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The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).
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Liquid chromatography-mass spectrometry (LC-MS) based metabolomics suffers from extended duty cycles and matrix-dependent quantitation. Chemical tags with 96 unique masses are reported, which alleviate the metabolomic workflow bottleneck and allow for absolute quantitation. A metabolic screen for carboxylic acids was performed on mammalian cells deprived of various nutrients and showed 24% RSD and analysis of 288 samples in 2 h.
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Metabolômica , Metabolômica/métodos , Humanos , Espectrometria de Massas , Marcação por Isótopo , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/análise , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga EscalaRESUMO
Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.
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Homocisteína , Espectrometria de Massas em Tandem , Homocisteína/análise , Homocisteína/metabolismo , Homocisteína/química , Espectrometria de Massas em Tandem/métodos , Metabolômica/métodos , Humanos , Tiazinas/químicaRESUMO
MAIN CONCLUSION: Genetic loci, particularly those with an effect in the independent panel, could be utilised to further reduce LMA expression when used with favourable combinations of genes known to affect LMA. Late maturity α-amylase (LMA) is a grain quality defect involving elevated α-amylase within the aleurone of wheat (Triticum aestivum L.) grains. The genes known to affect expression are the reduced height genes Rht-B1 (chromosome 4B) and Rht-D1 (chromosome 4D), and an ent-copalyl diphosphate synthase gene (LMA-1) on chromosome 7B. Other minor effect loci have been reported, but these are poorly characterised and further genetic understanding is needed. In this study, twelve F4-derived populations were created through single seed descent, genotyped and evaluated for LMA. LMA-1 haplotype C and the Rht-D1b allele substantially reduced LMA expression. The alternative dwarfing genes Rht13 and Rht18 had no significant effect on LMA expression. Additional quantitative trait loci (QTL) were mapped at 16 positions in the wheat genome. Effects on LMA expression were detected for four of these QTL in a large independent panel of Australian wheat lines. The QTL detected in mapping populations and confirmed in the large independent panel provide further opportunity for selection against LMA, especially if combined with Rht-D1b and/or favourable haplotypes of LMA-1.
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Triticum , alfa-Amilases , Austrália , Locos de Características Quantitativas , AlelosRESUMO
The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.
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Diferenciação Celular , Lisofosfolipídeos , Osteoblastos , Osteoclastos , Especificidade da Espécie , Esfingosina , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Humanos , Animais , Camundongos , Osteoclastos/metabolismo , Osteoclastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Osso e Ossos/metabolismo , Reabsorção Óssea/metabolismo , Células CultivadasRESUMO
According to a popular picture, criminal law lives up to the demands of its internal morality when its norms have counterparts with the same content in morality-when it conforms to what we call the mirror principle. This article argues that the popular picture must be redrawn by relying on a second principle, which we call the instrumental principle. Criminal law conforms to the instrumental principle when the existence of its norms helps to prevent or ameliorate moral wrongdoing. Our argument is that the instrumental principle forms part of the internal morality of criminal law, and supplies a justification for criminal laws that depart from the mirror principle. We further suggest that criminal law's internal morality is asymmetrical: though departures from the mirror principle are sometimes justified by the instrumental principle, departures from the latter are not justified by the former.
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Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.
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Fosfatidilcolinas , Espectrometria de Massas em Tandem , Humanos , Fosfatidilcolinas/química , Fosfatidilcolinas/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Ciclização , Linhagem Celular Tumoral , Células Hep G2 , Lisofosfatidilcolinas/análise , Lisofosfatidilcolinas/químicaRESUMO
Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.
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The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.
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Reabsorção Óssea , Osteoblastos , Osteogênese , Animais , Humanos , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Camundongos , Reabsorção Óssea/patologia , Reabsorção Óssea/metabolismo , Anabolizantes/farmacologia , Anabolizantes/uso terapêutico , Remodelação Óssea/efeitos dos fármacos , Osteoporose/patologia , Osteoporose/metabolismo , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Desenvolvimento Ósseo/efeitos dos fármacos , Osteoprotegerina/metabolismo , Feminino , Transdução de Sinais/efeitos dos fármacos , Peptídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologiaRESUMO
Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).
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Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos , Leucemia Mieloide Aguda , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Relação Estrutura-Atividade , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/farmacocinética , Descoberta de Drogas , Ratos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacocinética , Quinolonas/química , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Quinolonas/farmacocinética , Quinolonas/síntese química , Linhagem Celular Tumoral , Simulação de Acoplamento MolecularRESUMO
We introduce modifications to Monte Carlo simulations of the Feynman path integral that improve sampling of localized interactions. The algorithms generate trajectories in simple background potentials designed to concentrate them around the interaction region, reminiscent of importance sampling. This improves statistical sampling of the system and overcomes a long-time undersampling problem caused by the spatial diffusion inherent in Brownian motion. We prove the validity of our approach using previous analytic work on the distribution of values of the Wilson line over path integral trajectories and illustrate the improvements on some simple quantum mechanical systems.
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Respiratory syncytial virus (RSV) is the leading viral cause of childhood bronchiolitis and pneumonia causing over 3 million hospitalizations and 100,000 deaths in children under 5 years of age annually. Wastewater-based surveillance (WBS) has proven an effective early warning system for high-consequence pathogens, including SARS-CoV-2, polio, mpox, and influenza, but has yet to be fully leveraged for RSV surveillance. A model predicated on the Canadian province of Ontario demonstrates that implementation of a WBS system can potentially result in significant cost savings and clinical benefits when guiding an RSV preventive program with a long-acting monoclonal antibody. A network of integrated WBS initiatives offers the opportunity to help minimize the devastating global burden of RSV in children by optimizing the timing of preventive measures and we strongly advocate that its benefits continue to be explored.
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Infecções por Vírus Respiratório Sincicial , Humanos , Criança , Pré-Escolar , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vigilância Epidemiológica Baseada em Águas Residuárias , Vírus Sinciciais Respiratórios , Anticorpos Monoclonais , Ontário/epidemiologiaRESUMO
Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.
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This article was migrated. The article was marked as recommended. Context: We challenge the philosophical acceptability of the Angoff method, and propose an alternative method of standard setting based on how important it is for candidates to know the material each test item assesses, and not how difficult it is for a subgroup of candidates to answer each item. Methods: The practicalities of an alternative method of standard setting are evaluated here, for the first time, with direct comparison to an Angoff method. To negate bias due to any leading effects, a prospective cross-over design was adopted involving two groups of judges (n=7 and n=8), both of which set the standards for the same two 100 item multiple choice question tests, by the two different methods. Results: Overall, we found that the two methods took a similar amount of time to complete. The alternative method produced a higher cut-score (by 12-14%), and had a higher degree of variability between judges' cut-scores (by 5%). When using the alternative method, judges reported a small, but statistically significant, increase in their confidence to decide accurately the standard (by 3%). Conclusion: This is a new approach to standard setting where the quantitative differences are slight, but there are clear qualitative advantages associated with use of the alternative method.