ABSTRACT
Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.
Subject(s)
Anti-HIV Agents , HIV Integrase Inhibitors , HIV Integrase , HIV Integrase Inhibitors/pharmacology , HIV Integrase Inhibitors/chemistry , Allosteric Regulation , Catalytic Domain , HIV Integrase/metabolismABSTRACT
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Subject(s)
HIV Infections , HIV-1 , Humans , HIV Infections/drug therapy , Antiviral Agents/therapeutic use , Apoptosis , Cell Death , CD4-Positive T-Lymphocytes , Virus ReplicationABSTRACT
In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.
Subject(s)
Immunity, Cellular , Immunity, Humoral , Influenza Vaccines , Orthomyxoviridae Infections , mRNA Vaccines , Animals , Antibodies, Viral , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H1N1 Subtype , Influenza Vaccines/immunology , Mice , Nucleoproteins/genetics , Orthomyxoviridae Infections/prevention & control , Primates , Vaccines, Synthetic , mRNA Vaccines/immunologyABSTRACT
High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Progranulins/metabolism , Small Molecule Libraries/chemistry , Adaptor Proteins, Vesicular Transport/antagonists & inhibitors , Amides/chemistry , Amides/metabolism , Amino Acids/chemistry , Amino Acids/metabolism , Binding Sites , Crystallography, X-Ray , High-Throughput Screening Assays , Humans , Molecular Dynamics Simulation , Progranulins/antagonists & inhibitors , Protein Binding , Pyrazoles/chemistry , Pyrazoles/metabolism , Small Molecule Libraries/metabolism , Structure-Activity RelationshipABSTRACT
Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.
Subject(s)
Allosteric Regulation/drug effects , HIV Infections/drug therapy , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/drug effects , Cell Line , Drug Discovery , HIV Infections/metabolism , HIV Infections/virology , HIV-1/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Protein Interaction Maps/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.
Subject(s)
Cathepsin K/blood , Cysteine Proteinase Inhibitors/blood , Adolescent , Adult , Aged , Animals , Biomarkers/urine , Biphenyl Compounds/blood , Biphenyl Compounds/pharmacokinetics , Biphenyl Compounds/pharmacology , Biphenyl Compounds/urine , Blood Proteins/metabolism , Cathepsin K/antagonists & inhibitors , Collagen Type I/urine , Cysteine Proteinase Inhibitors/pharmacokinetics , Cysteine Proteinase Inhibitors/pharmacology , Cysteine Proteinase Inhibitors/urine , Dogs , Female , Humans , Macaca mulatta , Male , Middle Aged , Peptides/urine , Protein Binding , Pyrazoles/blood , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrazoles/urine , Rabbits , Sulfones/blood , Sulfones/pharmacokinetics , Sulfones/pharmacology , Sulfones/urine , Young AdultABSTRACT
During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.
Subject(s)
Cathepsin K/antagonists & inhibitors , Cathepsin K/metabolism , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Animals , Bone and Bones/enzymology , Drug Evaluation, Preclinical , Kinetics , RabbitsABSTRACT
We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.
Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Cation Transport Proteins/immunology , Opsonin Proteins/immunology , Staphylococcus aureus/immunology , Animals , Antibodies, Bacterial/genetics , Antibodies, Bacterial/metabolism , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Cation Transport Proteins/antagonists & inhibitors , Complement System Proteins/immunology , Disease Models, Animal , Heme/metabolism , Mice , Mice, Inbred BALB C , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutant Proteins/metabolism , Opsonin Proteins/metabolism , Phagocytosis , Protein Binding , Sepsis/immunology , Sepsis/prevention & control , Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Survival AnalysisABSTRACT
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.
Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal/immunology , Pichia/genetics , Receptor, ErbB-2/immunology , Recombinant Proteins/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antibody Affinity/immunology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Area Under Curve , Binding, Competitive/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Drug Evaluation, Preclinical , Fucose/metabolism , Genetic Engineering , Humans , Macaca fascicularis , Mice , Mice, Inbred C57BL , Pichia/metabolism , Polysaccharides/metabolism , Protein Binding/immunology , Receptors, IgG/immunology , Receptors, IgG/metabolism , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/pharmacology , Trastuzumab , Xenograft Model Antitumor AssaysABSTRACT
N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemi-glycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease (â¼20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The decreased binding affinities to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.
Subject(s)
Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Immunoglobulin G/isolation & purification , Antibody-Dependent Cell Cytotoxicity , Calorimetry, Differential Scanning , Chromatography, Liquid , Glycosylation , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Mass Spectrometry , TemperatureABSTRACT
Protein phosphorylation is frequently used as an indicator of cellular signaling activity. Elevated phosphorylation of tyrosine kinase receptors plays an important role in cancer pathogenesis. However, phosphoproteins are usually poorly preserved in clinical tissue samples that are routinely fixed in 10% formalin. Nonetheless, in oncology clinical trials, use of phosphoproteins as biomarkers has been considered to be of great value in evaluating the effectiveness of a given drug candidate. Therefore, it is worthy of investigating whether alternative fixatives would improve the preservation of phosphoproteins in tissue. We compared the IHC staining of a number of phosphoproteins in xenograft and human surgical tumor tissues fixed in three different fixatives: 10% formalin, 4% paraformaldehyde (PFA), and Streck's tissue fixative (STF). We found that STF significantly enhanced the staining intensity of phosphoproteins compared with 10% formalin or 4% PFA. STF fixative also showed superiority of preservation of phosphoproteins in human surgical samples. Our results indicate that the choice of fixative could significantly affect the usability of clinical tissue samples for evaluating phosphoprotein by IHC.
Subject(s)
Biomarkers, Tumor/analysis , Fixatives , Neoplasms/chemistry , Phosphoproteins/analysis , Animals , Breast Neoplasms/chemistry , Colonic Neoplasms/chemistry , Data Interpretation, Statistical , Female , Formaldehyde , Humans , Immunohistochemistry , Lung Neoplasms/chemistry , Mice , Mice, Nude , Neoplasm Transplantation , Polymers , Transplantation, HeterologousABSTRACT
We describe the biological evaluation of isothiazoloquinolones (ITQs) having structural modifications at the 6-, 7-, and 8-positions. Addition of a methoxy substituent to C-8 effected an increase in antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and a decrease in cytotoxic activity against Hep2 cells. Removal of fluorine from C-6 or replacement of the C-8 carbon with a nitrogen compromised anti-MRSA activity. When the groups attached at C-7 were compared, the anti-MRSA activity decreased in the order 6-isoquinolinyl > 4-pyridinyl > 5-dihydroisoindolyl > 6-tetrahydroisoquinolinyl. The compound with the most desirable in vitro biological profile was 9-cyclopropyl-6-fluoro-8-methoxy-7-(2-methylpyridin-4-yl)-9H-isothiazolo[5,4-b]quinoline-3,4-dione (7g). This ITQ demonstrated (i) strong in vitro anti-MRSA activity (MIC90 = 0.5 microg/mL), (ii) strong inhibitory activities against S. aureus DNA gyrase and topoisomerase IV, with weak activity against human topoisomerase II, (iii) weak cytotoxic activities against three cell lines, and (iv) efficacy in an in vivo murine thigh model of infection employing MRSA.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Quinolones/chemical synthesis , Staphylococcus aureus/drug effects , Thiazoles/chemical synthesis , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Line, Tumor , Drug Resistance, Multiple, Bacterial , Female , Humans , Methicillin Resistance , Mice , Quinolones/chemistry , Quinolones/pharmacology , Staphylococcus aureus/enzymology , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Topoisomerase II InhibitorsABSTRACT
We synthesized a diverse series of 9H-isothiazolo[5,4-b]quinoline-3,4-diones containing heteroaromatic groups at the 7-position via palladium-catalyzed cross-coupling. Many of these compounds demonstrated potent antistaphylococcal activity (MICs 2 microg/mL) against a multi-drug-resistant strain (ATCC 700699) and low cytotoxic activity (CC(50)>100 microM) against the human cell line Hep2 (laryngeal carcinoma).
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Azo Compounds/pharmacology , Methicillin Resistance , Quinolones/pharmacology , Staphylococcus aureus/drug effects , Sulfhydryl Compounds/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/toxicity , Azo Compounds/chemical synthesis , Azo Compounds/chemistry , Azo Compounds/toxicity , Cell Line, Tumor , Cyclization , Fluorine/chemistry , Humans , Molecular Structure , Quinolones/chemical synthesis , Quinolones/chemistry , Quinolones/toxicity , Staphylococcus aureus/physiology , Structure-Activity RelationshipABSTRACT
This report describes 9H-isothiazolo[5,4-b]quinoline-3,4-diones (ITQs) containing aromatic groups at the 7-position that were prepared using palladium-catalyzed cross-coupling and tested against a panel of susceptible and resistant bacteria. In general, these compounds were more effective against Gram-positive than Gram-negative organisms. Many of the ITQs were more potent than contemporary quinolones and displayed a particularly strong antistaphylococcal activity against a clinically important, multi-drug-resistant strain. In contrast with ITQs reported previously, several of the analogues described in this Letter demonstrated low cytotoxic activity against a human cell line.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Azo Compounds/chemical synthesis , Azo Compounds/toxicity , Hydrocarbons, Aromatic/chemistry , Quinolines/chemistry , Quinolines/pharmacology , Sulfhydryl Compounds/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/toxicity , Azo Compounds/chemistry , Azo Compounds/pharmacology , Cell Line , Escherichia coli/drug effects , Escherichia coli/enzymology , Humans , Microbial Sensitivity Tests , Molecular Structure , Quinolines/chemical synthesis , Quinolines/toxicity , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair beta-myosin heavy chain (beta MyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized beta MyHC proximal promoter elements contribute to NWB regulation.
Subject(s)
Chloramphenicol O-Acetyltransferase/metabolism , Muscle, Skeletal/metabolism , Myosin Heavy Chains/genetics , Nuclear Proteins , Promoter Regions, Genetic/physiology , Ventricular Myosins/genetics , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/genetics , DNA/metabolism , DNA-Binding Proteins/metabolism , Humans , Mice , Mice, Transgenic , Muscle, Skeletal/anatomy & histology , Mutation/physiology , Myosin Heavy Chains/metabolism , Nucleotides/physiology , Organ Size/physiology , Rats , Species Specificity , TEA Domain Transcription Factors , Transcription Factors/metabolism , Ventricular Myosins/metabolism , Weight-Bearing/physiologyABSTRACT
Insulin has long been assigned a key role in the regulation of growth and metabolism during fetal life. Our prior observations indicated that hepatic insulin signaling is attenuated in the late-gestation fetal rat. Therefore, we studied the perinatal ontogeny of hepatic insulin signaling extending from phosphatidylinositol 3-kinase (PI3K) to the ribosome. Initial studies demonstrated markedly decreased insulin-mediated activation of ribosomal protein S6 kinase 1 (S6K1) in the fetus. We found a similar pattern in the regulation of Akt, a kinase upstream from S6K1. Insulin produced minimal activation of insulin receptor substrate (IRS)-1-associated PI3K activity in fetal liver. A modest IRS-2-associated response was seen in the fetus. However, levels of both IRS-1 and IRS-2 were very low in fetal liver relative to adult liver. IRS-1 content and insulin responsiveness of PI3K, Akt, and S6K1 showed a transition to the adult phenotype during the first several postnatal weeks. Examination of downstream insulin signaling to the translational apparatus showed marked attenuation, relative to the adult, of fetal hepatic insulin-mediated phosphorylation of 4E-BP1, the regulatory protein for the eukaryotic initiation factor eIF4E, and ribosomal protein S6. The mammalian target of rapamycin (mTOR), a key integrator of nutritional and metabolic regulation of translation, was present in low amounts, was hypophosphorylated, and was not insulin sensitive in the fetus. Our results indicate that protein synthesis during late-gestation liver development may be mTOR and insulin independent. Reexamination of the role of insulin in fetal liver physiology may be warranted.