ABSTRACT
Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7â Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.
Subject(s)
Catalytic Domain , Protein-Arginine Deiminase Type 6 , Humans , Crystallography, X-Ray , Protein-Arginine Deiminase Type 6/metabolism , Protein-Arginine Deiminases/metabolism , Protein-Arginine Deiminases/chemistry , Protein-Arginine Deiminases/genetics , Protein Conformation , Hydrolases/chemistry , Hydrolases/metabolism , Models, Molecular , Calcium/metabolismABSTRACT
Molecular docking studies of quinoline and 2-chloroquinoline substrates at the active site of toluene dioxygenase (TDO), were conducted using Autodock Vina, to identify novel edge-to-face interactions and to rationalize the observed stereoselective cis-dihydroxylation of carbocyclic rings and formation of isolable cis-dihydrodiol metabolites. These in silico docking results of quinoline and pyridine substrates, with TDO, also provided support for the postulated cis-dihydroxylation of electron-deficient pyridyl rings, to give transient cis-dihydrodiol intermediates and the derived hydroxyquinolines. 2-Chloroquinoline cis-dihydrodiol metabolites were used as precursors in the chemoenzymatic synthesis of enantiopure arene oxide and arene dioxide derivatives of quinoline, in the context of its possible mammalian metabolism and carcinogenicity.
ABSTRACT
We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).
Subject(s)
Heterocyclic Compounds, 4 or More Rings/pharmacology , Heterocyclic Compounds, Bridged-Ring/pharmacology , Isoxazoles/pharmacology , Oxazepines/pharmacology , Oxazoles/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Active Transport, Cell Nucleus , Animals , Binding Sites , Cell Nucleus/metabolism , Dogs , HEK293 Cells , HeLa Cells , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, Bridged-Ring/chemical synthesis , Heterocyclic Compounds, Bridged-Ring/chemistry , Humans , Imidazoles/pharmacology , Isoxazoles/chemical synthesis , Isoxazoles/chemistry , Mice , NF-kappa B p50 Subunit/metabolism , NF-kappa B p52 Subunit/metabolism , Oxazepines/chemical synthesis , Oxazepines/chemistry , Oxazoles/chemical synthesis , Oxazoles/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Signal Transduction/drug effects , NF-kappaB-Inducing KinaseABSTRACT
The synthesis, structure-activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of compound 69 (BI 665915) that demonstrated an excellent cross-species drug metabolism and pharmacokinetics (DMPK) profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug-drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose-exposure relationship and a dose-dependent inhibition of LTB4 production.
Subject(s)
Acetamides/pharmacology , Arachidonate 5-Lipoxygenase/metabolism , Drug Discovery , Lipoxygenase Inhibitors/pharmacology , Oxadiazoles/pharmacology , Acetamides/chemical synthesis , Acetamides/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Lipoxygenase Inhibitors/chemical synthesis , Lipoxygenase Inhibitors/chemistry , Models, Molecular , Molecular Conformation , Oxadiazoles/chemical synthesis , Oxadiazoles/chemistry , Structure-Activity RelationshipABSTRACT
A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure-activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.
Subject(s)
Pyrimidines/chemistry , Receptors, Metabotropic Glutamate/chemistry , Styrenes/chemistry , Administration, Oral , Allosteric Regulation , Animals , Catalepsy/chemically induced , Catalepsy/drug therapy , High-Throughput Screening Assays , Humans , Models, Animal , Motor Activity/physiology , Pyrimidines/chemical synthesis , Pyrimidines/therapeutic use , Rats , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/metabolism , Structure-Activity Relationship , Styrenes/chemical synthesis , Styrenes/therapeutic useABSTRACT
We have been exploring the potential of 5-HT(2B) antagonists as a therapy for chronic heart failure. To assess the potential of this therapeutic approach, we sought compounds possessing the following attributes: (a) potent and selective antagonism of the 5-HT(2B) receptor, (b) low impact of serum proteins on potency, and (c) desirable pharmacokinetic properties. This Letter describes our investigation of a biphenyl benzimidazole class of compounds that resulted in 5-HT(2B) antagonists possessing the above attributes. Improving potency in a human serum albumin shift assay proved to be the most significant SAR discovery.
Subject(s)
Receptor, Serotonin, 5-HT2B/metabolism , Serotonin 5-HT2 Receptor Antagonists , Serotonin Antagonists/chemistry , Serotonin Antagonists/pharmacokinetics , Animals , Binding Sites , Male , Quantitative Structure-Activity Relationship , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptor, Serotonin, 5-HT2B/chemistry , Serotonin Antagonists/classificationABSTRACT
A range of seventeen quinoline alkaloids, involving several types of oxidations during their biosynthetic pathways, have been isolated from leaves of Choisya ternata. In addition to the nine known quinoline alkaloids, eight new members of the furoquinoline family, derived mainly from prenylation at C-5 (including two novel hydroperoxides), have been identified. The absolute configurations and enantiopurity values of all chiral quinoline alkaloids have been determined. One of the isolated alkaloids, 7-isopentenyloxy-gamma-fagarine, has been used as a precursor for the chemical asymmetric synthesis of the enantiopure alkaloids: evoxine, anhydroevoxine and evodine. The possible roles of oxygenase and other oxygen-atom-transfer enzymes, in the biosynthetic pathways of the C. ternata alkaloids, have been discussed.
Subject(s)
Alkaloids/chemistry , Alkaloids/chemical synthesis , Rutaceae/chemistry , Alkaloids/isolation & purification , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , StereoisomerismABSTRACT
Biotransformation of the parent furoquinoline alkaloid dictamnine and its 4-chlorofuroquinoline precursor, using the B8/36 bacterial mutant strain of Sphingomonas yanoikuyae, yielded, via biphenyl dioxygenase-catalysed dihydroxylation, the first isolable alkaloid cis-dihydrodiol metabolites; these metabolites were used in the chemoenzymatic synthesis of postulated arene oxide and phenol intermediates, and a range of derived furoquinoline alkaloids.
Subject(s)
Alkaloids/metabolism , Naphthalenes/metabolism , Oxides/metabolism , Phenols/metabolism , Quinolines/chemistry , Alkaloids/biosynthesis , Alkaloids/chemistry , Quinolines/metabolismABSTRACT
A combination method of ozonolysis and chiral stationary phase (CSP)-GC-MS analysis has been developed to determine the enantiopurity values and absolute configurations of a range of alkaloid and coumarin hemiterpenoids derived from C- and O-prenyl epoxides.