RESUMO
Histone deacetylases (HDACs) are a class of enzymes that cleave acyl groups from lysine residues of histone and non-histone proteins. There are 18 human HDAC isoforms with different cellular targets and functions. Among them, HDAC6 was found to be overexpressed in different types of cancer. However, when used in monotherapy, HDAC6 inhibition by selective inhibitors fails to show pronounced anti-cancer effects. The HDAC6 enzyme also addresses non-histone proteins like α-tubulin and cortactin, making it important for cell migration and angiogenesis. Recently, the NLRP3 inflammasome was identified as an important regulator of inflammation and immune responses and, importantly, HDAC6 is critically involved the activation of the inflammasome. We herein report the design, synthesis and biological evaluation of a library of selective HDAC6 inhibitors. Starting from the previously published crystal structure of MAIP-032 in complex with CD2 of zHDAC6, we performed docking studies to evaluate additional possible interactions of the cap group with the L1-loop pocket. Based on the results we synthesized 13 novel HDAC6 inhibitors via the Groebke-Blackburn-Bienaymé three component reaction as the key step. Compounds 8k (HDAC1 IC50: 5.87 µM; HDAC6 IC50: 0.024 µM; selectivity factor (SF1/6): 245) and 8m (HDAC1 IC50: 3.07 µM; HDAC6 IC50: 0.026 µM; SF1/6: 118) emerged as the most potent and selective inhibitors of HDAC6 and outperformed the lead structure MAIP-032 (HDAC1 IC50: 2.20 µM; HDAC6 IC50: 0.058 µM; SF1/6: 38) both in terms of inhibitory potency and selectivity. Subsequent immunoblot analysis confirmed the high selectivity of 8k and 8m for HDAC6 in a cellular environment. While neither 8k and 8m nor the selectivity HDAC6 inhibitor tubastatin A showed antiproliferative effects in the U-87 MG glioblastoma cell line, compound 8m attenuated cell migration significantly in wound healing assays in U-87 MG cells. Moreover, in macrophages compounds 8k and 8m demonstrated significant inhibition of LPS-induced IL1B mRNA expression and TNF release. These findings suggest that our imidazo[1,2-a]pyridine-capped HDAC6 inhibitors may serve as promising candidates for the development of drugs to effectively treat NLRP3 inflammasome-driven inflammatory diseases.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Neoplasias , Humanos , Desacetilase 6 de Histona , Inflamassomos , Inibidores de Histona Desacetilases/química , Anti-Inflamatórios/farmacologia , Neoplasias/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Multi-target drugs (MTDs) are emerging alternatives to combination therapies. Since both histone deacetylases (HDACs) and cyclooxygenase-2 (COX-2) are known to be overexpressed in several cancer types, we herein report the design, synthesis, and biological evaluation of a library of dual HDAC-COX inhibitors. The designed compounds were synthesized via an efficient parallel synthesis approach using preloaded solid-phase resins. Biological in vitro assays demonstrated that several of the synthesized compounds possess pronounced inhibitory activities against HDAC and COX isoforms. The membrane permeability and inhibition of cellular HDAC activity of selected compounds were confirmed by whole-cell HDAC inhibition assays and immunoblot experiments. The most promising dual inhibitors, C3 and C4, evoked antiproliferative effects in the low micromolar concentration range and caused a significant increase in apoptotic cells. In contrast to previous reports, the simultaneous inhibition of HDAC and COX activity by dual HDAC-COX inhibitors or combination treatments with vorinostat and celecoxib did not result in additive or synergistic anticancer activities.
Assuntos
Antineoplásicos , Inibidores de Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Ciclo-Oxigenase 2 , Proliferação de Células , Histona Desacetilases , Inibidores de Ciclo-Oxigenase/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Resposta ao Choque Térmico/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Animais , Antineoplásicos/química , Sítios de Ligação , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Mesilato de Imatinib/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Multimerização Proteica/efeitos dos fármacos , Análise Espectral , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer is the fifth leading cause of cancer deaths. Chemoresistance, particularly against platinum compounds, contributes to a poor prognosis. Histone deacetylase inhibitors (HDACi) and heat shock protein 90 inhibitors (HSP90i) are known to modulate pathways involved in chemoresistance. This study investigated the effects of HDACi (panobinostat, LMK235) and HSP90i (luminespib, HSP990) on the potency of cisplatin in ovarian cancer cell lines (A2780, CaOV3, OVCAR3 and cisplatin-resistant sub-clones). Preincubation with HDACi increased the cytotoxic potency of HSP90i, whereas preincubation with HSP90i had no effect. Preincubation with HSP90i or HDACi 48h prior to cisplatin enhanced the cisplatin potency significantly in all cell lines via apoptosis induction and affected the expression of apoptosis-relevant genes and proteins. For CaOV3CisR and A2780CisR, a preincubation with HDACi for 48-72 h led to complete reversal of cisplatin resistance. Furthermore, permanent presence of HDACi in sub-cytotoxic concentrations prevented the development of cisplatin resistance in A2780. However, triple combinations of HDACi, HSP90i and cisplatin were not superior to dual combinations. Overall, priming with HDACi sensitizes ovarian cancer cells to treatment with HSP90i or cisplatin and has an influence on the development of cisplatin resistance, both of which may contribute to an improved ovarian cancer treatment.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Neoplasias Ovarianas/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Carcinoma Epitelial do Ovário/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Humanos , Isoxazóis/farmacologia , Panobinostat/farmacologia , Piridonas/farmacologia , Pirimidinas/farmacologia , Resorcinóis/farmacologiaRESUMO
Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.
Assuntos
Benzamidas/toxicidade , Inibidores de Histona Desacetilases/toxicidade , Ácidos Hidroxâmicos/toxicidade , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Ensaio Cometa , Cricetinae , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Histonas/química , Histonas/metabolismo , Humanos , Testes para Micronúcleos , Fosforilação , Vorinostat/toxicidadeRESUMO
Inhibition of more than one cancer-related pathway by multi-target agents is an emerging approach in modern anticancer drug discovery. Here, based on the well-established synergy between histone deacetylase inhibitors (HDACi) and alkylating agents, we present the discovery of a series of alkylating HDACi using a pharmacophore-linking strategy. For the parallel synthesis of the target compounds, we developed an efficient solid-phase-supported protocol using hydroxamic acids immobilized on resins (HAIRs) as stable and versatile building blocks for the preparation of functionalized HDACi. The most promising compound, 3 n, was significantly more active in apoptosis induction, activation of caspase 3/7, and formation of DNA damage (γ-H2AX) than the sum of the activities of either active principle alone. Furthermore, to demonstrate the utility of our preloaded resins, the HAIR approach was successfully extended to the synthesis of a proof-of-concept proteolysis-targeting chimera (PROTAC), which efficiently degrades histone deacetylases.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Resinas Sintéticas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Estrutura MolecularRESUMO
Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.
Assuntos
Compostos de Dansil/farmacologia , Corantes Fluorescentes/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Peptoides/farmacologia , Acetilação/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Compostos de Dansil/síntese química , Compostos de Dansil/metabolismo , Compostos de Dansil/farmacocinética , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Histona Desacetilase 1/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacocinética , Histonas/química , Histonas/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Peptoides/síntese química , Peptoides/metabolismo , Peptoides/farmacocinética , Ligação ProteicaRESUMO
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.
Assuntos
Histona Desacetilases/biossíntese , Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/genética , Espasmos Infantis/genética , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Síndromes Epilépticas , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/genética , Humanos , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/fisiopatologia , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/patologia , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/patologiaRESUMO
α-Peptoids, or N-substituted glycine oligomers, are an important class of peptidomimetic foldamers with proteolytic stability. Nevertheless, the presence of cis/trans-amide bond conformers, which contribute to the high flexibility of α-peptoids, is considered as a major drawback. A modified peptoid backbone with an improved control of the amide bond geometry could therefore help to overcome this limitation. Herein, we have performed the first thorough analysis of the folding propensities of α-aminoxy peptoids (or N-substituted 2-aminoxyacetic acid oligomers). To this end, the amide bond geometry and the conformational properties of a series of model α-aminoxy peptoids were investigated by using 1D and 2D NMR experiments, X-ray crystallography, natural bond orbital (NBO) analysis, circular dichroism (CD) spectroscopy, and molecular dynamics (MD) simulations revealing a unique preference for cis-amide bonds even in the absence of cis-directing side chains. The conformational analysis based on the MD simulations revealed that α-aminoxy peptoids can adopt helical conformations that can mimic the spatial arrangement of peptide side chains in a canonical α-helix. Given their ease of synthesis and conformational properties, α-aminoxy peptoids represent a new member of the peptoid family capable of controlling the amide isomerism while maintaining the potential for side-chain diversity.
Assuntos
Peptoides/química , Amidas/química , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estereoisomerismo , TermodinâmicaRESUMO
Schistosoma mansoni histone deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Ftálicos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Esquistossomicidas/farmacologia , Animais , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases , Humanos , Simulação de Acoplamento Molecular , Ácidos Ftálicos/síntese química , Ácidos Ftálicos/química , Schistosoma mansoni/enzimologia , Esquistossomicidas/síntese química , Esquistossomicidas/química , Especificidade da EspécieRESUMO
Despite recent declines in mortality, malaria remains an important global health problem. New therapies are needed, including new drugs with novel modes of action compared to existing agents. Among new potential therapeutic targets for malaria, inhibition of parasitic histone deacetylases (HDACs) is a promising approach. Homology modeling of PfHDAC1, a known target of some anti-plasmodial HDAC inhibitors, revealed a unique threonine residue at the rim of the active site in close proximity to the location of the cap group of vorinostat-type HDAC inhibitors. Aiming to obtain HDAC inhibitors with potent and preferential anti-plasmodial activity, we synthesized a mini-library of alkoxyamide-based HDAC inhibitors containing hydrogen bond acceptors in the cap group. Using a 5-step synthetic route, 12 new inhibitors were synthesized and assayed against Plasmodium falciparum asexual blood stage parasites (clones 3D7 and Dd2) and human cells (HepG2). The most active compound 6h (Pf3D7 IC50 : 0.07 µM; PfDd2 IC50 : 0.07 µM) was 25-fold more toxic against the parasite versus human HepG2 cells. Selected compounds were shown to cause hyperacetylation of P. falciparum histone H4, indicating inhibition of one or more PfHDACs.
Assuntos
Álcoois/farmacologia , Amidas/farmacologia , Antimaláricos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Álcoois/síntese química , Álcoois/química , Amidas/síntese química , Amidas/química , Antimaláricos/síntese química , Antimaláricos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-AtividadeRESUMO
Epithelial-mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti-fibrotic effect in various diseases. However, the pathophysiological role of isoform-specific HDACs or class-selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK-2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor ß1 (TGF-ß1)-induced HK2 cells. However, restoration of TGF-ß1-induced E-cadherin down-regulation was only seen in HK-2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF-ß1-induced N-cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E-cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.
Assuntos
Caderinas/metabolismo , Células Epiteliais/metabolismo , Histona Desacetilases/metabolismo , Túbulos Renais Proximais/citologia , Antígenos CD , Benzamidas/farmacologia , Biomarcadores/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Piridinas/farmacologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Linhagem Celular Tumoral , Citocinas/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Marcação de Genes , Interferon gama/biossíntese , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α-aminoxy oligopeptides we used a straightforward combination of solution- and solid-phase-supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X-ray crystal structure of an α-aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right-handed 28 -helical conformation with precisely two residues per turn and a helical pitch of 5.8â Å. By 2Dâ ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 28 -helix as the predominant conformation in organic solvents. In aqueous solution, the α-aminoxy peptides exist in the 28 -helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α-aminoxy peptides have an increased propensity to take up a 28 -helical conformation in the presence of a model membrane. This indicates a correlation between the 28 -helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α-aminoxy peptides.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/síntese química , Solventes/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de ProteínaRESUMO
Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.
Assuntos
Acetilação/efeitos dos fármacos , Antimaláricos/farmacologia , Lisina/metabolismo , Plasmodium/efeitos dos fármacos , Plasmodium/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Flagelos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Plasmodium/crescimento & desenvolvimento , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
The straightforward synthesis of trispyrimidonamides as a new class of α-helix mimetics is reported. Because of the versatility of our synthetic protocol, a variety of side chains including aliphatic, basic, aromatic, and heteroaromatic residues were included. A comprehensive conformational analysis revealed that in polar solvents a trimeric compound adopts conformations that can lead to i, i + 4, i + 8, or i, i + 8 patterns of side chain orientation. This suggests that trispyrimidonamides could be promising α-helix mimetics to target hot spots that are distributed over a wider angular range of an α-helix interface than in the classical i, i + 4, i + 7 case.
Assuntos
Aminoácidos/química , Pirimidinonas/síntese química , Biomimética , Conformação Molecular , Estrutura Secundária de Proteína , Pirimidinonas/química , SolventesRESUMO
Histone deacetylase 6 (HDAC6) is an important target for the treatment of oncological and non-oncological diseases. Established HDAC6 inhibitors feature a hydroxamic acid as a zinc-binding group (ZBG) and thus possess mutagenic and genotoxic potential. Recently, the 2-(difluoromethyl)-1,3,4-oxadiazole (DFMO) group emerged as a novel ZBG. In this Viewpoint, we summarize the discovery of the mode of action of DFMOs. Additionally, we discuss opportunities and challenges in the journey toward the clinical development of DFMO-based drugs for the treatment of HDAC6-driven diseases.
RESUMO
Apicidins are a class of naturally occurring cyclic tetrapeptides produced by few strains within the Fusarium genus. These secondary metabolites have gained significant attention due to their antiprotozoal activity through HDAC inhibition, thereby highlighting their potential for the treatment of malaria. Predominantly, apicidins have been isolated from Fusarium semitectum, offering a deep insight into the biosynthetic pathway responsible for their formation. A similar biosynthetic gene cluster has also been identified in the rice pathogenic fungus F. fujikuroi, leading the discovery of three additional apicidins through genetic manipulation. Routine mass spectrometric screening of these compound-producing strains revealed another metabolite structurally related to previously studied apicidins. By optimizing culture conditions and developing an effective isolation method, we obtained a highly pure substance, whose chemical structure was fully elucidated using NMR and HRMS fragmentation. Further studies were conducted to determine cytotoxicity, antimalarial activity, and HDAC inhibitory activity of this new secondary metabolite alongside the previously known apicidins. This work not only expands the apicidin class with a new member but also provides extensive insights and comparative analysis of apicidin-like substances produced by F. fujikuroi.
RESUMO
Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.
Assuntos
Ferroptose , Inibidores de Histona Desacetilases , Peroxidação de Lipídeos , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Linhagem Celular Tumoral , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/antagonistas & inibidores , Relação Dose-Resposta a DrogaRESUMO
Our previously reported HDAC6 inhibitor (HDAC6i) Marbostat-100 (4) has provided many arguments for further clinical evaluation. By the substitution of the acidic hydrogen of 4 for different carbon residues, we were able to generate an all-carbon stereocenter, which significantly improves the hydrolytic stability of the inhibitor. Further asymmetric synthesis has shown that the (S)-configured inhibitors preferentially bind to HDAC6. This led to the highly selective and potent methyl-substituted derivative S-29b, which elicited a long-lasting tubulin hyperacetylation in MV4-11 cells. Finally, a crystal structure of the HDAC6/S-29b complex provided mechanistic explanation for the high potency and stereoselectivity of synthesized compound series.