RESUMO
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 2) discusses the recommendations for Hybrid LBA/LCMS and regulatory inputs from major global health authorities. Parts 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
Assuntos
Biomarcadores/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Anticorpos Anti-Idiotípicos/análise , Anticorpos Anti-Idiotípicos/imunologia , Conferências de Consenso como Assunto , Órgãos Governamentais , Humanos , Imunoensaio , Ligantes , Estudos de Validação como AssuntoRESUMO
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
Assuntos
Biomarcadores/análise , Ligantes , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Cromatografia Líquida de Alta Pressão , Conferências de Consenso como Assunto , Órgãos Governamentais , Humanos , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/imunologia , Substâncias Macromoleculares/farmacocinética , Espectrometria de Massas , Estudos de Validação como AssuntoAssuntos
Medicamentos Biossimilares/análise , Controle de Medicamentos e Entorpecentes , Anticorpos Neutralizantes/análise , Medicamentos Biossimilares/efeitos adversos , Controle de Medicamentos e Entorpecentes/métodos , Controle de Medicamentos e Entorpecentes/organização & administração , Humanos , Imunidade , Imunoensaio/métodos , Estados Unidos , United States Food and Drug AdministrationRESUMO
UNLABELLED: Leukemias expressing the constitutively activated tyrosine kinases (TK) BCR-ABL1 and FLT3/ITD activate signaling pathways that increase genomic instability through generation of reactive oxygen species (ROS), DNA double-strand breaks (DSB), and error-prone repair. The nonhomologous end-joining (NHEJ) pathway is a major pathway for DSB repair and is highly aberrant in TK-activated leukemias; an alternative form of NHEJ (ALT-NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and PARP1, increased frequency of large genomic deletions, and repair using DNA sequence microhomologies. This study, for the first time, demonstrates that the TK target c-MYC plays a role in transcriptional activation and subsequent expression of LIG3 and PARP1 and contributes to the increased error-prone repair observed in TK-activated leukemias. c-MYC negatively regulates microRNAs miR-150 and miR-22, which demonstrate an inverse correlation with LIG3 and PARP1 expression in primary and cultured leukemia cells and chronic myelogenous leukemia human patient samples. Notably, inhibition of c-MYC and overexpression of miR-150 and -22 decreases ALT-NHEJ activity. Thus, BCR-ABL1 or FLT3/ITD induces c-MYC expression, leading to genomic instability via augmented expression of ALT-NHEJ repair factors that generate repair errors. IMPLICATIONS: In the context of TK-activated leukemias, c-MYC contributes to aberrant DNA repair through downstream targets LIG3 and PARP1, which represent viable and attractive therapeutic targets.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligases/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Poli(ADP-Ribose) Polimerases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Poli(ADP-Ribose) Polimerase-1 , Proteínas de Ligação a Poli-ADP-Ribose , Transcrição Gênica , Proteínas de XenopusRESUMO
Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.
Assuntos
Apoptose , Aptâmeros de Peptídeos/farmacologia , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/patologia , Mutação/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética/genética , Animais , Aptâmeros de Peptídeos/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Estudos de Casos e Controles , Diferenciação Celular , Proliferação de Células , Dano ao DNA/genética , Reparo do DNA/genética , Epigenômica , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/prevenção & controle , Camundongos , Camundongos SCID , Modelos Moleculares , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos , RNA Mensageiro/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA decay pathway known as the 2'-5'-oligoadenylate (2-5A) system, whose established functions include antimicrobial and tumor suppressive activities. RNase-L activity requires binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3'-untranslated region (3' UTR), which exerts a strong negative effect on RNase-L levels. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity often in the 3' UTR. The miR-29 family acts as a tumor suppressor in several cancers, including acute and chronic myelogenous leukemia (CML), and has many oncogenic targets. We report that the miR-29 family represses RNase-L protein expression across several cell types. Using a luciferase reporter, we showed that miR-29 acts via 4 target sites within the RNASEL 3' UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L inhibits proliferation in vitro as well as tumor growth in a xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies.
Assuntos
Endorribonucleases/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Endorribonucleases/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Mutação , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23aâ¼miR-27aâ¼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.
Assuntos
Proteínas 14-3-3/genética , Leucemia/genética , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Proteínas 14-3-3/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/metabolismo , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.
Assuntos
Adenosina/metabolismo , Ácido Hialurônico/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Ácido Hialurônico/genética , Interleucina-12/imunologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptor A2A de Adenosina/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The balance between reactive oxygen species (ROS) and endogenous anti-oxidants is important in maintaining healthy tissues. Excessive ROS states occur in diseases such as ARDS and Idiopathic Pulmonary Fibrosis. Redox imbalance breaks down the extracellular matrix component hyaluronan (HA) into fragments that activate innate immune responses and perpetuate tissue injury. HA fragments, via a TLR and NF-kappaB pathway, induce inflammatory gene expression in macrophages and epithelial cells. NAC and DMSO are potent anti-oxidants which may help balance excess ROS states. METHODS: We evaluated the effect of H2O2, NAC and DMSO on HA fragment induced inflammatory gene expression in alveolar macrophages and epithelial cells. RESULTS: NAC and DMSO inhibit HA fragment-induced expression of TNF-alpha and KC protein in alveolar and peritoneal macrophages. NAC and DMSO also show a dose dependent inhibition of IP-10 protein expression, but not IL-8 protein, in alveolar epithelial cells. In addition, H2O2 synergizes with HA fragments to induce inflammatory genes, which are inhibited by NAC. Mechanistically, NAC and DMSO inhibit HA induced gene expression by inhibiting NF-kappaB activation, but NAC had no influence on HA-fragment-AP-1 mediated gene expression. CONCLUSION: ROS play a central role in a pathophysiologic "vicious cycle" of inflammation: tissue injury generates ROS, which fragment the extracellular matrix HA, which in turn synergize with ROS to activate the innate immune system and further promote ROS, HA fragment generation, inflammation, tissue injury and ultimately fibrosis. The anti-oxidants NAC and DMSO, by inhibiting the HA induced inflammatory gene expression, may help re-balance excessive ROS induced inflammation.
RESUMO
Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Czeta-, and NF-kappaB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2(null) mice.
Assuntos
Ácido Hialurônico/fisiologia , Mediadores da Inflamação/fisiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/metabolismo , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/metabolismo , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peso Molecular , Fator 88 de Diferenciação Mieloide , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/genética , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/fisiologiaRESUMO
Determination of protein oligomerization state can be technically challenging. We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states. We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins. The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET. Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization. These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/metabolismo , Escherichia coli/genética , Fluoresceína/química , Expressão Gênica , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Ligação Proteica , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Rodaminas/química , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Espectrometria de FluorescênciaRESUMO
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors.