Chemical Biology Core FacilityPublications
New 7-Methylguanine Derivatives Targeting the Influenza Polymerase PB2 Cap-Binding Domain.
Pautus, S., Sehr, P., Lewis, J., Fortune, A., Wolkerstorfer, A., Szolar, O., Guilligay, D., Lunardi, T., Decout, J.L. & Cusack, S.
J Med Chem. 2013 Nov 14;56(21):8915-30. doi: 10.1021/jm401369y. Epub 2013 Nov 6.
The heterotrimeric influenza virus polymerase performs replication and transcription of viral RNA in the nucleus of infected cells. Transcription by "cap-snatching" requires that host-cell pre-mRNAs are bound via their 5' cap to the PB2 subunit. Thus, the PB2 cap-binding site is potentially a good target for new antiviral drugs that will directly inhibit viral replication. Docking studies using the structure of the PB2 cap-binding domain suggested that 7-alkylguanine derivatives substituted at position N-9 and N-2 could be good candidates. Four series of 7,9-di- and 2,7,9-trialkyl guanine derivatives were synthesized and evaluated by an AlphaScreen assay in competition with a biotinylated cap analogue. Three synthesized compounds display potent in vitro activity with IC50 values lower than 10 muM. High-resolution X-ray structures of three inhibitors in complex with the H5N1 PB2 cap-binding domain confirmed the binding mode and provide detailed information for further compound optimization.
Tripolin A, a novel small-molecule inhibitor of aurora A kinase, reveals new regulation of HURP's distribution on microtubules.
Kesisova, I.A., Nakos, K.C., Tsolou, A., Angelis, D., Lewis, J., Chatzaki, A., Agianian, B., Giannis, A. & Koffa, M.D.
PLoS One. 2013;8(3):e58485. doi: 10.1371/journal.pone.0058485. Epub 2013 Mar 13.
Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development.
High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.
Fueller, F., Jehle, B., Putzker, K., Lewis, J.D. & Krauth-Siegel, R.L.
J Biol Chem. 2012 Mar 16;287(12):8792-802. doi: 10.1074/jbc.M111.338285. Epub2012 Jan 23.
In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 muM and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.
Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma.
Hose, D., Reme, T., Meissner, T., Moreaux, J., Seckinger, A., Lewis, J.D., Benes, V., Benner, A., Hundemer, M., Hielscher, T., Shaughnessy JD, J.r, Barlogie, B., Neben, K., Kramer, A., Hillengass, J., Bertsch, U., Jauch, A., De Vos, J., Rossi, J.F., Mohler, T., Blake, J., Zimmermann, J., Klein, B. & Goldschmidt, H.
Blood. 2009 Apr 30;113(18):4331-40. Epub 2009 Jan 26.
Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.
Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein.
Bartonova, V., Igonet, S., Sticht, J., Glass, B., Habermann, A., Vaney, M.C., Sehr, P., Lewis, J.D., Rey, F.A. & Krausslich, H.G.
J Biol Chem. 2008 Nov 14;283(46):32024-33. Epub 2008 Sep 4.
Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.
The structural basis for cap binding by influenza virus polymerase subunit PB2.
Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T., Sehr, P., Lewis, J.D., Ruigrok, R.W., Ortin, J., Hart, D.J. & Cusack, S.
Nat Struct Mol Biol. 2008 May;15(5):500-6. Epub 2008 May 4.
Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.
The E6 oncoprotein of human papillomavirus 16 as a therapeutic target
Dymalla, S; Butz, K; Lohrey, C; Scheffner, M; Sehr, P; Lewis, J; Hoppe-Seyler, F
Molecular Cancer Therapeutics Dec 2007 6(12)
Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen.
Sehr, P., Pawlita, M. & Lewis, J.
J Biomol Screen. 2007 Jun;12(4):560-7. Epub 2007 May 3.
Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins.
Thanatop: a novel 5-nitrofuran that is a highly active, cell-permeable inhibitor of topoisomerase II.
Polycarpou-Schwarz, M., Muller, K., Denger, S., Riddell, A., Lewis, J.D., Gannon, F. & Reid, G.
Cancer Res. 2007 May 1;67(9):4451-8.
A series of nitrofuran-based compounds were identified as inhibitors of estrogen signaling in a cell-based, high-throughput screen of a diverse library of small molecules. These highly related compounds were subsequently found to inhibit topoisomerase II in vitro at concentrations similar to that required for the inhibition of estrogen signaling in cells. The most potent nitrofuran discovered is approximately 10-fold more active than etoposide phosphate, a topoisomerase II inhibitor in clinical use. The nitrofurans also inhibit topoisomerase I activity, with approximately 20-fold less activity. Moreover, the nitrofurans, in contrast to etoposide, induce a profound cell cycle arrest in the G(0)-G(1) phase of the cell cycle, do not induce double-stranded DNA breaks, are not substrates for multidrug resistance protein-1 export from the cell, and are amenable to synthetic development. In addition, the nitrofurans synergize with etoposide phosphate in cell killing. Clonogenic assays done on a panel of human tumors maintained ex vivo in nude mice show that the most active compound identified in the screen is selective against tumors compared with normal hematopoietic stem cells. However, this compound had only moderate activity in a mouse xenograft model. This novel class of topoisomerase II inhibitor may provide additional chemotherapeutic strategies for the development of cytotoxic agents with proven clinical utility.
The role of the chemical biology core facility at EMBL: a vision for a European roadmap.
ACS Chem Biol. 2007 Jan 23;2(1):21-3. Europe PMC
Systematic discovery of new recognition peptides mediating protein interaction networks.
Neduva, V., Linding, R., Su-Angrand, I., Stark, A., de Masi, F., Gibson, T.J., Lewis, J.D., Serrano, L. & Russell, R.B.
PLoS Biol. 2005 Dec;3(12):e405. Epub 2005 Nov 15.
Many aspects of cell signalling, trafficking, and targeting are governed by interactions between globular protein domains and short peptide segments. These domains often bind multiple peptides that share a common sequence pattern, or "linear motif" (e.g., SH3 binding to PxxP). Many domains are known, though comparatively few linear motifs have been discovered. Their short length (three to eight residues), and the fact that they often reside in disordered regions in proteins makes them difficult to detect through sequence comparison or experiment. Nevertheless, each new motif provides critical molecular details of how interaction networks are constructed, and can explain how one protein is able to bind to very different partners. Here we show that binding motifs can be detected using data from genome-scale interaction studies, and thus avoid the normally slow discovery process. Our approach based on motif over-representation in non-homologous sequences, rediscovers known motifs and predicts dozens of others. Direct binding experiments reveal that two predicted motifs are indeed protein-binding modules: a DxxDxxxD protein phosphatase 1 binding motif with a KD of 22 microM and a VxxxRxYS motif that binds Translin with a KD of 43 microM. We estimate that there are dozens or even hundreds of linear motifs yet to be discovered that will give molecular insight into protein networks and greatly illuminate cellular processes.
Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES.
Boehringer, D., Thermann, R., Ostareck-Lederer, A., Lewis, J.D. & Stark, H.
Structure. 2005 Nov;13(11):1695-706.
Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.
The role of RNA interference in drug target validation: Application to Hepatitis C.
Ostareck-Lederer, A., Clauder-Münster, S., Thermann, R., Polycarpou-Schwartz, M., Gentzel, M., Wilm, M. & Lewis, J.D.
In "RNA Interference Technology", Appasani, K. (ed.), 2005, Cambridge University Press, pp. 318-330