Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following ...perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.
The Front Cover shows the synthesis of various quinoxalines in nothing but “hot water”. Aromatic quinoxalines can be synthesized in down to 10 min from the starting compounds in stoichiometric ratio ...in water at 230 °C through microwave‐assisted hydrothermal synthesis. The synthetic approach is tolerant to a broad array of functional groups and can be combined with protecting group strategies. A large‐scale computational comparison of all to date existing syntheses towards the presented quinoxalines shows that the hydrothermal approach is the greenest. More information can be found in the Full Paper by F. A. Amaya‐García et al.
Targeted protein degradation (TPD) is a new pharmacology based on small-molecule degraders that induce proximity between a protein of interest (POI) and an E3 ubiquitin ligase. Of the approximately ...600 E3s encoded in the human genome, only around 2% can be co-opted with degraders. This underrepresentation is caused by a paucity of discovery approaches to identify degraders for defined E3s. This hampers a rational expansion of the druggable proteome and stymies critical advancements in the field, such as tissue- and cell-specific degradation. Here, we focus on dynamic NEDD8 conjugation, a post-translational, regulatory circuit that controls the activity of 250 cullin RING E3 ligases (CRLs). Leveraging this regulatory layer enabled us to develop a scalable assay to identify compounds that alter the interactome of an E3 of interest by tracing their abundance after pharmacologically induced auto-degradation. Initial validation studies are performed for CRBN and VHL, but proteomics studies indicate broad applicability for many CRLs. Among amenable ligases, we select CRLDCAF15 for a proof-of-concept screen, leading to the identification of a novel DCAF15-dependent molecular glue degrader inducing the degradation of RBM23 and RBM39. Together, this strategy empowers the scalable identification of degraders specific to a ligase of interest.
Here, the hydrothermal synthesis (HTS) of 2,3‐diarylquinoxalines from 1,2‐diketones and o‐phenylendiamines (o‐PDAs) was achieved. The synthesis is simple, fast, and generates high yields, without ...requiring any organic solvents, strong acids or toxic catalysts. Reaction times down to <10 min without decrease in yield could be achieved through adding acetic acid as promoter, even for highly apolar biquinoxalines (yield >90 % in all cases). Moreover, it was shown that HTS has high compatibility: (i) hydrochlorides, a standard commercial form of amines, could be used directly as combined amine source and acidic catalyst, and (ii) Boc‐diprotected o‐PDA could be directly employed as substrate that underwent HT deprotection. A systematic large‐scale computational comparison of all reported syntheses of the presented quinoxalines from the same starting compounds showed that this method is more environmentally friendly and less toxic than all existing methods and revealed generic synthetic routes for improving reaction yields. Finally, the application of the synthesized compounds as fluorescent dyes for cell staining was explored.
In hot water: 2,3‐Diarylquinoxalines and biquinoxalines are synthesized in a rapid, efficient and green fashion in nothing but high‐temperature water. Through a large‐scale computational analysis, the herein reported reaction conditions are compared with all reported syntheses of the presented compounds.
The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of ...effective therapeutic strategies is an urgent clinical need.
We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC.
A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen.
Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line.
Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patient-derived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patient-derived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8).
The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care.
We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment.
Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a ...lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.
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Paralog-dependent isogenic cell assay exploits genetic interactions
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Isogenic cells with individual dependency on SLC16A1, SLC16A3, SLC16A7, and SLC16A8
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Chemical screening uncovering two series of compounds selective to SLC16A3
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Development of SLC16A3 chemical probe
Dvorak et al. report the development of a chemical probe targeting SLC16A3 using an assay system relying on a series of isogenic cell lines with engineered dependency on individual paralogs of SLC16A3. This system overcomes functional redundancies that hamper the development of specific cell-based screening assays.
Invited for this month′s cover is the group of Miriam Unterlass at the Technische Universität Wien and the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences. The image ...illustrates the synthesis of quinoxalines in “hot water” and the large‐scale computational comparison of all existing syntheses of these quinoxalines. The Full Paper itself is available at 10.1002/cssc.202100433.
“We love hydrothermal synthesis…” This and more about the story behind the research that inspired the Cover image is presented in the Cover Profile. Read the full text of the corresponding research at 10.1002/cssc.202100433. View the Front Cover here: 10.1002/cssc.202100608.
Molecular glue degraders (MGDs) are small molecules that degrade proteins of interest via the ubiquitin–proteasome system. While MGDs were historically discovered serendipitously, approaches for MGD ...discovery now include cell-viability-based drug screens or data mining of public transcriptomics and drug response datasets. These approaches, however, have target spaces restricted to the essential proteins. Here we develop a high-throughput workflow for MGD discovery that also reaches the nonessential proteome. This workflow begins with the rapid synthesis of a compound library by sulfur(VI) fluoride exchange chemistry coupled to a morphological profiling assay in isogenic cell lines that vary in levels of the E3 ligase CRBN. By comparing the morphological changes induced by compound treatment across the isogenic cell lines, we were able to identify FL2-14 as a CRBN-dependent MGD targeting the nonessential protein GSPT2. We envision that this workflow would contribute to the discovery and characterization of MGDs that target a wider range of proteins.
Chlamydia trachomatis (Ct) is the most common cause for bacterial sexually transmitted infections (STIs) worldwide with a tremendous impact on public health. With the aim to unravel novel targets of ...the chlamydia life cycle, we screen a compound library and identify 28 agents to significantly reduce Ct growth. The known anti-infective agent pentamidine—one of the top candidates of the screen—shows anti-chlamydia activity in low concentrations by changing the metabolism of host cells impairing chlamydia growth. Furthermore, it effectively decreases the Ct burden upon local or systemic application in mice. Pentamidine also inhibits the growth of Neisseria gonorrhea (Ng), which is a common co-infection of Ct. The conducted compound screen is powerful in exploring antimicrobial compounds against Ct in a medium-throughput format. Following thorough in vitro and in vivo assessments, pentamidine emerges as a promising agent for topical prophylaxis or treatment against Ct and possibly other bacterial STIs.
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•A compound screen identifies 28 non-antibiotics inhibiting Chlamydia trachomatis•Pentamidine inhibits chlamydia replication indirectly via the host cells•Systemic and intrauterine pentamidine treatment decreases chlamydia burden in mice•Pentamidine is a promising candidate for prophylaxis against bacterial STIs
As the numbers of sexually transmitted infections are rising, innovative prophylactic measures are needed. Knapp et al. performed a medium-throughput compound screen to identify new drugs inhibiting Chlamydia trachomatis growth in cell lines. The top hits were tested in a Chlamydia trachomatis mouse model for their ability to prevent infection.
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