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1.
Bioorg Med Chem ; 63: 116729, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35439688

ABSTRACT

In this article, we report the discovery of a series of pyrimidopyridones as inhibitors of IRAK4 kinase. From a previously disclosed 5-azaquinazoline series, we found that switching the pyridine ring for an N-substituted pyridone gave a novel hinge binding scaffold which retained potency against IRAK4. Importantly, introduction of the carbonyl established an internal hydrogen bond with the 4-NH, establishing a conformational lock and allowing truncation of the large basic substituent to a 1-methylcyclopyl group. Subsequent optimisation, facilitated by X-ray crystal structures, allowed identification of preferred substituents at both the pyridone core and pyrazole. Subsequent combinations of optimal groups allowed control of lipophilicity and identification of potent and selective inhibitors of IRAK4 with better in vitro permeability and lower clearance.


Subject(s)
Interleukin-1 Receptor-Associated Kinases , Pyridones , Molecular Conformation , Pyridones/pharmacology , Structure-Activity Relationship
2.
Bioorg Med Chem ; 28(23): 115815, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33091850

ABSTRACT

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.


Subject(s)
Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/metabolism , Quinazolines/chemistry , Aldehyde Oxidase/metabolism , Animals , Binding Sites , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Dogs , Drug Stability , Half-Life , Hepatocytes/metabolism , Humans , Interleukin-1 Receptor-Associated Kinases/metabolism , Mice , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinazolines/metabolism , Quinazolines/pharmacology , Rats , Structure-Activity Relationship
3.
J Biol Chem ; 293(15): 5492-5508, 2018 04 13.
Article in English | MEDLINE | ID: mdl-29414779

ABSTRACT

Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2, and the resulting NRTN-GFRa2 complex activates the transmembrane receptors rearranged during transfection (RET) or the neural cell adhesion molecule (NCAM). We report the crystal structure of NRTN, alone and in complex with GFRa2. This is the first crystal structure of a GFRa with all three domains and shows that domain 1 does not interact directly with NRTN, but it may support an interaction with RET and/or NCAM, via a highly conserved surface. In addition, biophysical results show that the relative concentration of GFRa2 on cell surfaces can affect the functional affinity of NRTN through avidity effects. We have identified a heparan sulfate-binding site on NRTN and a putative binding site in GFRa2, suggesting that heparan sulfate has a role in the assembly of the signaling complex. We further show that mutant NRTN with reduced affinity for heparan sulfate may provide a route forward for delivery of NRTN with increased exposure in preclinical in vivo models and ultimately to Parkinson's patients.


Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/chemistry , Heparitin Sulfate/chemistry , Multiprotein Complexes/chemistry , Neurturin/chemistry , Signal Transduction , Crystallography, X-Ray , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Heparitin Sulfate/metabolism , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Neurturin/genetics , Neurturin/metabolism , Protein Domains , Protein Structure, Quaternary
4.
Biochemistry ; 56(3): 458-467, 2017 01 24.
Article in English | MEDLINE | ID: mdl-28029774

ABSTRACT

N1-Acetylspermine oxidase (APAO) catalyzes the conversion of N1-acetylspermine or N1-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N1-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.


Subject(s)
Oxidoreductases/chemistry , Putrescine/chemistry , Spermidine/analogs & derivatives , Spermidine/chemistry , Spermine/analogs & derivatives , Aldehydes/chemistry , Aldehydes/metabolism , Animals , Catalytic Domain , Gene Expression , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Kinetics , Mice , Models, Molecular , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Propylamines/chemistry , Propylamines/metabolism , Protein Structure, Secondary , Putrescine/analogs & derivatives , Putrescine/metabolism , Spermidine/metabolism , Spermine/chemistry , Spermine/metabolism
5.
J Synchrotron Radiat ; 19(Pt 2): 288-9, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22338692

ABSTRACT

Currently there is no rack system for the long-term storage of SPINE pucks in spite of their commercial availability and heavy usage at the ESRF. The only way to store pucks is in transport dewar canisters which presents a number of limitations and drawbacks. Here a simple affordable rack for storing SPINE pucks is described, which we believe is accessible to not only synchrotrons but also both academic and industrial research laboratories.


Subject(s)
Equipment Design , Synchrotrons/instrumentation , Automation, Laboratory/instrumentation , Cold Temperature , Crystallography, X-Ray/instrumentation , Nitrogen
7.
ACS Med Chem Lett ; 12(2): 302-308, 2021 Feb 11.
Article in English | MEDLINE | ID: mdl-33603979

ABSTRACT

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

8.
J Med Chem ; 64(18): 13807-13829, 2021 09 23.
Article in English | MEDLINE | ID: mdl-34464130

ABSTRACT

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Inflammation/drug therapy , Isoindoles/therapeutic use , Orphan Nuclear Receptors/agonists , Sulfones/therapeutic use , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Dogs , Drug Inverse Agonism , Female , Humans , Imiquimod , Inflammation/chemically induced , Isoindoles/cerebrospinal fluid , Isoindoles/chemical synthesis , Isoindoles/pharmacokinetics , Male , Mice, Inbred C57BL , Molecular Structure , Rats, Wistar , Structure-Activity Relationship , Sulfones/cerebrospinal fluid , Sulfones/chemical synthesis , Sulfones/pharmacokinetics , Th17 Cells , Thymocytes/drug effects
9.
Nat Commun ; 10(1): 2915, 2019 07 02.
Article in English | MEDLINE | ID: mdl-31266946

ABSTRACT

The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding.


Subject(s)
Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Animals , Cell Line , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , Humans , Hydrogen Bonding , Ligands , Liver/metabolism , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred C57BL , Protein Binding , Protein Conformation, alpha-Helical , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism
10.
ACS Med Chem Lett ; 10(6): 972-977, 2019 Jun 13.
Article in English | MEDLINE | ID: mdl-31223457

ABSTRACT

The further optimization of a recently disclosed series of inverse agonists of the nuclear receptor RORC2 is described. Investigations into the left-hand side of compound 1, guided by X-ray crystal structures, led to the substitution of the 4-aryl-thiophenyl residue with the hexafluoro-2-phenyl-propan-2-ol moiety. This change resulted in to compound 28, which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement in vivo was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.

11.
J Med Chem ; 62(3): 1385-1406, 2019 02 14.
Article in English | MEDLINE | ID: mdl-30596500

ABSTRACT

The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.


Subject(s)
Homeostasis/drug effects , Mineralocorticoid Receptor Antagonists/pharmacology , Oxazines/pharmacology , Potassium/metabolism , Protective Agents/pharmacology , Sodium/metabolism , Animals , Heart/drug effects , Humans , Kidney/drug effects , Male , Mineralocorticoid Receptor Antagonists/chemical synthesis , Mineralocorticoid Receptor Antagonists/metabolism , Molecular Structure , Oxazines/chemical synthesis , Oxazines/metabolism , Potassium/urine , Protective Agents/chemical synthesis , Protective Agents/metabolism , Rats, Sprague-Dawley , Rats, Wistar , Receptors, Mineralocorticoid/metabolism , Renal Insufficiency, Chronic/drug therapy , Sodium/urine , Structure-Activity Relationship
12.
PLoS One ; 13(2): e0193380, 2018.
Article in English | MEDLINE | ID: mdl-29474466

ABSTRACT

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.


Subject(s)
Benzoates/pharmacology , Mineralocorticoid Receptor Antagonists/pharmacology , Oxazines/pharmacology , Administration, Oral , Aldosterone , Animals , Benzoates/chemistry , Benzoates/pharmacokinetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Eplerenone , Humans , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Male , Mice, Mutant Strains , Mineralocorticoid Receptor Antagonists/chemistry , Mineralocorticoid Receptor Antagonists/pharmacokinetics , Molecular Structure , Oxazines/chemistry , Oxazines/pharmacokinetics , Potassium/urine , Rats, Sprague-Dawley , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathology , Sodium/urine , Sodium, Dietary , Spironolactone/analogs & derivatives , Spironolactone/chemistry , Spironolactone/pharmacokinetics , Spironolactone/pharmacology
13.
J Med Chem ; 61(17): 7796-7813, 2018 09 13.
Article in English | MEDLINE | ID: mdl-30095900

ABSTRACT

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a small-molecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.


Subject(s)
Acetamides/pharmacology , Drug Design , Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Protein Conformation , Th17 Cells/drug effects , Th17 Cells/metabolism , Acetamides/administration & dosage , Acetamides/chemistry , Acetamides/pharmacokinetics , Administration, Oral , Animals , Binding Sites , Biological Availability , Cells, Cultured , Crystallography, X-Ray , Humans , Interleukin-17/metabolism , Models, Molecular , Molecular Structure , Nuclear Receptor Subfamily 1, Group F, Member 3/chemistry , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Protein Binding , Rodentia , Structure-Activity Relationship , Th17 Cells/immunology , Tissue Distribution
14.
Structure ; 13(2): 309-17, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15698574

ABSTRACT

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.


Subject(s)
Antigens/chemistry , Carboxypeptidases/antagonists & inhibitors , Cystatins/chemistry , Nerve Tissue Proteins/chemistry , Protease Inhibitors/chemistry , Tumor Suppressor Proteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Antigens/physiology , Crystallography, X-Ray , Humans , Inflammation/metabolism , Macrophages/metabolism , Membrane Proteins , Mice , Molecular Sequence Data , Protein Folding , Rats
15.
ChemMedChem ; 12(1): 50-65, 2017 01 05.
Article in English | MEDLINE | ID: mdl-27897427

ABSTRACT

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.


Subject(s)
Drug Design , Mineralocorticoid Receptor Antagonists/chemistry , Mineralocorticoid Receptor Antagonists/pharmacology , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/metabolism , Dose-Response Relationship, Drug , High-Throughput Screening Assays , Humans , Mineralocorticoid Receptor Antagonists/chemical synthesis , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Substrate Specificity
16.
ChemMedChem ; 11(2): 207-16, 2016 Jan 19.
Article in English | MEDLINE | ID: mdl-26553345

ABSTRACT

RORγt, an isoform of the retinoic acid-related orphan receptor gamma (RORc, RORγ), has been identified as the master regulator of T-helper 17 (TH 17) cell function and development, making it an attractive target for the treatment of autoimmune diseases. Validation for this target comes from antibodies targeting interleukin-17 (IL-17), the signature cytokine produced by TH 17 cells, which have shown impressive results in clinical trials. Through focused screening of our compound collection, we identified a series of N-sulfonylated benzoxazepines, which displayed micromolar affinity for the RORγ ligand-binding domain (LBD) in a radioligand binding assay. Optimization of these initial hits resulted in potent binders, which dose-dependently decreased the ability of the RORγ-LBD to interact with a peptide derived from steroid receptor coactivator 1, and inhibited the release of IL-17 secretion from isolated and cultured human TH 17 cells with nanomolar potency. A cocrystal structure of inverse agonist 15 (2-chloro-6-fluoro-N-(4-{[3-(trifluoromethyl)phenyl]sulfonyl}-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-yl)benzamide) bound to the RORγ-LBD illustrated that both hydrophobic interactions, leading to an induced fit around the substituted benzamide moiety of 15, as well as a hydrogen bond from the amide NH to His479 seemed to be important for the mechanism of action. This structure is compared with the structure of agonist 25 (N-(2-fluorophenyl)-4-[(4-fluorophenyl)sulfonyl]-2,3,4,5-tetrahydro-1,4-benzoxazepin-6-amine ) and structures of other known RORγ modulators.


Subject(s)
Interleukin-17/metabolism , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Oxazepines/pharmacology , Th17 Cells/drug effects , Th17 Cells/metabolism , Binding Sites/drug effects , Dose-Response Relationship, Drug , Humans , Interleukin-17/immunology , Models, Molecular , Molecular Structure , Oxazepines/chemical synthesis , Oxazepines/chemistry , Protein Binding/drug effects , Structure-Activity Relationship , Th17 Cells/immunology
17.
Sci Rep ; 6: 38644, 2016 12 20.
Article in English | MEDLINE | ID: mdl-27995962

ABSTRACT

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Protein Engineering/methods , Animals , Antibodies, Monoclonal/pharmacokinetics , Biophysical Phenomena , Chromatography, Gel , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Hydrogen , Mice , Mutation/genetics , Organ Specificity , Protein Binding , Protein Conformation , Protein Interaction Mapping , Protein Multimerization , Rats , Spectrometry, Mass, Electrospray Ionization , Surface Properties , Viscosity
18.
Biochim Biophys Acta ; 1555(1-3): 133-9, 2002 Sep 10.
Article in English | MEDLINE | ID: mdl-12206905

ABSTRACT

We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn(2+) the transition from the "peroxy" (P(R)) to the "ferryl" (F) intermediate has a time constant of approximately 100 micros and it is associated with proton transfer from the bulk solution with an intrinsic time constant of <<100 micros, but rate limited by the P(R)-->F transition. While in the presence of 100 microM Zn(2+) the P(R)-->F transition was slowed by a factor of approximately 2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn(2+)) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of approximately 2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn(2+) was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn(2+) was observed on the F-->O transition rate when measured in D(2)O, because in D(2)O, the transition is internally slowed to approximately 10 ms, which is already slower than with bound Zn(2+). Together with earlier results showing that both the P(R)-->F and F-->O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn(2+) binds to and blocks the entrance of the D-pathway.


Subject(s)
Electron Transport Complex IV/chemistry , Oxygen/chemistry , Protons , Zinc/chemistry , Binding Sites , Cations, Divalent , Deuterium Oxide , Electron Transport Complex IV/genetics , Models, Molecular , Mutation , Oxidation-Reduction
19.
Structure ; 23(12): 2280-2290, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26602186

ABSTRACT

Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.


Subject(s)
Conserved Sequence , Receptors, Glucocorticoid/chemistry , Receptors, Mineralocorticoid/chemistry , Amino Acid Sequence , Binding Sites , Evolution, Molecular , Humans , Molecular Sequence Data , Protein Binding , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism
20.
Biophys Chem ; 98(1-2): 105-14, 2002 Jul 10.
Article in English | MEDLINE | ID: mdl-12128193

ABSTRACT

Cytochrome c oxidase from Rhodobacter sphaeroides has two proton-input pathways leading from the protein surface towards the catalytic site, located within the membrane-spanning part of the enzyme. One of these pathways, the D-pathway, contains a highly conserved Glu residue [E(I-286)], which plays an important role in proton transfer through the pathway. In a recent study, we showed that a mutant enzyme in which E(I-286) was re-located to the opposite side of the D-pathway [EA(I-286)/IE(I-112) double mutant enzyme] was able to pump protons, although with a stoichiometry that was lower than that of the wild-type enzyme (approximately 0.6 H(+)/e(-)) (Aagaard et al. (2000) Biochemistry 39, 15847-15850). These results showed that the residue must not necessarily be located at a specific place in the amino-acid sequence, but rather at a specific location in space. In this study, we have investigated the effect of moving E(I-286) on the kinetics of specific reaction steps of the catalytic cycle in the pH range 6-11. Our results show that during the reaction of the four-electron reduced enzyme with O(2), the rates of the two first transitions (up to formation of the 'peroxy' intermediate, P(r)) are the same for the double mutant as for the wild-type enzyme, but formation of the oxo-ferryl (F) and fully oxidized (O) states, associated with proton uptake from the bulk solution, are slowed by factors of approximately 30 and approximately 400, respectively. Thus, in spite of the dramatically reduced transition rates, the proton-pumping stoichiometry is reduced only by approximately 40%. The apparent pK(a) values in the pH-dependencies of the rates of the P(R)-->F and F-->O transitions were >3 and approximately 2 units lower than those of the corresponding transitions in the wild-type enzyme, respectively. The relation between the modified pK(a)s, the transition rates between oxygen intermediates and the pumping stoichiometry is discussed.


Subject(s)
Electron Transport Complex IV/chemistry , Rhodobacter sphaeroides/enzymology , Binding Sites , Catalysis , Electron Transport , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Mutation , Oxidation-Reduction , Oxidoreductases/metabolism , Oxygen/chemistry , Oxygen/metabolism , Potentiometry , Protons , Spectrophotometry
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