RESUMO
Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.
Assuntos
Ácidos Nucleicos , Fosfatos Açúcares , Ácidos Nucleicos/química , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismo , Oligonucleotídeos/química , Conformação de Ácido NucleicoRESUMO
Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.
Assuntos
Arabidopsis/embriologia , Padronização Corporal , Feixe Vascular de Plantas/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Padronização Corporal/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Ácidos Indolacéticos/metabolismo , Feixe Vascular de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Elementos de Resposta/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
The biological activity of compounds directly depends on the three-dimensional arrangement of affinity fragments since a high degree of pharmacophore compliance with the binding site is required. 3-Benzylidene oxindoles are privileged structures due to their wide spectrum of biological activity, synthetic availability, and ease of modification. In particular, both kinase inhibitors and kinase activators can be found among 3-benzylidene oxindoles. In this work, we studied model compounds obtained via oxindole condensation with aldehydes and alkylphenones. These condensation products can exist in the form of E- and Z-isomers and also undergo isomerization in solutions. The factors causing isomeric transformation of these compounds were established. Comparative kinetic studies to obtain quantitative characteristics of UV-driven isomerization were first performed. The results obtained indicate dramatic differences in two subclasses, which should be considered when developing biologically active molecules.
Assuntos
Isomerismo , Oxindóis , CinéticaRESUMO
Tetrahydropyrazolo[1,5-a]pyrimidine (THPP) is an attractive scaffold for designing biologically active compounds. The most obvious way to obtain such compounds is to reduce pyrazolopyrimidines with complex hydrides, because the pyrimidine ring is reduced in the preference over the pyrazole ring. The presence of substituents at positions five and seven of pyrazolo[1,5-a]pyrimidines complicates the set of reaction products but makes it more attractive for medicinal chemistry because four possible stereoisomers can be formed during reduction. However, the formation of only syn-isomers has been described in the literature. This article is the first report on the formation of anti-configured isomers along with syn-isomers in the reduction of model 5,7-dimethylpyrazolo[1,5-a]pyrimidine, which was confirmed by NMR. The bicyclic core in the syn-configuration was shown to be conformationally stable, which was used to estimate the long-range interproton distances using NOESY data. At the same time, long-range dipole-dipole interactions corresponding to a distance between protons of more than 6 Å were first registered and quantified. In turn, the bicyclic core in the trans-configuration represents a conformationally labile system. For these structures, an analysis of conformations observed in solutions was carried out. Our results indicate the significant potential of trans-configured tetrahydropyrazolo[1,5-a]pyrimidines for the development of active small molecules. While possessing structural lability due to the low energy of the conformational transition, they have the ability to adjust to the active site of the desired target.
RESUMO
Targeting of MDM2-p53 protein-protein interaction is a current approach for the development of potent anticancer agents. The classical pharmacophore hypothesis for the design of such molecules describes the three point binding of a small molecule inhibitor to the MDM2 protein. However, this hypothesis is not confirmed when considering the activity of a number of known potent MDM2 inhibitors. Here we demonstrate the important role of the flexible N-terminal region of the MDM2 protein in the binding with small molecule compounds, which contributes to the transition from three point binding to four point binding during the development of new anticancer agents. To evaluate the contribution of the MDM2 N-terminal region to the structure-activity relationship of known MDM2 inhibitors, compounds of nutlin series, whose spatial configuration was shown to dramatically affect the target activity, were used as objects of the study. The key amino acid residues within the N-terminal region involved in the interaction with small molecule ligands were determined by means of molecular dynamics. The conformational stability of the flexible MDM2 fragment was simulated under different conditions. The effects of point mutations on the N-terminal region stability were also demonstrated.
Assuntos
Antineoplásicos/farmacologia , Imidazolinas/farmacologia , Domínios Proteicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/química , Desenho de Fármacos , Humanos , Imidazolinas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/químicaRESUMO
The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response cis-elements (AuxREs), but there is growing evidence that additional cis-elements occur in auxin-responsive regulatory regions. The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix-loop-helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.
Assuntos
Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Elementos de Resposta , Arabidopsis/metabolismo , Cromatina/metabolismo , Biologia Computacional , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/metabolismoRESUMO
Five lactam chemotypes amenable by the Castagnoli-Cushman reaction of imines and cyclic anhydrides have been investigated for their ability to activate p53 tumor suppressing transcription factor thus induce apoptosis in p53+ cancer cells. A virtual library of 1.07 million chemically diverse compounds based on these scaffolds was subjected to in silico screening first. The compounds displaying high docking score were visually prioritized to identify the best-fitting compounds, i. e. the ones which adequately mimic the interactions of clinical candidate inhibitor Nutlin-3a. These 38 compounds were synthesized and tested for apoptosis induction in p53+ H116 cancer cells to identify 9 potent apoptosis-inducers (two of them exceeding the activity of Nutlin-3a) which belonged to four different chemotypes. The activation of p53 involved in the proapoptotic activity observed was supported by effective induction of EGFP expression in human osteocarcinoma U2OS-pLV reporter cell line. Moreover, the two most potent apoptosis inducers displayed antiproliferative profile identical to several known advanced p53 activators: they inhibited the growth of p53+/+ HCT116 cells in much lower concentration range compared to p53-/- HCT116 cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Lactamas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetatos/farmacologia , Antineoplásicos/síntese química , Técnicas de Química Sintética/métodos , Desenho de Fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Lactamas/síntese química , Simulação de Acoplamento Molecular , Piperazinas/farmacologia , Piperidonas/farmacologia , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Compostos de Espiro/farmacologiaRESUMO
The absolute configurations of the diastereomers of novel amino acid ester derivatives of 2,3-substituted isoindolinones, which are known as apoptosis activators due to their ability to inhibit the MDM2-p53 PPI, were assigned using NMR and computational methods. Procedures for diastereomer separation and determining the absolute configuration were developed to perform the study. The high significance of N-benzyl fragment for the determination of the diastereomer absolute configuration by NMR methods was established; it is determined by a number of factors inherent in this fragment and the structural features of the studied substrates. Analysis of the individual isomer activity showed that the target inhibitory effect of S- and R-isoindolinone L-valinates differs by less than 20%. It can be explained by the presence of a flexible linker between the isoindolinone core and amino acid fragment, which provides the optimal arrangement of the molecule in the hydrophobic cavity of MDM2 for both isomers.
Assuntos
Aminoácidos/química , Ftalimidas/química , Ftalimidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Ftalimidas/isolamento & purificação , Ftalimidas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Prótons , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Ftalimidas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Ftalimidas/síntese química , Ftalimidas/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.
RESUMO
The use of viral protein inhibitors has shown to be insufficiently effective in the case of highly variable SARS-CoV-2. In this work, we examined the possibility of designing agents that bind to a highly conserved region of coronavirus (+)RNA. We demonstrated that while the design of antisense RNAs is based on the complementary interaction of nitrogenous bases, it is possible to use semirigid docking methods in the case of unnatural peptide nucleic acids. The transition from N-(2-aminoethyl)glycine chain to a more conformationally rigid piperidine-containing backbone allowed us to significantly increase the affinity of structures to the target RNA.
RESUMO
Ubiquitinylation of protein substrates results in various but distinct biological consequences, among which ubiquitin-mediated degradation is most well studied for its therapeutic application. Accordingly, artificially targeted ubiquitin-dependent degradation of various proteins has evolved into the therapeutically relevant PROTAC technology. This tethered ubiquitinylation of various targets coupled with a broad assortment of modifying E3 ubiquitin ligases has been made possible by rational design of bi-specific chimeric molecules that bring these proteins in proximity. However, forced ubiquitinylation inflicted by the binary warheads of a chimeric PROTAC molecule should not necessarily result in protein degradation but can be used to modulate other cellular functions. In this respect it should be noted that the ubiquitinylation of a diverse set of proteins is known to control their transport, transcriptional activity, and protein-protein interactions. This review provides examples of potential PROTAC usage based on non-degradable ubiquitinylation.
Assuntos
Proteólise , Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitinação , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , HumanosRESUMO
A small family composed of BRI1 KINASE INHIBITOR1 (BKI1) and MEMBRANE-ASSOCIATED KINASE REGULATORS (MAKRs) has recently captured the attention of plant biologists, due to their involvement in developmental processes downstream of hormones and Receptor-Like Kinases (RLK) signalling. BKI1/MAKRs are intrinsically disordered proteins (so-called unstructured proteins) and as such lack specific domains. Instead, they are defined by the presence of two conserved linear motifs involved in the interaction with lipids and proteins, respectively. Here, we first relate the discovery of the MAKR gene family. Then, we review the individual function of characterized family members and discuss their shared and specific modes of action. Finally, we explore and summarize the structural, comparative and functional genomics data available on this gene family. Together, this review aims at building a comprehensive reference about BKI1/MAKR protein function in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrinsicamente Desordenadas , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides , Regulação da Expressão Gênica de Plantas , Hormônios/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Desenvolvimento Vegetal/genética , Plantas/genética , Plantas/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/genéticaRESUMO
P-glycoprotein (P-gp) is found to be of considerable interest for the design of drugs capable of treating chemoresistant tumors. This transporter is an interesting target for which an efficient approach has not yet been developed in terms of computer simulation. In this work, we use a combination of docking, molecular dynamics, and metadynamics to fully explore the states that occur during the capture of a ligand and subsequent efflux by P-gp. The proposed approach allowed us to substantiate a number of experimentally established facts, as well as to develop a new criterion for identifying potential P-gp inhibitors.
RESUMO
Since the problem of transporter-mediated multidrug resistance of tumor cells is becoming increasingly important in cancer therapy, it is necessary to modulate the activity of efflux transporters of the ABC family, among which P-glycoprotein is the best known. We consider the nucleotide binding domain, a universal fragment of these transporters, as a target for the rational design of small molecule compounds capable of preventing ATP-dependent drug efflux. Using various ATP mimetics, we showed that they suppress the efflux of fluorescent substrates and paclitaxel from the cells due to suppressing the ATPase activity of the transporters. The combined use of paclitaxel and ATP mimetics significantly increases its antitumor efficacy, including in cells with the multidrug resistance phenotype. The considered compounds are promising agents for the development of therapeutic efflux modulators, since they are not toxic at the given concentrations and do not induce the transporter overexpression. Moreover, the compounds overcome not only P-gp-mediated but also BCRP-mediated resistance of tumor cells.
RESUMO
ABC transporters play an essential role in the development of multidrug resistance and thus are of interest in the context of anticancer therapy. However, MDR1, BCRP and MRP1 are involved in a number of key processes that maintain the viability of the body as a whole, as well as individual organs and cells. These transporters support protective properties of anatomical and histohematic barriers, determining the entry of both toxins and drugs into organs and tissues, as well as facilitating their elimination. This review discusses the main areas in which the use of modulators of the ABC exporter activity may be relevant due to either an initial imbalance in their activity or the need for the temporary change in the efflux rate for therapeutic purposes. Controlled modulation of the activity of the ABC family efflux transporters opens up broad prospects in the treatment of various diseases associated both with universal difficulties in the delivery of drugs that are transporter substrates and with the characteristics of individual patients caused by single nucleotide polymorphisms. Both activators and inhibitors of the transporters will find their application.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Resistencia a Medicamentos Antineoplásicos , Humanos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Neoplasias/metabolismo , Resistência a Múltiplos Medicamentos , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos MedicamentosRESUMO
The problem of chemoresistance development is an inescapable flipside of modern oncotherapy, in particular for Ñolorectal cancer patients. The search for or development of drugs effective against resistant tumors involves the use of model resistant cell lines in vitro. To obtain such lines, we reproduced the development of chemoresistance of human colon adenocarcinoma cells under the treatment with drugs of different mechanisms, a cytostatic (paclitaxel) and a targeted agent (Nutlin-3a, an inhibitor of p53-Mdm2 protein-protein interaction). In each case, we gradually increased the content of the substance in the medium, starting from effective concentrations that do not cause total cell death. When studying the lines resistant to the corresponding drug, we noted a reduced sensitivity to the drug of another mechanism of action. Analysis of the original and resistant lines showed that the cells use the universal efflux defense mechanism. The observed effect can be partially neutralized using inhibitors of the ABC transport proteins, including P-glycoprotein, known for its oncoprotective function. The role of the latter was confirmed by real-time RT-PCR and Western blotting.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/farmacologia , Paclitaxel/farmacologia , Piperazinas/farmacologia , Quinolinas/farmacologia , Vanadatos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidoresRESUMO
At the molecular level, response to an external factor or an internal condition causes reprogramming of temporal and spatial transcription. When an organism undergoes physiological and/or morphological changes, several signaling pathways are activated simultaneously. Examples of such complex reactions are the response to temperature changes, dehydration, various biologically active substances, and others. A significant part of the regulatory ensemble in such complex reactions remains unidentified. We developed metaRE, an R package for the systematic search for cis-regulatory elements enriched in the promoters of the genes significantly changed their transcription in a complex reaction. metaRE mines multiple expression profiling datasets generated to test the same organism's response and identifies simple and composite cis-regulatory elements systematically associated with differential expression of genes. Here, we showed metaRE performance for the identification of low-temperature-responsive cis-regulatory code in Arabidopsis thaliana and Danio rerio. MetaRE identified potential binding sites for known as well as unknown cold response regulators. A notable part of cis-elements was found in both searches discovering great conservation in low-temperature responses between plants and animals.
Assuntos
Reprogramação Celular/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica , Transcriptoma/genética , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Humanos , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Peixe-Zebra/genéticaRESUMO
Root stem cell niche functioning requires the formation and maintenance of the specific "auxin-rich domain" governed by directional auxin transport and local auxin production. Auxin maximum co-localizes with the WOX5 expression domain in the quiescent center that separates mitotically active proximal and distal root meristems. Here we unravel the interconnected processes happening under WOX5 overexpression by combining in vivo experiments and mathematical modeling. We showed that WOX5-induced TAA1-mediated auxin biosynthesis is the cause, whereas auxin accumulation, PIN transporters relocation, and auxin redistribution between proximal and distal root meristems are its subsequent effects that influence the formation of the well-described phenotype with an enlarged root cap. These findings helped us to clarify the role of WOX5, which serves as a local QC-specific regulator that activates biosynthesis of non-cell-autonomous signal auxin to regulate the distal meristem functioning. The mathematical model with WOX5-mediated auxin biosynthesis and auxin-regulated cell growth, division, and detachment reproduces the columella cells dynamics in both wild type and under WOX5 dysregulation.
RESUMO
AMP-activated protein kinase (AMPK) is currently the subject of intensive study and active discussions. AMPK performs its functions both at the cellular level, providing the switch between energy-consuming and energy-producing processes, and at the whole body level, particularly, regulating certain aspects of higher nervous activity and behavior. Control of such a 'main switch' compensates dysfunctions and associated diseases. In the present paper, we studied the binding of 3-benzylidene oxindoles to the kinase domain of the AMPK α-subunit, which is thought to prevent its interaction with the autoinhibitory domain and thus result in the AMPK activation. For this purpose, we developed the cellular test system based on the AMPKAR plasmid, which implements the FRET effect, synthesized a number of 3-benzylidene oxindole compounds and simulated their binding to various sites of the kinase domain. The most probable binding site for the studied compounds was established by the correlation of calculated and experimental data. The obtained results allow to analyze various classes of AMPK activators using virtual and high-content screening.