High-throughput Phenotypic Screening Research Articles

Abstract LB027: Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository

Abstract Second-generation androgen ablation therapies (ADT), enzalutamide, and abiraterone are initially effective on metastatic castration-resistant prostate cancer (mCRPC). However, nearly 30-40% of patients eventually become resistant to these treatments due to the presence of androgen-receptor variants (AR-V), which lack a ligand-binding domain. The grim reality that metastatic CRPC is virtually incurable for most patients poses a significant challenge for clinicians striving to enhance these individuals' quality of life and lifespan. Considering drug discovery efforts, we partnered with the National Cancer Institute's (NCI) Natural Product Repository, which possesses a diverse collection of natural product extracts derived from plant, marine, and microbial organisms. We developed high-throughput phenotypic screening assays to screen the crude natural product extract libraries on AR and AR-variants CRPC cell lines (C4-2B and 22Rv1). Several extracts inhibited the growth of both CRPC cell lines and were further confirmed for dose-dependent cell viability inhibition assays. Once we determined the IC50 dose, we performed a Western blot analysis that confirmed the inhibition of AR and AR-V7 expression in CRPC cells.In subsequent studies, we narrowed down by fractionating the most potent extracts and confirmed their ability to inhibit AR expression and signaling on CRPC cell lines. Currently, we are characterizing the potent compounds and their inhibitory activity on AR and AR-variants, in vitro and in vivo CRPC models. Many nutraceuticals have been shown to target AR signaling, synthesis, or degradation which effectively inhibited the growth of CRPC. Our collaborative efforts with NCI will permit us find N-terminal AR degrading molecules (AF-1), which are essential for the AR transcriptional activities of CRPC. In sum, discovery of ovel small molecules from our drug development program have the potential to translate these effective natural products into drugs with clinical benefits to treat CRPC. Citation Format: Neha Tyagi, Sepideh Hosseiniporgham, Ashish Tyagi, Balaji Chandrasekaran, Katherine Bonilla, Inna Kriger, James Saccchettini, Chendil Damodaran. Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB027.

Enhancing the Small-Scale Screenable Biological Space beyond Known Chemogenomics Libraries with Gray Chemical Matter─Compounds with Novel Mechanisms from High-Throughput Screening Profiles.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae

The current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections. We developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K.pneumoniae and A.baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using "eNTRy rules" criteria associated with increased likelihood of intracellular accumulation in Escherichia coli. We identified 72 compounds with confirmed activity against K.pneumoniae and/or drug-resistant A.baumannii. Two new chemical series with activity against XDR A.baumannii were identified meeting our criteria of potency (EC50≤50μM) and absence of cytotoxicity (HepG2 CC50≥100μM and red blood cell lysis HC50≥100μM). The activity of close analogues of the two chemical series was also determined against A.baumannii clinical isolates. This work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K.pneumoniae and A.baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects. BMBF and GARDP.

Abstract 3287: Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms

Abstract Development of resistance to targeted, chemotherapeutic, and immune-oncology treatments alike is a major barrier to long-term remission in advanced cancer patients. We developed an integrated computational-experimental platform to mitigate treatment resistance by identifying therapeutics that selectively target clinical resistance mechanisms. The key innovation is the computational framework that comprehensively maps tumor-specific mechanisms of response and resistance to a treatment based on clinicogenomic and/or preclinical pharmacogenomic data. It then screens for drugs or targets that are synthetically lethal with these resistance mechanisms. Here we present the identification of therapeutic options that mitigate resistance to CDK4/6 inhibitors as a case study. Leveraging a breast cancer-specific atlas of cellular architecture, our framework learned genetic mechanisms of CDK4/6 inhibitor resistance from a high-throughput phenotypic screen of ~700 cell lines treated with the clinical CDK4/6 inhibitor palbociclib. The learned mechanisms formed a parsimonious set of hierarchically linked protein complexes that coordinate G1-to-S transition (P=5.6 × 10−12). The mechanisms unified distinct resistant populations such as samples resistant due to CDK4/6-Rb checkpoint bypass and those resistant due to growth factor receptor activation. Applied to 70 ER+ metastatic breast cancer patients in a real-world dataset, the learned resistance mechanisms accurately predicted patient response to palbociclib (P=3 × 10−4, log-rank test). Computationally screening 20K clinical-grade small molecules and gene targets for therapeutic options synthetically lethal with the learned resistance mechanisms identified the known CDK4/6 resistance targets CDK2, CCNE1, and E2F. It also identified a target that, to our knowledge, has not previously been rigorously evaluated for its potential to mitigate CDK4/6 inhibitor resistance. In-vitro, monotherapy inhibition of the target by siRNA selectively decreased growth of breast cancer cell lines resistant to CDK4/6 inhibitors by 52% compared to CDK4/6i sensitive cell lines (P=0.023, Mann-Whitney U-test). As a combination therapy, inhibition of the target by siRNA selectively increased the sensitivity to palbociclib by nearly an order of magnitude in an otherwise resistant breast cancer cell line (expected vs observed IC50: 9.7 uM vs. 1.3 uM) while not having a substantial effect in a palbociclib-sensitive cell line (expected vs observed IC50: 0.57 uM vs. 0.56 uM). Finally, while monotherapy inhibition of the target and CDK4/6 induced cell cycle arrest, the combination strongly induced apoptosis in cell lines resistant to CDK4/6 inhibitors. These results highlight how computational deconvolution of tumor resistance mechanisms enable algorithmic identification of therapeutic options to target treatment-resistant cell populations. Citation Format: Adam Yaari, Eduardo Farias, Francisco Guedes, Oliver Priebe, Lee McDaniel, Tyler Earnest, Trey Ideker, Maxwell A. Sherman. Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3287.

Abstract 3095: A high-throughput phenotypic screen to identify small molecule inhibitors of Werner helicase

Abstract Werner helicase (WRN) is a RecQ DNA helicase involved in genome maintenance and DNA damage repair. It has recently been shown to be essential to cancer cells with high microsatellite instability (MSI-H). In MSI-H cancer cells, WRN depletion was shown to induce DNA damage and disrupt mitoses, resulting in a distinct phenotype of enlarged and fragmented nuclei. Here, we present a high-throughput phenotypic screen to identify compounds that selectively induce enlarged nuclei in MSI-H cancer cells. In HCT116, an MSI-H colorectal cancer cell line, approximately 40% of cells exhibited enlarged nuclei when WRN was depleted using siRNA. In contrast, fewer than 10% of control cells displayed this phenotype. In HT29, a microsatellite stable (MSS) cancer cell line, no such difference in nuclear morphology was observed, suggesting that the phenotype is specific to MSI-H cells. We used high content imaging to screen our in-house diversity library of approximately 175,000 compounds in 384-well format. 4,260 compounds induced enlarged nuclei in HCT116 cells, a 2.4% hit rate. After re-screening hits in HCT116 along with two additional MSI-H cancer cell lines, SW48 and SNU-407, and counter-screening in HT29 cells, 144 hits were confirmed to specifically enlarge nuclei in MSI-H but not MSS cells. Using the ADP-Glo Kinase Assay, two of these compounds were confirmed to inhibit WRN ATPase activity, suggesting WRN inhibition as a potential mechanism for their specific effects in MSI-H cells. This screen will also identify compounds which selectively kill MSI-H cells through mechanisms beyond direct inhibition of WRN, including interaction with WRN complex members or other members of DNA damage repair pathways. Validation studies including the assessment of cell viability and DNA damage in a panel of MSI-H and MSS cell lines will be performed. Our study proposes a phenotypic screening strategy for identifying small molecules with the potential to target WRN and uncover additional novel mechanisms for treating MSI-H cancers. Citation Format: Christine A. Moomau, Yipin Lu, Xiaobin Zhang, Xiang Zhai, Lina Gu, Qing Sheng, Fang Li, Yaoyu Chen. A high-throughput phenotypic screen to identify small molecule inhibitors of Werner helicase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3095.

Stress-Based Screening for Compounds That Inhibit β-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of β-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.

Phenotypic high-throughput screening identifies aryl hydrocarbon receptor agonism as common inhibitor of toxin-induced retinal pigment epithelium cell death.

The retinal pigment epithelium (RPE) is essential to maintain retinal function, and RPE cell death represents a key pathogenic stage in the progression of several blinding ocular diseases, including age-related macular degeneration (AMD). To identify pathways and compounds able to prevent RPE cell death, we developed a phenotypic screening pipeline utilizing a compound library and high-throughput screening compatible assays on the human RPE cell line, ARPE-19, in response to different disease relevant cytotoxic stimuli. We show that the metabolic by-product of the visual cycle all-trans-retinal (atRAL) induces RPE apoptosis, while the lipid peroxidation by-product 4-hydroxynonenal (4-HNE) promotes necrotic cell death. Using these distinct stimuli for screening, we identified agonists of the aryl hydrocarbon receptor (AhR) as a consensus target able to prevent both atRAL mediated apoptosis and 4-HNE-induced necrotic cell death. This works serves as a framework for future studies dedicated to screening for inhibitors of cell death, as well as support for the discussion of AhR agonism in RPE pathology.

NMJ Analyser: a novel method to quantify neuromuscular junction morphology in zebrafish.

Neuromuscular junction (NMJ) structural integrity is crucial for transducing motor neuron signals that initiate skeletal muscle contraction. Zebrafish has emerged as a simple and efficient model to study NMJ structural morphology and function in the context of developmental neurobiology and neuromuscular diseases. However, methods to quantify NMJ morphology from voluminous data of NMJ confocal images accurately, rapidly, and reproducibly are lacking. We developed an ImageJ macro called "NMJ Analyser" to automatically and unbiasedly analyse NMJ morphology in zebrafish. From the Z-stack of a zebrafish hemisomite, both presynaptic and postsynaptic fluorescently labeled termini at NMJs are extracted from background signal, with larger clusters of termini being segmented into individual termini using an unbiased algorithm. The program then determines whether each presynaptic terminus is co-localized with a postsynaptic terminus and vice versa, or whether it is orphaned, and tabulates the number of orphan and co-localized pre- and postsynaptic termini. The usefulness of this ImageJ macro plugin will be helpful to quantify NMJ parameters in zebrafish, particularly during development and in disease models of neuromuscular diseases. It can enable high-throughput NMJ phenotypic screens in the drug discovery process for neuromuscular diseases. It could also be further applied to the investigation of NMJ of other developmental systems. NMJ Analyser is available for download at https://github.com/PattenLab/NMJ-Analyser.git.

A Robust Phenotypic High-Throughput Antiviral Assay for the Discovery of Rabies Virus Inhibitors.

Rabies virus (RABV) causes severe neurological symptoms in mammals. The disease is almost inevitably lethal as soon as clinical symptoms appear. The use of rabies immunoglobulins (RIG) and vaccination in post-exposure prophylaxis (PEP) can provide efficient protection, but many people do not receive this treatment due to its high cost and/or limited availability. Highly potent small molecule antivirals are urgently needed to treat patients once symptoms develop. In this paper, we report on the development of a high-throughput phenotypic antiviral screening assay based on the infection of BHK-21 cells with a fluorescent reporter virus and high content imaging readout. The assay was used to screen a repurposing library of 3681 drugs (all had been studied in phase 1 clinical trials). From this series, salinomycin was found to selectively inhibit viral replication by blocking infection at the entry stage. This shows that a high-throughput assay enables the screening of large compound libraries for the purposes of identifying inhibitors of RABV replication. These can then be optimized through medicinal chemistry efforts and further developed into urgently needed drugs for the treatment of symptomatic rabies.

A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening

High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform’s potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.

Discovery of Zidovudine as a cardiomyocyte protectant for doxorubicin-induced toxicity through high-throughput phenotypic drug screening

Discovery of Zidovudine as a cardiomyocyte protectant for doxorubicin-induced toxicity through high-throughput phenotypic drug screening

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

A conserved metabolic signature associated with response to fast-acting anti-malarial agents.

In malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of anti-malarial drug candidates in the developmental pipeline at NITD. We show that NITD fast-acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug-exposed malaria parasites which is similar to another fast-acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to hemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify and inform on the mode of action of fast drug candidates of similar profiles in future drug discovery programs.

Variable staphyloxanthin production by Staphylococcus aureus drives strain-dependent effects on diabetic wound-healing outcomes

Strain-level variation in Staphylococcus aureus is a factor that contributes to disease burden and clinical outcomes in skin disorders and chronic wounds. However, the microbial mechanisms that drive these variable host responses are poorly understood. To identify mechanisms underlying strain-specific outcomes, we perform high-throughput phenotyping screens on S.aureus isolates cultured from diabetic foot ulcers. Isolates from non-healing wounds produce more staphyloxanthin, a cell membrane pigment. In murine diabetic wounds, staphyloxanthin-producing isolates delay wound closure significantly compared with staphyloxanthin-deficient isolates. Staphyloxanthin promotes resistance to oxidative stress and enhances bacterial survival in neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes reveals a mutation in the sigma B operon, resulting in marked differences in stress response gene expression. Our work illustrates a framework to identify traits that underlie strain-level variation in disease burden and suggests more precise targets for therapeutic intervention in S.aureus-positive wounds.

Characterization, Directed Evolution, and Targeting of DNA Virus-Encoded RNA Capping Enzymes Using Phenotypic Yeast Platforms.

The constant and the sudden emergence of zoonotic human and animal viruses is a significant threat to human health, the world economy, and the world food supply. This has necessitated the development of broad-spectrum therapeutic strategies to combat these emerging pathogens. Mechanisms that are essential for viral replication and propagation have been successfully targeted in the past to develop broad-spectrum therapeutics that can be readily repurposed to combat new zoonotic pathogens. Because of the importance of viral RNA capping enzymes to viral replication and pathogenesis, as well as their presence in both DNA and RNA viruses, these viral proteins have been a long-standing therapeutic target. Here, we use genome sequencing information and yeast-based platforms (YeRC0M) to identify, characterize, and target viral genome-encoded essential RNA capping enzymes from emerging strains of DNA viruses, i.e., Monkeypox virus and African Swine Fever Virus, which are a significant threat to human and domestic animal health. We first identified and biochemically characterized these viral RNA capping enzymes and their necessary protein domains. We observed significant differences in functional protein domains and organization for RNA capping enzymes from emerging DNA viruses in comparison to emerging RNA viruses. We also observed several differences in the biochemical properties of these viral RNA capping enzymes using our phenotypic yeast-based approaches (YeRC0M) as compared to the previous in vitro studies. Further, using directed evolution, we were able to identify inactivation and attenuation mutations in these essential viral RNA capping enzymes; these data could have implications on virus biocontainment as well as live attenuated vaccine development. We also developed methods that would facilitate high-throughput phenotypic screening to identify broad-spectrum inhibitors that selectively target viral RNA capping enzymes over host RNA capping enzymes. As demonstrated here, our approaches to identify, characterize, and target viral genome-encoded essential RNA capping enzymes are highly modular and can be readily adapted for targeting emerging viral pathogens as well as their variants that emerge in the future.

Advances in Malaria Pharmacology and the online Guide to MALARIA PHARMACOLOGY: IUPHAR Review X.

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology.

PHDs-seq: a large-scale phenotypic screening method for drug discovery through parallel multi-readout quantification

High-throughput phenotypic screening is a cornerstone of drug development and the main technical approach for stem cell research. However, simultaneous detection of activated core factors responsible for cell fate determination and accurate assessment of directional cell transition are difficult using conventional screening methods that focus on changes in only a few biomarkers. The PHDs-seq (Probe Hybridization based Drug screening by sequencing) platform was developed to evaluate compound function based on their transcriptional effects in a wide range of signature biomarkers. In this proof-of-concept demonstration, several sets of markers related to cell fate determination were profiled in adipocyte reprogramming from dermal fibroblasts. After validating the accuracy, sensitivity and reproducibility of PHDs-seq data in molecular and cellular assays, a panel of 128 signalling-related compounds was screened for the ability to induce reprogramming of keloid fibroblasts (KF) into adipocytes. Notably, the potent ATP-competitive VEGFR/PDGFR inhibitor compound, ABT869, was found to promote the transition from fibroblasts to adipocytes. This study highlights the power and accuracy of the PHDs-seq platform for high-throughput drug screening in stem cell research, and supports its use in basic explorations of the molecular mechanisms underlying disease development.

Spontaneously established syntrophic yeast communities improve bioproduction.

Nutritional codependence (syntrophy) has underexplored potential to improve biotechnological processes by using cooperating cell types. So far, design of yeast syntrophic communities has required extensive genetic manipulation, as the co-inoculation of most eukaryotic microbial auxotrophs does not result in cooperative growth. Here we employ high-throughput phenotypic screening to systematically test pairwise combinations of auxotrophic Saccharomyces cerevisiae deletion mutants. Although most coculture pairs do not enter syntrophic growth, we identify 49 pairs that spontaneously form syntrophic, synergistic communities. We characterized the stability and growth dynamics of nine cocultures and demonstrated that a pair of tryptophan auxotrophs grow by exchanging a pathway intermediate rather than end products. We then introduced a malonic semialdehyde biosynthesis pathway split between different pairs of auxotrophs, which resulted in increased production. Our results report the spontaneous formation of stable syntrophy in S. cerevisiae auxotrophs and illustrate the biotechnological potential of dividing labor in a cooperating intraspecies community.

High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds.

Entamoeba histolytica is a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection with E. histolytica is referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 81,664 compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound. The best compound in this series, JNJ001, showed excellent inhibition activity against E. histolytica trophozoites with EC50 values at 0.29 μM, which is better than the current approved treatment, metronidazole. Further experimentation confirmed the activity of this compound, as well as that of several structurally related compounds, originating from both the Janssen Jump-stARter library, and from chemical vendors, thus highlighting a new structure-activity relationship (SAR). In addition, we confirmed that the compound inhibited E. histolytica survival as rapidly as the current standard of care and inhibited transmissible cysts of the related model organism Entamoeba invadens. Together these results constitute the discovery of a novel class of chemicals with favorable in vitro pharmacological properties. The discovery may lead to an improved therapy against this parasite and in all of its life stages.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.