Publications by authors named "Anna Aagaard"

22 Publications

  • Page 1 of 1

AZD0284, a Potent, Selective, and Orally Bioavailable Inverse Agonist of Retinoic Acid Receptor-Related Orphan Receptor C2.

J Med Chem 2021 09 31;64(18):13807-13829. Epub 2021 Aug 31.

Pharmaron Beijing Co., Ltd., Taihe Road BDA, Beijing 100176, P. R. China.

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 . Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (), which combined potent inhibition of IL-17A secretion from primary human T17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical studies, compound 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, was progressed to phase 1 clinical studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.1c01197DOI Listing
September 2021

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

ACS Med Chem Lett 2021 Feb 27;12(2):302-308. Epub 2021 Jan 27.

Structure Biophysics & Fragments, Discovery Sciences, R&D, AstraZeneca, Gothenburg 431 83, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.0c00563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883464PMC
February 2021

Improving metabolic stability and removing aldehyde oxidase liability in a 5-azaquinazoline series of IRAK4 inhibitors.

Bioorg Med Chem 2020 12 15;28(23):115815. Epub 2020 Oct 15.

Discovery Sciences, R&D, AstraZeneca, Gothenburg, SE-431 83 Mölndal, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115815DOI Listing
December 2020

Molecular tuning of farnesoid X receptor partial agonism.

Nat Commun 2019 07 2;10(1):2915. Epub 2019 Jul 2.

Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Goethe University, Frankfurt, 60438, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10853-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606567PMC
July 2019

Discovery of Potent and Orally Bioavailable Inverse Agonists of the Retinoic Acid Receptor-Related Orphan Receptor C2.

ACS Med Chem Lett 2019 Jun 29;10(6):972-977. Epub 2019 May 29.

Medicinal Chemistry, DMPK, andBioscience, Research and Early Development, Respiratory, Inflammation and Autoimmune, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, SE-43183 Mölndal, Sweden.

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 , 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 , which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.9b00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580541PMC
June 2019

Identification of Mineralocorticoid Receptor Modulators with Low Impact on Electrolyte Homeostasis but Maintained Organ Protection.

J Med Chem 2019 02 30;62(3):1385-1406. Epub 2019 Jan 30.

Pharmaron Beijing Co., Ltd. , No. 6 Taihe Road, BDA , Beijing 100176 , P. R. China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01523DOI Listing
February 2019

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design.

J Med Chem 2018 09 27;61(17):7796-7813. Epub 2018 Aug 27.

Pharmaron Beijing Company, Ltd. , Taihe Road , BDA, Beijing 100176 , PR China.

Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of T17-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-T17-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00783DOI Listing
September 2018

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion.

PLoS One 2018 23;13(2):e0193380. Epub 2018 Feb 23.

Cardiovascular, Renal and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Gothenburg, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193380PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825103PMC
June 2018

Structure and biophysical characterization of the human full-length neurturin-GFRa2 complex: A role for heparan sulfate in signaling.

J Biol Chem 2018 04 2;293(15):5492-5508. Epub 2018 Feb 2.

Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg 43183, Sweden,

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 models and ultimately to Parkinson's patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA117.000820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900780PMC
April 2018

The Structure of Murine N-Acetylspermine Oxidase Reveals Molecular Details of Vertebrate Polyamine Catabolism.

Biochemistry 2017 01 12;56(3):458-467. Epub 2017 Jan 12.

Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development, AstraZeneca , Pepparedsleden 1, SE-431 83 Mölndal, Sweden.

N-Acetylspermine oxidase (APAO) catalyzes the conversion of N-acetylspermine or N-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 N-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.6b01140DOI Listing
January 2017

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Sci Rep 2016 12 20;6:38644. Epub 2016 Dec 20.

MedImmune Ltd, Granta Park, Cambridge, CB21 6GH, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep38644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5171805PMC
December 2016

Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

ChemMedChem 2017 01 15;12(1):50-65. Epub 2016 Dec 15.

Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, 43183, Mölndal, Sweden.

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 pK =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 pK =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201600529DOI Listing
January 2017

Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary Constraints.

Structure 2015 Dec 22;23(12):2280-2290. Epub 2015 Oct 22.

Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2015.09.012DOI Listing
December 2015

Benzoxazepines Achieve Potent Suppression of IL-17 Release in Human T-Helper 17 (TH 17) Cells through an Induced-Fit Binding Mode to the Nuclear Receptor RORγ.

ChemMedChem 2016 Jan 10;11(2):207-16. Epub 2015 Nov 10.

Department of Medicinal Chemistry, AstraZeneca, Respiratory, Inflammation and Autoimmunity iMed, Pepparedsleden 1, 43183, Mölndal, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201500432DOI Listing
January 2016

Synthesis and structure-activity relationship of 4-(1,3-benzothiazol-2-yl)-thiophene-2-sulfonamides as cyclin-dependent kinase 5 (cdk5)/p25 inhibitors.

Bioorg Med Chem Lett 2012 Sep 25;22(18):5919-23. Epub 2012 Jul 25.

Medicinal Chemistry, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden.

4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described. The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the hinge region via a water molecule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2012.07.068DOI Listing
September 2012

Solutions for the storage and handling of SPINE standard pucks.

J Synchrotron Radiat 2012 Mar 28;19(Pt 2):288-9. Epub 2012 Jan 28.

Discovery Sciences, AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0909049511054689DOI Listing
March 2012

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system.

J Struct Funct Genomics 2005 ;6(1):13-20

Institute for Molecular Bioscience, Australia.

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10969-005-0462-7DOI Listing
February 2006

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1.

Structure 2005 Feb;13(2):309-17

Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2004.12.013DOI Listing
February 2005

Subunit III of cytochrome c oxidase of Rhodobacter sphaeroides is required to maintain rapid proton uptake through the D pathway at physiologic pH.

Biochemistry 2003 Jun;42(24):7400-9

Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.

The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi0341298DOI Listing
June 2003

Intramolecular proton-transfer reactions in a membrane-bound proton pump: the effect of pH on the peroxy to ferryl transition in cytochrome c oxidase.

Biochemistry 2003 Feb;42(6):1488-98

Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.

In the membrane-bound redox-driven proton pump cytochrome c oxidase, electron- and proton-transfer reactions must be coupled, which requires controlled modulation of the kinetic and/or thermodynamic properties of proton-transfer reactions through the membrane-spanning part of the protein. In this study we have investigated proton-transfer reactions through a pathway that is used for the transfer of both substrate and pumped protons in cytochrome c oxidase from Rhodobacter sphaeroides. Specifically, we focus on the formation of the so-called F intermediate, which is rate limited by an internal proton-transfer reaction from a possible branching point in the pathway, at a glutamic-acid residue (E(I-286)), to the binuclear center. We have also studied the reprotonation of E(I-286) from the bulk solution. Evaluation of the data in terms of a model presented in this work gives a rate of internal proton transfer from E(I-286) to the proton acceptor at the catalytic site of 1.1 x 10(4) s(-1). The apparent pK(a) of the donor (E(I-286)), determined from the pH dependence of the F-formation kinetics, was found to be 9.4, while the pK(a) of the proton acceptor at the catalytic site is likely to be > or = 2.5 pH units higher. In the pH range up to pH 10 the proton equilibrium between the bulk solution and E(I-286) was much faster than 10(4) s(-1), while in the pH range above pH 10 the proton uptake from solution is rate limiting for the overall reaction. The apparent second-order rate constant for proton transfer from the bulk solution to E(I-286) is >10(13) M(-1) s(-1), which indicates that the proton uptake is assisted by a local buffer consisting of protonatable residues at the protein surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi026524oDOI Listing
February 2003

Inhibition of proton transfer in cytochrome c oxidase by zinc ions: delayed proton uptake during oxygen reduction.

Biochim Biophys Acta 2002 Sep;1555(1-3):133-9

Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0005-2728(02)00268-2DOI Listing
September 2002

Relocation of an internal proton donor in cytochrome c oxidase results in an altered pK(a) and a non-integer pumping stoichiometry.

Biophys Chem 2002 Jul;98(1-2):105-14

Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0301-4622(02)00088-1DOI Listing
July 2002
-->