A study comparing five different cAMP detection technologies in terms of sensitivity, robustness, and feasibility for HTS is presented. In this report, the following methods are described: a nonhomogeneous DELFIA, and the homogeneous methods based on time-resolved fluorescence (HTRF), luminescent singlet oxygen channeling or ALPHAScreen, FP, and high-affinity enzyme complementation. DELFIA had the highest sensitivity, whereas ALPHAScreen and HTRF shared several advantages, including high sensitivity, broad dynamic range, and minimal reagent addition steps. For G(s)-coupled antagonist screens, we found HTRF and ALPHAScreen the more sensitive and HTS-compatible techniques.

No abstract

High-content screening (HCS) is a powerful technique for monitoring phenotypic responses to treatments on a cellular and subcellular level. Cellular phenotypes can be characterized by multivariate image readouts such as shape, intensity, or texture. The corresponding feature vectors can thus be defined as HCS fingerprints that serve as a powerful biological compound descriptor. Therefore, clustering or classification of HCS fingerprints across compound treatments allows for the identification of similarities in protein targets or pathways. We developed an HCS-based profiling panel that serves as basis for characterizing the mode of action of compounds. This panel measures phenotypic effects in six different compartments of U-2OS cells, namely the nucleus, the cytoplasm, the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. We profiled a set of 2,725 well-annotated compounds and clustered their corresponding HCS fingerprints to establish links between predominant cellular phenotypes and cellular processes and protein targets. We found various different clusters enriched for individual targets (e.g., HDAC, HSP90, TOP1, HMGCR, TUB), signaling pathways (e.g., PIK3/AKT/mTOR), or gene sets associated with diseases (e.g., psoriasis, leukemia). Based on this clustering we were able to identify novel compound-target associations for selected compounds such as a submicromolar inhibitory activity of Silmitasertib (a casein kinase inhibitor) on PI3K and mTOR.

bSystemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

No abstract

Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.

No abstract

Tumor heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is well-known to affect the efficacy of anti-cancer drugs, most personalized treatment approaches do not account for intratumor heterogeneity. We addressed this issue by studying the heterogeneity of lymph node-derived B cell non-Hodgkin lymphoma (B-NHL) by single cell RNA-sequencing (scRNA-seq) and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subclones and compared their drug response and genomic profiles. Malignant subclones of the same patient responded strikingly different to anti-cancer drugs ex vivo, which recapitulated subclone-specific drug sensitivity during in vivo treatment. Tumor infiltrating T cells represented the majority of non-malignant cells, whose gene expression signatures were similar across all donors, whereas the frequencies of T cell subsets varied significantly between the donors. Our data provide new insights into the heterogeneity of B-NHL and highlight the relevance of intratumor heterogeneity for personalized cancer therapies.