Dominic Hoepfner

• Prof. Ph.D.
• Director at Novartis Biomedical Research

Metabolic dysfunction-associated steatotic liver disease (MASLD)-characterized by excess accumulation of fat in the liver-now affects one third of the world's population. As MASLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To...

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wid...

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcript...

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its t...

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory m...

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental 10 regulator that functions in mammalian stem cell reprogramming, brain development and 11 cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them 12 through molecular mechanisms that await identification. Here, we uncover that TRIM71 13 sile...

The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yea...

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic pur...

Genome-wide CRISPR phenotypic screens are clarifying many fundamental biological phenomena. While pooled screens can be used to study selectable features, arrayed CRISPR libraries extend the screening territory to cell-nonautonomous, biochemical and morphological phenotypes. Using a novel high-fidelity liquid-phase plasmid cloning technology, we ge...

Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, h...

BET bromodomain inhibitors hold promise as therapeutic agents in diverse indications, but their clinical progression has been challenging and none have received regulatory approval. Early clinical trials in cancer have shown heterogeneous clinical responses, development of resistance and adverse events. Increased understanding of their mechanism(s)...

Natural Products (NPs) are molecular' special equipment ' that impart survival benefits on their producers in nature. Due to their evolved functions to modulate biology these privileged metabolites are substantially represented in the drug market and are continuing to contribute to the discovery of innovative medicines such as the recently approved...

Human organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Due to the inherent multicellular complexity, interrogation by systematic genetic methodologies is challenging. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of genome editing, longi...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-con...

Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is sho...

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to...

Selective and specific inhibitors of Plasmodium falciparum lysyl‐tRNA synthetase represent promising therapeutic antimalarial avenues. Cladosporin was identified as a potent P. falciparum lysyl‐tRNA synthetase inhibitor, with an activity against parasite lysyl‐tRNA synthetase >100‐fold more potent than that of the activity registered against the hu...

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic re...

Gene knockout and knockdown strategies have been immensely successful probes of gene function, but small molecule inhibitors (SMIs) of gene products allow much greater time resolution and are particularly useful when the targets are essential for cell replication or survival. SMIs also serve as lead compounds for drug discovery. However, discovery...

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-res...

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl‐tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostl...

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compou...

Gossypol is an inhibitor of eukaryotic cells with an undetermined mode of action. Here we show that the chemogenomic profile of gossypol is strikingly similar to that of the iron chelators deferasirox and desferricoprogen. Iron import channels Fet1 and Fet3 are prominent in all three profiles. Furthermore, yeast inhibited by gossypol and deferasiro...

Members of the diazeniumdiolate class of natural compounds show potential for drug development because of their antifungal, antibacterial, antiviral, and antitumor activities. Yet, their biosynthesis has remained elusive to date. Here, we identify a gene cluster directing the biosynthesis of the diazeniumdiolate compound fragin in Burkholderia ceno...

Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the ident...

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...

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-ce...

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid...

The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization in vitro. Applying orthogonal genomic and proteomics approaches we now show for the first time that Chivosazole F exerts its effect by directly interacting with actin and demonstrate the cellular impact of Chivosazole F in an unb...

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue® (resazurin) or by direct optical measurement of...

Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound a...

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phos...

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target int...

Protein comparison 2. Alignment of ScSec14p, CnSec14-1p and CnSec14-2p.The CLUSTAL 2.1 alignment of S. cerevisiae Sec14p and C. neoformans CnSec14-1p and CnSec14-2p is shown. Red boxes represent conserved amino acids involved in NGx04 binding in the three proteins, blue boxes represent non-conserved amino acids. (EPS)

Protein comparison 1. Alignment of CnSec14-1p, ScSec14p and HsSec14-2p. The CLUSTAL 2.1 alignment of C. neoformans, S. cerevisiae and H. sapiens Sec14 proteins is shown and the NGx04 resistance conferring amino acids highlighted in red. (EPS)

Anti-fungal resistance testing. Inhibition of fluconazole resistant C. neoformans. NGx04 and close analogues were tested against the FLU-resistant C. neoformans BPY22 [45] in a 48 h proliferation assay and MIC-2 (50% inhibition) and MIC-0 (no visible fungal growth) determined. Values are given in μM as mean of tree replicates. Fluconazole was inclu...

Resistance mapping. Functional variomics screen for NGx04-resistant mutants. Statistics of the primary screen (genome-wide pool) and the secondary screen (SEC14 pool) are displayed. 1.5 OD600 of the corresponding pools were plated on a 15 cm SD agar dish containing 150 μM NGx04, resistant clones isolated and analyzed by sequencing of the expressed...

Effectiveness of growth inhibition. Fungicidal vs fungistatic effects of ergolines. Ergolines were tested at 16 μg/ml final concentration for 24 hrs. against FLU-sensitive (DSY4982) and FLU-resistant (BPY22 and BPY17) C. neoformans isolates [45] for fungicidal activity and Colony Forming Units in presence (NGxXX) and in absence (inoculum) of compou...

Evolutionary divergence. Complementation of yeast SEC14. A heterozygous SEC14/sec14 S. cerevisiae strain was transformed with the human SEC14 homologue (XP_011510441.1) under the control of the ADH1 promotor. Transformants were isolated, subjected to sporulation, tetrads dissected and analysed for viability. 18 tetrads are shown (horizontal). Only...

Physicochemical properties of ergolines. Rat liver microsome and artificial membrane permeability assay. NGx04 and close analogues were tested for metabolic stability (CYP MetCL) and artificial membrane permeability (logPAMPA) in duplicates. Value pairs are color coded and binning is indicated. (EPS)

Anti-fungal testing. Fungal pathogen assay. NGx04 and close analogues were tested against C. albicans (ATCC 24433), A. fumigatus (ATCC MYA-3627), R. oryzae (ATCC MYA-4621) and F. solani (ATCC MYA-3636) in a 72 h proliferation assay and MIC-2 (50% inhibition) and MIC-0 (no visible fungal growth) determined. Values are given in μM as mean of tree rep...

Limited detoxification capacity often directs aggregation-prone, potentially hazardous misfolded proteins to be deposited in designated cytosolic compartments known as "aggresomes". The roles of aggresomes as cellular quality control centers, and the cellular origin of the deposits contained within these structures, remain to be characterized. Here...

Drug development of promising hits from phenotypic screens is o en hampered due to a lack of informa on on the cellular target or the mode of ac on of the compound. An e cient method for target iden ca on is chemogenomic pro ling in the surrogate model Saccharomyces cerevisiae. Here we brie y review the progress of this technology, give some succes...

High-throughput screening (HTS) is an integral part of early drug discovery. Herein, we focused on those small molecules in a screening collection that have never shown biological activity despite having been exhaustively tested in HTS assays. These compounds are referred to as 'dark chemical matter' (DCM). We quantified DCM, validated it in qualit...

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falci...

Phenotypic screens are effective starting points to identify compounds with desirable activities. To find novel antifungals, we conducted a phenotypic screen in Saccharomyces cerevisiae and identified two discrete scaffolds with good growth inhibitory characteristics. Lack of broad-spectrum activity against pathogenic fungi called for directed chem...

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae-Erg26p, Homo sapiens-NSDHL (NAD(P) dependent steroid dehydrogenase-...

From the start of the pharmaceutical research natural products played a key role in drug discovery and development. Over time many discoveries of fundamental new biology were triggered by the unique biological activity of natural products. Unprecedented chemical structures, novel chemotypes, often pave the way to investigate new biology and to expl...

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-f...

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small...

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those o...

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively f...

We found two errors in our previous published paper [1]. Figure 4A has a mistake in the units in the labels, where it shows mM instead of micromolar (μM). A correctly labeled Figure 4A ensues. In Figures 2 and 4, the size bar scale is micrometers (μm). We apologize for the inconvenience caused to our readers.

Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the...

A chemicogenetic screen was performed in budding yeast mutants that have a weakened replication stress response. This identified an inhibitor of target of rapamycin (TOR) complexes 1 and 2 that selectively enhances the sensitivity of sgs1Δ cells to hydroxyurea and camptothecin. More importantly, the inhibitor has strong synthetic lethality in combi...

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeas...

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence thes...

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway, however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a n...

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae...

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel al...

Systemic 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...

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metaboli...

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the mod...

IP3 signaling pathway. IP3 and DAG are produced by the cleavage of PIP2 by Plc1. IP3 is released from the membrane and is the precursor of all other inositol phosphates (IPs). The four inositol polyphosphate kinases (Ipk2/Arg82, Ipk1, Ksc1, and VIP1) further process IP3 and constitute a nuclear signaling pathway. The major functions affected by the...

MHO1 expression analysis. A summary of the published MHO1 microarray data is presented. Conditions that increase or decrease MHO1 expression are indicated in red or green, respectively. The data were taken from http://transcriptome.ens.fr/ymgv/. MHO1 is upregulated upon Msn2 overexpression leading to the hypothesis that MHO1 is a stress response ge...

Expression levels of MHO1 and PLC1 in response to different stress conditions. Microarray data from [19] analyzing the gene expression of yeast cells in response to environmental stress was used to identify conditions that increase expression levels of MHO1. The PLC1 expression levels in response to the same conditions are also shown. (TIF)

Spotting assay of wild-type and mho1Δ strains on various compounds. A wild-type and a mho1Δ strain were serial diluted and spotted on YPD plates (A) or SD complete plates (B), containing 800 µM CoCl2, 100 µM benomyl, or 100 µM rapamycin (A and B) and 800 µM NaCl, or HU 100 µM (B). No differences in growth between the mho1Δ and the wild-type strains...

MHO1 promoter analysis. (A) An analysis of the MHO1 promoter region (−1 bp to −500 bp from the START codon) revealed that there are two potential binding sites for Msn2/Msn4 (shown in green) and a potential UASino (inositol-sensitive upstream activating sequence) binding site. The latter is often present in promoters of genes encoding phospholipid,...

Genes involved in the cAMP/PKA/PLC pathway, which were tested with the mho1Δ strain; none were SL with mho1Δ. (DOCX)

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver-and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood-and liver-stage prolif-eration. Cladosporin, a fungal secondary metabolit...

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we discovered an unexpected role for yeast dynein light chain (Dyn2) in the NPC. Dyn2 is a previously undescribed nucleoporin that functions as molecular glue to dimerize and stabilize the Nup82-Nsp1-Nup159 complex, a module of the cytop...

Eukaryotic cells contain functionally distinct, membrane enclosed compartments called organelles. Here we like to address two questions concerning this architectural lay out. How did this membrane complexity arise during evolution and how is this collection of organelles maintained in multiplying cells to ensure that new cells retain a complete set...

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we fol...