RESUMEN
Aryl hydrocarbon receptor (AHR) is an essential regulator of gut immunity and a promising therapeutic target for inflammatory bowel disease (IBD). Current AHR agonists are inadequate for clinical translation due to low activity, inadequate pharmacokinetics, or toxicity. We synthesized a structurally diverse library and used integrated computational and experimental studies to discover mechanisms governing ligand-receptor interaction and to design potent drug leads PY109 and PY108, which display physiochemical drug-likeness properties, desirable pharmacokinetic profiles, and low toxicity. In a murine model of dextran sulfate sodium-induced colitis, orally administered compounds increase interleukin-22 (IL-22) production and accelerate mucosal healing by modulating mucosal adaptive and innate lymphoid cells. AHR and IL-22 pathway induction was confirmed using RNA sequencing and characterization of the lymphocyte protein-protein interaction network. Significant induction of IL-22 was also observed using human T cells from patients with IBD. Our findings support rationally designed AHR agonists for IBD therapy.
Asunto(s)
Diseño de Fármacos , Inmunomodulación/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Receptores de Hidrocarburo de Aril/agonistas , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Colitis/etiología , Colitis/metabolismo , Colitis/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Expresión Génica , Humanos , Interleucinas/biosíntesis , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ligandos , Linfocitos/inmunología , Ratones , Modelos Moleculares , Conformación Molecular , Receptores de Hidrocarburo de Aril/química , Regeneración , Relación Estructura-Actividad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Cicatrización de Heridas/genética , Interleucina-22RESUMEN
Antizyme inhibitor (AZIN) stimulates cell proliferation by binding to and sequestering the cell cycle suppressor antizyme. Despite the important role of the antizyme-AZIN protein-protein interaction (PPI) in cell cycle regulation, there are no assays for directly measuring the binding of AZIN to antizyme that are amenable to high throughput screening. To address this problem, we developed and validated a novel antizyme-AZIN intramolecular FRET sensor using clover and mRuby2 fluorescent proteins. By introducing alanine mutations in the AZIN protein, we used this sensor to probe the PPI for key residues governing the binding interaction. We found that like many PPIs, the energy of the antizyme-AZIN binding interaction is distributed across many amino acid residues; mutation of individual residues did not have a significant effect on disrupting the PPI. We also examined the interaction between Clover-AZIN and antizyme-mRuby2 in cells. Evidence of a direct interaction between Clover-AZIN and antizyme-mRuby2 was observed within cells, validating the use of this FRET sensor for probing intracellular antizyme-AZIN PPI. In conclusion, we have developed and optimized a FRET sensor which can be adapted for high throughput screening of either in vitro or intracellular activity.
Asunto(s)
Proteínas Portadoras/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/química , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Proteínas Portadoras/metabolismo , Humanos , Ornitina Descarboxilasa/metabolismo , Unión Proteica/fisiología , Proteínas/metabolismoRESUMEN
Metastases account for more than 90% of all cancer deaths and respond poorly to most therapies. There remains an urgent need for new therapeutic modalities for the treatment of advanced metastatic cancers. The benzimidazole methylcarbamate drugs, commonly used as anti-helmitics, have been suggested to have anticancer activity, but progress has been stalled by their poor water solubility and poor suitability for systemic delivery to disseminated cancers. We synthesized and characterized the anticancer activity of novel benzimidazoles containing an oxetane or an amine group to enhance solubility. Among them, the novel oxetanyl substituted compound 18 demonstrated significant cytotoxicity toward a variety of cancer cell types including prostate, lung, and ovarian cancers with strong activity toward highly aggressive cancer lines (IC50: 0.9-3.8⯵M). Compound 18 achieved aqueous solubility of 361⯵M. In a mouse xenograft model of a highly metastatic human prostate cancer, compound 18 (30â¯mg/kg) significantly inhibited the growth of established tumors (T/C: 0.36) without noticeable toxicity.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , Carbamatos/farmacología , Agua/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Carbamatos/síntesis química , Carbamatos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Solubilidad , Relación Estructura-Actividad , Células Tumorales CultivadasAsunto(s)
Simulación por Computador , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Triptófano Oxigenasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Humanos , Inmunoterapia , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Triptófano Oxigenasa/inmunología , Triptófano Oxigenasa/metabolismoRESUMEN
The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC50 of the most potent ACL inhibitor A1 is 4.1µM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.
Asunto(s)
ATP Citrato (pro-S)-Liasa/antagonistas & inhibidores , Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Furanos/química , Ácidos Carboxílicos/química , Línea Celular , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
Aberrant cellular metabolism drives cancer proliferation and metastasis. ATP citrate lyase (ACL) plays a critical role in generating cytosolic acetyl CoA, a key building block for de novo fatty acid and cholesterol biosynthesis. ACL is overexpressed in cancer cells, and siRNA knockdown of ACL limits cancer cell proliferation and reduces cancer stemness. We characterized a new class of ACL inhibitors bearing the key structural feature of the natural product emodin. Structure-activity relationship (SAR) study led to the identification of 1d as a potent lead that demonstrated dose-dependent inhibition of proliferation and cancer stemness of the A549 lung cancer cell line. Computational modeling indicates this class of inhibitors occupies an allosteric binding site and blocks the entrance of the substrate citrate to its binding site.
Asunto(s)
ATP Citrato (pro-S)-Liasa/antagonistas & inhibidores , Diseño de Fármacos , Emodina/síntesis química , Emodina/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , ATP Citrato (pro-S)-Liasa/química , ATP Citrato (pro-S)-Liasa/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Emodina/química , Emodina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/efectos de los fármacos , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC50: 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC50: 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC50: 0.55µM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10µM, indicating specificity towards cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Chalconas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/genética , Antineoplásicos/síntesis química , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chalconas/síntesis química , Chalconas/química , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , MutaciónRESUMEN
The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome's chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Línea Celular , Colorantes Fluorescentes , Humanos , Estructura Molecular , Pliegue de Proteína , Conejos , Saccharomyces cerevisiae , Bibliotecas de Moléculas PequeñasRESUMEN
Inâ vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600-1000â nm). Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600â nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter.
Asunto(s)
Colorantes Fluorescentes/química , Péptidos/química , Proteínas Quinasas/química , Fosforilación , Transducción de SeñalRESUMEN
An anthraquinone-based fluorescent quencher is described that is applicable to fluorophores throughout the visible spectrum and into the near IR. This species has been used to construct a palate of multicolour sensors of proteolysis and photolysis.
Asunto(s)
Antraquinonas/química , Colorantes Fluorescentes/química , Técnicas Biosensibles , Fotólisis , Proteolisis , Espectrometría de Fluorescencia , Tripsina/análisis , Tripsina/metabolismoRESUMEN
The recently synthesized 3-tert-butyl-5-methyl-1,2,4-triazole reacted with KBH4 to give the new potassium tris(3-tert-butyl-5-methyl-1,2,4-triazolyl)borate K(Ttz(tBu,Me)) ligand. Ttz(tBu,Me) formed a four-coordinate (Ttz(tBu,Me))CoCl complex and five-coordinate (Ttz(tBu,Me))CoNO3 and (Ttz(tBu,Me))ZnOAc complexes. When these complexes were compared to their Tp(tBu,Me) analogues, it was found that Ttz(tBu,Me) resulted in negligible steric differences. K(Ttz(tBu,Me)) is more water-soluble than K(Tp(tBu,Me)), so bulky tris(triazolyl)borate ligands should lead to functional models for enzyme active sites in an aqueous environment and the creation of water-soluble analogues of Tp catalysts.
RESUMEN
Ligands of intermediate steric bulk were designed to mimic metalloenzymes with histidine and carboxlyate binding sites. The reaction between tris(3-isopropylpyrazolyl)methane and butyllithium followed by SO3NMe3 in THF yielded the new ligand lithium tris(3-isopropylpyrazolyl)methane sulfonate (LiTpmsiPr). Various metal salts reacted with LiTpmsiPr to give the octahedral complexes M(TpmsiPr)2 (M = Zn, Cu, Ni, Co, Fe) in which each ligand has N,N,O binding to the metal. In the reaction between LiTpmsiPr and ZnCl2, in addition to the major product Zn(TpmsiPr)2, [LiTpmsiPrZnCl2].2THF was also formed as a minor product with a tetrahedral zinc atom coordinated to either N,N,Cl,Cl in the solid phase or N,N,N,Cl in acetonitrile solution. Although TpmsiPr is coordinatively flexible and can act as a bipodal or tripodal ligand, it appears to favor the formation of octahedral L2M complexes.