RESUMO
Macrocyclic poly(glycidyl phenyl ether) (pGPE) synthesized via zwitterionic ring opening polymerization is typically contaminated by chains with linear and tadpole architecture. Although mass spectrometry (MS) analysis can readily confirm the presence of the linear byproduct, due to its unique mass, it is unable to differentiate between the cyclic and tadpole structures, which are constitutional isomers produced by backbiting reactions in monomeric or dimeric chains, respectively. To overcome this problem, ultraperformance reversed-phase liquid chromatography interfaced with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed. The separation achieved by UPLC revealed that the tadpole isomer elutes before the cyclic structure because of the increased polarity afforded by its distinctive substituents. The ratio of tadpole to cyclic species increased with the degree of polymerization, in agreement with the synthetic method used, as the potential for forming tadpole structures by backbiting is entropically favored in longer polymer chains. Once separated, the two isomers could be independently characterized by tandem mass spectrometry. The macrocyclic and tadpole species exhibit unique fragmentation patterns, including structurally diagnostic fragments for each structure.
Assuntos
Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Larva , Polímeros/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Ultraperformance liquid chromatography (UPLC) and ion mobility (IM) spectrometry were interfaced with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize a complex nonionic surfactant mixture. The surfactant was composed of a glycerol core, functionalized with poly(ethylene oxide) units (PEOn) that were partially esterified by caprylic and/or capric acid. Reversed-phase UPLC classified the blend based on polarity into four groups of eluates, corresponding to compounds with zero, one, two, or three fatty acid residues. Additional separation within each eluate group was achieved according to the length of the fatty acid chains. Coeluting molecules of similar polarity were dispersed in the gas phase by their collision cross section in the IM dimension. Performed in series, UPLC and IM allowed for the separation and detection of several isomeric and isobaric blend constituents, thereby enabling their isolation for conclusive MS/MS analysis to confirm or elucidate their primary structures and architectures (overall four-dimensional, 4D, characterization).
Assuntos
Cromatografia de Fase Reversa , Espectrometria de Massas em Tandem , Cromatografia Líquida , Espectrometria de Mobilidade Iônica , TensoativosRESUMO
Infarct expansion can occur after myocardial infarction (MI), which leads to adverse left ventricular (LV) remodeling and failure. An imbalance between matrix metalloproteinase (MMP) induction and tissue inhibitors of MMPs (TIMPs) can accelerate this process. Past studies have shown different biologic effects of TIMP-3, which may depend upon specific domains within the TIMP-3 molecule. This study tested the hypothesis that differential effects of direct myocardial injections of either a full-length recombinant TIMP-3 (F-TIMP-3) or a truncated form encompassing the N-terminal region (N-TIMP-3) could be identified post-MI. MI was induced in pigs that were randomized for MI injections (30 mg) and received targeted injections within the MI region of F-TIMP-3 (n = 8), N-TIMP-3 (n = 9), or saline injection (MI-only, n = 11). At 14 days post-MI, LV ejection fraction fell post-MI but remained higher in both TIMP-3 groups. Tumor necrosis factor and interleukin-10 mRNA increased by over 10-fold in the MI-only and N-TIMP-3 groups but were reduced with F-TIMP-3 at this post-MI time point. Direct MI injection of either a full-length or truncated form of TIMP-3 is sufficient to favorably alter the course of post-MI remodeling. The functional and differential relevance of TIMP-3 domains has been established in vivo since the TIMP-3 constructs demonstrated different MMP/cytokine expression profiles. These translational studies identify a unique and more specific therapeutic strategy to alter the course of LV remodeling and dysfunction after MI. SIGNIFICANCE STATEMENT: Using different formulations of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), when injected into the myocardial infarction (MI) region, slowed the progression of indices of left ventricular (LV) failure, suggesting that the N terminus of TIMP-3 is sufficient to attenuate early adverse functional events post-MI. Injections of full-length recombinant TIMP-3, but not of the N-terminal region of TIMP-3, reduced relative indices of inflammation at the mRNA level, suggesting that the C-terminal region affects other biological pathways. These unique proof-of-concept studies demonstrate the feasibility of using recombinant small molecules to selectively interrupt adverse LV remodeling post-MI.
Assuntos
Infarto do Miocárdio/patologia , Fragmentos de Peptídeos/farmacologia , Inibidor Tecidual de Metaloproteinase-3/química , Remodelação Ventricular/efeitos dos fármacos , Sequência de Aminoácidos , Colágeno/genética , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções , Metaloproteinases da Matriz/genética , Fragmentos de Peptídeos/química , Domínios Proteicos , RNA Mensageiro/genética , Inibidor Tecidual de Metaloproteinase-3/genéticaRESUMO
Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Sítio Alostérico/efeitos dos fármacos , Substituição de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Benzenoacetamidas/química , Benzenoacetamidas/metabolismo , Benzenoacetamidas/farmacologia , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutaminase/química , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/antagonistas & inibidores , Glutamina/química , Glutamina/metabolismo , Humanos , Ligação de Hidrogênio , Conformação Molecular , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfetos/química , Sulfetos/metabolismo , Sulfetos/farmacologia , Tiadiazóis/química , Tiadiazóis/metabolismo , Tiadiazóis/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 µM in the LAD2 cell assay.
Assuntos
Inibidores Enzimáticos/química , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/química , Lipocalinas/antagonistas & inibidores , Lipocalinas/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Prostaglandina D2/biossíntese , Prostaglandina D2/sangue , Prostaglandina H2/química , Prostaglandina H2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
Assuntos
Anticorpos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoconjugados/farmacologia , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Anticorpos/genética , Ligante CD27/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoconjugados/química , Maitansina/metabolismo , Camundongos , Camundongos SCID , Receptores Fc/genéticaRESUMO
A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.
Assuntos
Benzenoacetamidas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Benzenoacetamidas/química , Benzenoacetamidas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutaminase/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/metabolismo , Tiadiazóis/química , Tiadiazóis/metabolismoRESUMO
High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein-based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts-91% of which are successfully expressed and purified-are discussed. Mammalian expression combined with the use of solubility-promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult-to-produce proteins (Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80-fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult-to-produce protein reagents.
Assuntos
Mamíferos , Neurturina , Animais , Linhagem Celular , Proteínas Recombinantes de Fusão/genéticaRESUMO
Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.
RESUMO
The synergistic effect of the co-morbidities that comprise metabolic syndrome (MetS) is increasingly being recognised as an important contributor in the pathology of a broad spectrum of seemingly disparate conditions. However, in terms of male reproductive function, beyond erectile dysfunction, little is known about the influence of this cohort (collectively or separately) on spermatogenesis and sperm quality. The aims of this study were to assess the reproductive tract of a MetS animal model for detrimental changes, to determine whether a group of compounds (advanced glycation end products and their receptor) known to cause cell dysfunction and DNA damage was present and assess whether hypogonadotropic hypogonadism was the main contributing factor for the changes seen. Animals fed a high-fat diet were found to have significantly increased cholesterol, triglycerides, blood glucose, mean arterial pressure and visceral fat levels. Although serum testosterone was decreased, no changes were seen in either testicular or epididymal histology. Immunolocalisation of N(ε)-carboxymethyl-lysine and the receptor for advanced glycation end products was found in the testes, epididymides and sperm of the two treated groups of animals; however, ELISA did not show any difference in protein levels. Similarly, assessment of sperm nuclear DNA (nDNA) fragmentation by acridine orange test did not find significant differences in nDNA integrity. We conclude that the minimal effect on spermatogenesis and sperm quality seen in our model is probably due to the moderate increase of blood glucose rather than the hypogonadism.
Assuntos
Modelos Animais de Doenças , Hipogonadismo/etiologia , Síndrome Metabólica/fisiopatologia , Espermatogênese , Espermatozoides/metabolismo , Espermatozoides/patologia , Animais , Fragmentação do DNA , Gorduras na Dieta/efeitos adversos , Epididimo/metabolismo , Epididimo/patologia , Produtos Finais de Glicação Avançada/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Hiperglicemia/etiologia , Hipogonadismo/induzido quimicamente , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Coelhos , Distribuição Aleatória , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Análise do Sêmen , Espermatozoides/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Pamoato de Triptorrelina/toxicidadeRESUMO
Tissue Inhibitor of Metalloproteinase 3 (TIMP3) is a secreted protein that has a great utility to inhibit elevated metalloproteinase (MMP) activity in injured tissues including infarcted cardiac tissue, inflamed vessels, and joint cartilages. An imbalance between TIMP3 and active MMP levels in the local tissue area may cause worsening of disease progression. To counter balance elevated MMP levels, exogenous administration of TIMP3 appeared to be beneficial in preclinical studies. However, the current form of WT-TIMP3 molecule has a limitation to be a therapeutic candidate due to low production yield, short plasma half-life, injection site retention, and difficulty in delivery, etc. We have engineered TIMP3 molecules by adding extra glycosylation sites or fusing with albumin, Fc, and antibody to improve pharmacokinetic properties. In general, the C-terminal fusion of TIMP3 improved expression and production in mammalian cells and extended half-lives dramatically 5-20 folds. Of note, a site-specific glycosylation at K22S/F34N resulted in a higher level of expression and better cardiac function compared to other fusion proteins in the context of left ventricle ejection fraction (LVEF) changes in a rat myocardial infarction model. It appeared that cardiac efficacy depends on a high ECM binding affinity, in which K22S/F34N and N-TIMP3 showed a higher binding to the ECM compared to other engineered molecules. In conclusion, we found that the ECM binding and sustained residence of injected TIMP3 molecules are important for cardiac tissue localization and inhibition of adverse remodeling activity.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Metaloproteinases da Matriz/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Inibidor Tecidual de Metaloproteinase-3/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Proteína ADAM17/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos , Glicosilação , Humanos , Infusões Intravenosas , Injeções Intralesionais , Masculino , Mutação , Infarto do Miocárdio/etiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Inibidor Tecidual de Metaloproteinase-3/química , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/uso terapêutico , Resultado do TratamentoRESUMO
Dimeric interactions among anti- and pro-apoptotic members of the BCL-2 protein family are dynamically regulated and intimately involved in survival and death functions. We report the structure of a BCL-X(L) homodimers a 3D-domain swapped dimer (3DDS). The X-ray crystal structure demonstrates the mutual exchange of carboxy-terminal regions including BH2 (Bcl-2 homology 2) between monomer subunits, with the hinge region occurring at the hairpin turn between the fifth and sixth alpha helices. Both BH3 peptide-binding hydrophobic grooves are unoccupied in the 3DDS dimer and available for BH3 peptide binding, as confirmed by sedimentation velocity analysis. BCL-X(L) 3DDS dimers have increased pore-forming activity compared to monomers, suggesting that 3DDS dimers may act as intermediates in membrane pore formation. Chemical crosslinking studies of Cys-substituted BCL-X(L) proteins demonstrate that 3DDS dimers form in synthetic lipid vesicles.
Assuntos
Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteína bcl-X/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/fisiologia , Linfoma de Células B/patologia , Proteínas Oncogênicas/metabolismo , Biópsia , Proteínas de Ciclo Celular/genética , Morte Celular , Linhagem Celular Tumoral , Humanos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Oncogênicas/genética , Fases de Leitura Aberta , Plasmídeos , Tionucleotídeos/farmacologiaRESUMO
Cancer cells with elevated levels of BCL-2 and related survival proteins are broadly resistant to cytotoxic agents. Antisense oligodeoxynucleotides, and more recently small molecule ligands for BCL-2 and BCL-XL, are directly cytotoxic or synergistic with standard cytotoxic agents, and in some cases, may demonstrate selectivity for tumor cells. The usual issues for rational drug discovery are writ large upon BCL-2-targeted therapeutics. The molecular functions of BCL-2 are not well understood, such that validation of cytotoxic mechanisms related to BCL-2 as well as identification of surrogate markers for BCL-2 function are significant obstacles for drug development. Despite these problems, a substantial number of small molecules that bind to BCL-2 or BCL-XL are now available for pre-clinical testing; in turn, basic studies with these reagents should yield new insights about optimal strategies to disrupt BCL-2 survival functions.
Assuntos
Apoptose/genética , Genes bcl-2/genética , Neoplasias/genética , Neoplasias/terapia , Antineoplásicos/farmacologia , Sobrevivência Celular , Humanos , Ligantes , Neoplasias/fisiopatologia , Oligonucleotídeos Antissenso/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteína bcl-XRESUMO
The rational design of loops and turns is a key step towards creating proteins with new functions. We used a computational design procedure to create new backbone conformations in the second turn of protein L. The Protein Data Bank was searched for alternative turn conformations, and sequences optimal for these turns in the context of protein L were identified using a Monte Carlo search procedure and an energy function that favors close packing. Two variants containing 12 and 14 mutations were found to be as stable as wild-type protein L. The crystal structure of one of the variants has been solved at a resolution of 1.9 A, and the backbone conformation in the second turn is remarkably close to that of the in silico model (1.1 A RMSD) while it differs significantly from that of wild-type protein L (the turn residues are displaced by an average of 7.2 A). The folding rates of the redesigned proteins are greater than that of the wild-type protein and in contrast to wild-type protein L the second beta-turn appears to be formed at the rate limiting step in folding.
Assuntos
Proteínas de Bactérias/química , Simulação por Computador , Engenharia de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Cristalografia por Raios X , Bases de Dados de Proteínas , Cinética , Modelos Moleculares , Método de Monte Carlo , Mutação/genética , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Imunoconjugados/metabolismo , Maitansina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Linhagem Celular Tumoral , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Imunoconjugados/administração & dosagem , Lisossomos/metabolismo , Maitansina/administração & dosagemRESUMO
The Bcl-2 family of proteins provide the most unambiguous link between mitochondrial functions and apoptosis, as their only (or principal) functions appear to be as regulators of this cell death pathway. Rational drug design to manipulate the functions of these proteins has been hampered by the lack of a clear understanding of a biochemical or molecular function, with disruption of intra-family protein-protein interactions as the only known, but daunting, objective. There has been substantial progress in this task using molecular modeling and drug leads. The prospects are also good for development of chemical tools for functional analysis of the Bcl-2 proteins.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Antimicina A/metabolismo , Antimicina A/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Benzamidas/metabolismo , Benzamidas/farmacologia , Benzopiranos/metabolismo , Benzopiranos/farmacologia , Citocromos c/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Canais Iônicos/metabolismo , Mitocôndrias/fisiologia , Modelos Moleculares , Nitrilas/metabolismo , Nitrilas/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to the left ventricle (LV) remodeling that occurs after myocardial infarction (MI). However, translation of these observations into a clinically relevant, therapeutic strategy remains to be established. The present study investigated targeted TIMP augmentation through regional injection of a degradable hyaluronic acid hydrogel containing recombinant TIMP-3 (rTIMP-3) in a large animal model. MI was induced in pigs by coronary ligation. Animals were then randomized to receive targeted hydrogel/rTIMP-3, hydrogel alone, or saline injection and followed for 14 days. Instrumented pigs with no MI induction served as referent controls. Multimodal imaging (fluoroscopy/echocardiography/magnetic resonance imaging) revealed that LV ejection fraction was improved, LV dilation was reduced, and MI expansion was attenuated in the animals treated with rTIMP-3 compared to all other controls. A marked reduction in proinflammatory cytokines and increased smooth muscle actin content indicative of myofibroblast proliferation occurred in the MI region with hydrogel/rTIMP-3 injections. These results provide the first proof of concept that regional sustained delivery of an MMP inhibitor can effectively interrupt adverse post-MI remodeling.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Inibidor Tecidual de Metaloproteinase-3/administração & dosagem , Inibidor Tecidual de Metaloproteinase-3/uso terapêutico , Remodelação Ventricular/fisiologia , Animais , Modelos Animais de Doenças , Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Remodelação Ventricular/efeitos dos fármacosRESUMO
Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.