A MOZ-TIF2 leukemia mouse model displays KAT6-dependent H3K23 propionylation and overexpression of a set of active developmental genes.

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Synthesis of 3-Azabicyclo[3.1.0]hexane Derivates.

3-Azabicyclo[3.1.0]hexanes are an important class of nitrogen-containing heterocycles that have been found to be key structural features in a wide range of biologically active natural products, drugs, and agrochemicals. As a cutting-edge area, the synthesis of these derivatives has made spectacular progress in recent decades, with various transition-metal-catalyzed and transition-metal-free catalytic systems being developed. In this review, we provide an overview of recent advances in the efficient methods for the synthesis of 3-azabicyclo[3.1.0]hexane derivatives since 2010, emphasizing the scope of substrates and synthesis' applications, as well as the mechanisms of these reactions.

Visible-Light-Induced Synthesis of 3-Alkyl Chromones under Catalyst- and Additive-Free Conditions.

Herein, we reported an efficient and facile visible-light-induced 3-alkyl chromone synthesis from easily accessible o-hydroxyaryl enaminones and α-diazo esters. In this protocol, excellent yields were obtained with a broad substrate scope at room temperature, tolerating various functional groups. Of note is that this eco-friendly methodology features catalyst- and additive-free, mild reaction conditions, simple operation procedure, and easy scale-up, which affords a convenient pathway for the preparation of 3-alkyl chromones. Experimental results and density functional theory (DFT) computation analyses confirm the participation of carbene species and active cyclopropane intermediate.

Photocatalytic Benzylic C-H Oxidation/Cyclization of Enaminones to the Synthesis of Polysubstituted Oxazoles.

Photocatalytic benzylic C-H oxidation/cyclization of enaminones was efficiently achieved to afford oxazole derivatives under mild conditions. The oxygen in the oxazole ring originated from green oxidant molecular oxygen. The synthetic protocol features broad substrate scopes and good functional group tolerance. The combined experimental and theoretical studies reveal that in suit benzylic C-H oxidation/cyclization is involved in the reaction transformations.

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

Electrochemical Synthesis of Polysubstituted Sulfonated Pyrazoles via Cascade Intermolecular Condensation, Radical-Radical Cross Coupling Sulfonylation, and Pyrazole Annulation.

Electrochemical synthesis of polysubstituted sulfonated pyrazoles from enaminones and sulfonyl hydrazides was established under metal-free, exogenous-oxidant-free, and mild conditions. By judicious choice of different electrochemical reaction conditions, NH2-functionalized enaminones or N,N-disubstituted enaminones can react with aryl/alkyl sulfonyl hydrazides to afford tetra- or trisubstituted sulfonated pyrazoles in moderate to good yields, respectively. The gram-scale electrochemical transformation demonstrated the efficiency and practicability of this synthetic strategy. In addition, the sulfonated NH-pyrazole can be obtained via the dissociation of the N-tosyl group. Mechanistic studies reveal that the electrochemical cascade reaction synthesis of polysubstituted sulfonated pyrazoles proceeded via the sequence of intermolecular condensation, radical-radical cross coupling sulfonylation, and pyrazole annulation.

Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Synthesis of 2-phenyl-5,6,7,8-tetrahydroquinoxaline derivatives and screening for P2X1-purinoceptor antagonist activity in isolated preparations of rat vas deferens, for translation into a male contraceptive†.

Sympathetically mediated contractions of smooth muscle cells in the vasa deferentia are mediated by neuronally released adenosine 5'-triphosphate (ATP) and noradrenaline, which stimulate P2X1-purinoceptors and α1A-adrenoceptors, respectively. This process is crucial for sperm transport, as demonstrated in knockout mouse studies where simultaneous genetic deletion of P2X1-purinoceptors and α1A-adrenoceptors resulted in male infertility. We hypothesize that dual pharmacological antagonism of these two receptors could inhibit sperm transport sufficiently to provide a novel nonhormonal method of male contraception. To generate a suitable P2X1-purinoceptor antagonist, substituents were introduced on the phenyl moiety of 2-phenyl-5,6,7,8-tetrahydroquinoxaline to create a series of analogues that were tested for P2X1-purinoceptor antagonism in isolated preparations of rat vas deferens. Novel compounds were initially screened for their ability to attenuate contractile responses to electrical field stimulation (EFS: 60 V, 0.5 ms, 0.2 Hz). The addition of polar substituents to the meta, but not ortho, position markedly increased the inhibition of contractions, as did the addition of both polar and aliphatic substituents to the para position. Di-substituted compounds were also synthesized and tested, resulting in a compound 31 (2-hydroxy, 4-fluoro), which exhibited the greatest potency, with an IC50 of 14 µM (95% confidence limits: 12-16 µM). Additionally, compound 31 noncompetitively antagonized contractions mediated by exogenously administered αß-methylene ATP (10 nM-30 µM) but had no inhibitory effect on contractions mediated by exogenously administered noradrenaline (30 nM-100 µM) or acetylcholine (30 nM-100 µM). These results have contributed to a structure-activity relationship profile for the P2X1-purinoceptor that will inform future designs of more potent antagonists.

A Reusable CNT-Supported Single-Atom Iron Catalyst for the Highly Efficient Synthesis of C-N Bonds.

C-N bond formation is regarded as a very useful and fundamental reaction for the synthesis of nitrogen-containing molecules in both organic and pharmaceutical chemistry. Noble-metal and homogeneous catalysts have frequently been used for C-N bond formation, however, these catalysts have a number of disadvantages, such as high cost, toxicity, and low atom economy. In this work, a low-toxic and cheap iron complex (iron ethylene-1,2-diamine) has been loaded onto carbon nanotubes (CNTs) to prepare a heterogeneous single-atom catalyst (SAC) named Fe-Nx /CNTs. We employed this SAC in the synthesis of C-N bonds for the first time. It was found that Fe-Nx /CNTs is an efficient catalyst for the synthesis of C-N bonds starting from aromatic amines and ketones. Its catalytic performance was excellent, giving yields of up to 96 %, six-fold higher than the yields obtained with noble-metal catalysts, such as AuCl3 /CNTs and RhCl3 /CNTs. The catalyst showed efficacy in the reactions of thirteen aromatic amine substrates, without the need for additives, and seventeen enaminones were obtained. High-angle annular dark-field scanning transmission electron microscopy in combination with X-ray absorption spectroscopy revealed that the iron species were well dispersed in the Fe-Nx /CNTs catalyst as single atoms and that Fe-Nx might be the catalytic active species. This Fe-Nx /CNTs catalyst has potential industrial applications as it could be cycled seven times without any significant loss of activity.

I2-Promoted [4 + 2] cycloaddition of in situ generated azoalkenes with enaminones: facile and efficient synthesis of 1,4-dihydropyridazines and pyridazines.

A facile and efficient strategy for the synthesis of 1,4-dihydropyridazines and pyridazines through I2-promoted [4 + 2] cycloaddition of in situ generated azoalkenes with enaminones has been developed. The switch in selectivity is attributed to the judicious choice of different reaction temperatures. The key features of this work include controllable and selective synthesis, good functional group tolerance, good to excellent reaction yields, metal/base-free conditions, and also applicability to one-pot methodology.

pH-Responsive Polymers for Improving the Signal-to-Noise Ratio of Hypoxia PET Imaging with [18 F]Fluoromisonidazole.

To improve the signal-to-noise ratio of hypoxia positron emission tomography (PET) imaging, stimuli-responsive polymers are designed for the delivery of the hypoxia PET tracer fluorine-18 labeled fluoromisonidazole ([18 F]FMISO). Linear poly(N-(2-(hydroxypropyl)methacrylamide)) polymers are functionalized with hydrazide linkers that form pH-sensitive acyl hydrazone bonds after their conjugation to an [18 F]FMISO ketone analogue. The release of the [18 F]FMISO ketone analogue from the polymers is considerably faster at a lower pH and its uptake is significantly higher in cancer cells growing under acidic conditions. Additionally, the retention of the PET tracer is significantly higher in hypoxic cells compared to normoxic cells. The delivery of a PET tracer using stimuli-responsive polymers may be an attractive strategy to improve signal-to-noise ratios in PET imaging.

Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium.

Background: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives: To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. Methods: The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results: The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions: The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.

8-Mercaptoguanine Derivatives as Inhibitors of Dihydropteroate Synthase.

Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-µm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.

FAF-Drugs3: a web server for compound property calculation and chemical library design.

Drug attrition late in preclinical or clinical development is a serious economic problem in the field of drug discovery. These problems can be linked, in part, to the quality of the compound collections used during the hit generation stage and to the selection of compounds undergoing optimization. Here, we present FAF-Drugs3, a web server that can be used for drug discovery and chemical biology projects to help in preparing compound libraries and to assist decision-making during the hit selection/lead optimization phase. Since it was first described in 2006, FAF-Drugs has been significantly modified. The tool now applies an enhanced structure curation procedure, can filter or analyze molecules with user-defined or eight predefined physicochemical filters as well as with several simple ADMET (absorption, distribution, metabolism, excretion and toxicity) rules. In addition, compounds can be filtered using an updated list of 154 hand-curated structural alerts while Pan Assay Interference compounds (PAINS) and other, generally unwanted groups are also investigated. FAF-Drugs3 offers access to user-friendly html result pages and the possibility to download all computed data. The server requires as input an SDF file of the compounds; it is open to all users and can be accessed without registration at http://fafdrugs3.mti.univ-paris-diderot.fr.

New synthetic approaches towards analogues of bedaquiline.

Multi-drug resistant tuberculosis (MDR-TB) is of growing global concern and threatens to undermine increasing efforts to control the worldwide spread of tuberculosis (TB). Bedaquiline has recently emerged as a new drug developed to specifically treat MDR-TB. Despite being highly effective as a result of its unique mode of action, bedaquiline has been associated with significant toxicities and as such, safety concerns are limiting its clinical use. In order to access pharmaceutical agents that exhibit an improved safety profile for the treatment of MDR-TB, new synthetic pathways to facilitate the preparation of bedaquiline and analogues thereof have been discovered.