RESUMO
Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line.
Assuntos
Descoberta de Drogas , Proteoma , Proteômica , Espectrometria de Massas em Tandem , Proteômica/métodos , Humanos , Proteoma/análise , Descoberta de Drogas/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular TumoralRESUMO
Omics technologies focus on uncovering the complex nature of molecular mechanisms in cells and organisms, including biomarkers and drug targets discovery. Aiming at these tasks, we see that information extracted from omics data is still underused. In particular, characteristics of differentially regulated molecules can be combined in a single score to quantify the signaling pathway activity. Such a metric can be useful for comprehensive data interpretation to follow: (1) developing molecular responses in time; (2) potency of a drug on a certain cell culture; (3) ranking the signaling pathway activity in stimulated cells; and (4) integration of the omics data and assay-based measurements of cell viability, cytotoxicity, and proliferation. With recent advances in ultrafast mass spectrometry for quantitative proteomics allowing data collection in a few minutes, proteomics score for cellular response to stimuli can become a fast, accurate, and informative complement to bioassays. Here, we utilized an interquartile-based selection of differentially regulated features and a variety of schemes for quantifying cellular responses to come up with the quantitative metric for total cellular response and pathway activity. Validation was performed using antiproliferative and virus assays and label-free proteomics data collected for cancer cells subjected to drug stimulation.
Assuntos
Proteômica , Transdução de Sinais , Proteômica/métodos , BiomarcadoresRESUMO
Efficient protocols for the synthesis of triphenylcyclopentadienyl rhodium halides [(1,2,4-C5Ph3H2)RhX2]2 (1a,b: X = Cl, I) starting from 1,2,4-triphenylcyclopentadiene or the cyclooctadiene derivative (1,2,4-C5Ph3H2)Rh(cod) (2) were developed. Iodide abstraction from 1b with thallium or silver salts allowed us to prepare rhodocenium [(1,2,4-C5Ph3H2)RhCp]PF6 (3PF6) and mesitylene complex [(1,2,4-C5Ph3H2)Rh(mesitylene)](SbF6)2 (4(SbF6)2). Halides 1a,b (at 0.5 mol % loading) showed high catalytic activity in the construction of C-C, C-O, and C-N bonds via the C(sp2)-H activation approach. Their efficiency was demonstrated in the synthesis of more than 40 examples of polycyclic organic compounds (such as isocoumarins and naphthalenes, as well as isoquinolinium and dibenzo[a,f]quinolizinium salts). The protocols developed tolerate a wide range of functional groups. In particular, they were successfully used for the atom- and step-economical synthesis of hydroxy-substituted isocoumarins, including the natural product oospalactone 7fe. The 6- or 8-hydroxy-substituted isocoumarins showed moderate antiproliferative activity against several human cell lines in vitro.
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A series of novel organotin(IV) complexes on the base of 2-(N-3',5'-di-tert-butyl-4'-hydroxyphenyl)-iminomethylphenol (L) of formulae Me2SnBr2(L)2 (1), Bu2SnCl2(L)2(2), Ph2SnCl2(L) (3), Ph2SnCl2(L)2 (4) Ph3SnBr(L)2 (5) were synthesized and characterized by 1H, 13C, 119Sn NMR, IR, ESI-MS and elemental analysis. The crystal structures of initial L and complex 2 were determined by XRD method. It was found that L crystallizes in the orthorhombic syngony. The distorted octahedron geometry around Sn center is observed in the structure of complex 2. Intra- and inter-molecular hydrogen bonds were found in both structures. The antioxidant activity of new complexes as reducing agents, radical scavengers and lipoxygenase inhibitors was estimated spectrophotometrically in CUPRAC and DPPH tests (compounds 1 and 5 were found to be the most active in both methods), and in the process of enzymatic oxidation in vitro of linoleic acid under the action of lipoxygenase LOX 1-B (EC50 > 33.3 µM for complex 2). Furthermore, compounds 1-5 have been investigated for their antiproliferative activity in vitro towards HCT-116, MCF-7 and A-549 and non-malignant WI-38 human cell lines. Complexes 2 and 5 demonstrated the highest activity. The plausible mechanisms of the antiproliferative activity of compounds, including the influence on the polymerization of Tb+MAP, are discussed. Some of the synthesized compounds have also actively induced apoptosis and blocked proliferation in the cell cycle G2/M phase.
Assuntos
Antineoplásicos , Compostos Orgânicos de Estanho , Humanos , Antioxidantes/farmacologia , Compostos Orgânicos de Estanho/farmacologia , Compostos Orgânicos de Estanho/química , Bases de Schiff/farmacologia , Bases de Schiff/química , Cristalografia por Raios X , Antineoplásicos/farmacologiaRESUMO
The synthesis of the first conjugates of acridine with cobalt bis(dicarbollide) are reported. A novel 9-azido derivative of acridine was prepared through the reaction of 9-methoxyacridine with N3CH2CH2NH2, and its solid-state molecular structure was determined via single-crystal X-ray diffraction. The azidoacridine was used in a copper (I)-catalyzed azide-alkyne cycloaddition reaction with cobalt bis(dicarbollide)-based terminal alkynes to give the target 1,2,3-triazoles. DNA interaction studies via absorbance spectroscopy showed the weak binding of the obtained conjugates with DNA. The antiproliferative activity (IC50) of the boronated conjugates against a series of human cell lines was evaluated through an MTT assay. The results suggested that acridine derivatives of cobalt bis(dicarbollide) might serve as a novel scaffold for the future development of new agents for boron neutron capture therapy (BNCT).
Assuntos
Acridinas , Boro , Humanos , Boro/química , Estrutura Molecular , Acridinas/farmacologia , Cobalto/química , DNARESUMO
Protein quantitation in tissue cells or physiological fluids based on liquid chromatography/mass spectrometry is one of the key sources of information on the mechanisms of cell functioning during chemotherapeutic treatment. Information on significant changes in protein expression upon treatment can be obtained by chemical proteomics and requires analysis of the cellular proteomes, as well as development of experimental and bioinformatic methods for identification of the drug targets. Low throughput of whole proteome analysis based on liquid chromatography and tandem mass spectrometry is one of the main factors limiting the scale of these studies. The method of direct mass spectrometric identification of proteins, DirectMS1, is one of the approaches developed in recent years allowing ultrafast proteome-wide analyses employing minute-scale gradients for separation of proteolytic mixtures. Aim of this work was evaluation of both possibilities and limitations of the method for identification of drug targets at the level of whole proteome and for revealing cellular processes activated by the treatment. Particularly, the available literature data on chemical proteomics obtained earlier for a large set of onco-pharmaceuticals using multiplex quantitative proteome profiling were analyzed. The results obtained were further compared with the proteome-wide data acquired by the DirectMS1 method using ultrashort separation gradients to evaluate efficiency of the method in identifying known drug targets. Using ovarian cancer cell line A2780 as an example, a whole-proteome comparison of two cell lysis techniques was performed, including the freeze-thaw lysis commonly employed in chemical proteomics and the one based on ultrasonication for cell disruption, which is the widely accepted as a standard in proteomic studies. Also, the proteome-wide profiling was performed using ultrafast DirectMS1 method for A2780 cell line treated with lonidamine, followed by gene ontology analyses to evaluate capabilities of the method in revealing regulation of proteins in the cellular processes associated with drug treatment.
Assuntos
Neoplasias Ovarianas , Proteoma , Humanos , Feminino , Proteoma/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Neoplasias Ovarianas/tratamento farmacológico , Espectrometria de Massas em TandemRESUMO
Novel fluorescent 7-methoxy- and 7-(diethylamino)-coumarins modified with azido-group on the side chain have been synthesized. Their photophysical properties and single crystals structure characteristics have been studied. In order to demonstrate the possibilities of fluorescent labeling, obtained coumarins have been tested with closo-dodecaborate derivative bearing terminal alkynyl group. CuI catalyzed Huisgen 1,3-dipolar cycloaddition reaction has led to fluorescent conjugates formation. The absorption-emission spectra of the formed conjugates have been presented. The antiproliferative activity and uptake of compounds against several human cell lines were evaluated.
Assuntos
Química Click , Cumarínicos , Humanos , Cumarínicos/química , Compostos de Boro/química , Ânions , Azidas/químicaRESUMO
The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100â structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PLpro . In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PLpro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.
Assuntos
Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2 , SARS-CoV-2/efeitos dos fármacosRESUMO
A series of bifunctional Ru(III) complexes with lonidamine-modified ligands (lonidamine is a selective inhibitor of aerobic glycolysis in cancer cells) was described. Redox properties of Ru(III) complexes were characterized by cyclic voltammetry. An easy reduction suggested a perspective for these agents as their whole mechanism of action seems to be based on activation by metal atom reduction. New compounds demonstrated a more pronounced antiproliferative potency than the parental drug; individual new agents were more cytotoxic than cisplatin. Stability studies showed an increase in the stability of complexes along with the linker length. A similar trend was noted for antiproliferative activity, cellular uptake, apoptosis induction, and thioredoxin reductase inhibition. Finally, at concentrations that did not alter water solubility, the selected new complex evoked no acute toxicity in Balb/c mice.
Assuntos
Indazóis/química , Rutênio/química , Rutênio/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Oxirredução , Relação Estrutura-Atividade , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Nanoparticles based on biocompatible methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG113-b-P(D,L)LAn) copolymers as potential vehicles for the anticancer agent oxaliplatin were prepared by a nanoprecipitation technique. It was demonstrated that an increase in the hydrophobic PLA block length from 62 to 173 monomer units leads to an increase of the size of nanoparticles from 32 to 56 nm. Small-angle X-ray scattering studies confirmed the "core-corona" structure of mPEG113-b-P(D,L)LAn nanoparticles and oxaliplatin loading. It was suggested that hydrophilic oxaliplatin is adsorbed on the core-corona interface of the nanoparticles during the nanoprecipitation process. The oxaliplatin loading content decreased from 3.8 to 1.5% wt./wt. (with initial loading of 5% wt./wt.) with increasing PLA block length. Thus, the highest loading content of the anticancer drug oxaliplatin with its encapsulation efficiency of 76% in mPEG113-b-P(D,L)LAn nanoparticles can be achieved for block copolymer with short hydrophobic block.
Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Nanopartículas/química , Oxaliplatina/química , Poliésteres/química , Polietilenoglicóis/química , Polímeros/química , Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos/métodos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da PartículaRESUMO
Arylazoimidazoles are important dyes which were intensively studied in the past. In contrast, triarylazoimidazoles (derivatives which carry aryl substituents at the imidazole core) received almost no attention in the scientific literature. Here, we report a new family of simple and easily accessible triarylazoimidazole-group 12 metal complexes, which feature highly efficient photo-luminescence emission (Φ up to 0.44). Novel compounds exhibit bright red emission in solution, which could be excited with a visible light.
RESUMO
The dinuclear anticancer agents 1,n-bis{chlorido[3-(oxo-κO)-2-methyl-4-(1H)-pyridinonato-κO4](η6-p-cymene)-ruthenium(II)}alkane (PyRu 2n ) exhibit high antiproliferative activity in human cancer cell. Reactivity studies with DNA and protein revealed uncommon protein-DNA and DNA-DNA crosslinking ability. We report here studies on the reactions of the diruthenium organometallics PyRu 26 and PyRu 28 in comparison with a mononuclear analogue PyRu3 with amino acids using mass spectrometry and NMR spectroscopy. The compounds behave very similarly, independent of the spacer length between the metal center and of the nuclearity of the complexes. Incubation with L-cysteine (Cys) results in fast release of the pyridone ligand, with the Ru complexes able to form Cys adducts. In contrast, L-methionine forms, initially, adducts with the metal centers, but over time, the adducts decompose. Similar behavior was observed for the reaction with L-histidine with [Ru(η6-p-cymene)(L-histidine)] species detected.
Assuntos
Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Compostos Organometálicos/farmacologia , Rutênio/farmacologia , Aminoácidos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Rutênio/química , Relação Estrutura-AtividadeRESUMO
Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal-and in particular platinum compound-DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine-metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum-guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal-double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents.
Assuntos
Antineoplásicos/química , Reagentes de Ligações Cruzadas/química , Adutos de DNA/química , Compostos Organoplatínicos/química , Compostos de Platina/química , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/químicaRESUMO
Stability and narrow size distribution are among the main requirements that apply to drug formulations based on polymeric nanoparticles. In this study, we obtained a series of particles based on biodegradable poly(D,L-lactide)-b-poly(ethylene glycol) (P(D,L)LAn-b-PEG113) copolymers with varied hydrophobic P(D,L)LA block length n from 50 to 1230 monomer units stabilized by poly(vinyl alcohol) (PVA) by a simple "oil-in-water" emulsion method. We found that nanoparticles of P(D,L)LAn-b-PEG113 copolymers with relatively short P(D,L)LA block (n ≤ 180) are prone to aggregate in water. P(D,L)LAn-b-PEG113 copolymers with n ≥ 680 can form spherical unimodal particles with values of hydrodynamic diameter less than 250 nm and polydispersity less than 0.2. The aggregation behavior of P(D,L)LAn-b-PEG113 particles was elucidated in terms of tethering density and conformation of PEG chains at the P(D,L)LA core. Docetaxel (DTX) loaded nanoparticles based on P(D,L)LA680-b-PEG113 and P(D,L)LA1230-b-PEG113 copolymers were formulated and studied. It was observed that DTX-loaded P(D,L)LAn-b-PEG113 (n = 680, 1230) particles are characterized by high thermodynamic and kinetic stability in aqueous medium. The cumulative release of DTX from the P(D,L)LAn-b-PEG113 (n = 680, 1230) particles is sustained. An increase in P(D,L)LA block length results in a decrease in DTX release rate. The in vitro antiproliferative activity and selectivity studies revealed that DTX-loaded P(D,L)LA1230-b-PEG113 nanoparticles demonstrate better anticancer performance than free DTX. Favorable freeze-drying conditions for DTX nanoformulation based on P(D,L)LA1230-b-PEG113 particles were also established.
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The combination of one molecule of organic and metal-based fragments that exhibit antitumor activity is a modern approach in the search for new promising drugs. In this work, biologically active ligands based on lonidamine (a selective inhibitor of aerobic glycolysis used in clinical practice) were introduced into the structure of an antitumor organometallic ruthenium scaffold. Resistant to ligand exchange reactions, compounds were prepared by replacing labile ligands with stable ones. Moreover, cationic complexes containing two lonidamine-based ligands were obtained. Antiproliferative activity was studied in vitro by MTT assays. It was shown that the increase in the stability in ligand exchange reactions does not influence cytotoxicity. At the same time, the introduction of the second lonidamine fragment approximately doubles the cytotoxicity of studied complexes. The ability to induce apoptosis and caspase activation in tumour cell MCF7 was studied by employing flow cytometry.
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Anthracene derivatives of ruthenium(II) arene compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) or a sugar phosphite ligand, viz., 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-d-glucofuranoside, were prepared in order to evaluate their anticancer properties compared to the parent compounds and to use them as models for intracellular visualization by fluorescence microscopy. Similar IC(50) values were obtained in cell proliferation assays, and similar levels of uptake and accumulation were also established. The X-ray structure of [{Ru(η(6)-C(6)H(5)CH(2)NHCO-anthracene)Cl(2)(pta)] is also reported.
Assuntos
Antracenos/química , Antineoplásicos/química , Compostos Organometálicos/química , Compostos de Rutênio/química , Ligação de Hidrogênio , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Modelos Moleculares , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Difração de Raios XRESUMO
Using inductively coupled plasma mass spectrometry (in combination with ultrafiltration) and microemulsion electrokinetic chromatography, the drug properties of two new, potentially multi-targeting Ru(III) and Pt(IV) compounds, containing biologically active ligands, were evaluated. The ruthenium complex with bexarotene was shown to bind to albumin faster than to transferrin and exhibits much the same (to albumin) binding profile in human serum. The Pt(IV)-lonidamine complex interacts with albumin relatively slowly but possesses high stability and lipophilicity (log P 1.62), which makes it possible the cellular uptake in a free (of proteins) form. Although both examined compounds display a moderate solubility (below 10-4 M), this stands compatible with their nanomolar cytotoxic activities. The Ru(III) compound, whose active moiety is a complexed anion, is deemed promising to be loaded on nanoscale anion-exchangers with the aim of controlled delivery.
Assuntos
Antineoplásicos , Compostos Organometálicos , Rutênio , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Ligantes , Compostos Organometálicos/química , Preparações Farmacêuticas , Rutênio/química , Transferrina/química , Transferrina/metabolismoRESUMO
One concept of improving anticancer effects of conventional platinum-based antitumor drugs consists of conjugating these compounds with other biologically (antitumor) active agents, acting by a different mechanism. Here, we present synthesis, physicochemical characterization, biological effects, and mechanisms of action of four new analogs of conventional cisplatin, namely, cis-Pt(II) complexes containing either methyl or ethyl pyrazole N-donor ligands and chlorido or iodido ligands. It is noteworthy that while chlorido complexes display activity in a variety of cancer cell lines comparable to cisplatin, iodido complexes are considerably more potent due to their enhanced hydrophobicity and consequently enhanced cellular accumulation. Moreover, all of the studied Pt(II) alkylpyrazole complexes display a higher selectivity for tumor cells and effectively overcome the acquired resistance to cisplatin. Further results focused on the mechanism of action of the studied complexes and showed that in contrast to cisplatin and several platinum-based antitumor drugs, DNA damage by the investigated Pt(II)-alkylpyrazole complexes does not play a major role in their mechanism of action. Our findings demonstrate that inhibition of the tubulin kinesin Eg5, which is essential for forming a functional mitotic spindle, plays an important role in their mechanism of antiproliferative action.
RESUMO
This work studied the mechanism of action of a Pt(IV) complex 2 bearing two axial lonidamine ligands, which are selective inhibitors of aerobic glycolysis. The presence of two lonidamine ligands in 2 compared to the parent Pt(II) complex increased its antiproliferative activity, cellular accumulation, and changed its cell cycle profile and mechanism of cell death. In 3D cell culture, 2 showed exceptional antiproliferative activity with IC50 values as low as 1.6 µM in MCF7 cells. The study on the influence of the lonidamine ligands in the Pt complex on glycolysis showed only low potency of ligands to affect metabolic processes in cancer cells, making the investigated complex, not a dual- or multi-action prodrug. However, the Pt(IV) prodrug effectively delivers the cytotoxic Pt(II) complex into cancer cells.
Assuntos
Antineoplásicos , Pró-Fármacos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Indazóis , Ligantes , Pró-Fármacos/farmacologiaRESUMO
Organometallic ruthenium-arene compounds bearing a maltol ligand have been shown to be nearly inactive in in vitro anticancer assays, presumably due to the formation of dimeric Ru(II) species in aqueous solutions. In an attempt to stabilize such complexes, [Ru(eta(6)-p-cymene)(XY)Cl] (XY=pyrones or thiopyrones) complexes with different substitution pattern of the (thio)pyrone ligands have been synthesized, their structures characterized spectroscopically, and their aquation behavior investigated as well as their tumor-inhibiting potency. The aquation behavior of pyrone systems with electron-donating substituents and of thiopyrone complexes was found to be significantly different from that of the maltol-type complex reported previously. However, the formation of the dimer can be excluded as the primary reason for the inactivity of the complex because some of the stable compounds are not active in cancer cell lines either. In contrast, studies of their reactivity towards amino acids demonstrate different reactivities of the pyrone and thiopyrone complexes, and the higher stability of the latter probably renders them active against human tumor cells.