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1.
Comput Biol Chem ; 84: 107189, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31891900

RESUMO

A series of indole-derived methoxylated chalcones were described as anti-dermatophyte agents. The in vitro antifungal susceptibility testing against different dermatophytes revealed that most of compounds had potent activity against the dermatophyte strains. In particular, the 4-ethoxy derivative 4d with MIC values of 0.25-2 µg/ml was the most potent compound against Trichophyton interdigitale, Trichophyton veruccosum and Microsporum fulvum. Moreover, the 4-butoxy analog 4i displaying MIC values in the range of 1-16 µg/ml had the highest inhibitory activity against Trichophyton mentagrophytes, Microsporum canis, and Arthroderma benhamiae. To predict whether the synthesized compounds interact with tubulin binding site of dermatophytes, the 3D-structure of target protein was modeled by homology modeling and then used for molecular docking and molecular dynamics (MD) simulation studies. Docking simulation revealed that the promising compound 4d can properly bind with tubulin. The molecular dynamics analysis showed that interactions of compound 4d with the active site of target protein have binding stability throughout MD simulation. The results of this study could utilize in the design of more effective antifungal drugs with tubulin inhibition mechanism against keratinophilic fungi.


Assuntos
Antifúngicos/farmacologia , Chalconas/farmacologia , Indóis/farmacologia , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/metabolismo , Arthrodermataceae/efeitos dos fármacos , Sítios de Ligação , Chalconas/química , Chalconas/metabolismo , Humanos , Indóis/química , Indóis/metabolismo , Testes de Sensibilidade Microbiana , Fungos Mitospóricos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
2.
Eur J Med Chem ; 188: 111991, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31883490

RESUMO

Semisynthetic 18ß-glycyrrhetinic acid (GA) analogues bearing 1-en-2-cyano-3-oxo substitution on ring A have enhanced antitumor effects with reduced levels of HDAC3 and HDAC6 proteins. Aiming to inhibit both HDAC protein and activity, we developed a hybrid molecule by tethering active GA analogue methyl 2-cyano-3,11-dioxo-18ß-olean-1,12-dien-30-oate (CDODA-Me) and Vorinostat (SAHA). We tested the proper hybrid approaches of GA with hydroxamic acid and turned out that GA conjugated with SAHA by a piperazine linker was the best. The conjugate (15) of CDODA-Me and SAHA linked through a piperazine group was a potent cytotoxic agent against cancer cells with apoptosis induction. Compound 15 was more effective than the simple combination of CDODA-Me and SAHA to induce apoptosis. Mechanistic studies revealed that 15 was less effective than SAHA to inhibit HDAC activity, but was more effective than CDODA-Me to decrease the levels of HDAC3 and HDAC6 proteins with upregulated levels of acetylated H3 and acetylated α-tubulin. Compound 15 represents a new HDAC3 and HDAC6 inhibitor by reducing protein levels.


Assuntos
Antineoplásicos/farmacologia , Ácido Glicirretínico/análogos & derivados , Desacetilase 6 de Histona/metabolismo , Vorinostat/análogos & derivados , Vorinostat/farmacologia , Acetilação , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ácido Glicirretínico/farmacocinética , Ácido Glicirretínico/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Ratos Sprague-Dawley , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Vorinostat/farmacocinética
3.
Nat Chem Biol ; 15(12): 1183-1190, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740825

RESUMO

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.


Assuntos
Acetilesterase/química , Fatores de Despolimerização de Actina/química , Optogenética , Tubulina (Proteína)/química , Acetilação
4.
Soft Matter ; 15(40): 8137-8146, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31593193

RESUMO

Microtubules are stiff biopolymers that self-assemble via the addition of GTP-tubulin (αß-dimer bound to GTP), but hydrolysis of GTP- to GDP-tubulin within the tubules destabilizes them toward catastrophically-fast depolymerization. The molecular mechanisms and features of the individual tubulin proteins that drive such behavior are still not well-understood. Using molecular dynamics simulations of whole microtubules built from a coarse-grained model of tubulin, we demonstrate how conformational shape changes (i.e., deformations) in subunits that frustrate tubulin-tubulin binding within microtubules drive depolymerization of stiff tubules via unpeeling "ram's horns" consistent with experiments. We calculate the sensitivity of these behaviors to the length scales and strengths of binding attractions and varying degrees of binding frustration driven by subunit shape change, and demonstrate that the dynamic instability and mechanical properties of microtubules can be produced based on either balanced or imbalanced strengths of lateral and vertical binding attractions. Finally, we show how catastrophic depolymerization can be interrupted by small regions of the microtubule containing undeformed dimers, corresponding to incomplete lattice hydrolysis. The results demonstrate a mechanism by which microtubule rescue can occur.


Assuntos
Microtúbulos/química , Microtúbulos/metabolismo , Multimerização Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Termodinâmica
5.
Molecules ; 24(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540347

RESUMO

To elucidate interactions between the antifungal cyclic lipopeptides iturin A, fengycin, and surfactin produced by Bacillus bacteria and the microtubular protein ß-tubulin in plant pathogenic fungi (Fusarium oxysporum, Colletrotrichum gloeosporioides, Alternaria alternata, and Fusarium solani) in molecular docking and molecular dynamics simulations, we retrieved the structure of tubulin co-crystallized with taxol from the Protein Data Bank (PDB) (ID: 1JFF) and the structure of the cyclic lipopeptides from PubChem (Compound CID: 102287549, 100977820, 10129764). Similarity and homology analyses of the retrieved ß-tubulin structure with those of the fungi showed that the conserved domains shared 84% similarity, and the root mean square deviation (RMSD) was less than 2 Å. In the molecular docking studies, within the binding pocket, residues Pro274, Thr276, and Glu27 of ß-tubulin were responsible for the interaction with the cyclic lipopeptides. In the molecular dynamics analysis, two groups of ligands were formed based on the number of poses analyzed with respect to the RMSD. Group 1 was made up of 10, 100, and 500 poses with distances 0.080 to 0.092 nm and RMSDs of 0.10 to 0.15 nm. For group 2, consisting of 1000 poses, the initial and final distance was 0.1 nm and the RMSDs were in the range of 0.10 to 0.30 nm. These results suggest that iturin A and fengycin bind with higher affinity than surfactin to ß-tubulin. These two lipopeptides may be used as lead compounds to develop new antifungal agents or employed directly as biorational products to control plant pathogenic fungi.


Assuntos
Lipopeptídeos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Tubulina (Proteína)/química
6.
Eur J Med Chem ; 181: 111584, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419740

RESUMO

Developing new therapeutic strategies to overcome drug resistance of cancer cells is an ongoing endeavor. From among 2 million chemicals, we identified ethyl 4-oxo-2-phenyl-1,4-dihydroquinoline-6-carboxylate (AS1712) as a low-toxicity inhibitor of lung cancer cell proliferation and xenograft tumor growth. We show that AS1712 is active against broad cancer cell lines and is able to bind in the colchicine-binding pocket of ß-tubulin, thereby inhibiting microtubule assembly and, consequently, inducing mitotic arrest and apoptosis. Our cell-based structure-activity relationship study identified a new lead compound, RJ-LC-15-8, which had a greater anti-proliferative potency for H1975 cells than did AS1712, while maintaining a similar mechanism of action. Notably, AS1712 and RJ-LC-15-8 overcame P-glycoprotein efflux pump and ß-tubulin alterations that lead to acquired resistance against microtubule-targeting drugs of cancer cells. AS1712 and RJ-LC-15-8 may be lead compounds that overcome acquired resistance to microtubule-targeting agents of cancer cells.


Assuntos
Quinolonas/química , Quinolonas/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colchicina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tubulina (Proteína)/química
7.
BMC Mol Cell Biol ; 20(1): 41, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31464580

RESUMO

BACKGROUND: Microtubule proteins are able to produce electromagnetic fields and have an important role in memory formation, and learning. Therefore, microtubules have the potential to be affected by exogenous electromagnetic fields. This study aimed to examine the comparison of microtubule polymerization and its structural behavior in brain and sperm affected by 50 Hz extremely low-frequency electromagnetic field (ELEF). RESULTS: Twenties adult male rats were randomly and equally divided into control and experimental groups, to evaluate the effect of 50 Hz ELEF on the sperm and brain functions. Plus-maze, serum testosterone and corticosterone, and sperm evaluation were performed. Next, the semen and brain samples were obtained, and they were divided into four experimental groups for investigation of microtubule polymerization. There was no significant difference in testosterone and, corticosterone levels, anxiety behaviors, and sperm morphology between control and ELEF-exposure groups. The sperm viability, total and progressive motility were significantly higher in the ELEF-exposed group than that of the control group. The microtubule polymerization in sperm ELEF was significantly higher than in other groups. The secondary and tertiary structures of tubulins were significantly affected in the brain, and sperm ELEF groups. CONCLUSION: It seems that the polymerization of microtubules and conformational changes of tubulin dimers are improved by ELEF application.


Assuntos
Encéfalo/metabolismo , Campos Eletromagnéticos , Microtúbulos/química , Polimerização , Espermatozoides/metabolismo , Animais , Ansiedade/metabolismo , Comportamento Animal , Peso Corporal , Forma Celular , Sobrevivência Celular , Corticosterona/sangue , Fluorescência , Masculino , Microtúbulos/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos Wistar , Espermatozoides/citologia , Testosterona/sangue , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
8.
PLoS Comput Biol ; 15(8): e1007327, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31469822

RESUMO

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.


Assuntos
Tubulina (Proteína)/química , Fenômenos Biomecânicos , Biologia Computacional , Microscopia Crioeletrônica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura
9.
Adv Mater ; 31(39): e1903636, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31408579

RESUMO

Tubulin self-assembly into microtubules is a fascinating natural phenomenon. Its importance is not just crucial for functional and structural biological processes, but it also serves as an inspiration for synthetic nanomaterial innovations. The modulation of the tubulin self-assembly process without introducing additional chemical inhibitors/promoters or stabilizers has remained an elusive process. This work reports a versatile and vigorous strategy for controlling tubulin self-assembly by nanosecond electropulses (nsEPs). The polymerization assessed by turbidimetry is dependent on nsEPs dosage. The kinetics of microtubules formation is tightly linked to the nsEPs effects on structural properties of tubulin, and tubulin-solvent interface, assessed by autofluorescence, and the zeta potential. Moreover, the overall size of tubulin assessed by dynamic light scattering is affected as well. Additionally, atomic force microscopy imaging reveals the formation of different assemblies reflecting applied nsEPs. It is suggested that changes in C-terminal modification states alter tubulin polymerization-competent conformations. Although the assembled tubulin preserve their integral structure, they might exhibit a broad range of new properties important for their functions. Thus, these transient conformation changes of tubulin and their collective properties can result in new applications.


Assuntos
Eletricidade , Multimerização Proteica , Tubulina (Proteína)/química , Hidrodinâmica , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Tubulina (Proteína)/metabolismo
10.
Nat Commun ; 10(1): 3212, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324789

RESUMO

The C-terminus of α-tubulin undergoes a detyrosination/tyrosination cycle and dysregulation of this cycle is associated with cancer and other diseases. The molecular mechanisms of tubulin tyrosination are well studied, however it has remained unknown how tyrosine is cleaved from the tubulin tail. Here, we report the crystal structure of the long-sought detyrosination enzyme, the VASH2/SVBP heterodimer at 2.2 Å resolution and the structure of the tail/VASH2/SVBP complex at 2.5 Å resolution. VASH2 possesses a non-canonical Cys-His-Ser catalytic architecture for tyrosine cleavage. The dynamics of the α1- and α2- helices of VASH2 are related to the insolubility of VASH2. SVBP plays a chaperone-like role by extensively interacting with VASH2 and stabilizing these dynamic helices. A positively charged groove around the catalytic pocket and the α1- and α2- helices of VASH2 targets the tubulin tail for detyrosination. We provide insights into the mechanisms underlying the cycle of tubulin tyrosine cleavage and religation.


Assuntos
Proteínas Angiogênicas/química , Proteínas de Transporte/química , Tubulina (Proteína)/química , Proteínas Angiogênicas/genética , Animais , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Conformação Proteica , Conformação Proteica em alfa-Hélice , Células Sf9
11.
Int J Mol Sci ; 20(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315202

RESUMO

Platelets are anuclear cells with a short lifespan that play an essential role in many pathophysiological processes, including haemostasis, inflammation, infection, vascular integrity, and metastasis. Billions of platelets are produced daily from megakaryocytes (platelet precursors). Despite this high production, the number of circulating platelets is stable and, under resting conditions, they maintain their typical discoid shape thanks to cytoskeleton proteins. The activation of platelets is associated with dynamic and rapid changes in the cytoskeleton. Two cytoskeletal polymer systems exist in megakaryocytes and platelets: actin filaments and microtubules, based on actin, and α- and ß-tubulin heterodimers, respectively. Herein, we will focus on platelet-specific tubulins and their alterations and role of the microtubules skeleton in platelet formation (thrombopoiesis). During this process, microtubules mediate elongation of the megakaryocyte extensions (proplatelet) and granule trafficking from megakaryocytes to nascent platelets. In platelets, microtubules form a subcortical ring, the so-called marginal band, which confers the typical platelet discoid shape and is also responsible for changes in platelet morphology upon activation. Molecular alterations in the gene encoding ß1 tubulin and microtubules post-translational modifications may result in quantitative or qualitative changes in tubulin, leading to altered cytoskeleton reorganization that may induce changes in the platelet number (thrombocytopenia), morphology or function. Consequently, ß1-tubulin modifications may participate in pathological and physiological processes, such as development.


Assuntos
Plaquetas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Variação Genética , Humanos , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
12.
Comput Biol Chem ; 82: 37-43, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31255973

RESUMO

Tubulin protein is the fundamental unit of microtubules, and comprises of α and ß subunits arranged in an alternate manner forming protofilaments. These longitudinal protofilaments are made up of intra- (α-ß) and inter-dimer (ß-α) interactions. Literature review confirms that GTP hydrolysis results in considerable structural rearrangement within GTP binding site of ß-α dimer interface after the release of γ phosphate. In addition to this, the intra-dimer interface exhibits structural rigidity which needs further investigation. In this study, we explored the reasons for the flexibility and the rigidity of the ß-α dimer and the α-ß dimer respectively through molecular simulation and Anisotropic Normal Mode based analysis. As per the sequence alignment report, two glycine residues (Gly96 and Gly98) were observed in the T3 loop of the ß subunit which get substituted by Asp98 and Ala100 in the T3 loop of the α subunit. The higher mobility of glycine residues contributes to the flexibility of the T3 loop of inter-dimer when they come in direct contact with the GTPase Activating Protein (GAP) domain of the subunit. This was confirmed through RMSD, RMSF and Radius of Gyration based studies. Conversely, the intra-dimer exhibited a lower mobility in the absence of glycine residues. As per ANM based analysis, positive domain correlations were observed between T3 loop and GAP domain of intra- and inter- dimeric contact regions. However, these correlation motions were higher in the intra-dimer as compared to the inter-dimer interface. Thus on the basis of our findings, we hypothesize that the higher flexibility of T3 loop and the GAP domain of the inter-dimer is required for structural rearrangement and protofilament stability during hydrolysis. Furthermore, the slightly rigid nature of the T3 loop and the GAP domain of the intra-dimer assists in enhancing the monomer-monomer interaction through the higher positive domain correlation.


Assuntos
Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Anisotropia , Sítios de Ligação , Bovinos , Glicina/química , Simulação de Dinâmica Molecular , Mutação , Maleabilidade , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência , Tubulina (Proteína)/genética
13.
Inorg Chem ; 58(14): 9213-9224, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31241921

RESUMO

Four trimethoxy- and dimethoxyphenylamine-based Schiff base (L1-L4)-bearing RuII-p-cymene complexes (1-4) of the chemical formula [RuII(η6-p-cymene)(L)(Cl)] were synthesized, isolated in pure form, and structurally characterized using single-crystal X-ray diffraction and other analytical techniques. The complexes showed excellent in vitro antiproliferative activity against various forms of cancer that are difficult to cure, viz., triple negative human metastatic breast carcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. The 1H nuclear magnetic resonance data in the presence of 10% dimethylformamide-d7 or dimethyl sulfoxide-d6 in phosphate buffer (pD 7.4, containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h. Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5 molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, 1, exhibits an IC50 of approximately 10-15 µM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells. Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO ) revealed that 1 and 3 as well as L1 and L3 have a high affinity for the interface of the α and ß tubulin dimer in the colchicine binding site. The immunofluorescence studies showed that 1 and 3 strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC20 or IC50 dose for 12 h. The cell cycle analysis upon treatment with 1 showed that the complexes inhibit the mitotic phase because the arrest was observed in the G2/M phase. In summary, 1 and 3 are RuII half-sandwich complexes that are capable of disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the G2/M phase, and kill the cells by an apoptotic pathway.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos de Rutênio/síntese química , Compostos de Rutênio/farmacologia , Bases de Schiff/química , Tubulina (Proteína)/química , Anexinas/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzimidazóis , Carbocianinas , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular
14.
Nat Struct Mol Biol ; 26(7): 583-591, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235910

RESUMO

Microtubules are regulated by post-translational modifications of tubulin. The ligation and cleavage of the carboxy-terminal tyrosine of α-tubulin impact microtubule functions during mitosis, cardiomyocyte contraction and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1-SVBP alone, in complex with a tyrosine-derived covalent inhibitor and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α-tubulin tail by VASH1. The VASH1-SVBP-parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Tubulina (Proteína)/química
15.
Nat Struct Mol Biol ; 26(7): 571-582, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235911

RESUMO

Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.


Assuntos
Proteínas Angiogênicas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Angiogênicas/química , Animais , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Células Cultivadas , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Mapas de Interação de Proteínas , Tubulina (Proteína)/química
16.
Org Biomol Chem ; 17(25): 6201-6214, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31179474

RESUMO

We have recently reported computational models for prediction of cell-based anticancer activity using machine learning methods. Herein, we have developed an integrated strategy to discover new anticancer agents using a cascade of the established screening models. Application of this strategy identified 17 compounds with antitumor activity. Among these compounds, h2 (containing a pyrazolo[3,4-b]pyridin-6-one scaffold) exhibited anticancer activity against six tumor cell lines, including MDA-MB-231, HeLa, MCF-7, HepG2, CNE2 and HCT116, with IC50 values of 13.37, 13.04, 15.45, 7.05, 9.30 and 8.93 µM. Subsequently, a total of 61 h2 analogues were obtained by similarity searching and tested for their anticancer activities. I2 was identified as a novel anticancer agent having activity against MDA-MB-231, HeLa, MCF-7, HepG2, CNE2 and HCT116 tumor cell lines with IC50 values of 3.30, 5.04, 5.08, 3.71, 2.99 and 5.72 µM. I2 also showed potent cytotoxicity against adriamycin-resistant human breast and hepatocarcinoma cells. Further investigation revealed that I2 inhibited the microtubule polymerization by binding to the colchicine site, resulting in inhibition of cell migration, cell cycle arrest in the G2/M phase and apoptosis of cancer cells. Finally, molecular docking and molecular dynamics provided insights into the binding interactions of I2 with tubulin. This study identified I2 as a novel starting point for further development of anticancer agents that target tubulin.


Assuntos
Antineoplásicos/farmacologia , Pirazóis/farmacologia , Piridonas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/química , Piridonas/química , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia
17.
Mol Cell Biol ; 39(17)2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31208979

RESUMO

The cancer/testis antigen (CTA) genes were identified as human genes preferentially expressed in cancer cells and testis, but the contribution of CTAs to cancer and male germ cell development is unclear. In this study, we comprehensively examined mouse CTA functions and found that the majority of CTAs are involved in growth and/or survival of cancer cells. We focused on one mouse CTA gene, Tekt5, for its detailed functional analysis. Tekt5 knockdown (KD) in ovarian cancer cells caused G1 arrest and apoptosis, and p27kip1 was concomitantly upregulated. Tekt5 KD also resulted in decreased levels of acetylated α-tubulin and subsequent fragmentation of ß-III-tubulin, upregulation of HDAC6 that deacetylates α-tubulin, and nuclear accumulation of SMAD3 that induces p27kip1 expression. Because depolymerization of tubulin is known to cause translocation of SMAD3 to the nucleus, these results together suggested that TEKT5 negatively regulates Hdac6 expression and consequently maintains cell cycle via stabilization of tubulin. We also found that the number of spermatids was significantly decreased and acetylated α-tubulin levels were decreased in vivo by KD of Tekt5 in testis. Because acetylated α-tubulin is required for sperm morphogenesis, these results suggest that TEKT5 is necessary for spermiogenesis via maintenance of acetylated α-tubulin levels.


Assuntos
Proteínas dos Microtúbulos/genética , Neoplasias Ovarianas/genética , Espermatogênese , Espermatozoides/citologia , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , Masculino , Camundongos , Estabilidade Proteica , Proteína Smad3/metabolismo , Espermatozoides/metabolismo , Tubulina (Proteína)/química
18.
Curr Top Med Chem ; 19(15): 1289-1304, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31210108

RESUMO

Microtubules are essential for the mitotic division of cells and have been an attractive target for antitumour drugs due to the increased incidence of cancer and significant mitosis rate of tumour cells. In the past few years, tubulin-colchicine binding site, as one of the three binding pockets including taxol-, vinblastine- and colchicine-binding sites, has been focused on to design tubulin-destabilizing agents including inhibitors, antibody-drug conjugates and degradation agents. The present review is the first to cover a systemic and recent synopsis of tubulin-colchicine binding site agents. We believe that it would provide an increase in our understanding of receptor-ligand interaction pattern and consciousness of a series of challenges about tubulin target druggability.


Assuntos
Antineoplásicos/farmacologia , Colchicina/farmacologia , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação/efeitos dos fármacos , Colchicina/química , Colchicina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Mitose/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Tubulina (Proteína)/química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo
19.
Molecules ; 24(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31197105

RESUMO

Molecular hybridization has proven to be a successful multi-target strategy in the design and development of new antitumor agents. Based on this rational approach, we have planned hybrid molecules containing covalently linked pharmacophoric units, present individually in compounds acting as inhibitors of the cancer protein targets tubulin, human topoisomerase II and ROCK1. Seven new molecules, selected by docking calculation of the complexes with each of the proteins taken into consideration, have been efficiently synthesized starting from 2,3-dichloro-1,4-naphtoquinone or 6,7-dichloro-5,8-quinolinquinone. By screening the full National Cancer Institute (NCI) panel, including 60 human cancer cell lines, four molecules displayed good and sometimes better growth inhibition GI50 than the ROCK inhibitor Y-27632, the Topo II inhibitor podophyllotoxin and the tubulin inhibitor combretastatin A-4. The relative position of N,N heteroatoms in the structures of the tested compounds was crucial in affecting bioactivity and selectivity. Furthermore, compound 3 (2-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(3,4,5-trimethoxyphenoxy)naphthalene-1,4-dione) emerged as the most active in the series, showing a potent and selective inhibition of breast cancer BT-549 cells (GI50 < 10 nM).


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase II/farmacologia , Moduladores de Tubulina/farmacologia , Amidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Naftoquinonas/síntese química , Naftoquinonas/química , Neoplasias/genética , Podofilotoxina/farmacologia , Piridinas/farmacologia , Quinolinas/síntese química , Quinolinas/química , Estilbenos/farmacologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/química , Quinases Associadas a rho/genética
20.
Org Biomol Chem ; 17(25): 6184-6200, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31173031

RESUMO

Microtubules are a validated clinical target for the treatment of many cancers. We describe the design, synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain the CA-4 core structure with modifications to the stilbene linking group, and are predominantly piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC or Mukaiyama's reagent. All target compounds were screened for antiproliferative activity in MCF-7 breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC50 = 190 nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC50 values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise tubulin, induce G2/M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells, and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells. Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be developed further as novel, water-soluble molecules targeting microtubules.


Assuntos
Antineoplásicos/farmacologia , Piperazinas/farmacologia , Estilbenos/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Piperazinas/síntese química , Piperazinas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Estilbenos/síntese química , Estilbenos/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo
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