ABSTRACT
We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.
Subject(s)
NM23 Nucleoside Diphosphate Kinases/pharmacology , Stem Cells/drug effects , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Binding, Competitive , Biomarkers/metabolism , Cell Differentiation , Cells, Cultured , Culture Media, Conditioned/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Ligands , MicroRNAs/genetics , MicroRNAs/metabolism , Mucin-1/immunology , Mucin-1/metabolism , NM23 Nucleoside Diphosphate Kinases/chemistry , NM23 Nucleoside Diphosphate Kinases/metabolism , Protein Binding/drug effects , Protein Multimerization , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
A novel series of 4-aminophenylalanine and 4-aminocyclohexylalanine derivatives were designed and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4). The phenylalanine series afforded compounds such as 10 that were potent and selective (DPP-4, IC(50)=28nM), but exhibited limited oral bioavailability. The corresponding cyclohexylalanine derivatives such as 25 afforded improved PK exposure and efficacy in a murine OGTT experiment. The X-ray crystal structure of 25 bound to the DPP-4 active site is presented.
Subject(s)
Alanine/analogs & derivatives , Cyclohexanes/pharmacology , Dipeptidyl-Peptidase IV Inhibitors , Enzyme Inhibitors/pharmacology , Phenylalanine/analogs & derivatives , Administration, Oral , Alanine/chemistry , Alanine/pharmacology , Animals , Binding Sites , Biological Availability , Crystallography, X-Ray , Cyclohexanes/chemistry , Dipeptidyl Peptidase 4/chemistry , Enzyme Inhibitors/chemistry , Glucose Tolerance Test , Mice , Models, Molecular , Molecular Structure , Phenylalanine/chemistry , Phenylalanine/pharmacology , Structure-Activity RelationshipABSTRACT
The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.
Subject(s)
Liver/metabolism , Receptors, Glucagon/antagonists & inhibitors , Animals , CHO Cells , Carbon Radioisotopes , Cricetinae , Humans , Liver Glycogen/biosynthesis , Magnetic Resonance Spectroscopy/methods , Male , Mice , Mice, Transgenic , Molecular Structure , Pyruvic Acid/metabolism , Receptors, Glucagon/metabolism , Time FactorsABSTRACT
A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM) and moderate bioavailability (36% in mice).
Subject(s)
Receptors, Glucagon/antagonists & inhibitors , Thiophenes/chemical synthesis , Thiophenes/pharmacology , Humans , Molecular Structure , Structure-Activity Relationship , Thiophenes/classificationABSTRACT
HIV-1 protease inhibitors (PI) with an N-arylpyrrole moiety in the P(3) position afforded excellent antiviral potency and substantially improved aqueous solubility over previously reported variants. The rapid in vitro clearance of these compounds in human liver microsomes prompted oral coadministration with indinavir to hinder their metabolism by the cyctochrome P450 3A4 isozyme and allow for in vivo PK assessment.
Subject(s)
HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/drug effects , Indinavir/therapeutic use , Acquired Immunodeficiency Syndrome/drug therapy , Administration, Oral , Biotransformation , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Drug Design , Drug Resistance, Viral , Drug Therapy, Combination , HIV Protease Inhibitors/pharmacokinetics , Humans , Indinavir/pharmacology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Structure-Activity RelationshipABSTRACT
Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.
Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease/metabolism , HIV-1/drug effects , HIV-1/enzymology , Animals , Dogs , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/physiology , HIV Protease Inhibitors/administration & dosage , HumansABSTRACT
Substitution of the t-butylcarboxamide substituent in analogues of the HIV protease inhibitor (PI) Indinavir with a trifluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl substituent also affords a slower clearance rate in vivo (dogs); however, this may be due to more potent inhibition of at least two P450 isoforms.