Biology subjects

Hall, M. D.

Publications and source records attributed to Hall, M. D..

6 recordsLinked to original sources

bioRxiv2018

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, against four normal cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitutes a valuable resource for the scientific community and provides insight on the extent of cytotoxic compounds in screening libraries, identifying and avoiding compounds with cytotoxicity during high-throughput screening campaigns.

cell biology↗

bioRxiv2018

Resource-dependent dispersal and the irrelevance of extra-patch information for Daphnia carinata

Dispersal is fundamental to population dynamics and it is increasingly apparent that, despite most models treating dispersal as a constant, many organisms make dispersal decisions based upon information gathered from the environment. Ideally, organisms would make fully informed decisions, with knowledge of both intra-patch conditions (conditions in their current location) and extra-patch conditions (conditions in alternative locations). Acquiring information is energetically costly however, and extra-patch information will typically be costlier to obtain than intra-patch information. As a consequence, theory suggests that organisms will often make partially informed dispersal decisions, utilising intra-patch information only. We test this proposition in an experimental two-patch system using populations of the aquatic crustacean, Daphnia carinata. We manipulated conditions (food availability) in the populations home patch, and in its alternative patch. We found that D. carinata made use of intra-patch information (resource limitation in the home patch induced a ten-fold increase in dispersal probability) but made no use of extra-patch information (resource limitation in the alternative patch did not affect dispersal probability). Our work highlights the very large influence that information can have on dispersal probability, but also that dispersal decisions will often be made in only a partially informed manner. The magnitude of the response we observed also adds to the growing chorus that condition-dependence may be a significant driver of variation in dispersal.

ecology↗

bioRxiv2018

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a significant improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling both in vitro and in vivo assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells.

cancer biology↗

bioRxiv2017

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

cancer biology↗

bioRxiv2017

Procedures for enumerating and uniformly sampling transmission trees for a known phylogeny

One approach to the reconstruction of infectious disease transmission trees from pathogen genomic data has been to use a phylogenetic tree, reconstructed from pathogen sequences, and annotate its internal nodes to provide a reconstruction of which host each lineage was in at each point in time. If only one pathogen lineage can be transmitted to a new host (i.e. the transmission bottleneck is complete), this corresponds to partitioning the nodes of the phylogeny into connected regions, each of which represents evolution in an individual host. These partitions define the possible transmission trees that are consistent with a given phylogenetic tree. However, the mathematical properties of the transmission trees given a phylogeny remain largely unexplored. Here, we describe a procedure to calculate the number of possible transmission trees for a given phylogeny, and we show how to uniformly sample from these transmission trees. The procedure is outlined for situations where one sample is available from each host and trees do not have branch lengths, and we also provide extensions for incomplete sampling, multiple sampling, and the application to time trees in a situation where limits on the period during which each host could have been infected are known. The sampling algorithm is available as an R package (STraTUS).

evolutionary biology↗

bioRxiv2017

Assessing Inhibitors Of Mutant Isocitrate Dehydrogenase Using A Suite Of Pre-Clinical Discovery Assays

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation and secretion of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). Here, a panel of mIDH inhibitors were systematically profiled using biochemical, cell-based, and tier-one ADME techniques. We quantified the biochemical effect of each inhibitor on mIDH1 (R132H and R132C) and mIDH2 (R172Q). The effect of these inhibitors on 2-HG concentrations in seven cell lines representing five different IDH1 mutations in both 2D and 3D cell cultures was assessed. Target engagement of these inhibitors was analyzed utilizing cellular thermal shift assays (CETSA), the effects of inhibitors on reversing 2-HG-induced block on leukemic cellular differentiation. We conclude from our mIDH1 assay panel that AG-120 and a Novartis inhibitor exhibited excellent activity in all biochemical and most cellular assays. While AG-120 has superior DMPK properties, it lacks efficacy a leukemic differentiation model. In conclusion, we present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors.

cancer biology↗