RESUMO
Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.
Assuntos
Células Eritroides/citologia , Eritropoese , Histona Desmetilases/metabolismo , Células Mieloides/citologia , Animais , Linhagem Celular , Células Cultivadas , Células Eritroides/metabolismo , Deleção de Genes , Histona Desmetilases/genética , Humanos , Camundongos , Células Mieloides/metabolismoRESUMO
Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Furina/metabolismo , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Resultado do TratamentoRESUMO
Runt-related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age-related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor-alpha (TNF-α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF-α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF-α pathway inhibitors, we determined that in HRMECs, TNF-α-induced RUNX1 expression occurs via JNK activation, while NF-κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high D-glucose-stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP-1) and investigated the JNK-AP-1-RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF-α and D-glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti-VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis.
Assuntos
Neovascularização de Coroide/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Retinopatia da Prematuridade/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Degeneração Macular Exsudativa/metabolismo , Animais , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologia , Retina/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.
RESUMO
To study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E° = -0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Dano ao DNA/efeitos dos fármacos , Descoberta de Drogas , Células Hep G2 , Humanos , Imidazóis/metabolismo , Imidazóis/farmacocinética , Concentração Inibidora 50 , Camundongos , Testes de Sensibilidade Parasitária , Piridinas/metabolismo , Piridinas/farmacocinética , Albumina Sérica/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacocinéticaRESUMO
An antikinetoplastid pharmacomodulation study was done at position 8 of a previously identified pharmacophore in 3-nitroimidazo[1,2-a]pyridine series. Twenty original derivatives bearing an alkynyl moiety were synthesized via a Sonogashira cross-coupling reaction and tested in vitro, highlighting 3 potent (40 nM ≤ EC50 blood stream form≤ 70 nM) and selective (500 ≤ SI ≤ 1800) anti-T. brucei brucei molecules (19, 21 and 22), in comparison with four reference drugs. Among these hit molecules, compound 19 also showed the same level of activity against T. cruzi (EC50 amastigotes = 1.2 µM) as benznidazole and fexinidazole. An in vitro comet assay showed that nitroaromatic derivative 19 was not genotoxic. It displayed a low redox potential value (-0.68 V/NHE) and was shown to be bioactivated by type 1 nitroreductases both in Leishmania and Trypanosoma. The SAR study indicated that an alcohol function improved aqueous solubility while maintaining good activity and low cytotoxicity when the hydroxyl group was at position beta of the alkyne triple bond. Hit-compound 19 was also evaluated regarding in vitro pharmacokinetic data: 19 is BBB permeable (PAMPA assay), has a 16 min microsomal half-life and a high albumin binding (98.5%). Moreover, compound 19 was orally absorbed and was well tolerated in mouse after both single and repeated administrations at 100 mg/kg. Its mouse plasma half-life (10 h) is also quite encouraging, paving the way toward further efficacy evaluations in parasitized mouse models, looking for a novel antitrypanosomal lead compound.
Assuntos
Nitroimidazóis/farmacologia , Piridinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estrutura Molecular , Nitroimidazóis/síntese química , Nitroimidazóis/química , Testes de Sensibilidade Parasitária , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
Twenty nine original 3-nitroimidazo[1,2-a]pyridine derivatives, bearing a phenylthio (or benzylthio) moiety at position 8 of the scaffold, were synthesized. In vitro evaluation highlighted compound 5 as an antiparasitic hit molecule displaying low cytotoxicity for the human HepG2 cell line (CC50 > 100 µM) alongside good antileishmanial activities (IC50 = 1-2.1 µM) against L. donovani, L. infantum, and L. major; and good antitrypanosomal activities (IC50 = 1.3-2.2 µM) against T. brucei brucei and T. cruzi, in comparison to several reference drugs such as miltefosine, fexinidazole, eflornithine, and benznidazole (IC50 = 0.6 to 13.3 µM). Molecule 5, presenting a low reduction potential (E° = -0.63 V), was shown to be selectively bioactivated by the L. donovani type 1 nitroreductase (NTR1). Importantly, molecule 5 was neither mutagenic (negative Ames test), nor genotoxic (negative comet assay), in contrast to many other nitroaromatics. Molecule 5 showed poor microsomal stability; however, its main metabolite (sulfoxide) remained both active and nonmutagenic, making 5 a good candidate for further in vivo studies.
RESUMO
An antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki-Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (-0.56â V) than the initial hit (-0.45â V). Compound 21 displays micromolar antitrypanosomal activity (IC50 =1.5â µm) and low cytotoxicity on the human HepG2 cell line (CC50 =120â µm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L.â infantum and is not efficiently bioactivated by T.â brucei brucei typeâ I nitroreductase, which suggests the existence of an alternative mechanism of action.
Assuntos
Nitroquinolinas/farmacologia , Quinolonas/farmacologia , Tripanossomicidas/farmacologia , Catálise , Células Hep G2 , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Estrutura Molecular , Nitroquinolinas/síntese química , Nitroquinolinas/química , Nitroquinolinas/toxicidade , Paládio/química , Testes de Sensibilidade Parasitária , Quinolonas/síntese química , Quinolonas/química , Quinolonas/toxicidade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Based on a previously identified antileishmanial 6,8-dibromo-3-nitroimidazo[1,2-a]pyridine derivative, a Suzuki-Miyaura coupling reaction at position 8 of the scaffold was studied and optimized from a 8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridine substrate. Twenty-one original derivatives were prepared, screened in vitro for activity against L. infantum axenic amastigotes and T. brucei brucei trypomastigotes and evaluated for their cytotoxicity on the HepG2 human cell line. Thus, 7 antileishmanial hit compounds were identified, displaying IC50 values in the 1.1-3⯵M range. Compounds 13 and 23, the 2 most selective molecules (SI = >18 or >17) were additionally tested on both the promastigote and intramacrophage amastigote stages of L. donovani. The two molecules presented a good activity (IC50â¯=â¯1.2-1.3⯵M) on the promastigote stage but only molecule 23, bearing a 4-pyridinyl substituent at position 8, was active on the intracellular amastigote stage, with a good IC50 value (2.3⯵M), slightly lower than the one of miltefosine (IC50â¯=â¯4.3⯵M). The antiparasitic screening also revealed 8 antitrypanosomal hit compounds, including 14 and 20, 2 very active (IC50â¯=â¯0.04-0.16⯵M) and selective (SI = >313 to 550) molecules toward T. brucei brucei, in comparison with drug-candidate fexinidazole (IC50â¯=â¯0.6 & SIâ¯>â¯333) or reference drugs suramin and eflornithine (respective IC50â¯=â¯0.03 and 13.3⯵M). Introducing an aryl moiety at position 8 of the scaffold quite significantly increased the antitrypanosomal activity of the pharmacophore. Antikinetoplastid molecules 13, 14, 20 and 23 were assessed for bioactivation by parasitic nitroreductases (either in L. donovani or in T. brucei brucei), using genetically modified parasite strains that over-express NTRs: all these molecules are substrates of type 1 nitroreductases (NTR1), such as those that are responsible for the bioactivation of fexinidazole. Reduction potentials measured for these 4 hit compounds were higher than that of fexinidazole (-0.83â¯V), ranging from -0.70 to -0.64â¯V.
Assuntos
Antineoplásicos/farmacologia , Leishmania donovani/efeitos dos fármacos , Nitrorredutases/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Leishmania donovani/metabolismo , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
To study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-lifeâ¯>â¯40â¯min) and a 92% binding to human albumin.