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1.
Molecules ; 27(5)2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35268749

RESUMO

In this study, the effects of side-chain configurations of D-Ile residues of a retro-inverso (RI)-type inhibitor on the human T-cell leukemia virus type 1 (HTLV-1) protease containing a hydroxyethylamine dipeptide isostere were clarified. Prior to evaluation using the RI-type inhibitor, the effects of side-chain configurations of Ile residues of the substrate peptide on the HTLV-1 protease were examined to estimate the influence of side-chain configurations on enzyme activity. Based on the estimation of the influence of side-chain configurations on protease affinity, the RI-type inhibitors containing a D-allo-Ile residue in the corresponding substrate sequence, instead of a D-Ile residue, were synthesized via 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis. Refolded recombinant HTLV-1 protease (1-116, L40I) was used for the simple and short evaluation of the inhibitory activities of the synthesized RI-type inhibitors. The results clearly indicated that mimicking the whole topology, comprising both the main- and side-chain structures of the parent inhibitor, is effective for the design of potent RI-modified protease inhibitors.


Assuntos
Peptídeo Hidrolases
2.
Bioorg Med Chem ; 52: 116517, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34800875

RESUMO

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 ß-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1' position further improved the activity.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Reagentes de Ligações Cruzadas/farmacologia , Etilaminas/farmacologia , Compostos Macrocíclicos/farmacologia , Peptídeos/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Etilaminas/síntese química , Etilaminas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
3.
Bioorg Med Chem ; 50: 116459, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34700240

RESUMO

An aromatic substituent has been introduced into a known hydroxyethylamine (HEA)-type BACE1 inhibitor containing the superior substrate sequence to enhance inhibitory activity. The HEA-type isosteres bearing different hydroxyl group and methyl group configurations were prepared through a branched synthesis approach using intra- and inter-molecular epoxide opening reactions. The effect of their configuration was evaluated, showing that an R-configuration improved the inhibitory activity, while introduction of a methyl group on the isostere decreased the activity. Based on the non-substituted isostere with an R-configuration, 21 derivatives containing various substituents at the P1' site were synthesized. Our evaluation of the derivatives showed that the structure of the P1' site had a clear effect on activity, and highly potent inhibitor 40g, which showed sub-micromolar activity against recombinant BACE1 (rBACE1), was identified. The docking simulation of 40g with rBACE1 suggested that a carboxymethyl group at the para-position of the P1' benzene ring interacted with Lys285 in the S1' pocket.


Assuntos
Inibidores Enzimáticos/farmacologia , Etilaminas/farmacologia , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Etilaminas/síntese química , Etilaminas/química , Humanos , Estrutura Molecular , Proteínas Recombinantes , Relação Estrutura-Atividade
4.
Bioorg Med Chem ; 28(4): 115273, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926775

RESUMO

An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease. An alkyl or aryl substituent was also introduced at the 1-position of the octahydroisochromene scaffold and expected to introduce additional interactions with the protease. Sharpless-Katsuki asymmetric epoxidation and Sharpless asymmetric dihydroxylation were employed to construct the octahydroisochromene scaffold. The introductions of the P1 site His-al and the substituent at 1-position was achieved using successive reductive amination reactions. Our initial evaluations of the diastereo-isomeric mixtures (16a-d) revealed that the octahydroisochromene moiety functions as a core hydrophobic scaffold for the S2 pocket of the protease and the substituent at the 1-position may form additional interactions with the protease. The inhibitory activities of the diastereoisomerically-pure inhibitors (3a-d) strongly suggest that a specific stereo-isomer of the octahydroisochromene scaffold, (1S, 3S) 3b, directs the P1 site imidazole, the warhead aldehyde, and substituent at the 1-position of the fused ring to their appropriate pockets in the protease.


Assuntos
Benzopiranos/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Benzopiranos/síntese química , Benzopiranos/química , Proteases 3C de Coronavírus/metabolismo , Relação Dose-Resposta a Droga , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade
5.
Bioorg Chem ; 105: 104386, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33137556

RESUMO

Based on a structure-guided approach, aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles were designed to target metallo-ß-lactamases (MBLs), using Klebsiella pneumoniaeNDM-1 as a model. The in vitro MBLs inhibition showed remarkable inhibition constant for most of the designed compounds at a low micromolar range (1.5-16.4 µM) against NDM-1, IMP-1 and AIM-1 MBLs. Furthermore, all compounds showed promising antibacterial activity against (K+, K1-K9) resistant clinical isolates of K. pneumoniae and were able to re-sensitize resistant K. pneumoniae (K5) strain towards meropenem and cefalexin. Besides, in vivo toxicity testing exhibited that the most active compound was non-toxic and well tolerated by the experimental animals orally up to 350 mg/kg and up to 125 mg/kg parenterally. The docking experiments on NDM-1 and IMP-1 rationalized the observed in vitro MBLs inhibition activity. Generally, this work presents a fruitful matrix to extend the chemical space for MBLs inhibition. This aids in tackling drug-resistance issues in antibacterial treatment.


Assuntos
Antibacterianos/farmacologia , Hidrazonas/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Pirazóis/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Hidrazonas/síntese química , Hidrazonas/química , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/química , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/química
6.
Molecules ; 25(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867349

RESUMO

Three types of new coronaviruses (CoVs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and corona-virus disease 2019 (COVID-19). Neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. Each of the 3C-like (3CL) proteases of the three CoVs is essential for the proliferation of the CoVs, and an inhibitor of the 3CL protease (3CLpro) is thought to be an ideal therapeutic agent against SARS, MERS, or COVID-19. Among these, SARS-CoV is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. This article briefly reviews a series of studies on SARS-CoV, focusing on the development of inhibitors for the SARS-CoV 3CLpro based on molecular interactions with the 3CL protease. Our recent approach, based on the structure-based rational design of a novel scaffold for SARS-CoV 3CLpro inhibitor, is also included. The achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including SARS-CoV-2.


Assuntos
Antivirais/química , Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Inibidores de Proteases/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , COVID-19 , Domínio Catalítico , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
7.
Bioorg Med Chem ; 27(2): 425-435, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30558861

RESUMO

A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CLpro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CLpro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CLpro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CLpro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.


Assuntos
Antivirais/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Isoquinolinas/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Isoquinolinas/síntese química , Isoquinolinas/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Virais/química
8.
Chem Pharm Bull (Tokyo) ; 67(3): 253-257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828001

RESUMO

The plant alkaloids, iso-6-spectaline and spectaline, isolated from the Cassia or Senna genera contain a characteristic 2,6-disubstituted piperidin-3-ol scaffold. Although both natural products are reported to exhibit a variety of interesting biological activities, few stereo-selective schemes for the construction of the 2,6-disubstituted scaffold have been reported. Following our previous studies regarding the synthesis of (+)-spectaline, herein we report the first convergent synthesis of (-)-iso-6-spectaline using a cross-metathesis under thermal conditions where the cis-2,6-disubstituted piperidin-3-ol scaffold is condensed with a long alkyl chain containing a terminal olefin. The cis-2,6-disubstituted piperidin-3-ol used in the synthesis was prepared simply via Pd(II)-catalyzed diastereoselective cyclization. It was confirmed that (+)-spectaline, an epimer of (-)-iso-6-spectaline, was selectively synthesized by the cross-metathesis reaction under less intense thermal conditions starting from the same cis-2,6-disubstituted piperidin-3-ol derivative.


Assuntos
Paládio/química , Piperidinas/síntese química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Catálise , Cromatografia Líquida , Ciclização , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Piperidinas/química , Piperidinas/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Staphylococcus epidermidis/efeitos dos fármacos , Relação Estrutura-Atividade , Termodinâmica
9.
Inorg Chem ; 57(9): 5475-5485, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29634246

RESUMO

Fe(II)-coordinating hexapeptides containing three 2,2'-bipyridine moieties as side chains were designed and synthesized. A cyclic hexapeptide having three [(2,2'-bipyridin)-5-yl]-d-alanine (d-Bpa5) residues, in which d-Bpa5 and Gly are alternately arranged with 3-fold rotational symmetry, coordinated with Fe(II) to form a 1:1 octahedral Fe(II)-peptide complex with a single facial-Λ configuration of the metal-centered chirality. NMR spectroscopy and molecular dynamics simulations revealed that the Fe(II)-peptide complex has an apparent C3-symmetric conformations on the NMR time scale, while the peptide backbone is subject to dynamic conformational exchange between three asymmetric ß/γ conformations and one C3-symmetric γ/γ/γ conformation. The semirigid cyclic hexapeptide preferentially arranged these conformations of the small octahedral Fe(II)-bipyridine complex, as well as the Ru(II) congener, to underpin the single configuration of the metal-centered chirality.


Assuntos
2,2'-Dipiridil/química , Compostos Ferrosos/química , Compostos Macrocíclicos/química , Rutênio/química , 2,2'-Dipiridil/análogos & derivados , Ligantes , Compostos Macrocíclicos/síntese química , Estrutura Molecular , Estereoisomerismo
10.
Bioorg Med Chem Lett ; 27(12): 2746-2751, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28454669

RESUMO

Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC50 value of 43µM against SARS CoV 3CL R188I mutant protease.


Assuntos
Inibidores de Proteases/farmacologia , Serina/análogos & derivados , Proteínas Virais/antagonistas & inibidores , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Serina/síntese química , Serina/química , Serina/farmacologia , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
J Pept Sci ; 23(7-8): 581-586, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28378383

RESUMO

Effects of replacement and addition of an amino acid in a cyclic decapeptide 1 (cyclic-CYNPTTYQMC) for inhibitory activity to dimerization of human epidermal growth factor receptor (EGFR) were examined. By alanine scanning of 1 corresponding to the arm structure (residues 246-254) of a ß-hairpin loop sequence (residues 242-259) of EGFR, it was confirmed that replacement of any amino acid in the loop structure lowered the dimerization inhibitory activity of 1. Among the residues examined, Tyr at position 246 and Thr at 250 were found to be crucial for dimer formation. Addition of an amino acid to the N-terminus of 1 also affected the dimerization inhibitory activity. Addition of an amino acid containing a moderately hydrophilic side-chain increased the inhibitory activity. In contrast, an intramolecular hydrogen bond of 1 is not thought to be crucial for holding the dimer structure on the basis of the dimerization inhibitory activities of N-methylated analogues of 1. These results will be useful for the design and evaluation of a potent dimerization inhibitor as an anti-proliferation agent. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Aminoácidos/química , Receptores ErbB/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Dimerização , Humanos , Peptídeos/química , Conformação Proteica
12.
Biopolymers ; 106(4): 391-403, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26572934

RESUMO

Design of inhibitors against severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro) ) is a potentially important approach to fight against SARS. We have developed several synthetic inhibitors by structure-based drug design. In this report, we reveal two crystal structures of SARS 3CL(pro) complexed with two new inhibitors based on our previous work. These structures combined with six crystal structures complexed with a series of related ligands reported by us are collectively analyzed. To these eight complexes, the structural basis for inhibitor binding was analyzed by the COMBINE method, which is a chemometrical analysis optimized for the protein-ligand complex. The analysis revealed that the first two latent variables gave a cumulative contribution ratio of r(2) = 0.971. Interestingly, scores using the second latent variables for each complex were strongly correlated with root mean square deviations (RMSDs) of side-chain heavy atoms of Met(49) from those of the intact crystal structure of SARS-3CL(pro) (r = 0.77) enlarging the S2 pocket. The substantial contribution of this side chain (∼10%) for the explanation of pIC50 s was dependent on stereochemistry and the chemical structure of the ligand adapted to the S2 pocket of the protease. Thus, starting from a substrate mimic inhibitor, a design for a central scaffold for a low molecular weight inhibitor was evaluated to develop a further potent inhibitor. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 391-403, 2016.


Assuntos
Cisteína Endopeptidases , Peptidomiméticos/química , Inibidores de Proteases/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais , Proteases 3C de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química
13.
Bioorg Med Chem ; 24(6): 1241-54, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26879854

RESUMO

Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity.


Assuntos
Desenho de Fármacos , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Serina/análogos & derivados , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Proteases 3C de Coronavírus , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteases/química , Serina/síntese química , Serina/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
14.
J Pept Sci ; 22(7): 480-4, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27238594

RESUMO

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13-32 aggregated to a greater degree than native hCT under aqueous 2,2,2-trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine-T binding assays and atomic force microscopy suggest that the α-helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the ß-sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α-helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/síntese química , Calcitonina/química , Modelos Moleculares , Trifluoretanol/química , Água/química , Sequência de Aminoácidos , Benzotiazóis , Fluorenos/química , Humanos , Microscopia de Força Atômica , Agregados Proteicos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica , Técnicas de Síntese em Fase Sólida/métodos , Soluções , Espectrometria de Fluorescência , Tiazóis/química
15.
Biochim Biophys Acta ; 1844(4): 803-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24583237

RESUMO

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-ß-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.


Assuntos
Alquil e Aril Transferases/química , Proteínas Arqueais/química , Quitina/química , Modelos Moleculares , Pyrobaculum/química , Tiamina Monofosfato/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Quitina/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólise , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Pyrobaculum/enzimologia , Pyrobaculum/genética , Pyrococcus furiosus/química , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Tiamina Monofosfato/biossíntese
16.
Biochem Biophys Res Commun ; 456(3): 768-73, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25498500

RESUMO

Exosomes, the natural vehicles of various biological molecules, have been examined in several research fields including drug delivery. Although understanding of the biological functions of exosomes has increased, how exosomes are transported between cells remains unclear. We hypothesized that cell tropism is important for effective exosomal intercellular communication and that parental cells regulate exosome movement by modulating constituent exosomal molecules. Herein, we demonstrated the strong translocation of glioblastoma-derived exosomes (U251exo) into their parental (U251) cells, breast cancer (MDA-MB-231) cells, and fibrosarcoma (HT-1080). Furthermore, disruption of proteins of U251exo by enzymatic treatment did not affect their uptake. Therefore, we focused on lipid molecules of U251exo with the expectation that they are crucial for effective incorporation of U251exo by cancer cells. Phosphatidylethanolamine was identified as a unique lipid component of U251-MG cell-derived extracellular vesicles. From these results, valuable insight is provided into the targeting of U251exo to cancer cells, which will help to develop a cancer-targeted drug delivery system.


Assuntos
Sistemas de Liberação de Medicamentos , Exossomos/química , Exossomos/metabolismo , Neoplasias/metabolismo , Fosfatidiletanolaminas/análise , Comunicação Celular , Linhagem Celular Tumoral , Humanos
17.
Soft Matter ; 11(31): 6223-34, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26153461

RESUMO

Apolipoprotein A-I (apoA-I) is an amyloid-forming protein whose amyloidogenic properties are attributed mainly to its N-terminal fragment. Cell membranes are thought to be the primary target for the toxic amyloid aggregates. In the present study Förster resonance energy transfer (FRET) between the membrane fluorescent probe Laurdan as a donor and amyloid-specific dye Thioflavin T (ThT) as an acceptor was employed to explore the interactions of amyloid fibrils from apoA-I variants 1-83/G26R and 1-83/G26R/W@8 with the model membranes composed of phosphatidylcholine and its mixture with cholesterol. The changes in FRET efficiency upon fibril-lipid binding were found to correlate with the extent of protein fibrillization. AFM imaging revealed the presence of two polymorphic states of fibrillar 1-83/G26R/W@8 with the helical and twisted ribbon morphologies. The simulation-based analysis of the experimental FRET profiles provided the arguments in favor of untwisting of fibrillar assemblies upon their interaction with the model membranes. Evidence for the face-on orientation and superficial bilayer location of the membrane-bound fragments of 1-83/G26R/W@8 fibrils was obtained.


Assuntos
Amiloide/química , Apolipoproteína A-I/química , Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Membranas Artificiais
18.
Bioorg Med Chem Lett ; 25(22): 5127-32, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26459211

RESUMO

To identify a new non-peptidyl BACE1 inhibitor, we focused on the aminopyridine structure, which binds to the active sites of BACE1. Synthesis of aminopyridine derivatives and evaluation of inhibitory activity against rBACE1 are described. The 2-aminopyridine moiety and/or 3-methoxybenzaldehyde could be converted to terminal acetylene derivatives by the Sonogashira method. Sonogashira or Glaser cross-coupling reactions with the corresponding derivatives followed by hydrogenation could derive the designed compounds. Although inhibitory activities of the synthetic compounds against rBACE1 were weak, the aminopyridine motif has potential as a BACE1 inhibitor.


Assuntos
Aminopiridinas/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Aminopiridinas/síntese química , Secretases da Proteína Precursora do Amiloide/química , Animais , Ácido Aspártico Endopeptidases/química , Inibidores Enzimáticos/síntese química , Simulação de Acoplamento Molecular , Ratos
19.
Bioorg Med Chem ; 23(4): 876-90, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25614110

RESUMO

The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro)) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resolution, trans-decahydroisoquinolin derivatives were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CL(pro), which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CL(pro) inhibitor. X-ray crystallographic analyses of the SARS 3CL(pro) in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as additional interactions at the N-substituent of the inhibitor.


Assuntos
Quimases/antagonistas & inibidores , Isoquinolinas/química , Isoquinolinas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Antivirais/química , Antivirais/farmacologia , Quimases/química , Quimases/metabolismo , Cristalografia por Raios X , Humanos , Simulação de Acoplamento Molecular , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia
20.
Bioorg Med Chem ; 23(17): 5626-40, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26264846

RESUMO

A superior substrate sequence for BACE1 containing transition-state mimics at the scissile site was evaluated as a protease inhibitor. Hydroxymethylcarbonyl (HMC) and hydroxyethylamine (HEA) isosteres were selected as the transition state mimics, and incorporated into the scissile site of the superior sequence covering the P4 to P1' sites (Glu-Ile-Thi-Thi(*)Nva; (*)denotes the cleavage site). Isosteres having different absolute configurations of the hydroxyl group were synthesized separately, and the effect of the configuration was evaluated. Configuration of the hydroxyl group of each isostere showed a marked effect on the inhibitory activity; anti-configuration to the scissile site substituent had potent inhibitory activity in an HMC-type inhibitor, whereas anti-configuration of HEA-type inhibitors showed no inhibitory activity. Structural evaluations based on X-ray crystallographic analyses of recombinant BACE1 in complex with each inhibitor provided insights into the protein-ligand interactions, especially at the prime sites.


Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Peptídeos/química , Sítios de Ligação , Modelos Moleculares
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