Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development.

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Venetoclax and dinaciclib elicit synergistic preclinical efficacy against hypodiploid acute lymphoblastic leukemia.

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly nonexistent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-lineage ALL but with limitations as monotherapy. In this study, we set out to identify drugs enhancing the anti-leukemic effect of venetoclax in hypodiploid ALL. Using a highthroughput drug screen, we identified dinaciclib, a cyclin-dependent kinase inhibitor that worked synergistically with venetoclax to induce cell death in hypodiploid cell lines. This combination eradicated leukemic blasts within hypodiploid ALL patient-derived xenografts mice with low off-target toxicity. Our findings suggest that dual inhibition of BCL-2 (venetoclax) and CDK9/MCL-1 (dinaciclib) is a promising therapeutic approach in hypodiploid ALL, warranting further investigation to inform clinical trials in this high-risk patient population.

Small-Molecule Screening for Genetic Diseases.

The genetic determinants of many diseases, including monogenic diseases and cancers, have been identified; nevertheless, targeted therapy remains elusive for most. High-throughput screening (HTS) of small molecules, including high-content analysis (HCA), has been an important technology for the discovery of molecular tools and new therapeutics. HTS can be based on modulation of a known disease target (called reverse chemical genetics) or modulation of a disease-associated mechanism or phenotype (forward chemical genetics). Prominent target-based successes include modulators of transthyretin, used to treat transthyretin amyloidoses, and the BCR-ABL kinase inhibitor Gleevec, used to treat chronic myelogenous leukemia. Phenotypic screening successes include modulators of cystic fibrosis transmembrane conductance regulator, splicing correctors for spinal muscular atrophy, and histone deacetylase inhibitors for cancer. Synthetic lethal screening, in which chemotherapeutics are screened for efficacy against specific genetic backgrounds, is a promising approach that merges phenotype and target. In this article, we introduce HTS technology and highlight its contributions to the discovery of drugs and probes for monogenic diseases and cancer.

Single-step affinity purification for fungal proteomics.

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer.

The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.

Reimagining dots and dashes: Visualizing structure and function of organelles for high-content imaging analysis.

Organelles are responsible for biochemical and cellular processes that sustain life and their dysfunction causes diseases from cancer to neurodegeneration. While researchers are continuing to appreciate new roles of organelles in disease, the rapid development of specifically targeted fluorescent probes that report on the structure and function of organelles will be critical to accelerate drug discovery. Here, we highlight four organelles that collectively exemplify the progression of phenotypic discovery, starting with mitochondria, where many functional probes have been described, then continuing with lysosomes and Golgi and concluding with nascently described membraneless organelles. We introduce emerging probe designs to explore organelle-specific morphology and dynamics and highlight recent case studies using high-content analysis to stimulate further development of probes and approaches for organellar high-throughput screening.

Multiparametric High-Content Assays to Measure Cell Health and Oxidative Damage as a Model for Drug-Induced Liver Injury.

Drug-induced liver injury is an important cause of non-approval in drug development and the withdrawal of already approved drugs from the market. Screening human hepatic cell lines for toxicity has been used extensively to predict drug-induced liver injury in preclinical drug development. Assessing hepatic-cell health with more diverse markers will increase the value of in vitro assays and help predict the mechanism of toxicity. We describe three live cell-based assays using HepG2 cells to measure cell health parameters indicative of hepatotoxicity. The first assay measures cellular ATP levels using luciferase. The second and third assays are multiparametric high-content screens covering a panel of cell health markers including cell count, mitochondrial membrane potential and structure, nuclear morphology, vacuolar density, and reactive oxygen species and glutathione levels. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of cellular ATP content Basic Protocol 2: High-content analysis assay to assess cell count, mitochondrial membrane potential and structure, and reactive oxygen species Basic Protocol 3: High-content analysis assay to assess nuclear morphology, vacuoles, and glutathione content Support Protocol 1: Subculturing and maintaining HepG2 cells Support Protocol 2: Plating HepG2 cell line Support Protocol 3: Transferring compounds by pin tool Support Protocol 4: Generating dose-response curves.

Nup2 performs diverse interphase functions in Aspergillus nidulans.

The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket-associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.

Mitotic nuclear pore complex segregation involves Nup2 in Aspergillus nidulans.

Transport through nuclear pore complexes (NPCs) during interphase is facilitated by the nucleoporin Nup2 via its importin α- and Ran-binding domains. However, Aspergillus nidulans and vertebrate Nup2 also locate to chromatin during mitosis, suggestive of mitotic functions. In this study, we report that Nup2 is required for mitotic NPC inheritance in A. nidulans Interestingly, the role of Nup2 during mitotic NPC segregation is independent of its importin α- and Ran-binding domains but relies on a central targeting domain that is necessary for localization and viability. To test whether mitotic chromatin-associated Nup2 might function to bridge NPCs with chromatin during segregation, we provided an artificial link between NPCs and chromatin via Nup133 and histone H1. Using this approach, we bypassed the requirement of Nup2 for NPC segregation. This indicates that A. nidulans cells ensure accurate mitotic NPC segregation to daughter nuclei by linking mitotic DNA and NPC segregation via the mitotic specific chromatin association of Nup2.

PGE2 exerts dose-dependent opposite effects on net water and chloride absorption from the rat colon.

This work investigated the effect of different doses of PGE2 on net water and Cl- absorption from the rat colon, using an in situ perfusion technique. PGE2 exerted opposite effects at different concentrations. Net water and Cl- absorption was significantly reduced at low doses with a minimum at 0.4 microg/100g BW, and significantly elevated at high doses with an observed maximal effect at 21 microg/100g BW. At low doses, PGE2 increased in superficial cells, the activity of the Na+-K+ ATPase and the protein expression of the Na+K+2Cl- cotransporter, but reduced them in crypt cells. Thus, the reduction in net water and Cl- absorption was ascribed to an increase in secretion by surface cells that masked absorptive processes. At high doses, PGE2 increased significantly the activity of the Na+-K+ ATPase in superficial cells only, and was without any effect on the protein expression of the cotransporter and the pump in both surface and crypt cells. The observed increase in net water and Cl- absorption was attributed in this case to an increase in absorptive processes with no effect on secretion.

Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.

Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoid(tetO)-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.

Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region.

Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)-dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

Aniseed oil increases glucose absorption and reduces urine output in the rat.

Anise (Pimpinella anisum) has been used as a traditional aromatic herb in many drinks and baked foods because of the presence of volatile oils in its fruits commonly known as seeds. Hot water extracts of the seeds have been used also in folk medicine for their diuretic and laxative effect, expectorant and anti-spasmodic action, and their ability to ease intestinal colic and flatulence. The aim of this work was to study the effect of aniseed oil on transport processes through intestinal and renal epithelia and determine its mechanism of action. The essential oils were extracted from the seeds by hydrodistillation and analyzed by gas chromatography. Aniseed oil enhanced significantly glucose absorption from the rat jejunum and increased the Na+-K+ ATPase activity in a jejunal homogenate in a dose dependent manner. The oil, however, exerted no effect on water absorption from the colon and did not alter the activity of the colonic Na+-K+ ATPase. When added to drinking water, it reduced the volume of urine produced in the rat and increased the activity of the renal Na+-K+ ATPase even at extremely low concentrations. It was concluded that aniseed oil increases glucose absorption by increasing the activity of the Na+-K+ ATPase and consequently the sodium gradient needed for the sugar transport. Its anti-diuretic effect is also mediated through a similar mechanism in the kidney whereby a stimulation of the Na+-K+ pump increases tubular sodium reabsorption and osmotic water movement. The colonic Na+-K+ ATPase was however, resistant to the oil.

Tumor necrosis factor alpha down-regulates the Na+-K+ ATPase and the Na+-K+2Cl- cotransporter in the kidney cortex and medulla.

The effect of TNF-alpha on the renal Na+-K+ pump and the Na+-K+2Cl- cotransporter was investigated in the rat. Animals were injected with the cytokine, and 4h later, a homogenate from the cortical and medullary tissues was prepared and used to assay the activity of the Na+-K+ ATPase and the protein expression of the pump and symporter. TNF-alpha reduced the activity and expression of the pump in both cortex and medulla, and its effect disappeared when animals were pre-treated with indomethacin, suggesting that TNF-alpha acts via PGE2. Higher levels of PGE2 were detected by enzyme immunoassay, in kidney tissues isolated from rats treated with PGE2, thus confirming this hypothesis. The cytokine also down-regulated the Na+-K+2Cl- cotransporter but this effect was not abrogated by indomethacin. PGE2, injected into animals, exerted a dose-dependent effect. Low doses did not have any effect on the two transporters in the cortex while high doses inhibited and down-regulated the pump and up-regulated the cotransporter. In the medulla low doses increased the activity and expression of the pump but down-regulated the cotransporter while high doses exerted an exactly opposite effect on the two transporters. It was concluded that the effect of TNF-alpha on the pump is mediated via PGE2 which is released at relatively high doses. The effect of the cytokine on the cotransporter is, however, independent of PGE2.

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2.

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.