RESUMO
Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.
Assuntos
Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Testes Genéticos/métodos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Receptores de LDL/biossíntese , Bibliotecas de Moléculas Pequenas/administração & dosagem , Regulação para Cima/fisiologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos , Farnesil-Difosfato Farnesiltransferase/metabolismo , Humanos , Masculino , Camundongos , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.
Assuntos
Progressão da Doença , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Leucemia Mieloide Aguda , Inibidores de Prolil-Hidrolase , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Inibidores de Prolil-Hidrolase/uso terapêutico , Animais , Camundongos , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com PontesRESUMO
The human 2-oxoglutarate (2OG)- and Fe(ii)-dependent oxygenases factor inhibiting hypoxia-inducible factor-α (FIH) and HIF-α prolyl residue hydroxylases 1-3 (PHD1-3) regulate the response to hypoxia in humans via catalysing hydroxylation of the α-subunits of the hypoxia-inducible factors (HIFs). Small-molecule PHD inhibitors are used for anaemia treatment; by contrast, few selective inhibitors of FIH have been reported, despite their potential to regulate the hypoxic response, either alone or in combination with PHD inhibition. We report molecular, biophysical, and cellular evidence that the N-hydroxythiazole scaffold, reported to inhibit PHD2, is a useful broad spectrum 2OG oxygenase inhibitor scaffold, the inhibition potential of which can be tuned to achieve selective FIH inhibition. Structure-guided optimisation resulted in the discovery of N-hydroxythiazole derivatives that manifest substantially improved selectivity for FIH inhibition over PHD2 and other 2OG oxygenases, including Jumonji-C domain-containing protein 5 (â¼25-fold), aspartate/asparagine-ß-hydroxylase (>100-fold) and histone Nε-lysine demethylase 4A (>300-fold). The optimised N-hydroxythiazole-based FIH inhibitors modulate the expression of FIH-dependent HIF target genes and, consistent with reports that FIH regulates cellular metabolism, suppressed lipid accumulation in adipocytes. Crystallographic studies reveal that the N-hydroxythiazole derivatives compete with both 2OG and the substrate for binding to the FIH active site. Derivatisation of the N-hydroxythiazole scaffold has the potential to afford selective inhibitors for 2OG oxygenases other than FIH.
RESUMO
Studies on the inhibition of the human 2-oxoglutarate dependent oxygenase JMJD6, which is a cancer target, by 2-oxoglutarate mimics / competitors, including human drugs, drug candidates, and metabolites relevant to cancer are described. JMJD6 assays employed NMR to monitor inhibitor binding and use of mass spectrometry to monitor JMJD6-catalysed lysine hydroxylation. Notably, some clinically applied prolyl hydroxylase inhibitors also inhibit JMJD6. The results will help enable the development of inhibitors selective for human oxygenases, including JMJD6.
Assuntos
Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Ácidos Cetoglutáricos/farmacologia , Inibidores de Prolil-Hidrolase/farmacologia , Relação Dose-Resposta a Droga , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ácidos Cetoglutáricos/química , Estrutura Molecular , Inibidores de Prolil-Hidrolase/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-AtividadeRESUMO
Human prolyl-hydroxylases (PHDs) are hypoxia-sensing 2-oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia-inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia-related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late-stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain-penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active-site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π-π-stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking ß2-ß3, which are involved in dynamic substrate binding/product release.
Assuntos
Prolil Hidroxilases/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Pirazóis/farmacologia , Triazóis/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Prolil-Hidrolase/química , Pirazóis/química , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
The 2-oxoglutarate-dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on a promising class of 4-hydroxypyrimidine-containing PHD inhibitors.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Inibidores de Prolil-Hidrolase/farmacologia , Pirimidinonas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Prolil-Hidrolase/química , Pirimidinonas/química , Relação Estrutura-AtividadeRESUMO
The 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are targets for treatment of anaemia and other ischaemia related diseases. PHD inhibitors are in clinical trials; however, the number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on spiro[4.5]decanone containing PHD inhibitors. Together with other studies, our results reveal spiro[4.5]decanones as useful templates for generation of potent and selective 2OG oxygenase inhibitors.
RESUMO
Two studies examined whether use of dampening appraisals (e.g., thinking "this is too good to last") or amplifying appraisals (e.g., thinking "I deserve this") modulated affective experience when remembering (Study One) and anticipating (Study Two) positive events. Both studies used a mixed within-between participants design, with participants completing an uninstructed positive recall/anticipation task before being randomized to either control, dampening, or amplifying instructions during a second positive recall/anticipation task. During memory recall (Study One), instructed dampening increased dampening appraisals and led to a reduction in happiness and pleasantness and an increase in sadness, significantly differing from the control and amplifying conditions. While the amplifying condition significantly increased amplifying appraisals, it did not alter affective experience (relative to the control condition). During anticipation (Study Two), identical findings emerged for the dampening manipulation. The amplifying manipulation did not significantly increase amplifying appraisals, precluding conclusions being drawn about the impact of amplifying in this study. These results suggest that dampening appraisals contribute to altered affective experience when imagining and recalling positive activities and may account for why attempts to do so can have paradoxically negative effects in clinical populations. Moreover, the studies preliminarily validate a novel scale measuring state appraisal of positive experiences.
Assuntos
Afeto/fisiologia , Antecipação Psicológica/fisiologia , Felicidade , Memória Episódica , Tristeza/psicologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Rememoração Mental , Adulto JovemRESUMO
Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The 'clinical' PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
RESUMO
In human cells oxygen levels are 'sensed' by a set of ferrous iron and 2-oxoglutarate dependent dioxygenases. These enzymes regulate a broad range of cellular and systemic responses to hypoxia by catalysing the post-translational hydroxylation of specific residues in the alpha subunits of hypoxia inducible factor (HIF) transcriptional complexes. The HIF hydroxylases are now the subject of pharmaceutical targeting by small molecule inhibitors that aim to activate or augment the endogenous HIF transcriptional response for the treatment of anaemia and other hypoxic human diseases. Here we consider the rationale for this therapeutic strategy from the biochemical, biological and medical perspectives. We outline structural and mechanistic considerations that are relevant to the design of HIF hydroxylase inhibitors, including likely determinants of specificity, and review published reports on their activity in pre-clinical models and clinical trials.
Assuntos
Sistemas de Liberação de Medicamentos , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Anemia/tratamento farmacológico , Animais , Modelos Animais de Doenças , Eritropoetina/deficiência , Eritropoetina/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Inflamação/tratamento farmacológico , Isquemia/tratamento farmacológico , Oxigenases de Função Mista/metabolismo , Oxigênio/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Conformação Proteica , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.