Kendomycin Cytotoxicity against Bacterial, Fungal, and Mammalian Cells Is Due to Cation Chelation

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 shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.Peer reviewe

SpeedScreen, a Label-Free, Affinity-Based High-Throughput Screening Technology for the Discovery of Orphan Protein Ligands

A high-throughput screening method, tailored to the discovery of ligands for both known and orphan proteins, was developed and implemented into Novartis' lead discovery process. Neither labeling of target proteins nor labeling of screening compounds is required, as the ligands are affinity-selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small molecular weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in 96-well plate format. The protein fraction is then analyzed subsequently by liquid chromatography/mass spectrometry (LC/MS) for identification of the bound ligand(s). All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening (HTS) processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process. In addition to the screening of conventional compound libraries this technology can also be applied for the de-convolution of libraries originating from combinatorial chemistry efforts as well as complex natural extracts

Stendomycin selectively inhibits TIM23-dependent mitochondrial protein import

Stendomycin is an antifungal lipopeptide isolated from Streptomyces endus. In mammalian cells in culture (HeLa) Stendomycin triggers mitophagy causing significant and rapid loss of the mitochondrial marker Tom20. Counter screening suggests that this mitophagy is triggered by the uncoupling of mitochondrial membrane potential. Interestingly, we show that in S. cerevisiae Stendomycin inhibits Tim17 / Tim23-dependent, but not Tim22-dependent, protein import into mitochondria via the inner membrane translocon. Only TIM17 and TIM23 heterozygotes are significantly sensitive to Stendomycin in S. cerevisiae haploinsufficiency profiling. Stendomycin causes a rapid loss of normal mitochondrial morphology visualized with GFP-Cox4 or mitotracker. Selection of mutants resistant to Stendomycin in a growth assay reveals two point mutations in Tim17, Tim17G20D and Tim17L122W. These mutations increase significantly the IC50 of stendomycin from 0.8 uM to 2.4 uM (L122W) or 2.6 uM (G20S). The mitochondrial morphological effects of Stendomycin are abrogated in cells carrying Tim17G20D. In vitro assessment of protein import into intact purified mitochondria revealed that at a dose that had no effect on membrane potential the import of Tim17/Tim23 cargo, but not Tim22 cargo, was inhibited. The Tim17G20D mutant significantly abrogated the effects of Stendomycin on Tim17/Tim23-dependent import. In similar in vitro conditions membrane potential modulation was observed with doses 1000x higher than those required for complete Tim17/Tim23 import blockage. This effect on membrane potential is independent of the Tim17 compound resistant mutants. Thus Stendomycin has a dual action, first as a specific Tim17 / Tim23 inhibitor, and second as an mitochondrial membrane potential uncoupler apparently independent of Tim17 / Tim23

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands.

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process

SpeedScreen: The "missing link" between genomics and lead discovery.

SpeedScreen is a novel, label-free, in-solution, affinity-based selection methodology for high-throughput screening (HTS) developed at Novartis Pharma. The SpeedScreen protocol comprises in-solution affinity selection, followed by size exclusion chromatography in combination with microbore-liquid-chromatography/electrospray-ionization mass spectrometry (micro-LC/ESI-MS). The authors describe the basic concept behind assay development, HTS, and data analysis with the SpeedScreen technology. Advantages and limitations of SpeedScreen compared to alternative screening technologies are discussed, and an example is given from a SpeedScreen campaign applying this innovative affinity selection concept in HTS

The Target of Rapamycin Complex 2-dependent phosphorylation of Pan1 by Akl1 controls endocytosis dynamics.

The Target of Rapamycin Complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. To do this TORC2 regulates diverse processes including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization, and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here we report that Fpk1 and Fpk2 mediate TORC2 signals to actin polarization, but not endocytosis, via the amino-phospholipid flippases. To search for novel targets of the flippases kinases we exploited the fact that Fpk1 prefers to phosphorylate Ser residues found within the sequence RXS[L/Y] [D/E], which is present ~90 times in the yeast proteome. We observed that 25 of these are phosphorylated by Fpk1 in vitro. We focused on the Ark/Prk family kinase Akl1, as it has been previously implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to acutely inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which reduces phosphorylation of Pan1 and other endocytic coat proteins and ultimately contributes to an arrest of endocytosis. These results demonstrate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled by separate pathways that bifurcate at the level of the flippase kinases

Advantages and challenges of phenotypic screens: The identification of two novel antifungal geranylgeranyltransferase I inhibitors

Phenotypic screens are still the most effective starting points for compounds with desirable activities. To identify novel antifungal leads we have conducted a phenotypic screen in the yeast Saccharomyces cerevisiae and identified two different scaffolds with good growth inhibitory characteristics. Lack of broad spectrum antifungal activity against pathogenic fungi raised the question about the modulated target as required for directed chemical compound optimization. Chemogenomic profiling identified effects on geranylgeranyltransferase I (GGTase I), an enzyme that prenylates proteins involved in cell signaling like Cdc42p and Rho1p. Raising resistant mutants against both compounds confirmed the target hypothesis and allowed mapping of the compound binding site to the substrate binding pocket. Differential resistance conferring mutations and substrate competition for only one chemotype demonstrated a diverse binding mode for the two chemotypes. Exchange of the S.cerevisiae GGTase I complex by that of Candida albicans abolished growth inhibitory activity of both compounds thus confirming the identified target as well as the observed narrow antifungal spectrum. Reported lack of essentiality of this prenylation pathway in pathogenic species challenges the therapeutic value of these leads and demonstrates the importance of an integrated target identification platform following a phenotypic screen

Evidence for ligand-independent transcriptional activation of the human estrogen-related receptor alpha (ERRalpha): crystal structure of ERRalpha ligand binding domain in complex with peroxisome proliferator-activated receptor coactivator-1alpha.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor alpha (ERRalpha, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERRalpha LBD, solved to a resolution of 2.5 A, and the first structure of a PGC-1alpha complex. The putative ligand binding pocket (LBP) of ERRalpha is almost completely occupied by side chains, in particular with the bulky side chain of Phe328 (corresponding to Ala272 in ERRgamma and Ala350 in estrogen receptor alpha). Therefore, a ligand of a size equivalent to more than approximately 4 carbon atoms could only bind in the LBP, if ERRalpha would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERRalpha. The interactions of PGC-1alpha with ERRalpha also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1alpha peptide containing this nuclear box motif from the L3 site binds ERRalpha LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1alpha are present

TORC2 signaling pathway guarantees genome stability in the face of DNA strand breaks

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 combination with either the break-inducing antibiotic Zeocin or ionizing radiation, independent of the strain background. Lethality correlates with a rapid fragmentation of chromosomes that occurs only when TORC2, but not TORC1, is repressed. Genetic inhibition of Tor2 kinase, or its downstream effector kinases Ypk1/Ypk2, conferred similar synergistic effects in the presence of Zeocin. Given that Ypk1/Ypk2 controls the actin cytoskeleton, we tested the effects of actin modulators latrunculin A and jasplakinolide. These phenocopy TORC2 inhibition on Zeocin, although modulation of calcineurin-sensitive transcription does not. These results implicate TORC2-mediated actin filament regulation in the survival of low levels of DNA damage

Direct Interaction of Chivosazole F with Actin Elicits Cell Responses Similar to Latrunculin A but Distinct from Chondramide

The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization <i>in vitro</i>. 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 unbiased, genome-wide context in yeast and in mammalian cells. Furthermore, mutation-based resistance mapping identifies two SNPs located in the putative Chivosazole F binding site of actin. Comparing chemogenomic profiles and responses to the Chivosazole F-resistant SNPs shows a partially conserved mechanism of action for Chivosazole F and Latrunculin A, but clear divergence from Chondramide. In addition, C14orf80 is an evolutionarily highly conserved ORF, lacking any functional annotation. As editing of C14orf80 leads to Chivosazole F hyper-resistance, we propose a function for this gene product in counteracting perturbation of actin filaments