Publications by authors named "Grzegorz M Popowicz"

52 Publications

Deconstructing Noncovalent Kelch-like ECH-Associated Protein 1 (Keap1) Inhibitors into Fragments to Reconstruct New Potent Compounds.

J Med Chem 2021 Apr 5;64(8):4623-4661. Epub 2021 Apr 5.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.

Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound with a 220-380-fold stronger affinity ( = 16 μM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with values of 0.04-0.5 μM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02094DOI Listing
April 2021

Deep learning model predicts water interaction sites on the surface of proteins using limited-resolution data.

Chem Commun (Camb) 2020 Dec 25;56(98):15454-15457. Epub 2020 Nov 25.

Department of Bioinformatics, Wissenschaftszentrum Weihenstephan, Technische Universität München, Maximus-von-Imhof-Forum 3, 85354 Freising, Germany.

We develop a residual deep learning model, hotWater (https://pypi.org/project/hotWater/), to identify key water interaction sites on proteins for binding models and drug discovery. This is tested on new crystal structures, as well as cryo-EM and NMR structures from the PDB and in crystallographic refinement with promising results.
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http://dx.doi.org/10.1039/d0cc04383dDOI Listing
December 2020

Correction for Softley et al., "Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase".

Antimicrob Agents Chemother 2020 06 23;64(7). Epub 2020 Jun 23.

Biomolecular NMR and Center for Integrated Protein Science Munich at Department Chemie, Technical University of Munich, Garching, Germany

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http://dx.doi.org/10.1128/AAC.01077-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318002PMC
June 2020

Paramagnetic NMR in drug discovery.

J Biomol NMR 2020 Jul 10;74(6-7):287-309. Epub 2020 Jun 10.

Biomolecular NMR and Center for Integrated Protein Science Munich at Department Chemie, Technical University of Munich, Lichtenbergstraße 4, 85747, Garching, Germany.

The presence of an unpaired electron in paramagnetic molecules generates significant effects in NMR spectra, which can be exploited to provide restraints complementary to those used in standard structure-calculation protocols. NMR already occupies a central position in drug discovery for its use in fragment screening, structural biology and validation of ligand-target interactions. Paramagnetic restraints provide unique opportunities, for example, for more sensitive screening to identify weaker-binding fragments. A key application of paramagnetic NMR in drug discovery, however, is to provide new structural restraints in cases where crystallography proves intractable. This is particularly important at early stages in drug-discovery programs where crystal structures of weakly-binding fragments are difficult to obtain and crystallization artefacts are probable, but structural information about ligand poses is crucial to guide medicinal chemistry. Numerous applications show the value of paramagnetic restraints to filter computational docking poses and to generate interaction models. Paramagnetic relaxation enhancements (PREs) generate a distance-dependent effect, while pseudo-contact shift (PCS) restraints provide both distance and angular information. Here, we review strategies for introducing paramagnetic centers and discuss examples that illustrate the utility of paramagnetic restraints in drug discovery. Combined with standard approaches, such as chemical shift perturbation and NOE-derived distance information, paramagnetic NMR promises a valuable source of information for many challenging drug-discovery programs.
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http://dx.doi.org/10.1007/s10858-020-00322-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311382PMC
July 2020

A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells.

FEBS Lett 2020 08 23;594(15):2406-2420. Epub 2020 Jun 23.

Ridgeview Instruments AB, Uppsala, Sweden.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.
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http://dx.doi.org/10.1002/1873-3468.13847DOI Listing
August 2020

Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase.

Antimicrob Agents Chemother 2020 05 21;64(6). Epub 2020 May 21.

Biomolecular NMR and Center for Integrated Protein Science Munich at Department Chemie, Technical University of Munich, Garching, Germany

Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.
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http://dx.doi.org/10.1128/AAC.00123-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269475PMC
May 2020

Structure-activity relationship and cardiac safety of 2-aryl-2-(pyridin-2-yl)acetamides as a new class of broad-spectrum anticonvulsants derived from Disopyramide.

Bioorg Chem 2020 05 5;98:103717. Epub 2020 Mar 5.

Department of Drug Technology and Pharmaceutical Biotechnology, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland.

A series of 2-aryl-2-(pyridin-2-yl)acetamides were synthesized and screened for their anticonvulsant activity in animal models of epilepsy. The compounds were broadly active in the 'classical' maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests as well as in the 6 Hz and kindling models of pharmacoresistant seizures. Furthermore, the compounds showed good therapeutic indices between anticonvulsant activity and motor impairment. Structure-activity relationship (SAR) trends clearly showed the highest activity resides in unsubstituted phenyl derivatives or compounds having ortho- and meta- substituents on the phenyl ring. The 2-aryl-2-(pyridin-2-yl)acetamides were derived by redesign of the cardiotoxic sodium channel blocker Disopyramide (DISO). Our results show that the compounds preserve the capability of the parent compound to inhibit voltage gated sodium currents in patch-clamp experiments; however, in contrast to DISO, a representative compound from the series 1 displays high levels of cardiac safety in a panel of in vitro and in vivo experiments.
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http://dx.doi.org/10.1016/j.bioorg.2020.103717DOI Listing
May 2020

Structure-Activity Relationship in Pyrazolo[4,3-]pyridines, First Inhibitors of PEX14-PEX5 Protein-Protein Interaction with Trypanocidal Activity.

J Med Chem 2020 01 6;63(2):847-879. Epub 2020 Jan 6.

Center for Integrated Protein Science Munich at Chair of Biomolecular NMR, Department Chemie , Technische Universität München , Lichtenbergstrasse 4 , 85747 Garching , Germany.

protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against , including the human pathogen and parasites.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01876DOI Listing
January 2020

FSP1 is a glutathione-independent ferroptosis suppressor.

Nature 2019 11 21;575(7784):693-698. Epub 2019 Oct 21.

Department of Chemistry & Biomolecular Sciences, University of Ottawa, Ottawa, ON, Canada.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4) and radical-trapping antioxidants. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints and phospholipid composition contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q, CoQ): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.
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http://dx.doi.org/10.1038/s41586-019-1707-0DOI Listing
November 2019

Crystal Structure of Glucokinase (Glk1).

Int J Mol Sci 2019 Sep 28;20(19). Epub 2019 Sep 28.

Malopolska Centre of Biotechnology, Jagiellonian University, ul. Gronostajowa 7a, 30-387 Krakow, Poland.

Glucose phosphorylating enzymes are crucial in the regulation of basic cellular processes, including metabolism and gene expression. Glucokinases and hexokinases provide a pool of phosphorylated glucose in an adenosine diphosphate (ADP)- and ATP-dependent manner to shape the cell metabolism. The glucose processing enzymes from are poorly characterized despite the emerging contribution of this yeast strain to industrial and laboratory scale biotechnology. The first reports on glucokinase (Glk1) positioned the enzyme as an essential component required for glucose signaling. Nevertheless, no biochemical and structural information was available until now. Here, we present the first crystal structure of Glk1 together with biochemical characterization, including substrate specificity and enzyme kinetics. Additionally, comparative analysis of the presented structure and the prior structures of hexokinase (Hxk1) demonstrates the potential transitions between open and closed enzyme conformations upon ligand binding.
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http://dx.doi.org/10.3390/ijms20194821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801647PMC
September 2019

Structure and characterization of Aspergillus fumigatus lipase B with a unique, oversized regulatory subdomain.

FEBS J 2019 06 10;286(12):2366-2380. Epub 2019 Apr 10.

School of Food Science and Engineering, South China University of Technology, Guangzhou, China.

Fungal lipases are efficient and environment-friendly biocatalysts for many industrially relevant processes. One of the most widely applied lipases in the manufacturing industry is Candida antarctica lipase B (CALB). Here, we report the biochemical and structural characterization of a novel CALB-like lipase from an important human pathogen-Aspergillus fumigatus (AFLB), which has high sn-1,3-specificity toward triolein. AFLB crystal structure displays a CALB-like catalytic domain and hosts a unique tightly closed 'lid' domain that contains a disulfide bridge, as well as an extra N-terminal subdomain composed of residues 1-128 (including the helix α1-α5 located above the active site). To gain insight into the function of this novel lid and N-terminal subdomain, we constructed and characterized a series of mutants in these two domains. Deleting the protruding bulk lid's residues, replacing the bulk and tight lid with a small and loose lid from CALB, or breaking the disulfide bridge increased the affinity of CALB for glyceride substrates and improved its catalytic activity, along with the loss of enzyme fold stability and thermostability. N-terminal truncation mutants revealed that the N-terminal peptide (residues 1-59) is a strong inhibitor of AFLB binding to lipid films. This peptide thus limits AFLB's penetration power and specific activity, revealing a unique enzyme activity regulatory mechanism. Our findings on the functional and structural properties of AFLB provide a better understanding of the functions of the CALB-like lipases and pave the way for its future protein engineering. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6IDY.
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http://dx.doi.org/10.1111/febs.14814DOI Listing
June 2019

Come, sweet death: targeting glycosomal protein import for antitrypanosomal drug development.

Curr Opin Microbiol 2018 12 24;46:116-122. Epub 2018 Nov 24.

Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair of Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany. Electronic address:

Glycosomes evolved as specialized system for glycolysis in trypanosomatids. These organelle rely on protein import to maintain function. A machinery of peroxin (PEX) proteins is responsible for recognition and transport of glycosomal proteins to the organelle. Disruption of PEX-based import system was expected to be a strategy against trypanosomatids. Recently, a proof of this hypothesis has been presented. Here, we review current information about trypanosomatids' glycosomal transport components as targets for new trypanocidal therapies.
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http://dx.doi.org/10.1016/j.mib.2018.11.003DOI Listing
December 2018

HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs.

Nucleic Acids Res 2019 01;47(2):1011-1029

Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany.

HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.
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http://dx.doi.org/10.1093/nar/gky1138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344896PMC
January 2019

NMR fragment-based screening for development of the CD44-binding small molecules.

Bioorg Chem 2019 02 25;82:284-289. Epub 2018 Oct 25.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland. Electronic address:

The cell-surface protein CD44, a primary receptor for hyaluronic acid (HA), is one of the most promising targets for cancer therapies. It is prominently involved in the process of tumor growth and metastasis. The possibility of modulating the CD44-HA interaction with a pharmacological inhibitor is therefore of great importance, yet until now there are only few small molecules reported to bind to CD44. Here, we describe the results of the NMR fragment-based screening conducted against CD44 by which we found eight new hit compounds that bind to the receptor with the affinity in milimolar range. The NMR-based characterization revealed that there are two possible binding modes for these compounds, and for some of them the binding is no longer possible in the presence of hyaluronic acid. This could provide an interesting starting point for the development of new high-affinity ligands targeting the CD44-HA axis.
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http://dx.doi.org/10.1016/j.bioorg.2018.10.043DOI Listing
February 2019

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity.

J Biol Chem 2018 08 27;293(34):13191-13203. Epub 2018 Jun 27.

From the Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology,

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.
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http://dx.doi.org/10.1074/jbc.RA118.002649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109917PMC
August 2018

Identification of small-molecule inhibitors of USP2a.

Eur J Med Chem 2018 Apr 5;150:261-267. Epub 2018 Mar 5.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Krakow, Poland. Electronic address:

USP2a is a deubiquitinating protease that rescues its target proteins from destruction by the proteasome by reversing the process of protein ubiquitination. USP2a shows oncogenic properties in vivo and has been found to be a specific activator of cyclin D1. Many types of cancers are addicted to cyclin D1 expression. Targeting USP2a is a promising strategy for cancer therapy but little progress has been made in the field of inhibition of USP2a. Using NMR-based fragment screening and biophysical binding assays, we have discovered small molecules that bind to USP2a. Iterations of fragment combination and structure-driven design identified two 5-(2-thienyl)-3-isoxazoles as the inhibitors of the USP2a-ubiquitin protein-protein interaction. The affinity of these molecules for the catalytic domain of USP2a parallels their ability to interfere with USP2a binding to ubiquitin in vitro. Altogether, our results establish the 5-(2-thienyl)-3-isoxazole pharmacophore as an attractive starting point for lead optimization.
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http://dx.doi.org/10.1016/j.ejmech.2018.03.009DOI Listing
April 2018

Inhibitors of glycosomal protein import provide new leads against trypanosomiasis.

Microb Cell 2017 Jul 3;4(7):229-232. Epub 2017 Jul 3.

Institute of Biochemistry and Pathobiochemistry, Department of Systems Biochemistry, Faculty of Medicine, Ruhr University Bochum, 44780 Bochum, Germany.

Vector-borne trypanosomatid parasite infections in tropical and sub-tropical countries constitute a major threat to humans and livestock. parasites are transmitted by tsetse fly and lead to African sleeping sickness in humans and Nagana in cattle. In Latin American countries, infections spread by triatomine kissing bugs lead to Chagas disease. Various species of transmitted to humans by phlebotomine sandflies manifest in a spectrum of diseases termed Leishmaniasis. 20 million people are currently infected with trypanosomatid parasites, leading to over 30,000 deaths annually and half billion people at risk of the infection. It is estimated that 300,000 Chagas infected people reside in the United States and 100,000 in Europe. Glycosomes are peroxisome-like organelles found only in trypanosomatids. Glycolysis occurs in the cytosol in all other organisms, but glycolytic enzymes and other metabolic pathways are compartmentalized inside glycosomes in trypanosomatids. Glycosomes are essential for the parasite survival and hence thought to be an attractive drug target. Our recent study [Dawidowski Science (2017)] is the first to report small molecule inhibitors of glycosomal protein import. Using structure-based drug design, we developed small molecule inhibitors of the PEX5-PEX14 protein-protein interaction that disrupt glycosomal protein import and kill the parasites. Oral treatment of infected mice with PEX14 inhibitor significantly reduced the parasite levels with no adverse effect on mice. The study provides the grounds for further development of the glycosome inhibitors into clinical candidates and validates the parasite protein-protein interactions as drug targets.
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http://dx.doi.org/10.15698/mic2017.07.581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507685PMC
July 2017

Discovery of novel dual inhibitors against Mdm2 and Mdmx proteins by in silico approaches and binding assay.

Life Sci 2016 Jan 30;145:240-6. Epub 2015 Dec 30.

Department of Medicinal Chemistry, Faculty of Pharmacy, Drug Design and Development Research Center, Tehran University of Medical Sciences, 16 Azar Ave., Tehran, Iran. Electronic address:

Aims: The p53 protein, also called guardian of the genome, has a key role in cell cycle regulation. It is activated under stressful circumstances, such as DNA damage which results in permanent arrest or cell death. The protein is disabled in several types of human cancer due to over-expression of the two regulators, Mdm2 and Mdmx. As a result, inhibiting Mdm subtypes could reactivate p53 and bring about a promising therapeutic strategy in cancers.

Main Methods: Here a structure-based pharmacophore search and docking simulation are presented in order to filter our in-house library which contains 1035 compounds to find novel scaffolds that inhibit Mdm2 and Mdmx concomitantly. Afterwards, fluorescence polarization binding assay was used to obtain inhibition constant of final compounds.

Key Findings: Thirty two ligands were introduced to bioassay as a result of in-silico methods. Twelve of them inhibit both proteins with almost balanced Ki value ranging from 18 to 162μM for Mdm2 and 18 to 233μM for Mdmx. It was observed that all compounds fill Phe19 and Trp23 pockets of Mdm2/x binding sites and form a hydrogen bond with Trp23 pocket's neighbor amino acids in a manner similar to p53 protein. Additionally, it was concluded that Trp23 pocket of Mdmx has a bigger hydrophobic volume comparing with the one of Mdm2.

Significance: Three structure-activity relationship patterns are supposed which one of them presents usefulness features and can be used in future studies. This study presents first qualitative SAR for dual inhibitors against Mdm2/x.
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http://dx.doi.org/10.1016/j.lfs.2015.12.047DOI Listing
January 2016

Structural basis for the assembly of the Sxl-Unr translation regulatory complex.

Nature 2014 Nov 7;515(7526):287-90. Epub 2014 Sep 7.

1] Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, DE-85764, Germany [2] Center for Integrated Protein Science Munich at Biomolecular NMR Spectroscopy, Department Chemie, Technische Universität München, Lichtenbergstr. 4, DE-85747 Garching, Germany.

Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit. Here we report the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl-Unr-msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. Our results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.
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http://dx.doi.org/10.1038/nature13693DOI Listing
November 2014

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells.

J Mol Cell Biol 2014 Aug 28;6(4):312-23. Epub 2014 May 28.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.
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http://dx.doi.org/10.1093/jmcb/mju026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141198PMC
August 2014

Discovery of highly potent p53-MDM2 antagonists and structural basis for anti-acute myeloid leukemia activities.

ACS Chem Biol 2014 Mar 17;9(3):802-11. Epub 2014 Jan 17.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15261, United States.

The inhibition of p53-MDM2 interaction is a promising new approach to non-genotoxic cancer treatment. A potential application for drugs blocking the p53-MDM2 interaction is acute myeloid leukemia (AML) due to the occurrence of wild type p53 (wt p53) in the majority of patients. Although there are very promising preclinical results of several p53-MDM2 antagonists in early development, none of the compounds have yet proven the utility as a next generation anticancer agent. Herein we report the design, synthesis and optimization of YH239-EE (ethyl ester of the free carboxylic acid compound YH239), a potent p53-MDM2 antagonizing and apoptosis-inducing agent characterized by a number of leukemia cell lines as well as patient-derived AML blast samples. The structural basis of the interaction between MDM2 (the p53 receptor) and YH239 is elucidated by a co-crystal structure. YH239-EE acts as a prodrug and is the most potent compound that induces apoptosis in AML cells and patient samples. The observed superior activity compared to reference compounds provides the preclinical basis for further investigation and progression of YH239-EE.
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http://dx.doi.org/10.1021/cb400728eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985958PMC
March 2014

Transient protein states in designing inhibitors of the MDM2-p53 interaction.

Structure 2013 Dec 24;21(12):2143-51. Epub 2013 Oct 24.

Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.

Reactivation of p53 by release of the functional protein from its inhibition by MDM2 provides an efficient, nongenotoxic approach to a wide variety of cancers. We present the cocrystal structures of two complexes of MDM2 with inhibitors based on 6-chloroindole scaffolds. Both molecules bound to a distinct conformational state of MDM2 with nM-μM affinities. In contrast to other structurally characterized antagonists, which mimic three amino acids of p53 (Phe19, Trp23, and Leu26), the compounds induced an additional hydrophobic pocket on the MDM2 surface and unveiled a four-point binding mode. The enlarged interaction interface of the inhibitors resulted in extension of small molecules binding toward the "lid" segment of MDM2 (residues 19-23)--a nascent element that interferes with p53 binding. As supported by protein engineering and molecular dynamics studies, employing these unstable elements of MDM2 provides an efficient and yet unexplored alternative in development of MDM2-p53 association inhibitors.
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http://dx.doi.org/10.1016/j.str.2013.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104591PMC
December 2013

Structural and functional analysis of the natural JNK1 inhibitor quercetagetin.

J Mol Biol 2013 Jan 8;425(2):411-23. Epub 2012 Nov 8.

Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3',4',5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model. Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.
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http://dx.doi.org/10.1016/j.jmb.2012.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540140PMC
January 2013

Enabling large-scale design, synthesis and validation of small molecule protein-protein antagonists.

PLoS One 2012 12;7(3):e32839. Epub 2012 Mar 12.

Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Although there is no shortage of potential drug targets, there are only a handful known low-molecular-weight inhibitors of protein-protein interactions (PPIs). One problem is that current efforts are dominated by low-yield high-throughput screening, whose rigid framework is not suitable for the diverse chemotypes present in PPIs. Here, we developed a novel pharmacophore-based interactive screening technology that builds on the role anchor residues, or deeply buried hot spots, have in PPIs, and redesigns these entry points with anchor-biased virtual multicomponent reactions, delivering tens of millions of readily synthesizable novel compounds. Application of this approach to the MDM2/p53 cancer target led to high hit rates, resulting in a large and diverse set of confirmed inhibitors, and co-crystal structures validate the designed compounds. Our unique open-access technology promises to expand chemical space and the exploration of the human interactome by leveraging in-house small-scale assays and user-friendly chemistry to rationally design ligands for PPIs with known structure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032839PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299697PMC
August 2012

Structure of the stapled p53 peptide bound to Mdm2.

J Am Chem Soc 2012 Jan 14;134(1):103-6. Epub 2011 Dec 14.

Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 Å resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.
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http://dx.doi.org/10.1021/ja2090367DOI Listing
January 2012

Exhaustive fluorine scanning toward potent p53-Mdm2 antagonists.

ChemMedChem 2012 Jan 27;7(1):49-52. Epub 2011 Sep 27.

Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA.

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http://dx.doi.org/10.1002/cmdc.201100428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772510PMC
January 2012

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.

Nature 2011 May 25;474(7350):173-8. Epub 2011 May 25.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.
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http://dx.doi.org/10.1038/nature10143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587138PMC
May 2011

The structure-based design of Mdm2/Mdmx-p53 inhibitors gets serious.

Angew Chem Int Ed Engl 2011 Mar 21;50(12):2680-8. Epub 2011 Feb 21.

Max Planck Institute for Biochemistry, Martinsried, Germany.

The p53 protein is the cell's principal bastion of defense against tumor-associated DNA damage. Commonly referred as a "guardian of the genome", p53 is responsible for determining the fate of the cell when the integrity of its genome is damaged. The development of tumors requires breaching this defense line. All known tumor cells either mutate the p53 gene, or in a similar number of cases, use internal cell p53 modulators, Mdm2 and Mdmx proteins, to disable its function. The release of functional p53 from the inhibition by Mdm2 and Mdmx should in principle provide an efficient, nongenotoxic means of cancer therapy. In recent years substantial progress has been made in developing novel p53-activating molecules thanks to several reported crystal structures of Mdm2/x in complex with p53-mimicking peptides and nonpeptidic drug candidates. Understanding the structural attributes of ligand binding holds the key to developing novel, highly effective, and selective drug candidates. Two low-molecular-weight compounds have just recently progressed into early clinical studies.
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http://dx.doi.org/10.1002/anie.201003863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113661PMC
March 2011

The p53-MDM2/MDMX axis - A chemotype perspective.

Medchemcomm 2011 ;2:246-260

University of Pittsburgh, Department of Pharmaceutical Science, Drug Discovery Institute, Pittsburgh, PA, USA.

The protein-protein interaction (PPI) of the tumor suppressor p53 and its negative regulator MDM2 consists of the most intense studied PPI with a group of small molecular weight antagonists described and many more disclosed in patent literature. Due to the Å-level structural insight into p53 interaction with MDM2 there is a reasonable understanding of the requirements of the molecules to bind. In contrast and despite the very close homology and 3-D similarity no potent MDMX antagonist has been disclosed up to date. The current review summarizes the different disclosed chemotypes for MDM2 including a discussion of the cocrystal structures. Structures and approaches to reconstitute functional p53 from mutated p53 are presented. Finally new screening methods and recent biotech deals based on p53 are discussed.
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http://dx.doi.org/10.1039/C0MD00248HDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898590PMC
January 2011