Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.

In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.

Pyruvate anaplerosis is a targetable vulnerability in persistent leukaemic stem cells.

Deregulated oxidative metabolism is a hallmark of leukaemia. While tyrosine kinase inhibitors (TKIs) such as imatinib have increased survival of chronic myeloid leukaemia (CML) patients, they fail to eradicate disease-initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays, we generate multi-omics datasets that offer unique insight into metabolic adaptation and nutrient fate in patient-derived CML LSCs. We demonstrate that LSCs have increased pyruvate anaplerosis, mediated by increased mitochondrial pyruvate carrier 1/2 (MPC1/2) levels and pyruvate carboxylase (PC) activity, in comparison to normal counterparts. While imatinib reverses BCR::ABL1-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics reveals that deregulated pyruvate anaplerosis is not affected by imatinib. Encouragingly, genetic ablation of pyruvate anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that MSDC-0160, a clinical orally-available MPC1/2 inhibitor, inhibits pyruvate anaplerosis and targets imatinib-resistant CML LSCs in robust pre-clinical CML models. Collectively these results highlight pyruvate anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient-derived samples.

Arginine dependency is a therapeutically exploitable vulnerability in chronic myeloid leukaemic stem cells.

To fuel accelerated proliferation, leukaemic cells undergo metabolic deregulation, which can result in specific nutrient dependencies. Here, we perform an amino acid drop-out screen and apply pre-clinical models of chronic phase chronic myeloid leukaemia (CML) to identify arginine as a nutrient essential for primary human CML cells. Analysis of the Microarray Innovations in Leukaemia (MILE) dataset uncovers reduced ASS1 levels in CML compared to most other leukaemia types. Stable isotope tracing reveals repressed activity of all urea cycle enzymes in patient-derived CML CD34+ cells, rendering them arginine auxotrophic. Thus, arginine deprivation completely blocks proliferation of CML CD34+ cells and induces significantly higher levels of apoptosis when compared to arginine-deprived cell lines. Similarly, primary CML cells, but not normal CD34+ samples, are particularly sensitive to treatment with the arginine-depleting enzyme, BCT-100, which induces apoptosis and reduces clonogenicity. Moreover, BCT-100 is highly efficacious in a patient-derived xenograft model, causing > 90% reduction in the number of human leukaemic stem cells (LSCs). These findings indicate arginine depletion to be a promising and novel strategy to eradicate therapy resistant LSCs.

Palladium-Catalyzed Aryldifluoromethylation of Aryl Halides with Aryldifluoromethyl Trimethylsilanes.

Diaryl difluoromethanes are valuable targets for medicinal chemistry because they are bioisosteres of diaryl ethers and can function as replacements for diaryl methane, ketone, and sulfone groups. However, methods to prepare diaryl difluoromethanes are scarce, especially methods starting from abundant aryl halides. We report the Pd-catalyzed aryldifluoromethylation of aryl halides with aryldifluoromethyl trimethylsilanes (TMSCF2 Ar). The reaction occurs when the catalyst contains a simple, but unusual, dialkylaryl phosphine ligand that promotes transmetallation of the silane. Computational studies show that reductive elimination following transmetallation occurs with a low barrier, despite the fluorine atoms on the α-carbon, due to coordination of the difluorobenzyl π-system to palladium. The co-development of a cobalt-catalyzed synthesis of the silanes broadened the scope of the process including several applications to the synthesis biologically relevant diaryl difluoromethanes.

ULK1 inhibition promotes oxidative stress-induced differentiation and sensitizes leukemic stem cells to targeted therapy.

Inhibition of autophagy has been proposed as a potential therapy for individuals with cancer. However, current lysosomotropic autophagy inhibitors have demonstrated limited efficacy in clinical trials. Therefore, validation of novel specific autophagy inhibitors using robust preclinical models is critical. In chronic myeloid leukemia (CML), minimal residual disease is maintained by persistent leukemic stem cells (LSCs), which drive tyrosine kinase inhibitor (TKI) resistance and patient relapse. Here, we show that deletion of autophagy-inducing kinase ULK1 (unc-51­like autophagy activating kinase 1) reduces growth of cell line and patient-derived xenografted CML cells in mouse models. Using primitive cells, isolated from individuals with CML, we demonstrate that pharmacological inhibition of ULK1 selectively targets CML LSCs ex vivo and in vivo, when combined with TKI treatment. The enhanced TKI sensitivity after ULK1-mediated autophagy inhibition is driven by increased mitochondrial respiration and loss of quiescence and points to oxidative stress­induced differentiation of CML LSCs, proposing an alternative strategy for treating patients with CML.

Direct Observation of Diastereomeric α-C-Bound Enolates during Enantioselective α-Arylations: Synthesis, Characterization, and Reactivity of Arylpalladium Fluorooxindole Complexes.

The Pd-catalyzed asymmetric α-arylation of carbonyl compounds is a valuable strategy to form benzylic stereocenters. However, the origin of the stereoselectivity of these reactions is poorly understood, and little is known about the reactivity of the putative diastereomeric arylpalladium enolate intermediates. To this end, we report the synthesis and characterization of a series of diphosphine-ligated arylpalladium fluoroenolate complexes, including complexes bearing a metal-bound, stereogenic carbon and an enantioenriched chiral diphosphine ligand. These complexes reductively eliminate to form chiral α-aryl-α-fluorooxindoles with enantioselectivities and rates that are relevant to those of the catalytic process with SEGPHOS as the ancillary ligand. Kinetic studies showed that the rate of reductive elimination is slightly slower than the rate of epimerization of the intermediate, causing the reductive elimination step to impart the greatest influence on the enantioselectivity. DFT calculations of these processes are consistent with these experimental rates and suggest that the minor diastereomer forms the major enantiomer of the product. The rates of reductive elimination from complexes containing a variety of electronically varied aryl ligands revealed the unusual trend that complexes bearing more electron-rich aryl ligands react faster than those bearing more electron-poor aryl ligands. Noncovalent Interaction (NCI) and Natural Bond Orbital (NBO) analyses of the transition-state structures for reductive elimination from the SEGPHOS-ligated complexes revealed key donor-acceptor interactions between the Pd center and the fluoroenolate fragment. These interactions stabilize the pathway to the major product enantiomer more strongly than they stabilize that to the minor enantiomer.

Deazapurine Nucleoside Analogues for the Treatment of Trichomonas vaginalis.

Trichomoniasis is the most common nonviral sexually transmitted disease in humans, but treatment options are limited. Here, we report a resorufin-based drug sensitivity assay for high-throughput microplate-based screening under hypoxic conditions. A 5203-compound enamine kinase library and several specialized compound series were tested for the inhibition of Trichomonas growth at 10 µM with Z' values of >0.5. Hits were rescreened in serial dilution to establish an IC50 concentration. A series of 7-substituted 7-deazaadenosine analogues emerged as highly potent anti-T. vaginalis agents, with EC50 values in the low double digit nanomolar range. These analogues exhibited excellent selectivity indices. Follow-up medicinal chemistry efforts identified an optimal ribofuranose and C7 substituent. Several nucleosides rapidly cleared cultures of T. vaginalis at a concentrations of just 2 × EC50. Preliminary in vivo evaluation in a murine trichomoniasis model (Tritrichomonas foetus) revealed promising activity upon topical administration, validating purine nucleoside analogues as a new class of antitrichomonal agents.

Unusual Electronic Effects of Ancillary Ligands on the Perfluoroalkylation of Aryl Iodides and Bromides Mediated by Copper(I) Pentafluoroethyl Complexes of Substituted Bipyridines.

Several perfluoroalkylcopper compounds have been reported previously that serve as reagents or catalysts for the perfluoroalkylation of aryl halides. However, the relationships between the reactivity of such complexes and the electronic properties of the ancillary ligands are unknown, and such relationships are not well-known in general for copper complexes that mediate or catalyze cross coupling. We report the synthesis and characterization of a series of pentafluoroethylcopper(I) complexes ligated by bipyridine ligands possessing varied electronic properties. In contrast to the limited existing data on the reactivity of L2Cu(I)-X complexes bearing amine and pyridine-type ligands in Ullmann-type aminations with aryl halides, the reactions of aryl halides with pentafluoroethylcopper(I) complexes bearing systematically varied bipyridine ligands were faster for complexes bearing less electron-donating bipyridines than for complexes bearing more electron-donating bipyridines. Analysis of the rates of reaction and the relative populations of the neutral complexes [(R2bpy)CuC2F5] and ionic complexes [(R2bpy)2Cu][Cu(C2F5)2] formed by these reagents in solution suggests that this effect of electronics on the reaction rate results from an unusual trend of faster oxidative addition of aryl halides to [(R2bpy)CuC2F5] complexes containing less electron-donating R2bpy ligands than to those containing more electron-donating R2bpy ligands.

An in vitro model for studying CNS white matter: functional properties and experimental approaches.

The normal development and maintenance of CNS white matter, and its responses to disease and injury, are defined by synergies between axons, oligodendrocytes, astrocytes and microglia, and further influenced by peripheral components such as the gut microbiome and the endocrine and immune systems. Consequently, mechanistic insights, therapeutic approaches and safety tests rely ultimately on in vivo models and clinical trials. However, in vitro models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs).  Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this in vitro tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding in vivo approaches on experimental animals.

Copper-Mediated C-N Coupling of Arylsilanes with Nitrogen Nucleophiles.

A method for the oxidative coupling of arylsilanes with nitrogen nucleophiles is reported. This method occurs with a broad range of heptamethyltrisiloxylarenes and nitrogen nucleophiles, proceeds with the arylsilane as limiting reagent, and does not require a fluoride activator with electron-poor arylsilanes. The combination of this method with C-H silylation generates arylamines from unactivated arenes with site selectivity controlled by steric effects. This combination of steps gives direct access to many compounds that cannot be accessed via alternative C-H functionalization methods, including direct C-H amination or the combination of C-H borylation and amination.

Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach.

Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1×10¹¹ vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting.

Spatially segregated SNARE protein interactions in living fungal cells.

The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, from fungi to mammals. We describe the isolation of the snc1, sso1, and sso2 genes encoding exocytic SNARE proteins from the filamentous fungus Trichoderma reesei. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in subapical but not apical hyphal regions. The vesicle SNARE SNCI also localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips. Using fluorescence lifetime imaging microscopy and Foerster resonance energy transfer analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of ternary SNARE complex formation between SNCI and SSOI or SSOII, respectively, is spatially segregated. SNARE complex formation could be detected between SNCI and SSOI in subapical hyphal compartments along the plasma membrane, but surprisingly, not in growing hyphal tips, previously thought to be the main site of exocytosis. In contrast, SNCI.SSOII complexes were found exclusively in growing apical hyphal compartments. These findings demonstrate spatially distinct sites of plasma membrane SNARE complex formation in fungi and the existence of multiple exocytic SNAREs, which are functionally and spatially segregated. This is the first demonstration of spatially regulated SNARE interactions within the same membrane.

Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

Does endocytosis occur in fungal hyphae?

The evidence and arguments for and against the occurrence of endocytosis in fungal hyphae are summarized. The balance of evidence is in favour of the existence of endocytosis. This is supported by an analysis of the recently sequenced Neurospora genome which strongly suggests that this fungus possesses the complex protein machinery required to conduct endocytosis.