Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists.

ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed 'ERBBprofiler', in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib's preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.

Prieurianin/endosidin 1 is an actin-stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana.

Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.

Natural product-inspired cascade synthesis yields modulators of centrosome integrity.

In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1.

IFNα primes cancer cells for Fusicoccin-induced cell death via 14-3-3 PPI stabilization.

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.

Psoromic acid is a selective and covalent Rab-prenylation inhibitor targeting autoinhibited RabGGTase.

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 µM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.

Small-molecule inhibition of APT1 affects Ras localization and signaling.

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

Insight into Targeting Exon20 Insertion Mutations of the Epidermal Growth Factor Receptor with Wild Type-Sparing Inhibitors.

Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.

Anti-leukemic effect of CDK9 inhibition in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy characterized by chemotherapy resistance and a median survival of less than 2 years. Here, we investigated the pharmacological effects of the novel highly specific cyclin-dependent kinase 9 (CDK9) inhibitor LDC526 and its clinically used derivate atuveciclib employing primary T-PLL cells in an ex vivo drug sensitivity testing platform. Importantly, all T-PLL samples were sensitive to CDK9 inhibition at submicromolar concentrations, while conventional cytotoxic drugs were found to be largely ineffective. At the cellular level LDC526 inhibited the phosphorylation at serine 2 of the RNA polymerase II C-terminal domain resulting in decreased de novo RNA transcription. LDC526 induced apoptotic leukemic cell death through down-regulating MYC and MCL1 both at the mRNA and protein level. Microarray-based transcriptomic profiling revealed that genes down-modulated in response to CDK9 inhibition were enriched for MYC and JAK-STAT targets. By contrast, CDK9 inhibition increased the expression of the tumor suppressor FBXW7, which may contribute to decreased MYC and MCL1 protein levels. Finally, the combination of atuvecliclib and the BCL2 inhibitor venetoclax exhibited synergistic anti-leukemic activity, providing the rationale for a novel targeted-agent-based treatment of T-PLL.

Dual Inhibition of GSK3ß and CDK5 Protects the Cytoskeleton of Neurons from Neuroinflammatory-Mediated Degeneration In Vitro and In Vivo.

Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3ß is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3ß. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3ß in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.

Natural product-derived modulators of cell cycle progression and viral entry by enantioselective oxa Diels-Alder reactions on the solid phase.

The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired alpha,beta-unsaturated delta-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity.

Gasdermin D plays a vital role in the generation of neutrophil extracellular traps.

The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach.

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.

Development of selective, potent RabGGTase inhibitors.

Members of the Ras superfamily of small GTPases are frequently mutated in cancer. Therefore, inhibitors have been developed to address the acitivity of these GTPases by inhibiting their prenylating enzymes FTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I, only a handful of RabGGTase inhibitors have been developed. The most active RabGGTase inhibitor known until recently was an FTase inhibitor which hit RabGGTase as an off-target. We recently reported our efforts to tune the selectivity of these inhibitors toward RabGGTase. Here we describe an extended set of selective inhibitors. The requirements for selective RabGGTase inhibitors are described in detail, guided by multiple crystal structures. In order to relate in vitro and cellular activity, a high-throughput assay system to detect the attachment of [(3)H]geranylgeranyl groups to Rab was used. Selective RabGGTase inhibition allows the establishment of novel drug discovery programs aimed at the development of anticancer therapeutics.