Publications by authors named "Michael Albers"

28 Publications

  • Page 1 of 1

Discovery and optimization of substituted oxalamides as novel heme-displacing IDO1 inhibitors.

Bioorg Med Chem Lett 2021 02 15;33:127744. Epub 2020 Dec 15.

Phenex Pharmaceuticals AG, Waldhofer Str. 104, 69123 Heidelberg, Germany.

Since the advent of antibody checkpoint inhibitors as highly efficient drugs for cancer treatment, the development of immunomodulating small molecules in oncology has gained great attention. Drug candidates targeting IDO1, a key enzyme in tryptophan metabolism, are currently under clinical investigation in combination with PD-1/PD-L1 agents as well as with other established anti-tumor therapeutics. A ligand based design approach from hydroxyamidine 4 that aimed at heme-binding IDO1 inhibitors resulted in new compounds with moderate IDO1 potency. A hybrid structure design that made use of the linrodostat structure (2) led to oxalamide derived, heme-displacing IDO1 inhibitors with high cell-based IDO1 potency and a favorable ADME/PK profile.
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http://dx.doi.org/10.1016/j.bmcl.2020.127744DOI Listing
February 2021

Discovery of highly potent heme-displacing IDO1 inhibitors based on a spirofused bicyclic scaffold.

Bioorg Med Chem Lett 2021 02 11;33:127738. Epub 2020 Dec 11.

Phenex Pharmaceuticals AG, Waldhofer Str. 104, 69123 Heidelberg, Germany.

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1).
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http://dx.doi.org/10.1016/j.bmcl.2020.127738DOI Listing
February 2021

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Nat Chem 2020 08 6;12(8):732-739. Epub 2020 Jul 6.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technical University of Munich, Garching, Germany.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
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http://dx.doi.org/10.1038/s41557-020-0484-6DOI Listing
August 2020

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

Bioorg Med Chem Lett 2020 06 21;30(12):127205. Epub 2020 Apr 21.

Janssen Research and Development, LLC, San Diego, CA 92121, USA. Electronic address:

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.
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http://dx.doi.org/10.1016/j.bmcl.2020.127205DOI Listing
June 2020

Discovery and optimization of new oxadiazole substituted thiazole RORγt inverse agonists through a bioisosteric amide replacement approach.

Bioorg Med Chem Lett 2020 06 7;30(12):127174. Epub 2020 Apr 7.

Janssen Research and Development, LLC, San Diego, CA 92121, USA. Electronic address:

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.
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http://dx.doi.org/10.1016/j.bmcl.2020.127174DOI Listing
June 2020

3-Substituted Quinolines as RORγt Inverse Agonists.

Bioorg Med Chem Lett 2019 06 12;29(12):1463-1470. Epub 2019 Apr 12.

Discovery Product Development and Supply, Janssen Research and Development, Welsh and McKean Roads, Spring House, PA 19477, United States.

We have previously reported the syntheses of a series of 3,6-disubstituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). These molecules are potent binders but are high molecular weight and they exhibited poor solubility at pH 2 and pH 7. This manuscript details our efforts at improving physical chemical properties for this series of compounds by increasing the diversity at the 3-position (i.e. introducing heteroatoms and lowering the molecular weight). These efforts have led to molecules which are potent binders with improved solubility.
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http://dx.doi.org/10.1016/j.bmcl.2019.04.021DOI Listing
June 2019

Photoactivated Colibactin Probes Induce Cellular DNA Damage.

Angew Chem Int Ed Engl 2019 01 27;58(5):1417-1421. Epub 2018 Dec 27.

Department of Chemistry, Umeå University, 90187, Umeå, Sweden.

Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative "warhead" and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.
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http://dx.doi.org/10.1002/anie.201812326DOI Listing
January 2019

Identification and biological evaluation of thiazole-based inverse agonists of RORγt.

Bioorg Med Chem Lett 2018 05 3;28(9):1446-1455. Epub 2018 Apr 3.

Phenex Pharmaceuticals AG, Waldhofer Strasse 104, 69123 Heidelberg, Germany.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.093DOI Listing
May 2018

Regulation of IL-22BP in psoriasis.

Sci Rep 2018 03 23;8(1):5085. Epub 2018 Mar 23.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.
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http://dx.doi.org/10.1038/s41598-018-23510-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865214PMC
March 2018

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

Sci Rep 2016 12 1;6:37977. Epub 2016 Dec 1.

Janssen Research &Development, La Jolla, California, United States.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.
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http://dx.doi.org/10.1038/srep37977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131364PMC
December 2016

Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway.

PLoS Pathog 2015 Dec 3;11(12):e1005307. Epub 2015 Dec 3.

Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.
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http://dx.doi.org/10.1371/journal.ppat.1005307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669118PMC
December 2015

The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila.

Mol Microbiol 2016 Feb 27;99(4):778-93. Epub 2015 Nov 27.

Max von Pettenkofer Institute, Ludwig-Maximilians University, Pettenkoferstraße 9a, 80336, Munich, Germany.

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.
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http://dx.doi.org/10.1111/mmi.13265DOI Listing
February 2016

Covalent Protein Labeling by Enzymatic Phosphocholination.

Angew Chem Int Ed Engl 2015 Aug 3;54(35):10327-30. Epub 2015 Jul 3.

Chemical Biology Center (KBC), Institute of Chemistry, Umeå University, 90187 Umeå (Sweden).

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.
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http://dx.doi.org/10.1002/anie.201502618DOI Listing
August 2015

Exploring adenylylation and phosphocholination as post-translational modifications.

Chembiochem 2014 Jan 31;15(1):19-26. Epub 2013 Oct 31.

Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany).

Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.
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http://dx.doi.org/10.1002/cbic.201300508DOI Listing
January 2014

Mechanism of the tissue-specific action of the selective androgen receptor modulator S-101479.

Biol Pharm Bull 2013 ;36(3):442-51

Central Research Laboratories, Kaken Pharmaceutical Co., Ltd, 14 Shinomiya, Minamigawara-cho, Yamashina-ku, Kyoto 607–8042, Japan.

Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce anabolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimulation of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction assays revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, β-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tissues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues.
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http://dx.doi.org/10.1248/bpb.b12-00885DOI Listing
August 2013

Amino acid building blocks for Fmoc solid-phase synthesis of peptides phosphocholinated at serine, threonine, and tyrosine.

J Org Chem 2013 Mar 13;78(6):2715-9. Epub 2013 Feb 13.

Department of Chemical Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn Strasse 11, D-44227 Dortmund, Germany.

Phosphocholination of eukaryotic host cell proteins has recently been identified as a novel post-translational modification important for bacterial pathogenesis. Here, we describe the first straightforward synthetic strategy for peptides containing phosphocholinated serine, threonine, or tyrosine residues using preformed functional amino acid building blocks, fully compatible with standard Fmoc solid-phase peptide synthesis.
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http://dx.doi.org/10.1021/jo302587gDOI Listing
March 2013

Amino acid building blocks for efficient Fmoc solid-phase synthesis of peptides adenylylated at serine or threonine.

Org Lett 2011 Nov 26;13(22):6014-7. Epub 2011 Oct 26.

Department of Chemical Biology, Max-Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, D-44227, Dortmund, Germany.

The first straightforward building block based (non-interassembly) synthesis of peptides containing adenylylated serine and threonine residues is described. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The described Thr-AMP SPPS-building block has been employed in the synthesis of the Thr-adenylylated sequence of human GTPase CDC42 (Ac-SEYVP-T(AMP)-VFDNYGC-NH(2)). Further, we demonstrate proof-of-concept for the synthesis of an Ser-adenylylated peptide (Ac-GSGA-S(AMP)-AGSGC-NH(2)) from the corresponding adenylylated serine building block.
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http://dx.doi.org/10.1021/ol2024696DOI Listing
November 2011

Efficient synthesis and applications of peptides containing adenylylated tyrosine residues.

Angew Chem Int Ed Engl 2011 Sep 25;50(39):9200-4. Epub 2011 Aug 25.

Max-Planck-Institut für molekulare Physiologie, Abt. Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

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http://dx.doi.org/10.1002/anie.201103203DOI Listing
September 2011

Neurotrophic estrogens: essential profile and endpoints for drug discovery.

Drug Discov Today 2008 Sep 15;13(17-18):734-47. Epub 2008 May 15.

Global Drug Discovery, Bayer Schering Pharma AG, Berlin, Germany.

Criteria for the early recognition of selective neurotrophic action are crucial for the discovery of estrogens for supplementation therapy. The comparative characterization of 'tool' compounds in different paradigms demonstrates that estrogen-mediated CNS effects are discernible before the manifestation of changes in primary target organs. Agonist activity at, and recruitment of the coactivator SRC-1 by, the estrogen receptor alpha accurately reflect peripheral, but not neurotrophic, efficacy. Interaction with, and SRC-1 recruitment at, the estrogen receptor beta appears to be an essential prerequisite for pronounced CNS effects. Monitoring of the hypothalamo-pituitary-adrenal axis activity and the differential organ-specific induction of estrogen-responsive proteins are helpful for early delineation of CNS efficacy. Behavioral and antioxidant efficacy are useful confirmatory readouts, with limited roles in lead selection. Finally, an algorithm for the identification of estrogens with a neurotrophic profile can be generated by assigning 'performance grades' in a multifarious test array.
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http://dx.doi.org/10.1016/j.drudis.2008.03.009DOI Listing
September 2008

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas).

Biochim Biophys Acta 2007 Nov 28;1774(11):1422-30. Epub 2007 Aug 28.

Institut für Biochemie, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany.

An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).
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http://dx.doi.org/10.1016/j.bbapap.2007.08.013DOI Listing
November 2007

Panning for SNuRMs: using cofactor profiling for the rational discovery of selective nuclear receptor modulators.

Drug Discov Today 2007 Oct 19;12(19-20):860-9. Epub 2007 Sep 19.

Phenex Pharmaceuticals AG, Gebaeude J542N, Werksgelaende BASF, D-67056 Ludwigshafen, Germany.

Drugs that target nuclear receptors are clinically, as well as commercially, successful. Their widespread use, however, is limited by an inherent propensity of nuclear receptors to trigger beneficial, as well as adverse, pharmacological effects upon drug activation. Hence, selective drugs that display reduced adverse effects, such as the selective estrogen receptor modulator (SERM) Raloxifene, have been developed by guidance through classical cell culture assays and animal trials. Full agonist and selective modulator nuclear receptor drugs, in general, differ by their ability to recruit certain cofactors to the receptor protein. Hence, systematic cofactor profiling is advancing into an approach for the rationally guided identification of selective NR modulators (SNuRMs) with improved therapeutic ratio.
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http://dx.doi.org/10.1016/j.drudis.2007.07.025DOI Listing
October 2007

Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

Nucleic Acids Res 2007 20;35(5):1673-86. Epub 2007 Feb 20.

Cell Biology Section, Division of Intramural Research, National Institute of Enironmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ERalpha polyubiquitination, increases the level of ERalpha protein, and enhances ERalpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERalpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERalpha, but eliminates the effects of RAP80 on ERalpha polyubiquitination, the level of ERalpha protein, and ERalpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERalpha as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERalpha activity.
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http://dx.doi.org/10.1093/nar/gkl1112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865050PMC
April 2007

A novel principle for partial agonism of liver X receptor ligands. Competitive recruitment of activators and repressors.

J Biol Chem 2006 Feb 13;281(8):4920-30. Epub 2005 Dec 13.

PheneX Pharmaceuticals AG, 67056 Ludwigshafen, Germany and Department of Vascular and Metabolic Diseases, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland.

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.
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http://dx.doi.org/10.1074/jbc.M510101200DOI Listing
February 2006

Automated yeast two-hybrid screening for nuclear receptor-interacting proteins.

Mol Cell Proteomics 2005 Feb 15;4(2):205-13. Epub 2004 Dec 15.

PheneX Pharmaceuticals AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.
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http://dx.doi.org/10.1074/mcp.M400169-MCP200DOI Listing
February 2005

Computer-assisted generation of a protein-interaction database for nuclear receptors.

Mol Endocrinol 2003 Aug 8;17(8):1555-67. Epub 2003 May 8.

LION Bioscience AG, 69123 Heidelberg, Germany.

With the increasing amount of biological data available, automated methods for information retrieval become necessary. We employed computer-assisted text mining to retrieve all protein-protein interactions for nuclear receptors from MEDLINE in a systematic way. A dictionary of protein names and of terms denoting interactions was generated, and trioccurrences of two protein names and one interaction term in one sentence were retrieved. Abstracts containing at least one such trioccurrence were manually checked by biologists to select the relevant interactions out of the automatically extracted data. In total, 4360 abstracts were retrieved containing data on protein interactions for nuclear receptors. The resulting database contains all reported protein interactions involving nuclear receptors from 1966 to September 2001. Remarkably, the annual increase in number of reported interactors for nuclear receptors has been following an exponential growth curve in the years 1991 to 2001. Apparent in the data set is the high complexity of protein interactions for nuclear receptors. The number of interactions correlates with the number of published papers for a given receptor, suggesting that the number of reported interactors is a reflection of the intensity of research dedicated to a given receptor. Indeed, comparison of the retrieved data to a systematic yeast two-hybrid-based interaction analysis suggests that most NRs are similar with respect to the number of interacting proteins. The data set obtained serves as a source for information on NR interactions, as well as a reference data set for the improvement of advanced text-mining methods.
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http://dx.doi.org/10.1210/me.2002-0424DOI Listing
August 2003

Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors.

RNA 2003 Jan;9(1):138-50

Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK.

Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p. We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro. We demonstrate that Prp45p and Prp46p are spliceosome associated throughout the splicing process and both are essential for pre-mRNA splicing. Under nonsplicing conditions they also associate in coprecipitation assays with low levels of the U2, U5, and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a postsplicing snRNP complex.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370377PMC
http://dx.doi.org/10.1261/rna.2119903DOI Listing
January 2003

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol.

Mol Cell Biol 2003 Feb;23(3):864-72

LION Bioscience AG, 69120 Heidelberg, Germany.

Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRbeta as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRbeta is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRalpha and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRbeta. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRbeta as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140718PMC
http://dx.doi.org/10.1128/mcb.23.3.864-872.2003DOI Listing
February 2003

Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.

J Biol Chem 2002 May 22;277(18):16179-88. Epub 2002 Jan 22.

Exploratory Research, LION Bioscience Ktiengesellschaft, D-69120 Heidelberg, Germany.

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.
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http://dx.doi.org/10.1074/jbc.M107793200DOI Listing
May 2002