Publications by authors named "Norbert Sträter"

79 Publications

Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

J Med Chem 2021 01 5;64(1):845-860. Epub 2021 Jan 5.

Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, California 94545, United States.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A and A). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of , which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01835DOI Listing
January 2021

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis.

Nat Commun 2020 08 25;11(1):4243. Epub 2020 Aug 25.

Department of Internal Medicine, Division of Rheumatology, Leipzig University, Liebigstraße 19, 04103, Leipzig, Germany.

Increased extracellular Ca concentrations ([Ca]) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca]. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1β release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1β release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.
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http://dx.doi.org/10.1038/s41467-020-17749-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447633PMC
August 2020

Discovery of AB680: A Potent and Selective Inhibitor of CD73.

J Med Chem 2020 10 20;63(20):11448-11468. Epub 2020 Jul 20.

Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, California 94545, United States.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent ( = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00525DOI Listing
October 2020

Structural Studies on the Inhibitory Binding Mode of Aromatic Coumarinic Esters to Human Kallikrein-Related Peptidase 7.

J Med Chem 2020 06 21;63(11):5723-5733. Epub 2020 May 21.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01806DOI Listing
June 2020

Membrane Phospholipids and Polyphosphates as Cofactors and Binding Molecules of SERPINA12 (vaspin).

Molecules 2020 Apr 24;25(8). Epub 2020 Apr 24.

Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, 04103 Leipzig, Germany.

Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (K: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in β-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.
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http://dx.doi.org/10.3390/molecules25081992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221550PMC
April 2020

Ribosomal Target-Binding Sites of Antimicrobial Peptides Api137 and Onc112 Are Conserved among Pathogens Indicating New Lead Structures To Develop Novel Broad-Spectrum Antibiotics.

Chembiochem 2020 Sep 30;21(18):2628-2634. Epub 2020 Jun 30.

Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Deutscher Platz 5, 04103, Leipzig, Germany.

Proline-rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter-based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high-priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (K ) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad-spectrum antibiotics. Api137 bound weaker with K values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the K values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off-target binding.
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http://dx.doi.org/10.1002/cbic.202000109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540576PMC
September 2020

Discovery of Potent and Selective Non-Nucleotide Small Molecule Inhibitors of CD73.

J Med Chem 2020 04 8;63(8):3935-3955. Epub 2020 Apr 8.

Arcus Biosciences, Inc., 3928 Point Eden Way, Hayward, California 94545, United States.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2-indazol-6-yl)-1-1,2,3-benzotriazol-6-yl]-1-pyrazol-1-yl}methyl)benzonitrile (, IC = 12 nM) and 4-({5-[4-chloro-1-(2-indazol-6-yl)-1-1,2,3-benzotriazol-6-yl]-1-pyrazol-1-yl}methyl)benzonitrile (, IC = 19 nM). Cocrystallization of with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01713DOI Listing
April 2020

2-Substituted α,β-Methylene-ADP Derivatives: Potent Competitive Ecto-5'-nucleotidase (CD73) Inhibitors with Variable Binding Modes.

J Med Chem 2020 03 28;63(6):2941-2957. Epub 2020 Feb 28.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, D-04103 Leipzig, Germany.

CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure-activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,β-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo- and 2-chloro-adenosine-5'--[(phosphonomethyl)phosphonic acid] (, ) being the most potent inhibitors with values toward human CD73 of 3-6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP () was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky -substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01611DOI Listing
March 2020

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle.

J Mol Biol 2019 07 28;431(15):2747-2761. Epub 2019 May 28.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany. Electronic address:

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.
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http://dx.doi.org/10.1016/j.jmb.2019.05.027DOI Listing
July 2019

Crystallization of ectonucleotide phosphodiesterase/pyrophosphatase-3 and orientation of the SMB domains in the full-length ectodomain.

Acta Crystallogr F Struct Biol Commun 2018 Nov 16;74(Pt 11):696-703. Epub 2018 Oct 16.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.

Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI cells, purified using two chromatographic steps and crystallized. Its structure at 2.77 Å resolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not interact with the PDE domain but extends further away from the PDE domain. Deletion of the SMB domains resulted in crystals that diffracted to 2.4 Å resolution and are suitable for substrate-binding studies.
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http://dx.doi.org/10.1107/S2053230X18011111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213977PMC
November 2018

Crystal structure and substrate binding mode of ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3).

Sci Rep 2018 07 18;8(1):10874. Epub 2018 Jul 18.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, Leipzig, 04103, Germany.

Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3) is a membrane-bound glycoprotein that regulates extracellular levels of nucleotides. NPP3 is known to contribute to the immune response on basophils by hydrolyzing ATP and to regulate the glycosyltransferase activity in Neuro2a cells. Here, we report on crystal structures of the nuclease and phosphodiesterase domains of rat NPP3 in complex with different substrates, products and substrate analogs giving insight into details of the catalytic mechanism. Complex structures with a phosphate ion, the product AMP and the substrate analog AMPNPP provide a consistent picture of the coordination of the substrate in which one zinc ion activates the threonine nucleophile whereas the other zinc ion binds the phosphate group. Co-crystal structures with the dinucleotide substrates Ap4A and UDPGlcNAc reveal a binding pocket for the larger leaving groups of these substrates. The crystal structures as well as mutational and kinetic analysis demonstrate that the larger leaving groups interact only weakly with the enzyme such that the substrate affinity is dominated by the interactions of the first nucleoside group. For this moiety, the nucleobase is stacked between Y290 and F207 and polar interactions with the protein are only formed via water molecules thus explaining the limited nucleobase selectivity.
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http://dx.doi.org/10.1038/s41598-018-28814-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052110PMC
July 2018

Kallikrein-related peptidase 14 is the second KLK protease targeted by the serpin vaspin.

Biol Chem 2018 09;399(9):1079-1084

Institute of Biochemistry, Faculty of Life Sciences, Leipzig University, Brüderstrasse 34, D-04103 Leipzig, Germany.

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multi-specific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.
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http://dx.doi.org/10.1515/hsz-2018-0108DOI Listing
September 2018

Glycosylation of human vaspin (SERPINA12) and its impact on serpin activity, heparin binding and thermal stability.

Biochim Biophys Acta Proteins Proteom 2017 Sep 29;1865(9):1188-1194. Epub 2017 Jun 29.

Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, 04103 Leipzig, Germany. Electronic address:

Vaspin is a glycoprotein with three predicted glycosylation sites at asparagine residues located in proximity to the reactive center loop and close to domains that play important roles in conformational changes underlying serpin function. In this study, we have investigated the glycosylation of human vaspin and its effects on biochemical properties relevant to vaspin function. We show that vaspin is modified at all three sites and biochemical data demonstrate that glycosylation does not hinder inhibition of the target protease kallikrein 7. Although binding affinity to heparin is slightly decreased, the protease inhibition reaction is still significantly accelerated in the presence of heparin. Glycosylation did not affect thermal stability.
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http://dx.doi.org/10.1016/j.bbapap.2017.06.020DOI Listing
September 2017

Amylose recognition and ring-size determination of amylomaltase.

Sci Adv 2017 Jan 13;3(1):e1601386. Epub 2017 Jan 13.

Institut für Bioanalytische Chemie, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

Starch is a major carbon and energy source throughout all kingdoms of life. It consists of two carbohydrate polymers, branched amylopectin and linear amylose, which are sparingly soluble in water. Hence, the enzymatic breakdown by glycoside hydrolases (GHs) is of great biological and societal importance. Amylomaltases (AMs) are GHs specialized in the hydrolysis of α-1,4-linked sugar chains such as amylose. They are able to catalyze an intramolecular transglycosylation of a bound sugar chain yielding polymeric sugar rings, the cycloamyloses (CAs), consisting of 20 to 100 glucose units. Despite a wealth of data on short oligosaccharide binding to GHs, no structural evidence is available for their interaction with polymeric substrates that better represent the natural polysaccharide. We have determined the crystal structure of AM in complex with a 34-meric CA-one of the largest carbohydrates resolved by x-ray crystallography and a mimic of the natural polymeric amylose substrate. In total, 15 glucose residues interact with the protein in an extended crevice with a length of more than 40 Å. A modified succinimide, derived from aspartate, mediates protein-sugar interactions, suggesting a biological role for this nonstandard amino acid. The structure, together with functional assays, provides unique insights into the interaction of GHs with their polymeric substrate and reveals a molecular ruler mechanism for minimal ring-size determination of CA products.
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http://dx.doi.org/10.1126/sciadv.1601386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235332PMC
January 2017

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).

J Biol Chem 2017 01 9;292(3):994-1004. Epub 2016 Dec 9.

From the Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology,

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (K = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.
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http://dx.doi.org/10.1074/jbc.M116.748020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247670PMC
January 2017

Understanding the Structural Basis of Adhesion GPCR Functions.

Handb Exp Pharmacol 2016 ;234:67-82

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Unlike conventional G-protein-coupled receptors (GPCRs), adhesion GPCRs (aGPCRs) have large extracellular regions that are autoproteolytically cleaved from their membrane-embedded seven-pass transmembrane helices. Autoproteolysis occurs within the conserved GPCR-Autoproteolysis INducing (GAIN) domain that is juxtaposed to the transmembrane domain and cleaves the last beta strand of the GAIN domain. The other domains of the extracellular region are variable and specific to each aGPCR and are likely involved in adhering to various ligands. Emerging evidence suggest that extracellular regions may modulate receptor function and that ligand binding to the extracellular regions may induce receptor activation via multiple mechanisms. Here, we summarize current knowledge about the structural understanding for the extracellular regions of aGPCRs and discuss their possible functional roles that emerge from the available structural information.
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http://dx.doi.org/10.1007/978-3-319-41523-9_4DOI Listing
June 2017

Regulatory Function of Hexokinase 2 in Glucose Signaling in Saccharomyces cerevisiae.

J Biol Chem 2016 08;291(32):16477

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, 04103 Leipzig, Germany.

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http://dx.doi.org/10.1074/jbc.L116.735514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974362PMC
August 2016

Proline-rich Antimicrobial Peptides Optimized for Binding to Escherichia coli Chaperone DnaK.

Protein Pept Lett 2016 ;23(12):1061-1071

Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA.

The bacterial protein DnaK promotes folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted refolding is especially important under stress conditions induced by antibiotic therapies reducing the desired bactericidal effects. DnaK is supposedly targeted by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli ΔdnaK mutants and wild type strains are equally susceptible indicating further intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK- complexes revealed forward and reverse binding modes at the substrate binding domain. Here, we used these ligand-target structures for the first time to rationally optimize peptides using molecular modeling and docking leading to the prediction of four-residue long sequences for improved binding to DnaK. When these sequences were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants. However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.
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http://dx.doi.org/10.2174/0929866523666160719124712DOI Listing
March 2017

Characterization of the Domain Orientations of E. coli 5'-Nucleotidase by Fitting an Ensemble of Conformers to DEER Distance Distributions.

Structure 2016 Jan 24;24(1):43-56. Epub 2015 Dec 24.

Center for Structural Biology, Vanderbilt University, Nashville, TN 37212, USA; Department of Chemistry, Vanderbilt University, Nashville, TN 37212, USA; Vanderbilt University, 7330 Stevenson Center, Station B 351822, Nashville, TN 37235, USA. Electronic address:

Escherichia coli 5'-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate).
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http://dx.doi.org/10.1016/j.str.2015.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706771PMC
January 2016

Crystal structure of cleaved vaspin (serpinA12).

Biol Chem 2016 Jan;397(2):111-23

The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1' (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.
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http://dx.doi.org/10.1515/hsz-2015-0229DOI Listing
January 2016

Crystal structure of human platelet phosphofructokinase-1 locked in an activated conformation.

Biochem J 2015 Aug 11;469(3):421-32. Epub 2015 Jun 11.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany

Phosphofructokinase-1 (Pfk) acts as the main control point of flux through glycolysis. It is involved in complex allosteric regulation and Pfk mutations have been linked to cancer development. Whereas the 3D structure and structural basis of allosteric regulation of prokaryotic Pfk has been studied in great detail, our knowledge about the molecular basis of the allosteric behaviour of the more complex mammalian Pfk is still very limited. To characterize the structural basis of allosteric regulation, the subunit interfaces and the functional consequences of modifications in Tarui's disease and cancer, we analysed the physiological homotetramer of human platelet Pfk at up to 2.67 Å resolution in two crystal forms. The crystallized enzyme is permanently activated by a deletion of the 22 C-terminal residues. Complex structures with ADP and fructose-6-phosphate (F6P) and with ATP suggest a role of three aspartates in the deprotonation of the OH-nucleophile of F6P and in the co-ordination of the catalytic magnesium ion. Changes at the dimer interface, including an asymmetry observed in both crystal forms, are the primary mechanism of allosteric regulation of Pfk by influencing the F6P-binding site. Whereas the nature of this conformational switch appears to be largely conserved in bacterial, yeast and mammalian Pfk, initiation of these changes differs significantly in eukaryotic Pfk.
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http://dx.doi.org/10.1042/BJ20150251DOI Listing
August 2015

A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

Biochem J 2015 Sep 21;470(3):357-67. Epub 2015 Jul 21.

Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, 04103 Leipzig, Germany

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.
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http://dx.doi.org/10.1042/BJ20150643DOI Listing
September 2015

α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective ecto-5'-Nucleotidase (CD73) Inhibitors.

J Med Chem 2015 Aug 30;58(15):6248-63. Epub 2015 Jul 30.

†PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00802DOI Listing
August 2015

Structural basis of the stereospecificity of bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase.

J Biol Chem 2015 Apr 26;290(15):9727-37. Epub 2015 Feb 26.

From the Department of Environmental Microbiology, Helmholtz Centre for Environmental Research (UFZ), 04318 Leipzig and

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.
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http://dx.doi.org/10.1074/jbc.M115.645689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392272PMC
April 2015

Protein surface charge of trypsinogen changes its activation pattern.

BMC Biotechnol 2014 Dec 28;14:109. Epub 2014 Dec 28.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Universität Leipzig, 04103, Leipzig, Germany.

Background: Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein-protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein-protein interaction.

Results: Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.

Conclusion: The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein-protein interactions, as shown here for human trypsinogen.
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http://dx.doi.org/10.1186/s12896-014-0109-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299543PMC
December 2014

Insect-derived proline-rich antimicrobial peptides kill bacteria by inhibiting bacterial protein translation at the 70S ribosome.

Angew Chem Int Ed Engl 2014 Nov 12;53(45):12236-9. Epub 2014 Sep 12.

Institut für Bioanalytische Chemie, Biotechnologisch-Biomedizinisches Zentrum (BBZ), Universität Leipzig, Deutscher Platz 5, 04103 Leipzig (Germany).

Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently in vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240 nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.
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http://dx.doi.org/10.1002/anie.201407145DOI Listing
November 2014

Posttranslational incorporation of noncanonical amino acids in the RNase S system by semisynthetic protein assembly.

Methods Mol Biol 2014 ;1216:71-87

Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany,

The unique ribonuclease S (RNase S) system, derived from proteolytic cleavage of bovine ribonuclease A (RNase A), consists of a tight complex formed by a peptide (amino acids 1-20) and a protein (21-124) part. These fragments, designated as S-peptide and S-protein, can be separated by two purification steps. By addition of synthetic S-peptide derivatives to the S-protein, semisynthetic RNase S is reassembled with high efficiency. Based on this peptide-protein complementation noncanonical amino acids can be easily introduced into a protein host. Here we describe the preparation of the S-protein from RNase A as well as the characterization of the reassembled semisynthetic RNase S complex. Complex formation can be monitored by RNase activity, circular dichroism, or fluorescence polarization. Structure-based enzyme design of the RNase S scaffold is possible based on high-resolution crystal structures of RNase S and its semisynthetic variants.
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http://dx.doi.org/10.1007/978-1-4939-1486-9_4DOI Listing
May 2015

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis.

Acta Crystallogr F Struct Biol Commun 2014 May 25;70(Pt 5):578-82. Epub 2014 Apr 25.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and group B-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.
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http://dx.doi.org/10.1107/S2053230X14008723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014322PMC
May 2014

Fluorine-containing 6,7-dialkoxybiaryl-based inhibitors for phosphodiesterase 10 A: synthesis and in vitro evaluation of inhibitory potency, selectivity, and metabolism.

ChemMedChem 2014 Jul 11;9(7):1476-87. Epub 2014 Apr 11.

Institut für Pharmazie, Universität Leipzig, Brüderstr. 34, 04103 Leipzig (Germany), Fax: (+49) 341 9736889.

Based on the potent phosphodiesterase 10 A (PDE10A) inhibitor PQ-10, we synthesized 32 derivatives to determine relationships between their molecular structure and binding properties. Their roles as potential positron emission tomography (PET) ligands were evaluated, as well as their inhibitory potency toward PDE10A and other PDEs, and their metabolic stability was determined in vitro. According to our findings, halo-alkyl substituents at position 2 of the quinazoline moiety and/or halo-alkyloxy substituents at positions 6 or 7 affect not only the compounds' affinity, but also their selectivity toward PDE10A. As a result of substituting the methoxy group for a monofluoroethoxy or difluoroethoxy group at position 6 of the quinazoline ring, the selectivity for PDE10A over PDE3A increased. The same result was obtained by 6,7-difluoride substitution on the quinoxaline moiety. Finally, fluorinated compounds (R)-7-(fluoromethoxy)-6-methoxy-4-(3-(quinoxaline-2-yloxy)pyrrolidine-1-yl)quinazoline (16 a), 19 a-d, (R)-tert-butyl-3-(6-fluoroquinoxalin-2-yloxy)pyrrolidine-1-carboxylate (29), and 35 (IC50 PDE10A 11-65 nM) showed the highest inhibitory potential. Further, fluoroethoxy substitution at position 7 of the quinazoline ring improved metabolic stability over that of the lead structure PQ-10.
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http://dx.doi.org/10.1002/cmdc.201300522DOI Listing
July 2014

Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca.

Appl Microbiol Biotechnol 2014 Sep 13;98(18):7815-23. Epub 2014 Apr 13.

Institut für Bioanalytische Chemie, Fakultät für Chemie und Mineralogie, Universität Leipzig, Leipzig, Germany,

Bacterial cutinases are promising catalysts for the modification and degradation of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. We have crystallized and structurally characterized a cutinase from Thermobifida fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with our analysis of the thermal stability and modelling studies, we suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from Thermobifida alba AHK119 and propose a model for the binding of the enzyme towards its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.
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http://dx.doi.org/10.1007/s00253-014-5672-0DOI Listing
September 2014