RESUMEN
BACKGROUND: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. OBJECTIVE: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. METHODS: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model. RESULTS: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with ß-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. CONCLUSIONS: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.
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Eosinófilos/inmunología , Hipersensibilidad/tratamiento farmacológico , Pulmón/inmunología , Nanopartículas/uso terapéutico , Péptidos/uso terapéutico , Receptores CCR3/antagonistas & inhibidores , Hipersensibilidad Respiratoria/tratamiento farmacológico , Alérgenos/inmunología , Línea Celular , Movimiento Celular , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Transducción de SeñalRESUMEN
NMR allows assessment of protein structure in solution. Unlike conventional X-ray crystallography that provides snapshots of protein conformations, all conformational states are simultaneously accessible to analysis by NMR. This is a significant advantage for discovery and characterization of allosteric effects. These effects are observed when binding at one site of the protein affects another distinct site through conformational transitions. Allosteric regulation of proteins has been observed in multiple physiological processes in health and disease, providing an opportunity for the development of allosteric inhibitors. These compounds do not directly interact with the orthosteric site of the protein but influence its structure and function. In this book chapter, we provide an overview on how NMR methods are utilized to identify allosteric sites and to discover novel inhibitors, highlighting examples from the field. We also describe how NMR has contributed to understanding of allosteric mechanisms and propose that it is likely to play an important role in clarification and further development of key concepts of allostery.
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Sitio Alostérico , Descubrimiento de Drogas , Ligandos , Espectroscopía de Resonancia Magnética , Regulación Alostérica , Sitios de Unión , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Conformación ProteicaRESUMEN
The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein-protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein-protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.
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Variación Genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Humanos , Unión Proteica , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas p21(ras)/química , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In ß-arrestin recruitment assays, ubiquitin did not sufficiently recruit ß-arrestin2 to CXCR4 (EC50 > 10 µM), whereas the EC50 for CXCL12 was 4.6 nM (95% confidence interval 3.1-6.1 nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.
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Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxis , Ensayo de Inmunoadsorción Enzimática , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Receptores CXCR4/agonistas , Receptores CXCR4/química , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.
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Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.
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Neovascularización Coroidal , Degeneración Macular Húmeda , Ratones , Animales , Células Endoteliales/metabolismo , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Degeneración Macular Húmeda/tratamiento farmacológico , Degeneración Macular Húmeda/metabolismoRESUMEN
The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.
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Marcadores de Afinidad/química , Benzamidas/química , Diseño de Fármacos , Histona Desacetilasa 2/química , Inhibidores de Histona Desacetilasas/síntesis química , Modelos Moleculares , Biotina/química , Dominio Catalítico , Línea Celular Tumoral , Histona Desacetilasa 1/química , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/metabolismo , Humanos , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an 'upside-down' fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC(50) of 28 µM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.
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Quelantes , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Western Blotting , Línea Celular , Línea Celular Tumoral , Quelantes/síntesis química , Quelantes/metabolismo , Quelantes/farmacología , Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Ligandos , Estructura Molecular , Unión Proteica/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Zinc/químicaRESUMEN
Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.
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Calmodulina , Neoplasias , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Humanos , Mutación , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras) , Transducción de SeñalRESUMEN
End-binding proteins (EBs) associate with the growing microtubule plus ends to regulate microtubule dynamics as well as the interaction with intracellular structures. EB3 contributes to pathological vascular leakage through interacting with the inositol 1,4,5-trisphosphate receptor 3 (IP3R3), a calcium channel located at the endoplasmic reticulum membrane. The C-terminal domain of EB3 (residues 200-281) is functionally important for this interaction because it contains the effector binding sites, a prerequisite for EB3 activity and specificity. Structural data for this domain is limited. Here, we report the backbone chemical shift assignments for the human EB3 C-terminal domain and computationally explore its EB3 conformations. Backbone assignments, along with computational models, will allow future investigation of EB3 structural dynamics, interactions with effectors, and will facilitate the development of novel EB3 inhibitors.
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Proteínas Asociadas a Microtúbulos/química , Resonancia Magnética Nuclear Biomolecular , Humanos , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.
RESUMEN
Acute lung injury (ALI) is a lethal inflammatory lung disorder whose incidence is on the rise. Alveolar macrophages normally act to resolve inflammation, but when dysregulated they can provoke ALI. We demonstrate that monocyte-derived macrophages (CD11b+ macrophages) recruited into the airspace upregulate the anti-inflammatory function of alveolar macrophages by suppressing their stimulator of type 1 interferon gene (STING) signaling. Depletion of CD11b+ macrophages in mice (macrophagedep mice) after endotoxin or after Pseudomonas aeruginosa causes expansion of the inflammatory alveolar macrophage population, leading to neutrophil accumulation, irreversible loss of lung vascular barrier function, and lethality. We show that CD11b+ macrophages suppress alveolar macrophage-STING signaling via sphingosine kinase-2 (SPHK2) generation of sphingosine-1-phosphate (S1P). Thus, adoptive transfer of wild-type (WT) or STING-/-, but not SPHK2-/-, CD11b monocytes from murine bone marrow into injured macrophagedep mice rescue anti-inflammatory alveolar macrophages and reverse lung vascular injury. SPHK2-induced S1P generation in CD11b+ macrophages has the potential to educate alveolar macrophages to resolve ALI.
Asunto(s)
Antígeno CD11b/metabolismo , Inflamación/patología , Lisofosfolípidos/metabolismo , Macrófagos Alveolares/metabolismo , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Traslado Adoptivo , Animales , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/irrigación sanguínea , Pulmón/patología , Macrófagos Alveolares/microbiología , Ratones Endogámicos C57BL , Nucleótidos Cíclicos/metabolismo , Pseudomonas aeruginosa/fisiología , Transducción de Señal , Esfingosina/metabolismo , Células U937RESUMEN
Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.
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Neoplasias de la Mama/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas p21 Activadas/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Activación Enzimática , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/genética , Ratones SCID , Fosforilación , Serina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas p21 Activadas/química , Quinasas p21 Activadas/genética , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
[This corrects the article DOI: 10.1021/acsmedchemlett.7b00126.].
RESUMEN
We observed in PRESTO-Tango ß-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and ß1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in ß-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin-4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Receptores CXCR4/agonistas , Receptores CXCR/agonistas , Sitios de Unión , Células Cultivadas , Quimiocina CXCL12/antagonistas & inhibidores , Quimiotaxis/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Resonancia Magnética Nuclear Biomolecular , Prazosina/farmacología , Quinazolinas/farmacología , Quinoxalinas/farmacología , Receptores CXCR/química , Receptores CXCR4/química , beta-Arrestinas/metabolismoRESUMEN
Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
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Tolerancia a Medicamentos , Proteínas de Unión al GTP/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/química , Transducción de Señal , Animales , Bencilaminas , Células CHO , Quimiocina CXCL12/metabolismo , Quimiotaxis , Cricetinae , Cricetulus , Ciclamas , Endocitosis , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Ligandos , Ratones , Fosforilación , Dominios Proteicos , Células THP-1 , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismoRESUMEN
Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson's disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe's disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.
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Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patología , Psicosina/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Galactosilceramidasa/metabolismo , Humanos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
Histone deacetylases (HDACs) are promising drug targets for a variety of therapeutic applications. Herein we describe the design, synthesis, biological evaluation in cellular models of cancer, and preliminary drug metabolism and pharmacokinetic studies (DMPK) of a series of secondary and tertiary N-substituted 7-aminoheptanohydroxamic acid-based HDAC inhibitors. Introduction of an amino group with one or two surface binding groups (SBGs) yielded a successful strategy to develop novel and potent HDAC inhibitors. The secondary amines were found to be generally more potent than the corresponding tertiary amines. Docking studies suggested that the SBGs of tertiary amines cannot be favorably accommodated at the gorge region of the binding site. The secondary amines with naphthalen-2-ylmethyl, 5-phenylthiophen-2-ylmethyl, and 1H-indol-2-ylmethyl (2 j) substituents exhibited the highest potency against classâ I HDACs: HDAC1 IC50 39-61â nm, HDAC2 IC50 260-690â nm, HDAC3 IC50 25-68â nm, and HDAC8 IC50 320-620â nm. The cytotoxicity of a representative set of secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors against HT-29, SH-SY5Y, and MCF-7 cancer cells correlated with their inhibition of HDAC1, 2, and 3 and was found to be similar to or better than that of suberoylanilide hydroxamic acid (SAHA). Compounds in this series increased the acetylation of histones H3 and H4 in a time-dependent manner. DMPK studies indicated that secondary amine 2 j is metabolically stable and has plasma and brain concentrations >23- and >1.6-fold higher than the IC50 value for classâ I HDACs, respectively. Overall, the secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors exhibit excellent lead- and drug-like properties and therapeutic capacity for cancer applications.
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Aminas/farmacología , Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Aminas/síntesis química , Aminas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Histone deacetylase 8 (HDAC8) is a promising drug target for multiple therapeutic applications. Here, we describe the modeling, design, synthesis, and biological evaluation of a novel series of C1-substituted tetrahydroisoquinoline (TIQ)-based HDAC8 inhibitors. Minimization of entropic loss upon ligand binding and use of the unique HDAC8 "open" conformation of the binding site yielded a successful strategy for improvement of both HDAC8 potency and selectivity. The TIQ-based 3g and 3n exhibited the highest 82 and 55 nM HDAC8 potency and 330- and 135-fold selectivity over HDAC1, respectively. Selectivity over other class I isoforms was comparable or better, whereas inhibition of HDAC6, a class II HDAC isoform, was below 50% at 10 µM. The cytotoxicity of 3g and 3n was evaluated in neuroblastoma cell lines, and 3n displayed concentration-dependent cytotoxicity similar to or better than that of PCI-34051. The selectivity of 3g and 3n was confirmed in SH-SY5Y cells as both did not increase the acetylation of histone H3 and α-tubulin. Discovery of the novel TIQ chemotype paves the way for the development of HDAC8 selective inhibitors for therapeutic applications.
RESUMEN
Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four typeâ I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3â µM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.