RESUMO
The inhibition of kynurenine production is considered a promising target for cancer immunotherapy. In this study, an amino acid derivative, compound 1 was discovered using a cell-based assay with our screening library. Compound 1 suppressed kynurenine production without inhibiting indoleamine 2,3-dioxygenase 1 (IDO1) activity. The activity of 1 was derived from the inhibition of IDO1 by a metabolite of 1, O-benzylhydroxylamine (OBHA, 2a). A series of N-substituted 2a derivatives that exhibit potent activity in cell-based assays may represent effective prodrugs. Therefore, we synthesized and evaluated novel N,O-substituted hydroxylamine derivatives. The structure-activity relationships revealed that N,O-substituted hydroxylamine 2c inhibits kynurenine production in a cell-based assay. We conducted an in vivo experiment with 2c, although the effectiveness of O-substituted hydroxylamine derivatives in vivo has not been previously reported. The results indicate that N,O-substituted hydroxylamine derivatives are promising IDO1 inhibitors.
Assuntos
Hidroxilamina , Indolamina-Pirrol 2,3,-Dioxigenase , Cinurenina , Cinurenina/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Relação Estrutura-Atividade , Humanos , Hidroxilamina/química , Hidroxilamina/farmacologia , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Estrutura Molecular , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Relação Dose-Resposta a DrogaRESUMO
An Fe-catalyzed unprotected hydroxylamine mediated Heck-type coupling between sulfinic acids and alkenes for the regioselective synthesis of (E)-vinyl sulfones has been developed. Mechanism studies indicated for the first time that a radical process may be involved and that hydroxylamines play multiple roles, including those of a mild oxidant and an in situ base. It was found for the first time that this transformation not only realizes C-S bond construction promoted by unprotected hydroxylamines, but also provides a practical and complementary method for the preparation of structurally important (E)-vinyl sulfones.
Assuntos
Hidroxilaminas , Ferro , Hidroxilaminas/química , Ferro/química , Catálise , Sulfonas/químicaRESUMO
A novel series of 1-aryl-N-[4-phenyl-5-(arylazo)thiazol-2-yl)methanimines has been synthesized via the condensation of 2-amino-4-phenyl-5-arylazothiazole with various aromatic aldehydes. The synthesized imines were characterized by spectroscopic techniques, namely 1H and 13C-NMR, FTIR, MS, and Elemental Analysis. A molecular comparative docking study for 3a-f was calculated, with reference to two approved drugs, Molnupiravir and Remdesivir, using 7BQY (Mpro; PDB code 7BQY; resolution: 1.7 A°) under identical conditions. The binding scores against 7BQY were in the range of -7.7 to -8.7 kcal/mol for 3a-f. The high scores of the compounds indicated an enhanced binding affinity of the molecules to the receptor. This is due to the hydrophobic interactions and multi-hydrogen bonds between 3a-f ligands and the receptor's active amino acid residues. The main aim of using in silco molecular docking was to rank 3a-f with respect to the approved drugs, Molnupiravir and Remdesivir, using free energy methods as greener pastures. A further interesting comparison presented the laydown of the ligands before and after molecular docking. These results and other supporting statistical analyses suggested that ligands 3a-f deserve further investigation in the context of potential therapeutic agents for COVID-19. Free-cost, PASS, SwissADME, and Way2drug were used in this research paper to determine the possible biological activities and cytotoxicity of 3a-f.
Assuntos
Antivirais/química , Tratamento Farmacológico da COVID-19 , Iminas/química , Tiazóis/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Alanina/análogos & derivados , Alanina/química , Antivirais/síntese química , Antivirais/farmacocinética , Antivirais/toxicidade , Sítios de Ligação , Simulação por Computador , Proteases 3C de Coronavírus/química , Citidina/análogos & derivados , Citidina/química , Hidroxilaminas/química , Iminas/síntese química , Iminas/farmacocinética , Iminas/toxicidade , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , Tiazóis/síntese química , Tiazóis/farmacocinética , Tiazóis/toxicidadeRESUMO
It has been proposed that a mitochondrial switch involving a high mitochondrial superoxide production is associated with cancer metastasis. We here report an EPR analysis of ROS production using cyclic hydroxylamines in superinvasive SiHa-F3 compared with less invasive SiHa wild-type human cervix cancer cells. Using the CMH probe, no significant difference was observed in the overall level of ROS between SiHa and SiHa-F3 cells. However, using mitochondria-targeted cyclic hydroxylamine probe mitoTEMPO-H, we detected a significantly higher mitochondrial ROS content in SiHa-F3 compared with the wild-type SiHa cells. To investigate the nature of mitochondrial ROS, we overexpressed superoxide dismutase 2, a SOD isoform exclusively localized in mitochondria, in SiHa-F3 superinvasive cells. A significantly lower signal was detected in SiHa-F3 cells overexpressing SOD2 compared with SiHa-F3. Despite some limitations discussed in the paper, our EPR results suggest that mitochondrial ROS (at least partly superoxide) are produced to a larger extent in superinvasive cancer cells compared with less invasive wild-type cancer cells.
Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Linhagem Celular Tumoral , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Humanos , Hidroxilaminas/química , Invasividade Neoplásica , Compostos Organofosforados/química , Piperidinas/química , Superóxido Dismutase/metabolismo , Superóxidos/análiseRESUMO
Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.
Assuntos
Ácido Aconítico/análise , Extratos Celulares/análise , Succinatos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Técnicas Biossensoriais , Butilaminas/química , Cromatografia Líquida de Alta Pressão , Formiatos/química , Hidroxilaminas/química , Interferon gama/química , Limite de Detecção , Lipopolissacarídeos/química , Macrófagos/química , Camundongos , Células RAW 264.7 , Sensibilidade e EspecificidadeRESUMO
The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.
Assuntos
Peptídeos/química , Técnicas de Síntese em Fase Sólida/métodos , Compostos de Sulfidrila/química , Ubiquitina/química , Ubiquitinação , Cisteína/química , Escherichia coli/genética , Ésteres/química , Expressão Gênica , Hidroxilaminas/química , Imidas/química , Inteínas , Espectroscopia de Ressonância Magnética , Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina/biossíntese , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/biossíntese , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/isolamento & purificação , Ubiquitinas/biossíntese , Ubiquitinas/química , Zinco/metabolismoRESUMO
Proton-coupled electron transfer (PCET) is fundamental to many important biological reactions, including solar energy conversion and DNA synthesis. For example, class Ia ribonucleotide reductases (RNRs) contain a tyrosyl radical-diiron cofactor with one aspartate ligand, D84. The tyrosyl radical, Y122â¢, in the ß2 subunit acts as a radical initiator and oxidizes an active site cysteine in the α2 subunit. A transient quaternary α2/ß2 complex is induced by substrate and effector binding. The hydroxamic acid, hydroxyurea (HU), reduces Y122⢠in a PCET reaction involving an electron and proton. This reaction is associated with the loss of activity, a conformational change at Y122, and a change in hydrogen bonding to the Fe1 ligand, D84. Here, we use isotopic labeling, solvent isotope exchange, proton inventories, and reaction-induced Fourier transform infrared (RIFT-IR) spectroscopy to show that the PCET reactions of hydroxamic acids are associated with a characteristic spectrum, which is assignable to electrostatic changes at nonligating aspartate residues. Notably, RIFT-IR spectroscopy reveals this characteristic spectrum when the effects of HU, hydroxylamine, and N-methylhydroxylamine are compared. A large solvent isotope effect is observed for each of the hydroxamic acid reactions, and proton inventories predict that the reactions are associated with the transfer of multiple protons in the transition state. The reduction of Y122⢠with 4-methoxyphenol does not lead to these characteristic carboxylate shifts and is associated with only a small solvent isotope effect. In addition to studies of the effects of hydroxamic acids on ß2 alone, the reactions involving the quaternary α2ß2 complex were also investigated. HU treatment of the quaternary complex, α2/ß2/ATP/CDP, leads to a similar carboxylate shift spectrum, as observed with ß2 alone. The use of globally labeled 13C chimeras (13C α2, 13C ß2) confirms the assignment. Because the spectrum is sensitive to 13C ß2 labeling, but not 13C α2 labeling, the quaternary complex spectrum is assigned to electrostatic changes in ß2 carboxylate groups. Examination of the ß2 X-ray structure reveals a hydrogen-bonded network leading from the protein surface to Y122. This predicted network includes nonligating aspartates, glutamate ligands to the iron cluster, and predicted crystallographically resolved water molecules. The network is similar when class Ia RNR structures from Escherichia coli, human, and mouse are compared. We propose that the PCET reactions of hydroxamic acids are mediated by a hydrogen-bonded proton wire in the ß2 subunit.
Assuntos
Hidroxilamina/química , Hidroxilaminas/química , Hidroxiureia/química , Prótons , Ribonucleotídeo Redutases/química , Tirosina/química , Animais , Elétrons , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Humanos , Ligação de Hidrogênio , Camundongos , Oxirredução , Subunidades Proteicas/químicaRESUMO
Primary hyperoxalurias (PH) are inborn errors of glyoxylate metabolism characterized by an increase in endogenous oxalate production. Oxalate overproduction may cause calcium-oxalate crystal formation leading to kidney stones, nephrocalcinosis, and ultimately kidney failure. Twenty-four hour urine oxalate excretion is an inaccurate measure for endogenous oxalate production in PH patients and not applicable in those with kidney failure. Treatment efficacy cannot be assessed with this measure during clinical trials. We describe the development and validation of a gas chromatography-tandem mass spectrometry method to analyze the samples obtained following a stable isotope infusion protocol of 13C2-oxalate and 1-13C-glycolate in both healthy individuals and PH patients. Isotopic enrichments of plasma oxalate, glycolate, and glyoxylate were measured on a gas chromatography-triple quadrupole mass spectrometry system using ethylhydroxylamine and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) for analyte derivatization. Method precision was good for oxalate and glycolate (coefficients of variation [CV] were <6.3% and <4.2% for inter- and intraday precision, respectively) and acceptable for glyoxylate (CV <18.3% and <6.7% for inter- and intraday precision, respectively). The enrichment curves were linear over the specified range. Sensitivity was sufficient to accurately analyze enrichments. This new method allowed calculation of kinetic features of these metabolites, thus enabling a detailed analysis of the various pathways involved in glyoxylate metabolism. The method will further enhance the investigation of the metabolic PH derangements, provides a tool to accurately assess the therapeutic efficacy of new promising therapeutic interventions for PH, and could serve as a clinical tool to improve personalized therapeutic strategies.
Assuntos
Glicolatos/sangue , Glioxilatos/sangue , Hiperoxalúria Primária/metabolismo , Oxalatos/sangue , Acetamidas/química , Isótopos de Carbono/química , Fluoracetatos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glicolatos/química , Glicolatos/metabolismo , Glioxilatos/química , Glioxilatos/metabolismo , Humanos , Hidroxilaminas/química , Hiperoxalúria Primária/sangue , Marcação por Isótopo , Compostos de Organossilício/química , Oxalatos/química , Oxalatos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Polipose Intestinal/tratamento farmacológico , Triptofano/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Animais , Antineoplásicos/química , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Polipose Intestinal/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ultrasound-responsive chemistry was exploited in manufacture of drug delivery nanoparticulates for pursuit of on-demand ultrasound-stimulated drug release function. In principle, the ultrasound-labile oxyl-alkylhydroxylamine (-oa-) linkage was tailored between the segments of amphiphiles. Consequently, the hydrophobic chemotherapeutic doxorubicin could be readily assembled with the hydrophobic segments of amphiphiles into interior compartments, whereas the hydrophilic segments represented as the external surroundings. Upon ultrasonication, the proposed phase-segregated self-assemblies were determined to be subjected to evident structural rearrangement as a consequence of -oa- cleavage. Simultaneously, the release rate of doxorubicin payloads appeared to accelerate due to the ultrasound-induced structural destabilization, consequently eliciting potent cytotoxic efficacy at the affected cells. Another noteworthy characteristic of the proposed self-assemblies was poly (lactobionamidoethyl methacrylate) (pLAMA) as the hydrophilic components of the amphiphiles, characterized to possess galactosylated residues. In view of the specific affinity of galactosylated residues (and lactosylated residues) to asialoglycoprotein receptors (overexpressed on the surface of intractable hepatocellular carcinoma), the proposed self-assemblies were determined to impart preferential affinities to hepatocellular carcinoma. Together with the strategic ultrasound-stimulated drug release property, our proposed drug delivery system demonstrated appreciably pharmaceutical efficacy on hepatocellular carcinoma.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Portadores de Fármacos/química , Hidroxilaminas/química , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/química , Ácidos Polimetacrílicos/química , Ondas Ultrassônicas , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Liberação Controlada de Fármacos , Galactose/química , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , PolimerizaçãoRESUMO
This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 µl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); 5α-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, DHT (33 pM-33 nM); 11-ketodihydrotestosterone, 11KDHT (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11ß-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11ß-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like DHT, DHEA and P5, and a panel of 11-ketosteroids.
Assuntos
Estradiol/análise , Hidroxilaminas/análise , Cetosteroides/análise , Próstata/química , Cromatografia Líquida de Alta Pressão/métodos , Estradiol/sangue , Humanos , Hidroxilaminas/sangue , Hidroxilaminas/química , Marcação por Isótopo/métodos , Cetosteroides/sangue , Cetosteroides/química , Masculino , Espectrometria de Massas em Tandem/métodosRESUMO
The first direct synthesis of 3-N-methyl-9-formylcytisine via electrophylic formylation is described. It is established, that Vilsmeier-Haack and Gatterman variants of this reaction are unsuccessful in the case with 3-substituted (-)-cytisine derivatives, but Duff procedure (with hexamethylenetetramine in trifluoroacetic acid) gives a possibility to obtain the target pseudo aromatic aldehyde with the 69% yield. Convenient precursors for [4 + 2]- or [3 + 2]-cycloaddition reactions are obtained by means of condensation of synthesized 3-N-methyl-9-formylcytisine with acetone, nitromethane and phosphorous ylides with yields from 70 to 87%. Alternative aprroach to alkenyl products and to 9-alkynyl-3-methylcytisine is realized using the Heck and Sonogashira cross-coupling reactions of methyl vinyl ketone, cyclohexenone or trimethylsilylacetylene with 9-bromo-3-methylcytisine (55, 70 and 60% accordingly). It is shown, that interaction of 3-N-methyl-9-formylcytisine with hydroxylamines leads to corresponding nitrone (93%) and oxime (70%). All individual compounds are isolated by column chromatography and completely characterized on the basis of NMR spectroscopy data.
Assuntos
Alcaloides/química , Piridonas/química , Aldeídos , Azocinas , Formiatos/química , Hidroxilaminas/química , Espectroscopia de Ressonância Magnética , Óxidos de Nitrogênio/síntese química , Oximas/síntese química , Quinolizinas/químicaRESUMO
We report amperometric detection of formaldehyde (HCHO) using hydroxylamine hydrochloride and single-walled carbon nanotubes (SWCNTs). Hydroxylamine hydrochloride reacts with HCHO to emit HCl vapor, which injects a hole carrier into semiconducting SWCNTs. The increase of conductivity in SWCNTs is easily monitored using an ohmmeter. The debundling of SWCNTs with a metallo-supramolecular polymer (MSP) increased the active surface area in the SWCNTs network, leading to excellent sensitivity to HCHO with a limit of detection (LoD) of 0.016 ppm. The response of sensor is reversible, and the sensor is reusable. The selectivity to HCHO is 105-106 times higher than interferences with other volatiles such as water, methanol, and toluene. Moreover, false-positive responses caused by a significant variation of humidity and/or temperature are successfully discriminated from true-positive responses by using two sensors, one with and the other without hydroxylamine hydrochloride, in a referenced system.
Assuntos
Técnicas de Química Analítica/métodos , Formaldeído/análise , Hidroxilaminas/química , Nanotubos de Carbono/química , Tensoativos/química , Técnicas de Química Analítica/instrumentação , Formaldeído/química , Limite de Detecção , Padrões de ReferênciaRESUMO
IDO1 (indoleamine 2, 3-dioxygenase 1), a well characterized immunosuppressive enzyme, has attracted growing attention as a potential target for cancer immunotherapy. Hydroxylamidine compounds INCB024360 and INCB14943 (INCB024360 analogue) are highly effective IDO1 inhibitors. INCB024360 is undergoing clinical trials for treatment of various types of human cancer. Here, we determined the co-crystal structure of IDO1 and INCB14943, and elucidate the detailed binding mode. INCB14943 binds to heme iron in IDO1 protein through the oxime nitrogen. Further analysis also reveals that a halogen bonding interaction between the chlorine atom (3-Cl) of INCB14943 and the sulphur atom of C129 significantly improves the inhibition activity against IDO1. Comparing with the other reported inhibitors, the oxime nitrogen and halogen bond interaction are identified as the unique features of INCB14943 among the IDO1 inhibitors. Thus, our study provides novel insights into the interaction between a small molecule inhibitor INCB14943 and IDO1 protein. The structural information will facilitate future IDO1 inhibitor design.
Assuntos
Hidroxilaminas/química , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/ultraestrutura , Oxidiazóis/química , Oximas/química , Sítios de Ligação , Ativação Enzimática , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In order to create a soft tissue surplus, implantable volume expanders are often utilized in dental surgery. Implanted tissue expanders should gradually increase their volume, exerting a constant pressure on the surrounding tissue for weeks. Current tissue expanders are based predominantly on externally inflatable balloons or on osmotically active tissue expanders that use soft hydrogels wrapped in perforated plastic coatings, which limit fluid entry and swelling. We have designed and examined tissue expanders based on the controlled rate expansive hydrogels synthesized from copolymers of selected methacrylates and N-vinylpyrrolidone, cross-linked with a combination of non-degradable (glycol dimethacrylates) and hydrolytically degradable (N,O-dimethacryloylhydroxylamine) cross-linkers. These copolymers have close-to-linear volume expansion rates (up to 6-9 times their original volume) and exert an increasing swelling pressure in vitro. The anesthetic benzocaine has been incorporated into the hydrogels, and kinetic release experiments have shown that most of the drug (90%) was released within 48 h. Our proposed hydrogel expanders are homogeneous and have suitable mechanical properties, thus simplifying the surgical manipulations required. Further studies will be needed to completely evaluate their biocompatibility and tissue response to the implants.
Assuntos
Hidrogéis/química , Metacrilatos/química , Medicina Bucal/métodos , Polímeros/química , Dispositivos para Expansão de Tecidos , Anestésicos/administração & dosagem , Materiais Biocompatíveis/química , Reagentes de Ligações Cruzadas/química , Humanos , Hidroxilaminas/química , Cinética , PressãoRESUMO
An optimized protocol for the masking of α-ketoacids with acid-labile cyclic acetal protecting groups is reported. Unlike prior approaches, these new conditions allow the synthesis of protected α-ketoacids bearing aromatic, hindered alkyl, and protected polar side chains. Attachment to a Wang-type linker and solid support provides a resin that delivers fully unprotected C-terminal peptide α-ketoacids upon resin cleavage. These peptides are the key starting materials for chemical protein synthesis using the α-ketoacid-hydroxylamine ligation.
Assuntos
Hidroxilaminas/química , Cetoácidos/síntese química , Peptídeos/síntese química , Cetoácidos/química , Estrutura Molecular , Peptídeos/químicaRESUMO
Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.
Assuntos
Técnicas de Química Sintética/métodos , Hidroxilaminas/química , Proteínas/síntese química , Fluorenos/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Hormônios Peptídicos/síntese química , Hormônios Peptídicos/química , Proteínas/químicaRESUMO
Indoleamine 2,3-dioxygenase-1 (IDO1) is a promising therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. Recently important advances have been made in understanding IDO1's catalytic mechanism. Although much remains to be discovered, there is strong evidence that the mechanism proceeds through a heme-iron bound alkylperoxy transition or intermediate state. Accordingly, we explored stable structural mimics of the alkylperoxy species and provide evidence that such structures do mimic the alkylperoxy transition or intermediate state. We discovered that O-benzylhydroxylamine, a commercially available compound, is a potent sub-micromolar inhibitor of IDO1. Structure-activity studies of over forty derivatives of O-benzylhydroxylamine led to further improvement in inhibitor potency, particularly with the addition of halogen atoms to the meta position of the aromatic ring. The most potent derivatives and the lead, O-benzylhydroxylamine, have high ligand efficiency values, which are considered an important criterion for successful drug development. Notably, two of the most potent compounds demonstrated nanomolar-level cell-based potency and limited toxicity. The combination of the simplicity of the structures of these compounds and their excellent cellular activity makes them quite attractive for biological exploration of IDO1 function and antitumor therapeutic applications.
Assuntos
Desenho de Fármacos , Hidroxilaminas/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Hidroxilaminas/síntese química , Hidroxilaminas/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Here we demonstrate a type of pH and reduction dual-sensitive biodegradable micelles, which were self-assembled by a cationic polymer in an aqueous solution. Due to tumor cells or tissues showing low pH and high reduction concentration, these micelles possessed specific tumor targetability and maximal drug-release controllability inside tumor cells upon changes in physical and chemical environments, but presented good stability at physiological conditions. CCK-8 assay showed that the DOX-loaded micelles had a similar cytotoxicity for MCF-7 tumor cells as free DOX, and blank micelles had a very low cytotoxicity to the cells. Fluorescent microscopy observation revealed that the drug-loaded micelles could be quickly internalized by endosomes to inhibit cancer cell growth. These results indicated these biodegradable micelles, as a novel and effective pH- and redox-responsive nanocarrier, have a potential to improve drug delivery and enhance the antitumor efficacy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/farmacologia , Liberação Controlada de Fármacos/fisiologia , Hidroxilaminas/química , Micelas , Neoplasias/tratamento farmacológico , Biodegradação Ambiental , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , OxirreduçãoRESUMO
The potential of RNA interference (RNAi)-based therapeutics for cancer has received much attention; however, delivery of RNAi effectors, such as small interfering RNA (siRNA), remains an obstacle to clinical translation. Non-viral delivery vectors have been used extensively to enhance siRNA delivery. Recently, the potential of gold nanoparticles (AuNPs) for transporting drugs, proteins and genetic materials has been demonstrated. Previously, our laboratory synthesised positively charged, surfactant-free AuNPs in water by the reduction of gold (III) chloride (AuCl3) using hydroxylamine hydrochloride (NH2OH·HCl) in the presence of L-cysteine methyl ester hydrochloride (HSCH2CH(NH2)COOCH3·HCl) as a capping agent. These AuNPs, which achieve higher cell viability in comparison to cetyl trimethyl ammonium bromide (CTAB, a surfactant)-capped counterparts, have demonstrated potential for siRNA delivery. However, it is well known that systemic administration of cationic delivery systems without biological stablising moieties causes non-specific binding with negatively charged serum proteins, resulting in particle aggregation and opsonisation. Consequently, highly stable AuNPs capped with l-cysteine methyl ester hydrochloride conjugated to poly(ethylene glycol) (PEG) were synthesised in this study. PEGylation enhanced the biocompatibility of the AuNPs by reducing toxicity in a range of cell types, by inhibiting interaction with serum proteins thus avoiding aggregation, and, by providing protection against degradation by nucleases. Moreover, these PEGylated AuNPs formed nanoparticles (NPs) with siRNA (which was first compacted with protamine), and had a diameter within the nanoscale range (â¼ 250 nm) and a near neutral surface charge (â¼ 10 mV). In the future a bifunctional PEG chain on the AuNPs (i.e., SH-PEG-NH2, SH-PEG-COOH) will be used to facilitate conjugation of a targeting ligand to enhance cell specific uptake.