplay between iron chelation, zinc homeostasis, and retromer function in the bioactivity of an ethanol adduct of the feijoa fruit-derived ellagitannin vescalagin.

Nature has been a rich source of pharmaceutical compounds, producing 80% of our currently prescribed drugs. The feijoa plant, Acca sellowiana, is classified in the family Myrtaceae, native to South America, and currently grown worldwide to produce feijoa fruit. Feijoa is a rich source of bioactive compounds with anticancer, anti-inflammatory, antibacterial, and antifungal activities; however, the mechanism of action of these compounds is largely not known. Here, we used chemical genetic analyses in the model organism Saccharomyces cerevisiae to investigate the mechanism of action of a feijoa-derived ethanol adduct of vescalagin (EtOH-vescalagin). Genome-wide barcode sequencing analysis revealed yeast strains lacking genes in iron metabolism, zinc metabolism, retromer function, or mitochondrial function were hypersensitive to 0.3 µM EtOH-vescalagin. This treatment increased expression of iron uptake proteins at the plasma membrane, which was a compensatory response to reduced intracellular iron. Likewise, EtOH-vescalagin increased expression of the Cot1 protein in the vacuolar membrane that transports zinc into the vacuole to prevent cytoplasmic accumulation of zinc. Each individual subunit in the retromer complex was required for the iron homeostatic mechanism of EtOH-vescalagin, while only the cargo recognition component in the retromer complex was required for the zinc homeostatic mechanism. Overexpression of either retromer subunits or high-affinity iron transporters suppressed EtOH-vescalagin bioactivity in a zinc-replete condition, while overexpression of only retromer subunits increased EtOH-vescalagin bioactivity in a zinc-deficient condition. Together, these results indicate that EtOH-vescalagin bioactivity begins with extracellular iron chelation and proceeds with intracellular transport of zinc via the retromer complex. More broadly, this is the first report of a bioactive compound to further characterize the poorly understood interaction between zinc metabolism and retromer function.

Identification of mebendazole as an ethylene signaling activator reveals a role of ethylene signaling in the regulation of lateral root angles.

The lateral root angle or gravitropic set-point angle (GSA) is an important trait for root system architecture (RSA) that determines the radial expansion of the root system. The GSA therefore plays a crucial role for the ability of plants to access nutrients and water in the soil. Only a few regulatory pathways and mechanisms that determine GSA are known. These mostly relate to auxin and cytokinin pathways. Here, we report the identification of a small molecule, mebendazole (MBZ), that modulates GSA in Arabidopsis thaliana roots and acts via the activation of ethylene signaling. MBZ directly acts on the serine/threonine protein kinase CTR1, which is a negative regulator of ethylene signaling. Our study not only shows that the ethylene signaling pathway is essential for GSA regulation but also identifies a small molecular modulator of RSA that acts downstream of ethylene receptors and that directly activates ethylene signaling.

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy.

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

Chemical-genetics refines transcription factor regulatory circuits.

Transcriptional dysregulation is a key step in oncogenesis, but our understanding of transcriptional control has relied on genetic approaches that are slow and allow for compensation. Chemical-genetic approaches have shortened the time frame for the analysis of transcription factors from days or weeks to minutes. These studies show that while DNA-binding proteins bind to thousands of sites, they are directly required to regulate only a small cadre of genes. Moreover, these transcriptional control networks are far more distinct, with much less overlap and interconnectivity than predicted from DNA binding. The identified direct targets can then be used to dissect the mechanism of action of these factors, which could identify ways to therapeutically manipulate these oncogenic transcriptional control networks.

Chemoproteomics, A Broad Avenue to Target Deconvolution.

As a vital project of forward chemical genetic research, target deconvolution aims to identify the molecular targets of an active hit compound. Chemoproteomics, either with chemical probe-facilitated target enrichment or probe-free, provides a straightforward and effective approach to profile the target landscape and unravel the mechanisms of action. Canonical methods rely on chemical probes to enable target engagement, enrichment, and identification, whereas click chemistry and photoaffinity labeling techniques improve the efficiency, sensitivity, and spatial accuracy of target recognition. In comparison, recently developed probe-free methods detect protein-ligand interactions without the need to modify the ligand molecule. This review provides a comprehensive overview of different approaches and recent advancements for target identification and highlights the significance of chemoproteomics in investigating biological processes and advancing drug discovery processes.

Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Plants and Small Molecules: An Up-and-Coming Synergy.

The emergence of Arabidopsis thaliana as a model system has led to a rapid and wide improvement in molecular genetics techniques for studying gene function and regulation. However, there are still several drawbacks that cannot be easily solved with molecular genetic approaches, such as the study of unfriendly species, which are of increasing agronomic interest but are not easily transformed, thus are not prone to many molecular techniques. Chemical genetics represents a methodology able to fill this gap. Chemical genetics lies between chemistry and biology and relies on small molecules to phenocopy genetic mutations addressing specific targets. Advances in recent decades have greatly improved both target specificity and activity, expanding the application of this approach to any biological process. As for classical genetics, chemical genetics also proceeds with a forward or reverse approach depending on the nature of the study. In this review, we addressed this topic in the study of plant photomorphogenesis, stress responses and epigenetic processes. We have dealt with some cases of repurposing compounds whose activity has been previously proven in human cells and, conversely, studies where plants have been a tool for the characterization of small molecules. In addition, we delved into the chemical synthesis and improvement of some of the compounds described.

Network Analysis Reveals the Molecular Bases of Statin Pleiotropy That Vary with Genetic Background.

Many approved drugs are pleiotropic: for example, statins, whose main cholesterol-lowering activity is complemented by anticancer and prodiabetogenic mechanisms involving poorly characterized genetic interaction networks. We investigated these using the Saccharomyces cerevisiae genetic model, where most genetic interactions known are limited to the statin-sensitive S288C genetic background. We therefore broadened our approach by investigating gene interactions to include two statin-resistant genetic backgrounds: UWOPS87-2421 and Y55. Networks were functionally focused by selection of HMG1 and BTS1 mevalonate pathway genes for detection of genetic interactions. Networks, multilayered by genetic background, were analyzed for key genes using network centrality (degree, betweenness, and closeness), pathway enrichment, functional community modules, and Gene Ontology. Specifically, we found modification genes related to dysregulated endocytosis and autophagic cell death. To translate results to human cells, human orthologues were searched for other drug targets, thus identifying candidates for synergistic anticancer bioactivity. IMPORTANCE Atorvastatin is a highly successful drug prescribed to lower cholesterol and prevent cardiovascular disease in millions of people. Though much of its effect comes from inhibiting a key enzyme in the cholesterol biosynthetic pathway, genes in this pathway interact with genes in other pathways, resulting in 15% of patients suffering painful muscular side effects and 50% having inadequate responses. Such multigenic complexity may be unraveled using gene networks assembled from overlapping pairs of genes that complement each other. We used the unique power of yeast genetics to construct genome-wide networks specific to atorvastatin bioactivity in three genetic backgrounds to represent the genetic variation and varying response to atorvastatin in human individuals. We then used algorithms to identify key genes and their associated FDA-approved drugs in the networks, which resulted in the distinction of drugs that may synergistically enhance the known anticancer activity of atorvastatin.

Dynamic metabolome profiling uncovers potential TOR signaling genes.

Although the genetic code of the yeast Saccharomyces cerevisiae was sequenced 25 years ago, the characterization of the roles of genes within it is far from complete. The lack of a complete mapping of functions to genes hampers systematic understanding of the biology of the cell. The advent of high-throughput metabolomics offers a unique approach to uncovering gene function with an attractive combination of cost, robustness, and breadth of applicability. Here, we used flow-injection time-of-flight mass spectrometry to dynamically profile the metabolome of 164 loss-of-function mutants in TOR and receptor or receptor-like genes under a time course of rapamycin treatment, generating a dataset with >7000 metabolomics measurements. In order to provide a resource to the broader community, those data are made available for browsing through an interactive data visualization app hosted at https://rapamycin-yeast.ethz.ch. We demonstrate that dynamic metabolite responses to rapamycin are more informative than steady-state responses when recovering known regulators of TOR signaling, as well as identifying new ones. Deletion of a subset of the novel genes causes phenotypes and proteome responses to rapamycin that further implicate them in TOR signaling. We found that one of these genes, CFF1, was connected to the regulation of pyrimidine biosynthesis through URA10. These results demonstrate the efficacy of the approach for flagging novel potential TOR signaling-related genes and highlight the utility of dynamic perturbations when using functional metabolomics to deliver biological insight.

A Nucleophilic Activity-Based Probe Enables Profiling of PLP-Dependent Enzymes.

PLP-dependent enzymes represent an important class of highly "druggable" enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. Hence, we developed a probe that contains an N-hydroxyalanine warhead that acts as a covalent inhibitor of PLP-dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP-dependent enzymes in vitro as well as look at whole-cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP-dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.

Interplay of phytohormones and epigenetic regulation: A recipe for plant development and plasticity.

Both phytohormone signaling and epigenetic mechanisms have long been known to play crucial roles in plant development and plasticity in response to ambient stimuli. Indeed, diverse signaling pathways mediated by phytohormones and epigenetic processes integrate multiple upstream signals to regulate various plant traits. Emerging evidence indicates that phytohormones and epigenetic processes interact at multiple levels. In this review, we summarize the current knowledge of the interplay between phytohormones and epigenetic processes from the perspective of phytohormone biology. We also review chemical regulators used in epigenetic studies and propose strategies for developing novel regulators using multidisciplinary approaches.

Substrate-selective small-molecule modulators of enzymes: Mechanisms and opportunities.

Small-molecule inhibitors of enzymes are widely used tools in reverse chemical genetics to probe biology and explore therapeutic opportunities. They are often compared with genetic knockdown or knockout and are expected to produce phenotypes similar to the genetic perturbations. This review aims to highlight that small molecule inhibitors of enzymes and genetic perturbations may not necessarily produce the same phenotype due to the possibility of substrate-selective or substrate-dependent effects of the inhibitors. Examples of substrate-selective inhibitors and the mechanisms for the substrate-selective effects are discussed. Substrate-selective modulators of enzymes have distinct advantages and cannot be easily replaced with biologics. Thus, they present an exciting opportunity for chemical biologists and medicinal chemists.

Cellular Thermal Shift Assay for the Detection of Small Molecule-Target Interactions in Arabidopsis Cells.

Chemical genetics takes advantage of small molecule-protein interactions to explore various biological processes. Although an attractive alternative to classical genetics in plants, the identification of small-molecule targets remains a challenge and limits the broad use of the compounds. The cellular thermal shift assay (CETSA), based on the principle that binding of small molecules could affect the thermal stability of proteins, has been applied for target validation in plant cells. Combined with high-resolution mass spectrometry, CETSA can detect small-molecule targets by monitoring the changes in the protein thermal stability caused by the interactions with small molecules at the proteome level. Here we describe the small-molecule target validation as well as the target identification with mass spectrometry by means of CETSA.

Activation of the RMTg Nucleus by Chemogenetic Techniques Alleviates the Learning and Memory Impairment in APP/PS1 Mice.

Objective: There is a relationship between non-rapid eye movement (NREM) sleep and Alzheimer's disease (AD). The rostromedial tegmental nucleus (RMTg) is activated can enhance NREM. Therefore, our experiment was designed to investigate the effects of activation of RMTg by chemical genetic techniques on APP/PS1 mice learning and memory. Materials and Methods: After the AAV-hSyn-hM3Dq-mCherry virus was injected into the RMTg nucleus, CNO solution was intraperitoneally injected to activate RMTg. The new object test and Morris water maze were used to determine the learning and memory level; T2-weighted imaging (T2WI) scanning was performed to analyze the volume of hippocampus and entorhinal cortex of each group; The virus transfection status was determined by laser confocal microscope and use immunohistochemical detection to observe the deposition of Beta Amyloid 1-42 (Aß42). Results: Activation of RMTg by chemical genetic techniques can reduce the escape latency and increase discrimination index (RI) and the number of crossing platform; Activation of RMTg by chemical genetic techniques reduced the atrophy of the entorhinal cortex. Aß42 deposition in the brain was decreased after activation of RMTg. Conclusion: Activation of the RMTg nucleus by chemogenetic techniques can improve the learning and memory impairment in APP/PS1 mice.

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation.

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.