High-throughput Compound Screening Research Articles

Generating and Characterizing Adipose Spheroids from Adipose-Derived Stromal/Stem Cells.

Advances in technology and automation over the past several decades have made it feasible to perform high-throughput compound screening with cell spheroids, a valuable approach for drug discovery. It is entirely feasible to generate multiple 384-well plates containing adipose spheroids from cryopreserved, single-donor, adipose stem cells, thus incorporating genetic diversity into the discovery stages of research. In this protocol, we describe our method for isolating primary humanadipose stem cells and synthesizing cell spheroids comprised of mature adipocytes and stromal cells. Also included are representative outcome measurements useful for characterizing adipocyte metabolism and health. Wherever possible, we describe technologies that can be used to automate characterization and increase throughput.

High-throughput screening of natural compounds for prophage induction in controlling pathogenic bacteria in food

High-throughput screening of natural compounds for prophage induction in controlling pathogenic bacteria in food

High-throughput screening of compounds targeting RNA editing in Trypanosoma brucei: Novel molecular scaffolds with broad trypanocidal effects

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.

Characterization of critical parameters using an air-liquid interface model with RPMI 2650 cells for permeability studies of small molecules.

The field of nasal drug delivery gained enormously on interest over the past decade. Performing nasal in vivo studies is expensive and time-consuming, but also unfeasible for an initial high-throughput compound and formulation screening. Therefore, the development of fast and high-throughput in vitro models to screen compounds for their permeability through the nasal epithelium and mucosa is constantly expanding. Yet, the protocols used for nasal in vitro permeability studies are varying, which limits the comparability and reproducibility of generated data. This project aimed to elucidate the influence of different culture and assay parameters of RPMI 2650 cells grown under air-liquid interface (ALI) conditions on the transepithelial electrical resistance (TEER) and apparent permeability (Papp) values of five selected reference compounds, covering the range of low to moderate to high permeability. The influence of the passage number, seeding density, and timepoint of airlift was minimal in our approach, while the substrate pore density had a significant influence on the Papp values of carbamazepine, propranolol, and metoprolol, classified as highly permeable compounds, but not on atenolol and aciclovir. Elevation of the experimental concentration of carbamazepine, propranolol, and metoprolol in the donor compartment had an increasing effect on the Papp values, while prolonging the assay time did not have a significant influence. Based on the results reported here, RPMI 2650 cells cultured under ALI conditions offer the possibility of a standardized high-throughput screening model for small molecules and their formulations for in vitro drug permeation studies to predict and select optimal conditions for their nasal delivery.

Deciphering melting behaviors of energetic compounds using interpretable Machine learning for melt-castable applications

Melting behaviors of energetic compounds determine their thermo-responsive properties, which affects the manufacturing operations and structural stabilities. However, the decisive physicochemical mechanisms for melting behaviors of energetic compounds are absent, which hurdles the acceptancy and generalization of prediction models for rational molecular design. Herein, an interpretable machine learning (IML) approach was proposed to construct accurate prediction models for melting point values and melt-castable potentials of energetic compounds. A high-quality melting-point dataset comprising 239 neutral CHNO compounds was obtained, and the data was collected with strict criteria involving chemical formulations, crystal structures and experimental measurement operations. Tailored descriptors, which involve the priori insights for melting behaviors, were adopted for IML algorithms. The extreme gradient boosting model presents the best prediction accuracy (85 %) for the classification of melting point values between 273 K and 650 K; while 87 % accuracy is achieved for the classification of melt-castable property. Importantly, model interpretation assists to reveal that the close contacts between OC and NC, dipole, oxygen balance and the cohesive energy are decisive features for melting point prediction; while enthalpy of sublimation and polar surface area are paramount for the identification of melt-castable property. The ability to determine melting behaviors using IML will broaden the high-throughput screening of energetic compounds and offer valuable guidance towards molecular design.

EQUIBIND: A geometric deep learning-based protein-ligand binding prediction method.

Structure-based virtual screening plays a critical role in drug discovery. However, numerous docking programs, such as AutoDock Vina and Glide, are time-consuming due to the necessity of generating numerous molecular conformations and executing steps like scoring, ranking, and refinement for the ligand-receptor complexes. Consequently, achieving rapid and reliable virtual screening remains a noteworthy challenge. Recently, a team of researchers from Massachusetts Institute of Technology, led by Stärk et al., developed an SE(3)-equivariant geometric deep learning based protein-ligand binding prediction approach, EQUIBIND. In comparison to conventional docking methods, EQUIBIND has the capacity to predict the binding modes of small molecules with target proteins rapidly and precisely. It presents an innovative resolution for high-throughput screening of drug-like compounds.

Detergent-free parasite transformation and replication assay for drug screening against intracellular Leishmania amastigotes

Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse effects, variable efficacy, the development of resistant parasites, high cost, parenteral administration and lack of availability in endemic areas. Therefore, active searching for new antileishmanial drugs has been done for years, mainly by academia. Drug screening techniques have been a challenge since the intracellular localization of Leishmania amastigotes implies that the host cell may interfere with the quantification of the parasites and the final estimation of the effect. One of the procedures to avoid host cell interference is based on its detergent-mediated lysis and subsequent transformation of viable amastigotes into promastigotes, their proliferation and eventual quantification as an axenic culture of promastigotes. However, the use of detergent involves additional handling of cultures and variability. In the present work, cultures of intracellular amastigotes were incubated for 72 h at 26 °C after exposure to the test compounds and the transformation and proliferation of parasites took place without need of adding any detergent. The assay demonstrated clear differentiation of negative and positive controls (average Z´ = 0.75) and 50% inhibitory concentrations of compounds tested by this method and by the gold standard enumeration of Giemsa-stained cultures were similar (p = 0.5002) and highly correlated (r = 0.9707). This simplified procedure is less labor intensive, the probability of contamination and the experimental error are reduced, and it is appropriate for the automated high throughput screening of compounds.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

High throughput compound screening in neuronal cells identifies statins as activators of ataxin 3 expression

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. SCA3 is the most common form, caused by the expansion of CAG repeats within the ataxin 3 (ATXN3) gene. The mutation results in the expression of an abnormal protein, containing long polyglutamine (polyQ) stretches. The polyQ stretch confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. Thus, modulators of ATXN3 expression could potentially ameliorate the pathology in SCA3 patients. Therefore, we generated a CRISPR/Cas9 modified ATXN3-Exon4-Luciferase (ATXN3-LUC) genomic fusion- and control cell lines to perform a reporter cell line-based high-throughput screen comprising 2640 bioactive compounds, including the FDA approved drugs. We found no unequivocal inhibitors of, but identified statins as activators of the LUC signal in the ATXN3-LUC screening cell line. We further confirmed that Simvastatin treatment of wild type SK-N-SH cells increases ATXN3 mRNA and protein levels which likely results from direct binding of the activated sterol regulatory element binding protein 1 (SREBP1) to the ATXN3 promotor. Finally, we observed an increase of normal and expanded ATXN3 protein levels in a patient-derived cell line upon Simvastatin treatment, underscoring the potential medical relevance of our findings.

Development of assays to support identification and characterization of modulators of DExH-box helicase DHX9

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3′ to 5′ directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions. Assay development efforts revealed DHX9 can use double stranded RNA with 18-mer poly(U) 3′ overhangs and as well as significantly shorter overhangs at the 5′ or 3′ end as substrates. The enzymatic assays are augmented by a robust SPR assay for compound validation. A mechanism-derived inhibitor, GTPγS, was characterized as part of the validation of these assays and a crystal structure of GDP bound to cat DHX9 has been solved. In addition to enabling drug discovery efforts for DHX9, these assays may be extrapolated to other RNA helicases providing a valuable toolkit for this important target class.

Identification and characterization of topoisomerase III beta poisons

We designed and carried out a high-throughput screen for compounds that trap topoisomerase III beta (TOP3B poisons) by developing a Comparative Cellular Cytotoxicity Screen. We found a bisacridine compound NSC690634 and a thiacyanine compound NSC96932 that preferentially sensitize cell lines expressing TOP3B, indicating that they target TOP3B. These compounds trap TOP3B cleavage complex (TOP3Bcc) in cells and in vitro and predominately act on RNA, leading to high levels of RNA-TOP3Bccs. NSC690634 also leads to enhanced R-loops in a TOP3B-dependent manner. Preliminary structural activity studies show that the lengths of linkers between the two aromatic moieties in each compound are critical; altering the linker length completely abolishes the trapping of TOP3Bccs. Both of our lead compounds share a similar structural motif, which can serve as a base for further modification. They may also serve in anticancer, antiviral, and/or basic research applications.

A high-throughput screening assay for silencing established HIV-1 macrophage infection identifies nucleoside analogs that perturb H3K9me3 on proviral genomes.

HIV-infected macrophages are long-lived cells that represent a barrier to functional cure. Additionally, low-level viral expression by central nervous system (CNS) macrophages contributes to neurocognitive deficits that develop despite antiretroviral therapy (ART). We recently identified H3K9me3 as an atypical epigenetic mark associated with chronic HIV infection in macrophages. Thus, strategies are needed to suppress HIV-1 expression in macrophages, but the unique myeloid environment and the responsible macrophage/CNS-tropic strains require cell/strain-specific approaches. Here, we generated an HIV-1 reporter virus from a CNS-derived strain with intact auxiliary genes expressing destabilized luciferase. We employed this reporter virus in polyclonal infection of primary human monocyte-derived macrophages (MDM) for a high-throughput screen (HTS) to identify compounds that suppress virus expression from established macrophage infection. Screening ~6,000 known drugs and compounds yielded 214 hits. A secondary screen with 10-dose titration identified 24 meeting criteria for HIV-selective activity. Using three replication-competent CNS-derived macrophage-tropic HIV-1 isolates and viral gene expression readout in MDM, we confirmed the effect of three purine analogs, nelarabine, fludarabine, and entecavir, showing the suppression of HIV-1 expression from established macrophage infection. Nelarabine inhibited the formation of H3K9me3 on HIV genomes in macrophages. Thus, this novel HTS assay can identify suppressors of HIV-1 transcription in established macrophage infection, such as nucleoside analogs and HDAC inhibitors, which may be linked to H3K9me3 modification. This screen may be useful to identify new metabolic and epigenetic agents that ameliorate HIV-driven neuroinflammation in people on ART or prevent viral recrudescence from macrophage reservoirs in strategies to achieve ART-free remission. IMPORTANCE Macrophages infected by HIV-1 are a long-lived reservoir and a barrier in current efforts to achieve HIV cure and also contribute to neurocognitive complications in people despite antiretroviral therapy (ART). Silencing HIV expression in these cells would be of great value, but the regulation of HIV-1 in macrophages differs from T cells. We developed a novel high-throughput screen for compounds that can silence established infection of primary macrophages, and identified agents that downregulate virus expression and alter provirus epigenetic profiles. The significance of this assay is the potential to identify new drugs that act in the unique macrophage environment on relevant viral strains, which may contribute to adjunctive treatment for HIV-associated neurocognitive disorders and/or prevent viral rebound in efforts to achieve ART-free remission or cure.

High-Throughput Screening and Proteomic Characterization of Compounds Targeting Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery.

Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

Flight for fish in drug discovery: a review of zebrafish-based screening of molecules.

Human disease and biological practices are modelled in zebrafish (Danio rerio) at various phases of drug development as well as toxicity evaluation. The zebrafish is ideal for in vivo pathological research and high-resolution investigation of disease progress. Zebrafish has an advantage over other mammalian models, it is cost-effective, it has external development and embryo transparency, easy to apply genetic manipulations, and open to both forward and reverse genetic techniques. Drug screening in zebrafish is suitable for target identification, illness modelling, high-throughput screening of compounds for inhibition or prevention of disease phenotypes and developing new drugs. Several drugs that have recently entered the clinic or clinical trials have their origins in zebrafish. The sophisticated screening methods used in zebrafish models are expected to play a significant role in advancing drug development programmes. This review highlights the current developments in drug discovery processes, including understanding the action of drugs in the context of disease and screening novel candidates in neurological diseases, cardiovascular diseases, glomerulopathies and cancer. Additionally, it summarizes the current techniques and approaches for the selection of small molecules and current technical limitations on the execution of zebrafish drug screening tests.

Comparing the value of mono- vs coculture for high-throughput compound screening in hematological malignancies

AbstractLarge-scale compound screens are a powerful model system for understanding variability of treatment response and discovering druggable tumor vulnerabilities of hematological malignancies. However, as mostly performed in a monoculture of tumor cells, these assays disregard modulatory effects of the in vivo microenvironment. It is an open question whether and to what extent coculture with bone marrow stromal cells could improve the biological relevance of drug testing assays over monoculture. Here, we established a high-throughput platform to measure ex vivo sensitivity of 108 primary blood cancer samples to 50 drugs in monoculture and coculture with bone marrow stromal cells. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and 36% of compounds in acute myeloid leukemia (AML), including chemotherapeutics, B–cell receptor inhibitors, proteasome inhibitors, and Bromodomain and extraterminal domain inhibitors. Only the JAK inhibitors ruxolitinib and tofacitinib exhibited increased efficacy in AML and CLL stromal coculture. We further confirmed the importance of JAK-STAT signaling for stroma-mediated resistance by showing that stromal cells induce phosphorylation of STAT3 in CLL cells. We genetically characterized the 108 cancer samples and found that drug-gene associations strongly correlated between monoculture and coculture. However, effect sizes were lower in coculture, with more drug-gene associations detected in monoculture than in coculture. Our results justify a 2-step strategy for drug perturbation testing, with large-scale screening performed in monoculture, followed by focused evaluation of potential stroma-mediated resistances in coculture.

Using a Quantitative High-Throughput Screening Platform to Identify Molecular Targets and Compounds as Repurposing Candidates for Endometriosis

Endometriosis, defined as the growth of hormonally responsive endometrial-like tissue outside of the uterine cavity, is an estrogen-dependent, chronic, pro-inflammatory disease that affects up to 11.4% of women of reproductive age and gender-diverse people with a uterus. At present, there is no long-term cure, and the identification of new therapies that provide a high level of efficacy and favourable long-term safety profiles with rapid clinical access are a priority. In this study, quantitative high-throughput compound screens of 3517 clinically approved compounds were performed on patient-derived immortalized human endometrial stromal cell lines. Following assay optimization and compound criteria selection, a high-throughput screening protocol was developed to enable the identification of compounds that interfered with estrogen-stimulated cell growth. From these screens, 23 novel compounds were identified, in addition to their molecular targets and in silico cell-signalling pathways, which included the neuroactive ligand–receptor interaction pathway, metabolic pathways, and cancer-associated pathways. This study demonstrates for the first time the feasibility of performing large compound screens for the identification of new translatable therapeutics and the improved characterization of endometriosis molecular pathophysiology. Further investigation of the molecular targets identified herein will help uncover new mechanisms involved in the establishment, symptomology, and progression of endometriosis.

Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation

ABSTRACT The main protease (3-chymotrypsin-like protease, 3CLpro) of SARS-CoV-2 has become a focus of anti-coronavirus research. Despite efforts, drug development targeting 3CLpro has been hampered by limitations in the currently available activity assays. Additionally, the emergence of 3CLpro mutations in circulating SARS-CoV-2 variants has raised concerns about potential resistance. Both emphasize the need for a more reliable, sensitive, and facile 3CLpro assay. Here, we report an orthogonal dual reporter-based gain-of-signal assay for measuring 3CLpro activity in living cells. It builds on the finding that 3CLpro induces cytotoxicity and reporter expression suppression, which can be rescued by its inhibitor or mutation. This assay circumvents most limitations in previously reported assays, especially false positives caused by nonspecific compounds and signal interference from test compounds. It is also convenient and robust for high throughput screening of compounds and comparing the drug susceptibilities of mutants. Using this assay, we screened 1789 compounds, including natural products and protease inhibitors, with 45 compounds that have been reported to inhibit SARS-CoV-2 3CLpro among them. Except for the approved drug PF-07321332, only five of these inhibit 3CLpro in our assays: GC376; PF-00835231; S-217622; Boceprevir; and Z-FA-FMK. The susceptibilities of seven 3CLpro mutants prevalent in circulating variants to PF-07321332, S-217622, and GC376 were also assessed. Three mutants were identified as being less susceptible to PF-07321322 (P132H) and S-217622 (G15S, T21I). This assay should greatly facilitate the development of novel 3CLpro-targeted drugs and the monitoring of the susceptibility of emerging SARS-CoV-2 variants to 3CLpro inhibitors.

Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.