RESUMEN

[This corrects the article DOI: 10.1039/D3CB00126A.].

RESUMEN

Quite recently we discovered that copper(II) complexes with isomeric morpholine-thiosemicarbazone hybrid ligands show good cytotoxicity in cancer cells and that the molecular target responsible for this activity might be tubulin. In order to obtain better lead drug candidates, we opted to exploit the power of coordination chemistry to (i) assemble structures with globular shape to better fit the colchicine pocket and (ii) vary the metal ion. We report the synthesis and full characterization of bis-ligand cobalt(III) and iron(III) complexes with 6-morpholinomethyl-2-formylpyridine 4N-(4-hydroxy-3,5-dimethylphenyl)-3-thiosemicarbazone (HL1), 6-morpholinomethyl-2-acetylpyridine 4N-(4-hydroxy-3,5-dimethylphenyl)-3-thiosemicarbazone (HL2), and 6-morpholinomethyl-2-formylpyridine 4N-phenyl-3-thiosemicarbazone (HL3), and mono-ligand nickel(II), zinc(II) and palladium(II) complexes with HL1, namely [CoIII(HL1)(L1)](NO3)2 (1), [CoIII(HL2)(L2)](NO3)2 (2), [CoIII(HL3)(L3)](NO3)2 (3), [FeIII(L2)2]NO3 (4), [FeIII(HL3)(L3)](NO3)2 (5), [NiII(L1)]Cl (6), [Zn(L1)Cl] (7) and [PdII(HL1)Cl]Cl (8). We discuss the effect of the metal identity and metal complex stoichiometry on in vitro cytotoxicity and antitubulin activity. The high antiproliferative activity of complex 4 correlated well with inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity were supported by experimental results and molecular docking calculations.

Asunto(s)

Colchicina , Complejos de Coordinación , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Complejos de Coordinación/síntesis química , Colchicina/química , Colchicina/metabolismo , Colchicina/farmacología , Humanos , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/síntesis química , Sitios de Unión , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Polimerizacion , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Estructura Molecular , Proliferación Celular/efectos de los fármacos

RESUMEN

Ovarian cancer is among the most prevalent causes of mortality among women. Despite improvements in diagnostic methods, non-specific symptoms and delayed gynecological exams can lead to late-stage ovarian tumor discovery. In this study, the effect of an anti-cancer compound, 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (Compound 1), was examined. The impacts of cytotoxicity, apoptosis, and metabolomic changes in ovarian cancer cell lines SK-OV-3 and OVCAR-3, as well as glycosphingolipid (GSL) expression, on cancer stem cells (CSCs), marked as CD49f+, and non-CSCs (CD49f-) were explored. Treatment with Compound 1 reduced the percentage of CSCs compared to non-treated cells (p < 0.001). The functional impact of eight GSLs on CSCs and non-CSCs was examined using flow cytometry. The glycophenotype changed in both cell lines, with increases or decreases in its expression, after the treatment. These findings raise the possibility of specifically targeting CSCs in ovarian cancer therapy. Additionally, treatment with Compound 1 resulted in statistically meaningful increased apoptosis, including both early and late apoptosis (p < 0.001), suggesting a pivotal role in initiating programmed cell death by the apoptotic pathway. The analysis revealed that the metabolic activity of treated cancer cells was lower compared to those of the control group (p < 0.001).

Asunto(s)

Apoptosis , Glicoesfingolípidos , Metabolómica , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Glicoesfingolípidos/metabolismo , Línea Celular Tumoral , Metabolómica/métodos , Antineoplásicos/farmacología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Metaboloma/efectos de los fármacos , Piridinas/farmacología

RESUMEN

Biocatalysis has played a limited role in the early stages of drug discovery. This is often attributed to the limited substrate scope of enzymes not affording access to vast areas of novel chemical space. Here, we have shown a promiscuous nitroreductase enzyme (NR-55) can be used to produce a panel of functionalised anilines from a diverse panel of aryl nitro starting materials. After screening on analytical scale, we show that sixteen substrates could be scaled to 1 mmol scale, with several poly-functional anilines afforded with ease under the standard conditions. The aniline products were also screened for activity against several cell lines of interest, with modest activity observed for one compound. This study demonstrates the potential for nitroreductase biocatalysis to provide access to functional fragments under benign conditions.

RESUMEN

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

Asunto(s)

Senescencia Celular , Mitocondrias , Mitofagia , Ubiquitina-Proteína Ligasas , Mitofagia/efectos de los fármacos , Humanos , Senescencia Celular/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Quinasas/metabolismo , Fenotipo , Autofagia/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Superóxidos/metabolismo , Proteínas de Unión al ARN

RESUMEN

The development of copper(II) thiosemicarbazone complexes as potential anticancer agents, possessing dual functionality as inhibitors of R2 ribonucleotide reductase (RNR) and tubulin polymerization by binding at the colchicine site, presents a promising avenue for enhancing therapeutic effectiveness. Herein, we describe the syntheses and physicochemical characterization of four isomeric proligands H2L3-H2L6, with the methylmorpholine substituent at pertinent positions of the pyridine ring, along with their corresponding Cu(II) complexes 3-6. Evidently, the position of the morpholine moiety and the copper(II) complex formation have marked effects on the in vitro antiproliferative activity in human uterine sarcoma MES-SA cells and the multidrug-resistant derivative MES-SA/Dx5 cells. Activity correlated strongly with quenching of the tyrosyl radical (Y•) of mouse R2 RNR protein, inhibition of RNR activity in the cancer cells, and inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity, supported by experimental results and molecular modeling calculations, are presented.

Asunto(s)

Antineoplásicos , Cobre , Morfolinas , Ribonucleótido Reductasas , Tiosemicarbazonas , Tubulina (Proteína) , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/síntesis química , Humanos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Morfolinas/farmacología , Morfolinas/química , Morfolinas/síntesis química , Cobre/química , Ratones , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Estructura-Actividad , Polimerizacion/efectos de los fármacos , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Piridinas/farmacología , Piridinas/química , Piridinas/síntesis química , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Ensayos de Selección de Medicamentos Antitumorales , Modelos Moleculares

RESUMEN

Deoxycholic acid derivatives containing various heterocyclic functional groups at C-3 on the steroid scaffold were designed and synthesized as promising dual tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) inhibitors, which are potential targets to potentiate topoisomerase poison antitumor therapy. The methyl esters of DCA derivatives with benzothiazole or benzimidazole moieties at C-3 demonstrated promising inhibitory activity in vitro against TDP1 with IC50 values in the submicromolar range. Furthermore, methyl esters 4d-e, as well as their acid counterparts 3d-e, inhibited the phosphodiesterase activity of both TDP1 and TDP2. The combinations of compounds 3d-e and 4d-e with low-toxic concentrations of antitumor drugs topotecan and etoposide showed significantly greater cytotoxicity than the compounds alone. The docking of the derivatives into the binding sites of TDP1 and TDP2 predicted plausible binding modes of the DCA derivatives.

Asunto(s)

Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/metabolismo , Modelos Moleculares , Ácido Desoxicólico/farmacología , Relación Estructura-Actividad

RESUMEN

Upon undergoing mucoid conversion within the lungs of cystic fibrosis patients, the pathogenic bacterium Pseudomonas aeruginosa synthesises copious quantities of the virulence factor and exopolysaccharide alginate. The enzyme guanosine diphosphate mannose dehydrogenase (GMD) catalyses the rate-limiting step and irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Since there is no corresponding enzyme in humans, strategies that could prevent its mechanism of action could open a pathway for new and selective inhibitors to disrupt bacterial alginate production. Using virtual screening, a library of 1447 compounds within the Known Drug Space parameters were evaluated against the GMD active site using the Glide, FRED and GOLD algorithms. Compound hit evaluation with recombinant GMD refined the panel of 40 potential hits to 6 compounds which reduced NADH production in a time-dependent manner; of which, an usnic acid derivative demonstrated inhibition six-fold stronger than a previously established sugar nucleotide inhibitor, with an IC50 value of 17 µM. Further analysis by covalent docking and mass spectrometry confirm a single site of GMD alkylation.

RESUMEN

Phosphatidylcholine-specific phospholipase C (PC-PLC) is an enzyme that catalyzes the formation of the important secondary messengers phosphocholine and diacylglycerol (DAG) from phosphatidylcholine. Although PC-PLC has been linked to the progression of many pathological conditions, including cancer, atherosclerosis, inflammation and neuronal cell death, studies of PC-PLC on the protein level have been somewhat neglected with relatively scarce data. To date, the human gene expressing PC-PLC has not yet been found, and the only protein structure of PC-PLC that has been solved was from Bacillus cereus (PC-PLCBc). Nonetheless, there is evidence for PC-PLC activity as a human functional equivalent of its prokaryotic counterpart. Additionally, inhibitors of PC-PLCBc have been developed as potential therapeutic agents. The most notable classes include 2-aminohydroxamic acids, xanthates, N,N'-hydroxyureas, phospholipid analogues, 1,4-oxazepines, pyrido[3,4-b]indoles, morpholinobenzoic acids and univalent ions. However, many medicinal chemistry studies lack evidence for their cellular and in vivo effects, which hampers the progression of the inhibitors towards the clinic. This review outlines the pathological implications of PC-PLC and highlights current progress and future challenges in the development of PC-PLC inhibitors from the literature.

Asunto(s)

Fosfatidilcolinas , Fosfolipasas de Tipo C , Humanos , Fosfatidilcolinas/metabolismo

RESUMEN

A series of four indolo[2,3-e]benzazocines HL1-HL4 and two indolo[2,3-f]benzazonines HL5 and HL6, as well as their respective copper(II) complexes 1-6, were synthesized and characterized by 1H and 13C NMR spectroscopy, ESI mass spectrometry, single crystal X-ray diffraction (SC-XRD) and combustion analysis (C, H, N). SC-XRD studies of precursors Vd, VIa·0.5MeOH, of ligands HL4 and HL6·DCM, and complexes 2·2DMF, 5·2DMF, 5'·iPrOH·MeOH provided insights into the energetically favored conformations of eight- and nine-membered heterocycles in the four-ring systems. In addition, proton dissociation constants (pKa) of HL1, HL2 and HL5, complexes 1, 2 and 5, overall stability constants (log ß) of 1, 2 and 5 in 30% (v/v) DMSO/H2O at 298 K, as well as thermodynamic solubility of HL1-HL6 and 1-6 in aqueous solution at pH 7.4 were determined by UV-vis spectroscopy. All compounds were tested for antiproliferative activity against Colo320, Colo205 and MCF-7 cell lines and showed IC50 values in the low micromolar to sub-micromolar concentration range, while some of them (HL1, HL5 and HL6, 1, 2 and 6) showed remarkable selectivity towards malignant cell lines. Ethidium bromide displacement studies provided evidence that DNA is not the primary target for these drugs. Rather, inhibition of tubulin assembly is likely the underlying mechanism responsible for their antiproliferative activity. Tubulin disassembly experiments showed that HL1 and 1 are effective microtubule destabilizing agents binding to the colchicine site. This was also confirmed by molecular modelling investigations. To the best of our knowledge, complex 1 is the first reported transition metal complex to effectively bind to the tubulin-colchicine pocket.

Asunto(s)

Antineoplásicos , Complejos de Coordinación , Compuestos Heterocíclicos , Cobre/química , Tubulina (Proteína) , Modelos Moleculares , Complejos de Coordinación/química , Espectroscopía de Resonancia Magnética , Microtúbulos , Antineoplásicos/química , Cristalografía por Rayos X , Ligandos

RESUMEN

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is an important enzyme in the DNA repair system. The ability of the enzyme to repair DNA damage induced by a topoisomerase 1 poison such as the anticancer drug topotecan makes TDP1 a promising target for complex antitumor therapy. In this work, a set of new 5-hydroxycoumarin derivatives containing monoterpene moieties was synthesized. It was shown that most of the conjugates synthesized demonstrated high inhibitory properties against TDP1 with an IC50 in low micromolar or nanomolar ranges. Geraniol derivative 33a was the most potent inhibitor with IC50 130 nM. Docking the ligands to TDP1 predicted a good fit with the catalytic pocket blocking access to it. The conjugates used in non-toxic concentration increased cytotoxicity of topotecan against HeLa cancer cell line but not against conditionally normal HEK 293A cells. Thus, a new structural series of TDP1 inhibitors, which are able to sensitize cancer cells to the topotecan cytotoxic effect has been discovered.

Asunto(s)

Antineoplásicos , Topotecan , Humanos , Topotecan/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/química , Relación Estructura-Actividad , Hidrolasas Diéster Fosfóricas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral

RESUMEN

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a promising target for antitumor therapy; the use of TDP1 inhibitors with a topoisomerase 1 poison such as topotecan is a potential combination therapy. In this work, a novel series of 3,5-disubstituted thiazolidine-2,4-diones was synthesized and tested against TDP1. The screening revealed some active compounds with IC50 values less than 5 µM. Interestingly, compounds 20d and 21d were the most active, with IC50 values in the submicromolar concentration range. None of the compounds showed cytotoxicity against HCT-116 (colon carcinoma) and MRC-5 (human lung fibroblasts) cell lines in the 1-100 µM concentration range. Finally, this class of compounds did not sensitize cancer cells to the cytotoxic effect of topotecan.

Asunto(s)

Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Tiazolidinedionas , Humanos , Modelos Moleculares , Monoterpenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Topotecan/farmacología , Tiazolidinedionas/farmacología

RESUMEN

Due to the role of cancer stem cells (CSCs) in tumor resistance and glycosphingolipid (GSL) involvement in tumor pathogenesis, we investigated the effect of a newly synthesized compound (3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide 1 on the percentage of CSCs and the expression of six GSLs on CSCs and non-CSCs on breast cancer cell lines (MDA-MB-231 and MCF-7). We also investigated the effect of 1 on the metabolic profile of these cell lines. The MTT assay was used for cytotoxicity determination. Apoptosis and expression of GSLs were assessed by flow cytometry. A GC-MS-coupled system was used for the separation and identification of metabolites. Compound 1 was cytotoxic for both cell lines, and the majority of cells died by treatment-induced apoptosis. The percentage of CSCs was significantly lower in the MDA-MB-231 cell line. Treatment with 1 caused a decrease of CSC IV6Neu5Ac-nLc4Cer+ MDA-MB-231 cells. In the MCF-7 cell line, the percentage of GalNAc-GM1b+ CSCs was increased, while the expression of Gg3Cer was decreased in both CSC and non-CSC. Twenty-one metabolites were identified by metabolic profiling. The major impact of the treatment was in glycolysis/gluconeogenesis, pyruvate and inositol metabolism. Compound 1 exhibited higher potency in MBA-MB-231 cells, and it deserves further examination.

Asunto(s)

Antineoplásicos , Neoplasias de la Mama , Quinolinas , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Glucosa/metabolismo , Glicoesfingolípidos/metabolismo , Humanos , Inositol/farmacología , Células Madre Neoplásicas/metabolismo , Piridinas/metabolismo , Piridinas/farmacología , Piruvatos/metabolismo , Quinolinas/farmacología

RESUMEN

Alzheimer's disease (AD) is a neurodegenerative disease associated with memory impairment and other central nervous system (CNS) symptoms. Two myrtenal-adamantane conjugates (MACs) showed excellent CNS potential against Alzheimer's models. Adamantane is a common pharmacophore for drug design, and myrtenal (M) demonstrated neuroprotective effects in our previous studies. The aim of this study is to evaluate the MACs' neuroprotective properties in dementia. METHODS: Scopolamine (Scop) was applied intraperitoneally in Wistar rats for 11 days, simultaneously with MACs or M as a referent, respectively. Brain acetylcholine esterase (AChE) activity, noradrenaline and serotonin levels, and oxidative brain status determination followed behavioral tests on memory abilities. Molecular descriptors and docking analyses for AChE activity center affinity were performed. RESULTS: M derivatives have favorable physicochemical parameters to enter the CNS. Both MACs restored memory damaged by Scop, showing significant AChE-inhibitory activity in the cortex, in contrast to M, supported by the modeling analysis. Moderate antioxidant properties were manifested by glutathione elevation and catalase activity modulation. MACs also altered noradrenaline and serotonin content in the hippocampus. CONCLUSION: For the first time, neuroprotective properties of two MACs in a rat dementia model were observed. They were stronger than the natural M effects, which makes the substances promising candidates for AD treatment.

Asunto(s)

Adamantano , Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Acetilcolinesterasa/metabolismo , Adamantano/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Monoterpenos Bicíclicos , Aprendizaje por Laberinto , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Norepinefrina , Estrés Oxidativo , Ratas , Ratas Wistar , Escopolamina/farmacología , Serotonina/metabolismo

RESUMEN

Tyrosyl-DNA phosphodiesterase 1(TDP1) is a promising target for a new therapy in oncological disease as an adjunct to topoisomerase 1 (TOP1) drugs. In this paper, novel thiazolidin-4-one derivatives with a benzyl and monoterpene substituents were synthesized. Compounds with a monoterpene fragment attached via a phenyloxy linker were active against TDP1 with IC50 values in the 1 ÷ 3 µM range, while direct attachment of monoterpene moiety to the thiazolidin-4-one fragment had no activity. Molecular modelling predicted two plausible binding modes of the active compounds both effectively blocking access to the catalytic site of TDP. At non-toxic concentrations the active ligands potentiated the efficacy of the TOP1 poison topotecan in human cervical cancer HeLa cells, but not in non-cancerous HEK293A cells.

Asunto(s)

Inhibidores de Fosfodiesterasa , Hidrolasas Diéster Fosfóricas , Esterasas/metabolismo , Células HeLa , Humanos , Monoterpenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Relación Estructura-Actividad

RESUMEN

Inhibiting tyrosyl-DNA phosphodiesterase 1 (TDP1) is a promising strategy for increasing the effectiveness of existing antitumor therapy since it can remove the DNA lesions caused by anticancer drugs, which form covalent complexes with topoisomerase 1 (TOP1). Here, new adamantane-monoterpene conjugates with a 1,2,4-triazole or 1,3,4-thiadiazole linker core were synthesized, where (+)-and (-)-campholenic and (+)-camphor derivatives were used as monoterpene fragments. The campholenic derivatives 14a-14b and 15a-b showed activity against TDP1 at a low micromolar range with IC50 ~5-6 µM, whereas camphor-containing compounds 16 and 17 were ineffective. Surprisingly, all the compounds synthesized demonstrated a clear synergy with topotecan, a TOP1 poison, regardless of their ability to inhibit TDP1. These findings imply that different pathways of enhancing topotecan toxicity other than the inhibition of TDP1 can be realized.

Asunto(s)

Adamantano , Antineoplásicos , Adamantano/farmacología , Antineoplásicos/farmacología , Alcanfor , Monoterpenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Topotecan/farmacología

RESUMEN

A series of latonduine and indoloquinoline derivatives HL1-HL8 and their copper(II) complexes (1-8) were synthesized and comprehensively characterized. The structures of five compounds (HL6, [CuCl(L1)(DMF)]·DMF, [CuCl(L2)(CH3OH)], [CuCl(L3)]·0.5H2O, and [CuCl2(H2L5)]Cl·2DMF) were elucidated by single crystal X-ray diffraction. The copper(II) complexes revealed low micro- to sub-micromolar IC50 values with promising selectivity toward human colon adenocarcinoma multidrug-resistant Colo320 cancer cells as compared to the doxorubicin-sensitive Colo205 cell line. The lead compounds HL4 and 4 as well as HL8 and 8 induced apoptosis efficiently in Colo320 cells. In addition, the copper(II) complexes had higher affinity to DNA than their metal-free ligands. HL8 showed selective inhibition for the PIM-1 enzyme, while 8 revealed strong inhibition of five other enzymes, i.e., SGK-1, PKA, CaMK-1, GSK3ß, and MSK1, from a panel of 50 kinases. Furthermore, molecular modeling of the ligands and complexes showed a good fit to the binding pockets of these targets.

Asunto(s)

Complejos de Coordinación/química , Cobre/química , Compuestos Heterocíclicos con 3 Anillos/química , Inhibidores de Proteínas Quinasas/química , Quinolinas/química , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sitios de Unión , Bovinos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/metabolismo , Complejos de Coordinación/farmacología , Complejos de Coordinación/uso terapéutico , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Humanos , Indoles/química , Conformación Molecular , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Solubilidad , Relación Estructura-Actividad

RESUMEN

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Asunto(s)

Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Trifosfato/metabolismo , Sitios de Unión/efectos de los fármacos , Descubrimiento de Drogas , Proteínas HSP70 de Choque Térmico/química , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Dominios Proteicos/efectos de los fármacos

RESUMEN

The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor-positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive, triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative, as well as estrogen receptor-positive, breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells, suggesting its mechanism of action differs from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds, as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.

Asunto(s)

Antineoplásicos , Neoplasias de la Mama/tratamiento farmacológico , Carbocianinas , Citotoxinas , Indoles , Piperazinas , Piridinas , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Células CHO , Carbocianinas/química , Carbocianinas/farmacología , Cricetulus , Citotoxinas/química , Citotoxinas/farmacología , Femenino , Células HEK293 , Humanos , Indoles/química , Indoles/farmacología , Piperazinas/química , Piperazinas/farmacología , Piridinas/química , Piridinas/farmacología

Asunto(s)

Inhibidores Enzimáticos/química , Nucleótidos/biosíntesis , Aspergillus/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Deshidrogenasas de Carbohidratos/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Galactosa/análogos & derivados , Galactosa/biosíntesis , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/metabolismo , Azúcares de Nucleósido Difosfato/biosíntesis , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/metabolismo , Pseudomonas aeruginosa/enzimología , Uridina Difosfato/análogos & derivados , Uridina Difosfato/biosíntesis