In vivo quantitative high-throughput screening for drug discovery and comparative toxicology.

Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk and, increasingly, for academic probe discovery. Phenotypic high-throughput screening assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. Caenorhabditis elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here, a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser-scanning cytometry is described, in which GFP-expressing organisms revealing phenotype-modifying structure-activity relationships guide subsequent life-stage and proteomic analyses, and Escherichia coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles, mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in seven-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations with a physiological impact. We anticipate that this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.

Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for PMP22 Gene-Dosage Disorder Drug Discovery.

Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.

Structure-activity relationship of ipglycermide binding to phosphoglycerate mutases.

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure-activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase-phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

High-Throughput Screening to Identify Inhibitors of the Type I Interferon-Major Histocompatibility Complex Class I Pathway in Skeletal Muscle.

Immunosuppressants used to treat autoimmunity are often not curative and have many side effects. Our purpose was to identify therapeutics for autoimmunity of the skeletal muscle termed idiopathic inflammatory myopathies (myositis). Recent evidence shows that the pro-inflammatory type I interferons (IFN) and a downstream product major histocompatibility complex (MHC) class I are pathogenic in myositis. We conducted quantitative high-throughput screening on >4500 compounds, including all approved drugs, through a series of cell-based assays to identify those that inhibit the type I IFN-MHC class I pathway in muscle precursor cells (myoblasts). The primary screen utilized CRISPR/Cas9 genome-engineered human myoblasts containing a pro-luminescent reporter HiBit fused to the C-terminus of endogenous MHC class I. Active compounds were counter-screened for cytotoxicity and validated by MHC class I immunofluorescence, Western blot, and RT-qPCR. Actives included Janus kinase inhibitors, with the most potent being ruxolitinib, and epigenetic/transcriptional modulators like histone deacetylase inhibitors and the hypoxia-inducible factor 1 inhibitor echinomycin. Testing in animal models and clinical trials is necessary to translate these therapies to myositis patients. These robust assay technologies can be further utilized to interrogate the basic mechanisms of the type I IFN-MHC class I pathway, identify novel molecular probes, and elucidate possible environmental triggers that may lead to myositis.

Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.

CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.

A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening.

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gαs gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC®1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.

Diverse captive non-human primates with phytanic acid-deficient diets rich in plant products have substantial phytanic acid levels in their red blood cells.

BACKGROUND: Humans and rodents with impaired phytanic acid (PA) metabolism can accumulate toxic stores of PA that have deleterious effects on multiple organ systems. Ruminants and certain fish obtain PA from the microbial degradation of dietary chlorophyll and/or through chlorophyll-derived precursors. In contrast, humans cannot derive PA from chlorophyll and instead normally obtain it only from meat, dairy, and fish products. RESULTS: Captive apes and Old world monkeys had significantly higher red blood cell (RBC) PA levels relative to humans when all subjects were fed PA-deficient diets. Given the adverse health effects resulting from PA over accumulation, we investigated the molecular evolution of thirteen PA metabolism genes in apes, Old world monkeys, and New world monkeys. All non-human primate (NHP) orthologs are predicted to encode full-length proteins with the marmoset Phyh gene containing a rare, but functional, GA splice donor dinucleotide. Acox2, Scp2, and Pecr sequences had amino acid positions with accelerated substitution rates while Amacr had significant variation in evolutionary rates in apes relative to other primates. CONCLUSIONS: Unlike humans, diverse captive NHPs with PA-deficient diets rich in plant products have substantial RBC PA levels. The favored hypothesis is that NHPs can derive significant amounts of PA from the degradation of ingested chlorophyll through gut fermentation. If correct, this raises the possibility that RBC PA levels could serve as a biomarker for evaluating the digestive health of captive NHPs. Furthermore, the evolutionary rates of the several genes relevant to PA metabolism provide candidate genetic adaptations to NHP diets.

The gene expression profiles of induced pluripotent stem cells from individuals with childhood cerebral adrenoleukodystrophy are consistent with proposed mechanisms of pathogenesis.

INTRODUCTION: X-linked adrenoleukodystrophy (X-ALD) is a complex disorder with variable expressivity that affects the nervous, adrenocortical and male reproductive systems. Although ABCD1 mutations are known to provide the genetic basis for X-ALD, its pathogenesis is not fully elucidated. While elevated very long chain fatty acid (VLCFA) levels in blood and reduced VLCFA catabolic activity in cultured fibroblasts are biomarkers used to identify ABCD1 mutation carriers, the roles peroxisomal lipid metabolism play in disease etiology are unknown. METHODS: Primary skin fibroblasts from two male patients with the childhood cerebral form of the disease (CCALD) caused by ABCD1 frameshift or missense mutations and three healthy donors were transduced with retroviral vectors expressing the OCT4, SOX2, KLF4 and c-MYC factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, DNA copy number variation, and genotyping analysis and tested for pluripotency through in vitro differentiation and teratoma formation. Saturated VLCFA (sVLCFA) and plasmalogen levels in primary fibroblasts and iPSCs from healthy donors as well as CCALD patients were determined through mass spectroscopy. RESULTS: Skin fibroblasts from CCALD patients and healthy donors were reprogrammed into validated iPSCs. Unlike fibroblasts, CCALD patient iPSCs show differentially expressed genes (DEGs) relevant to both peroxisome abundance and neuroinflammation. Also, in contrast to fibroblasts, iPSCs from patients showed no significant difference in sVLCFA levels relative to those from controls. In all cell types, the plasmalogen levels tested did not correlate with ABCD1 mutation status. CONCLUSION: Normal ABCD1 gene function is not required for reprogramming skin fibroblasts into iPSCs or maintaining pluripotency. Relative to DEGs found in fibroblasts, DEGs uncovered in comparisons of CCALD patient and control iPSCs are more consistent with major hypotheses regarding disease pathogenesis. These DEGs were independent of differences in sVLCFA levels, which did not vary according to ABCD1 mutation status. The highlighted genes provide new leads for pathogenic mechanisms that can be explored in animal models and human tissue specimens. We suggest that these iPSC resources will have applications that include assisting efforts to identify genetic and environmental modifiers and screening for therapeutic interventions tailored towards affected cell populations and patient genotypes.

Nonsense suppressor therapies rescue peroxisome lipid metabolism and assembly in cells from patients with specific PEX gene mutations.

Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies.

mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft.

BACKGROUND: Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted rho0 counterparts grown in culture and as tumor xenografts in immune-deficient mice. RESULTS: Cultured A549 rho0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways. CONCLUSION: We have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells.

Synthesis and anticancer properties of water-soluble zinc ionophores.

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 micromol/kg (300 micromol/m(2)) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1-responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents.

Biochemical and genetic evaluation of the role of AMP-activated protein kinase in polysaccharide storage myopathy in Quarter Horses.

OBJECTIVE: To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses. ANIMALS: 30 PSSM-affected and 30 unaffected (control) Quarter Horses. PROCEDURES: By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK gamma subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA. RESULTS: No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK gamma subunit genes were also not associated with PSSM. CONCLUSIONS AND CLINICAL RELEVANCE: These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds.

OBJECTIVE: To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation. ANIMALS: 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed. PROCEDURES: Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype. RESULTS: Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait. CONCLUSIONS AND CLINICAL RELEVANCE: It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.

Inheritance of recurrent exertional rhabdomyolysis in thoroughbreds.

OBJECTIVE: To develop a diagnostic test for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds that relied on in vitro contracture of muscle biopsy specimens and determine whether the inheritance pattern of RER diagnosed on the basis of this contracture test was consistent with an autosomal dominant trait. DESIGN: Clinical trial. ANIMALS: 8 adult horses with RER and 16 control adult horses for development of the contracture test; 23 foals for inheritance of RER. PROCEDURE: External intercostal muscle biopsy specimens from the 24 adult horses were tested for contracture in response to halothane and caffeine, and criteria for a positive test result were determined. These criteria were then applied to results for the 23 foals to determine whether they had RER. Simple segregation analysis was performed to determine whether results were consistent with a dominant pattern of inheritance. RESULTS: Results of the contracture test were positive for 5 of the 12 colts and 4 of the 11 fillies. Results of segregation analysis were consistent with an auto-. somal dominant pattern of inheritance. Two sires with RER produced colts with RER, supporting the hypothesis that RER had an autosomal, rather than an X-linked, inheritance pattern. In addition, in 1 instance, an unaffected colt was produced by 2 affected parents, which was not consistent with a recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: Although the expression of the RER trait is influenced by sex, temperament, and diet, among other factors, results from the in vitro muscle contracture test and this breeding trial suggest that RER in Thoroughbreds can be modeled as a genetic trait with an autosomal dominant pattern of inheritance.