Sharp, S.Y., Boxall, K., Rowlands, M., Prodromou, C., Roe, S.M., Maloney, A., Powers, M., Clarke, P.A., Box, G., Sanderson, S., et al.
(2019). Correction: In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors. Cancer res,
Martino, F., Pal, M., Muñoz-Hernández, H., Rodríguez, C.F., Núñez-Ramírez, R., Gil-Carton, D., Degliesposti, G., Skehel, J.M., Roe, S.M., Prodromou, C., et al.
(2018). RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex. Nature communications,
Martino, F., Pal, M., Muñoz-Hernández, H., Rodríguez, C.F., Núñez-Ramírez, R., Gil-Carton, D., Degliesposti, G., Skehel, J.M., Roe, S.M., Prodromou, C., et al.
(2018). Author Correction: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex. Nature communications,
Alt, A., Dang, H.Q., Wells, O.S., Polo, L.M., Smith, M.A., McGregor, G.A., Welte, T., Lehmann, A.R., Pearl, L.H., Murray, J.M., et al.
(2017). Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nature communications,
Li, Z., Zhou, L., Prodromou, C., Savic, V. & Pearl, L.H.
(2017). HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients. Cell reports,
Hallett, S.T., Pastok, M.W., Morgan, R.M., Wittner, A., Blundell, K.L., Felletar, I., Wedge, S.R., Prodromou, C., Noble, M.E., Pearl, L.H., et al.
(2017). Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System. Cell reports,
Jeggo, P.A., Pearl, L.H. & Carr, A.M.
(2016). DNA repair, genome stability and cancer: a historical perspective. Nature reviews cancer,
Densham, R.M., Garvin, A.J., Stone, H.R., Strachan, J., Baldock, R.A., Daza-Martin, M., Fletcher, A., Blair-Reid, S., Beesley, J., Johal, B., et al.
(2016). Human BRCA1–BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nature structural & molecular biology,
Schulze, A., Beliu, G., Helmerich, D.A., Schubert, J., Pearl, L.H., Prodromou, C. & Neuweiler, H.
(2016). Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism. Nature chemical biology,
Grundy, G.J., Rulten, S.L., Arribas-Bosacoma, R., Davidson, K., Kozik, Z., Oliver, A.W., Pearl, L.H. & Caldecott, K.W.
(2016). The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins. Nature communications,
Grundy, G.J., Polo, L.M., Zeng, Z., Rulten, S.L., Hoch, N.C., Paomephan, P., Xu, Y., Sweet, S.M., Thorne, A.W., Oliver, A.W., et al.
(2016). PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2. Nature communications,
Smith, J.R., de Billy, E., Hobbs, S., Powers, M., Prodromou, C., Pearl, L., Clarke, P.A. & Workman, P.
(2015). Restricting direct interaction of CDC37 with HSP90 does not compromise chaperoning of client proteins. Oncogene,
Morgan, R.M., Pal, M., Roe, S.M., Pearl, L.H. & Prodromou, C.
(2015). Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes. Acta crystallographica section d biological crystallography,
Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90)2–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes..
Smith, J.R., de Billy, E., Hobbs, S., Powers, M., Prodromou, C., Pearl, L., Clarke, P.A. & Worknnan, P.
(2015). Restricting direct interaction of CDC37 with HSP90 does not compromise chaperoning of client proteins. Oncogene,
Pearl, L.H., Schierz, A.C., Ward, S.E., Al-Lazikani, B. & Pearl, F.M.
(2015). Therapeutic opportunities within the DNA damage response. Nature reviews cancer,
Ali, A.A., Timinszky, G., Arribas-Bosacoma, R., Kozlowski, M., Hassa, P.O., Hassler, M., Ladurner, A.G., Pearl, L.H. & Oliver, A.W.
(2015). Erratum: Corrigendum: The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks. Nature structural & molecular biology,
Baldock, R.A., Day, M., Wilkinson, O.J., Cloney, R., Jeggo, P.A., Oliver, A.W., Watts, F.Z. & Pearl, L.H.
(2015). ATM Localization and Heterochromatin Repair Depend on Direct Interaction of the 53BP1-BRCT 2 Domain with γH2AX. Cell reports,
Pal, M., Morgan, M., Phelps, S.E., Roe, S.M., Parry-Morris, S., Downs, J.A., Polier, S., Pearl, L.H. & Prodromou, C.
(2014). Structural Basis for Phosphorylation-Dependent Recruitment of Tel2 to Hsp90 by Pih1. Structure,
Walker, S., Meisenberg, C., Bibby, R.A., Askwith, T., Williams, G., Rininsland, F.H., Pearl, L.H., Oliver, A.W., El-Khamisy, S., Ward, S., et al.
(2014). Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Analytical biochemistry,
Qu, M., Rappas, M., Wardlaw, C.P., Garcia, V., Ren, J.-., Day, M., Carr, A.M., Oliver, A.W., Du, L.-. & Pearl, L.H., et al.
(2013). Phosphorylation-Dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1. Molecular cell,
Chambers, A.L., Pearl, L.H., Oliver, A.W. & Downs, J.A.
(2013). The BAH domain of Rsc2 is a histone H3 binding domain. Nucleic acids research,
Fugel, W., Oberholzer, A.E., Gschloessl, B., Dzikowski, R., Pressburger, N., Preu, L., Pearl, L.H., Baratte, B., Ratin, M., Okun, I., et al.
(2013). 3,6-Diamino-4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridine-5-carbonitriles Are Selective Inhibitors of Plasmodium falciparum Glycogen Synthase Kinase-3. Journal of medicinal chemistry,
Meier, C., Brookings, D.C., Ceska, T.A., Doyle, C., Gong, H., McMillan, D., Saville, G.P., Mushtaq, A., Knight, D., Reich, S., et al.
(2012). Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting. Journal of structural biology,
Spagnolo, L., Barbeau, J., Curtin, N.J., Morris, E.P. & Pearl, L.H.
(2012). Visualization of a DNA-PK/PARP1 complex. Nucleic acids research,
Ali, A.A., Timinszky, G., Arribas-Bosacoma, R., Kozlowski, M., Hassa, P.O., Hassler, M., Ladurner, A.G., Pearl, L.H. & Oliver, A.W.
(2012). The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks. Nature structural & molecular biology,
Tahtouh, T., Elkins, J.M., Filippakopoulos, P., Soundararajan, M., Burgy, G., Durieu, E., Cochet, C., Schmid, R.S., Lo, D.C., Delhommel, F., et al.
(2012). Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B. Journal of medicinal chemistry,
Rappas, M., Oliver, A.W. & Pearl, L.H.
(2011). Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1. Nucleic acids research,
Ali, M.M., Bagratuni, T., Davenport, E.L., Nowak, P.R., Silva-Santisteban, M.C., Hardcastle, A., McAndrews, C., Rowlands, M.G., Morgan, G.J., Aherne, W., et al.
(2011). Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response. Embo j,
Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1 alpha bound to ADP, revealing the 'phosphoryl-transfer' competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase 'active' conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1 alpha is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1 alpha as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies. The EMBO Journal (2011) 30, 894-905. doi: 10.1038/emboj.2011.18; Published online 11 February 2011.
Dekker, C., Roe, S.M., McCormack, E.A., Beuron, F., Pearl, L.H. & Willison, K.R.
(2011). The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. Embo j,
The cytosolic chaperonin CCT is a 1-MDa protein-folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell-cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8-membered rings stacked back-to-back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 angstrom resolution, revealing the subunit organisation and the location of discrete patches of co-evolving 'signature residues' that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP-binding heterogeneity and subunit-subunit interactions. The location of the evolutionarily conserved N-terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins. The EMBO Journal (2011) 30, 3078-3090. doi:10.1038/emboj.2011.208; Published online 24 June 2011.
Mollapour, M., Tsutsumi, S., Truman, A.W., Xu, W., Vaughan, C.K., Beebe, K., Konstantinova, A., Vourganti, S., Panaretou, B., Piper, P.W., et al.
(2011). Threonine 22 Phosphorylation Attenuates Hsp90 Interaction with Cochaperones and Affects Its Chaperone Activity. Molecular cell,
Maclagan, K., Tommasi, R., Laurine, E., Prodromou, C., Driscoll, P.C., Pearl, L.H., Reich, S. & Savva, R.
(2011). A combinatorial method to enable detailed investigation of protein–protein interactions. Future medicinal chemistry,
Morris, E.P., Rivera-Calzada, A., da Fonseca, P.C., Llorca, O., Pearl, L.H. & Spagnolo, L.
(2011). Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic acids res,
The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break..
Boos, D., Sanchez-Pulido, L., Rappas, M., Pearl, L.H., Oliver, A.W., Ponting, C.P. & Diffley, J.F.
(2011). Regulation of DNA Replication through Sld3-Dpb11 Interaction Is Conserved from Yeast to Humans. Current biology,
Garces, F., Pearl, L.H. & Oliver, A.W.
(2011). The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase. Molecular cell,
Millson, S.H., Chua, C.-., Roe, S.M., Polier, S., Solovieva, S., Pearl, L.H., Sim, T.-., Prodromou, C. & Piper, P.W.
(2011). Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90. The faseb journal,
Anderson, V.E., Walton, M.I., Eve, P.D., Boxall, K.J., Antoni, L., Caldwell, J.J., Aherne, W., Pearl, L.H., Oliver, A.W., Collins, I., et al.
(2011). CCT241533 Is a Potent and Selective Inhibitor of CHK2 that Potentiates the Cytotoxicity of PARP Inhibitors. Cancer res,
CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC50 of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 mu mol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors. Cancer Res; 71(2); 463-72. (C) 2011 AACR..
Day, J.E., Sharp, S.Y., Rowlands, M.G., Aherne, W., Hayes, A., Raynaud, F.I., Lewis, W., Roe, S.M., Prodromou, C., Pearl, L.H., et al.
(2011). Targeting the Hsp90 Molecular Chaperone with Novel Macrolactams Synthesis, Structural, Binding, and Cellular Studies. Acs chemical biology,
Cuomo, M.E., Platt, G.M., Pearl, L.H. & Mittnacht, S.
(2011). Cyclin-Cyclin-dependent Kinase Regulatory Response Is Linked to Substrate Recognition. J biol chem,
Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins..
Kumar, S., Hinks, J.A., Maman, J., Ravirajan, C.T., Pearl, L.H. & Isenberg, D.A.
(2011). p185, an Immunodominant Epitope, Is an Autoantigen Mimotope. Journal of biological chemistry,
Caldwell, J.J., Welsh, E.J., Matijssen, C., Anderson, V.E., Antoni, L., Boxall, K., Urban, F., Hayes, A., Raynaud, F.I., Rigoreau, L.J., et al.
(2011). Structure-Based Design of Potent and Selective 2-(Quinazolin-2-yl)phenol Inhibitors of Checkpoint Kinase 2. J med chem,
Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46 (IC50 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533)..
Mollapour, M., Tsutsumi, S., Donnelly, A.C., Beebe, K., Tokita, M.J., Lee, M.-., Lee, S., Morra, G., Bourboulia, D., Scroggins, B.T., et al.
(2010). Swe1Wee1-Dependent Tyrosine Phosphorylation of Hsp90 Regulates Distinct Facets of Chaperone Function. Molecular cell,
Rowlands, M., McAndrew, C., Prodromou, C., Pearl, L., Kalusa, A., Jones, K., Workman, P. & Aherne, W.
(2010). Detection of the ATPase Activity of the Molecular Chaperones Hsp90 and Hsp72 Using the Transcreener (TM) AdP Assay Kit. J biomol screen,
The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and atpase activity is essential for their function. inhibition of Hsp90 atpase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. several small-molecule inhibitors of the atpase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. a number of methods for the measurement of atpase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. the authors have evaluated the use of commercial reagents (Transcreener (TM) ADP) for the measurement of atpase activity of both yeast and human Hsp90 (ATP K-m similar to 500 mu M) and human Hsp72 (ATP K-m similar to 1 mu M). the low atpase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). the potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener (TM) reagents and compared well to that determined using other assay formats. (Journal of Biomolecular Screening 2010: 279-286).
Woodbine, L., Grigoriadou, S., Goodarzi, A.A., Riballo, E., Tape, C., Oliver, A.W., van Zelm, M.C., Buckland, M.S., Davies, E.G. & Pearl, L.H., et al.
(2010). An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity. Dna repair,
Zhang, M., Kadota, Y., Prodromou, C., Shirasu, K. & Pearl, L.H.
(2010). Structural Basis for Assembly of Hsp90-Sgt1-CHORD Protein Complexes: Implications for Chaperoning of NLR Innate Immunity Receptors. Molecular cell,
Hilton, S., Naud, S., Caldwell, J.J., Boxall, K., Burns, S., Anderson, V.E., Antoni, L., Allen, C.E., Pearl, L.H., Oliver, A.W., et al.
(2010). Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2. Bioorgan med chem,
5-(Hetero)aryl-3-(4-carboxamidophenyl)-2-aminopyridine inhibitors of CHK2 were identified from high throughput screening of a kinase-focussed compound library. Rapid exploration of the hits through straightforward chemistry established structure-activity relationships and a proposed ATP-competitive binding mode which was verified by X-ray crystallography of several analogues bound to CHK2. Variation of the 5-(hetero)aryl substituent identified bicyclic dioxolane and dioxane groups which improved the affinity and the selectivity of the compounds for CHK2 versus CHK1. The 3-(4-carboxamidophenyl) substituent could be successfully replaced by acyclic omega-aminoalkylamides, which made additional polar interactions within the binding site and led to more potent inhibitors of CHK2. Compounds from this series showed activity in cell-based mechanistic assays for inhibition of CHK2. (C) 2009 Elsevier Ltd. All rights reserved..
Day, J.E., Sharp, S.Y., Rowlands, M.G., Aherne, W., Lewis, W., Roe, S.M., Prodromou, C., Pearl, L.H., Workman, P. & Moody, C.J., et al.
(2010). Inhibition of Hsp90 with Resorcylic Acid Macrolactones: Synthesis and Binding Studies. Chem-eur j,
A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho-toluic acid dianion, esterification, followed by a ring-closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following de-protection of the two phenolic groups. Co-crystallization of one of the new macrolactones with the N-terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90..
Richardson, C.J., Gao, Q., Mitsopoulous, C., Zvelebil, M., Pearl, L.H. & Pearl, F.M.
(2009). MoKCa database - mutations of kinases in cancer. Nucleic acids res,
Members of the protein kinase family are amongst the most commonly mutated genes in human cancer, and both mutated and activated protein kinases have proved to be tractable targets for the development of new anticancer therapies The MoKCa database (Mutations of Kinases in Cancer, http://strubiol.icr.ac.uk/extra/mokca) has been developed to structurally and functionally annotate, and where possible predict, the phenotypic consequences of mutations in protein kinases implicated in cancer. Somatic mutation data from tumours and tumour cell lines have been mapped onto the crystal structures of the affected protein domains. Positions of the mutated amino-acids are highlighted on a sequence-based domain pictogram, as well as a 3D-image of the protein structure, and in a molecular graphics package, integrated for interactive viewing. The data associated with each mutation is presented in the Web interface, along with expert annotation of the detailed molecular functional implications of the mutation. Proteins are linked to functional annotation resources and are annotated with structural and functional features such as domains and phosphorylation sites. MoKCa aims to provide assessments available from multiple sources and algorithms for each potential cancer-associated mutation, and present these together in a consistent and coherent fashion to facilitate authoritative annotation by cancer biologists and structural biologists, directly involved in the generation and analysis of new mutational data..
Vaughan, C.K., Piper, P.W., Pearl, L.H. & Prodromou, C.
(2009). A common conformationally coupled ATPase mechanism for yeast and human cytoplasmic HSP90s. Febs journal,
Prodromou, C., Nuttall, J.M., Millson, S.H., Roe, S.M., Sim, T.S., Tan, D., Workman, P., Pearl, L.H. & Piper, P.W.
(2009). Structural Basis of the Radicicol Resistance Displayed by a Fungal Hsp90. Acs chem biol,
Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually tow binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself..
Ali, A.A., Jukes, R.M., Pearl, L.H. & Oliver, A.W.
(2009). Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. Nucleic acids research,
Doré, A.S., Kilkenny, M.L., Rzechorzek, N.J. & Pearl, L.H.
(2009). Crystal Structure of the Rad9-Rad1-Hus1 DNA Damage Checkpoint Complex—Implications for Clamp Loading and Regulation. Molecular cell,
Oliver, A.W., Swift, S., Lord, C.J., Ashworth, A. & Pearl, L.H.
(2009). Structural basis for recruitment of BRCA2 by PALB2. Embo rep,
The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy terminal beta-propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein protein interaction and explains the effects of both cancer associated truncating mutants in PALB2 and missense mutations in the amino terminal region of BRCA2..
Nilapwar, S., Williams, E., Fu, C., Prodromou, C., Pearl, L.H., Williams, M.A. & Ladbury, J.E.
(2009). Structural–Thermodynamic Relationships of Interactions in the N-Terminal ATP-Binding Domain of Hsp90. Journal of molecular biology,
Recuero-Checa, M.A., Doré, A.S., Arias-Palomo, E., Rivera-Calzada, A., Scheres, S.H., Maman, J.D., Pearl, L.H. & Llorca, O.
(2009). Electron microscopy of Xrcc4 and the DNA ligase IV–Xrcc4 DNA repair complex. Dna repair,
Bunney, T.D., Opaleye, O., Roe, S.M., Vatter, P., Baxendale, R.W., Walliser, C., Everett, K.L., Josephs, M.B., Christow, C., Rodrigues-Lima, F., et al.
(2009). Structural Insights into Formation of an Active Signaling Complex between Rac and Phospholipase C Gamma 2. Mol cell,
Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLC gamma(2)). Based on this structure, we illustrate distinct requirements for PLC gamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLC gamma(2), but not the related PLC beta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTP gamma S. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors..
Kilkenny, M.L., Dore, A.S., Roe, S.M., Nestoras, K., Ho, J.C., Watts, F.Z. & Pearl, L.H.
(2008). Structural and functional analysis of the Crb2-BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair. Genes & development,
Martin, C.J., Gaisser, S., Challis, I.R., Carletti, I., Wilkinson, B., Gregory, M., Prodromou, C., Roe, S.M., Pearl, L.H., Boyd, S.M., et al.
(2008). Molecular Characterization of Macbecin as an Hsp90 Inhibitor#. Journal of medicinal chemistry,
Pearl, L.H., Prodromou, C. & Workman, P.
(2008). The Hsp90 molecular chaperone: an open and shut case for treatment. Biochem j,
The molecular chaperone Hsp90 (90 kDa heat-shock protein) is a remarkably versatile protein involved in the stress response and in normal homoeostatic control mechanisms. It interacts with 'client proteins', including protein kinases, transcription factors and others, and either facilitates their stabilization and activation or directs them for proteasomal degradation. By this means, Hsp90 displays a multifaceted ability to influence signal transduction, chromatin remodelling and epigenetic regulation, development and morphological evolution. Hsp90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis at the N-terminus. The cycle is also regulated by a group of co-chaperones and accessory proteins. Here we review the biology of the Hsp90 molecular chaperone, emphasizing recent progress in our understanding of structure-function relationships and the identification of new client proteins. In addition we describe the exciting progress that has been made in the development of Hsp90 inhibitors, which are now showing promise in the clinic for cancer treatment. We also identify the gaps in our current understanding and highlight important topics for future research..
Eccles, S.A., Massey, A., Raynaud, F.I., Sharp, S.Y., Box, G., Valenti, M., Patterson, L., Brandon, A.D., Gowan, S., Boxall, F., et al.
(2008). NVP-AUY922: A novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis. Cancer res,
We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K-d = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1 alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials..
Millson, S.H., Vaughan, C.K., Zhai, C., Ali, M.M., Panaretou, B., Piper, P.W., Pearl, L.H. & Prodromou, C.
(2008). Chaperone ligand-discrimination by the TPR-domain protein Tah1. Biochemical journal,
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein–protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90α and Hsp90β proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins..
Pike, A.C., Rellos, P., Niesen, F.H., Turnbull, A., Oliver, A.W., Parker, S.A., Turk, B.E., Pearl, L.H. & Knapp, S.
(2008). Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites. The embo journal,
Zhang, M., Botër, M., Li, K., Kadota, Y., Panaretou, B., Prodromou, C., Shirasu, K. & Pearl, L.H.
(2008). Structural and functional coupling of Hsp90- and Sgt1-centred multi-protein complexes. The embo journal,
Brough, P.A., Aherne, W., Barril, X., Borgognoni, J., Boxall, K., Cansfield, J.E., Cheung, K.M., Collins, I., Davies, N.G., Drysdale, M.J., et al.
(2008). 4,5-diarylisoxazole HSP90 chaperone inhibitors: Potential therapeutic agents for the treatment of cancer. J med chem,
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI(50) averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by similar to 50%..
Zhang, M.-., Gaisser, S., Nur-E-Alam, M., Sheehan, L.S., Vousden, W.A., Gaitatzis, N., Peck, G., Coates, N.J., Moss, S.J., Radzom, M., et al.
(2008). Optimizing Natural Products by Biosynthetic Engineering: Discovery of Nonquinone Hsp90 Inhibitors†. Journal of medicinal chemistry,
Vaughan, C.K., Mollapour, M., Smith, J.R., Truman, A., Hu, B., Good, V.M., Panaretou, B., Neckers, L., Clarke, P.A., Workman, P., et al.
(2008). Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol cell,
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Serl 3, but little is known about the regulation of this essential posttranslational modification. We show that Serl 3 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSerl 3-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Pptl), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Pptl associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Pptl regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes..
Sharp, S.Y., Boxall, K., Rowlands, M., Prodromou, C., Roe, S.M., Maloney, A., Powers, M., Clarke, P.A., Box, G., Sanderson, S., et al.
(2007). In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors. Cancer res,
The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90 beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH2-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mu mol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development..
Rivera-Calzada, A., Spagnolo, L., Pearl, L.H. & Llorca, O.
(2007). Structural model of full-length human Ku70–Ku80 heterodimer and its recognition of DNA and DNA-PKcs. Embo reports,
Sharp, S.Y., Prodromou, C., Boxall, K., Powers, M.V., Holmes, J.L., Box, G., Matthews, T.P., Cheung, K.M., Kalusa, A., Janmes, K., et al.
(2007). Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues. Mol cancer ther,
Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K-d) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC50 of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors..
Oliver, A.W., Knapp, S. & Pearl, L.H.
(2007). Activation segment exchange: a common mechanism of kinase autophosphorylation?. Trends in biochemical sciences,
Hassler, M., Singh, S., Yue, W.W., Luczynski, M., Lakbir, R., Sanchez-Sanchez, F., Bader, T., Pearl, L.H. & Mittnacht, S.
(2007). Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture. Mol cell,
The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell-cycle progression via interactions involving the central "pocket" and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved and harbors missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a globular entity formed by two rigidly connected cyclin-like folds. The similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation..
Yue, W.W., Hassler, M., Roe, S.M., Thompson-Vale, V. & Pearl, L.H.
(2007). Insights into histone code syntax from structural and biochemical studies of CARM1 methyltransferase. The embo journal,
Ali, M.M., Roe, S.M., Vaughan, C.K., Meyer, P., Panaretou, B., Piper, P.W., Prodromou, C. & Pearl, L.H.
(2006). Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex. Nature,
Spagnolo, L., Rivera-Calzada, A., Pearl, L.H. & Llorca, O.
(2006). Three-Dimensional Structure of the Human DNA-PKcs/Ku70/Ku80 Complex Assembled on DNA and Its Implications for DNA DSB Repair. Molecular cell,
Jennings, B.H., Pickles, L.M., Wainwright, S.M., Roe, S.M., Pearl, L.H. & Ish-Horowicz, D.
(2006). Molecular Recognition of Transcriptional Repressor Motifs by the WD Domain of the Groucho/TLE Corepressor. Molecular cell,
Doré, A.S., Kilkenny, M.L., Jones, S.A., Oliver, A.W., Roe, S.M., Bell, S.D. & Pearl, L.H.
(2006). Structure of an archaeal PCNA1–PCNA2–FEN1 complex: elucidating PCNA subunit and client enzyme specificity. Nucleic acids research,
Vaughan, C.K., Gohlke, U., Sobott, F., Good, V.M., Ali, M.M., Prodromou, C., Robinson, C.V., Saibil, H.R. & Pearl, L.H.
(2006). Structure of an Hsp90-Cdc37-Cdk4 Complex. Molecular cell,
Reich, S., Puckey, L.H., Cheetham, C.L., Harris, R., Ali, A.A., Bhattacharyya, U., Maclagan, K., Powell, K.A., Prodromou, C., Pearl, L.H., et al.
(2006). Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications. Protein science,
Proisy, N., Sharp, S.Y., Boxall, K., Connelly, S., Roe, S.M., Prodromou, C., Slawin, A.M., Pearl, L.H., Workman, P. & Moody, C.J., et al.
(2006). Inhibition of Hsp90 with synthesis macrolactones: Synthesis and structural and biological evaluation of ring and conformational analogs of radicicol. Chem biol,
A series of benzo-macrolactones of varying ring size and conformation has been prepared by chemical synthesis and evaluated by structural and biological techniques. Thus, 12- to 16-membered lactones were obtained by concise routes, involving ring-closing metathesis as a key step. In enzyme assays, the 13-, 15-, and 16-membered analogs are good inhibitors, suggesting that they can adoptthe required conformation to fit in the ATP-binding site. This was confirmed by cocrystallization of 13-, 14-, and 15-membered lactones with the N-terminal domain of yeast Hsp90, showing that they bind similarly to the "natural" 14-membered radicicol. The most active compounds in the ATPase assays also showed the greatest growth-inhibitory potency in HCT116 human colon cancer cells and the established molecular signature of Hsp90 inhibition, i.e., depletion of client proteins with upregulation of Hsp70..
Truman, A.W., Millson, S.H., Nuttall, J.M., King, V., Mollapour, M., Prodromou, C., Pearl, L.H. & Piper, P.W.
(2006). Expressed in the YeastSaccharomyces cerevisiae, Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase. Eukaryotic cell,
ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in theslt2ΔSaccharomyces cerevisiaemutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of theslt2Δ yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5..
Oliver, A.W., Paul, A., Boxall, K.J., Barrie, S.E., Aherne, G.W., Garrett, M.D., Mittnacht, S. & Pearl, L.H.
(2006). Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange. Embo j,
The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation..
Bunney, T.D., Harris, R., Gandarillas, N.L., Josephs, M.B., Roe, S.M., Sorli, S.C., Paterson, H.F., Rodrigues-Lima, F., Esposito, D., Ponting, C.P., et al.
(2006). Structural and mechanistic insights into ras association domains of phospholipase C epsilon. Mol cell,
Ras proteins signal to a number of distinct pathways by interacting with diverse effectors. Studies of ras/effect or interactions have focused on three classes, Raf kinases, ral guanylnucleotide-exchange factors, and phosphatidylinositol-3-kinases. Here we describe ras interactions with another effector, the recently identified phospholipase C epsilon (PLC epsilon). We solved structures of PLC epsilon RA domains (RA1 and RA2) by NMR and the structure of the RA2/ras complex by X-ray crystallography. Although the similarity between ubiqultin-like folds of RA1 and RA2 proves that they are homologs, only RA2 can bind ras. Some of the features of the RA2/ras interface are unique to PLC epsilon, while the ability to make contacts with both switch I and II regions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLC epsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLC epsilon, has a role in membrane targeting with further regulatory impact on PLC activity..
Ioannou, Y., Giles, I., Lambrianides, A., Richardson, C., Pearl, L., Latchman, D., Isenberg, D. & Rahman, A.
(2006). . Bmc biotechnology,
Kumar, S., Nagl, S., Kalsi, J.K., Ravirajan, C.T., Athwal, D., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2005). Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody. Molecular immunology,
Oliver, A.W., Jones, S.A., Roe, S.M., Matthews, S., Goodwin, G.H. & Pearl, L.H.
(2005). Crystal structure of the proximal BAH domain of the polybromo protein. Biochemical journal,
The BAH domain (bromo-associated homology domain) was first identified from a repeated motif found in the nuclear protein polybromo – a large (187 kDa) modular protein comprising six bromodomains, two BAH domains and an HMG box. To date, the BAH domain has no ascribed function, although it is found in a wide range of proteins that contain additional domains involved in either transcriptional regulation (e.g. SET, PHD and bromodomain) and/or DNA binding (HMG box and AT hook). The molecular function of polybromo itself also remains unclear, but it has been identified as a key component of an SWI/SNF (switching/sucrose non-fermenting)-related, ATP-dependent chromatin-remodelling complex PBAF (polybromo, BRG1-associated factors; also known as SWI/SNF-B or SWI/SNFβ). We present in this paper the crystal structure of the proximal BAH domain from chicken polybromo (BAH1), at a resolution of 1.6 Å (1 Å=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein–protein interactions..
(2005). Hsp90 and Cdc37 – a chaperone cancer conspiracy. Current opinion in genetics & development,
Rivera-Calzada, A., Maman, J.P., Spagnolo, L., Pearl, L.H. & Llorca, O.
(2005). Three-Dimensional Structure and Regulation of the DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs). Structure,
Kumar, S., Bunting, K.A., Kalsi, J., Hinks, J.A., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2005). Lupus autoantibodies to native DNA preferentially bind DNA presented on PolIV. Immunology,
Hu, B., Liao, C., Millson, S.H., Mollapour, M., Prodromou, C., Pearl, L.H., Piper, P.W. & Panaretou, B.
(2005). Qri2/Nse4, a component of the essential Smc5/6 DNA repair complex. Molecular microbiology,
Millson, S.H., Truman, A.W., King, V., Prodromou, C., Pearl, L.H. & Piper, P.W.
(2005). A Two-Hybrid Screen of the Yeast Proteome for Hsp90 Interactors Uncovers a Novel Hsp90 Chaperone Requirement in the Activity of a Stress-Activated Mitogen-Activated Protein Kinase, Slt2p (Mpk1p). Eukaryotic cell,
The Hsp90 chaperone cycle catalyzes the final activation step of several important eukaryotic proteins (Hsp90 “clients”). Although largely a functional form of Hsp90, an Hsp90-Gal4p DNA binding domain fusion (Hsp90-BD) displays no strong interactions in the yeast two-hybrid system, consistent with a general transience of most Hsp90-client associations. Strong in vivo interactions are though detected when the E33A mutation is introduced into this bait, a mutation that should arrest Hsp90-client complexes at a stage where the client is stabilized, yet prevented from attaining its active form. This E33A mutation stabilized the two-hybrid interactions of the Hsp90-BD fusion with ∼3% of theSaccharomyces cerevisiaeproteome in a screen of the 6,000 yeast proteins expressed as fusions to the Gal4p activation domain (AD). Among the detected interactors were the two stress-activated mitogen-activated protein (MAP) kinases of yeast, Hog1p and Slt2p (Mpk1p). Column retention experiments using wild-type and mutant forms of Hsp90 and Slt2p MAP kinase, as well as quantitative measurements of the effects of stress on the two-hybrid interaction of mutant Hsp90-BD and AD-Slt2p fusions, revealed that Hsp90 binds exclusively to the dually Thr/Tyr-phosphorylated, stress-activated form of Slt2p [(Y-P,T-P)Slt2p] and also to the MAP kinase domain within this (Y-P,T-P)Slt2p. Phenotypic analysis of a yeast mutant that expresses a mutant Hsp90 (T22Ihsp82) revealed that Hsp90 function is essential for this (Y-P,T-P)Slt2p to activate one of its downstream targets, the Rlm1p transcription factor. The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p..
Zhang, M., Windheim, M., Roe, S.M., Peggie, M., Cohen, P., Prodromou, C. & Pearl, L.H.
(2005). Chaperoned Ubiquitylation—Crystal Structures of the CHIP U Box E3 Ubiquitin Ligase and a CHIP-Ubc13-Uev1a Complex. Molecular cell,
Cheung, K.M., Matthews, T.P., James, K., Rowlands, M.G., Boxall, K.J., Sharp, S.Y., Maloney, A., Roe, S.M., Prodromou, C., Pearl, L.H., et al.
(2005). The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors. Bioorg med chem lett,
High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 mu M) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90. (c) 2005 Elsevier Ltd. All rights reserved..
Roe, S.M., Ali, M.M., Meyer, P., Vaughan, C.K., Panaretou, B., Piper, P.W., Prodromou, C. & Pearl, L.H.
(2004). The Mechanism of Hsp90 Regulation by the Protein Kinase-Specific Cochaperone p50cdc37. Cell,
Harris, R., Esposito, D., Sankar, A., Maman, J.D., Hinks, J.A., Pearl, L.H. & Driscoll, P.C.
(2004). The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR). Journal of molecular biology,
Polychronopoulos, P., Magiatis, P., Skaltsounis, A.-., Myrianthopoulos, V., Mikros, E., Tarricone, A., Musacchio, A., Roe, S.M., Pearl, L., Leost, M., et al.
(2004). Structural Basis for the Synthesis of Indirubins as Potent and Selective Inhibitors of Glycogen Synthase Kinase-3 and Cyclin-Dependent Kinases. Journal of medicinal chemistry,
Kumar, S., Kalsi, J., Bunting, K., Ravirajan, C.T., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2004). Fine binding characteristics of human autoantibodies—partial molecular characterization. Molecular immunology,
Siligardi, G., Hu, B., Panaretou, B., Piper, P.W., Pearl, L.H. & Prodromou, C.
(2004). Co-chaperone Regulation of Conformational Switching in the Hsp90 ATPase Cycle. Journal of biological chemistry,
Meyer, P., Prodromou, C., Liao, C., Hu, B., Mark Roe, S., Vaughan, C.K., Vlasic, I., Panaretou, B., Piper, P.W. & Pearl, L.H., et al.
(2004). Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. The embo journal,
Llorca, O. & Pearl, L.H.
(2004). Electron microscopy studies on DNA recognition by DNA-PK. Micron,
Rowlands, M.G., Newbatt, Y.M., Prodromou, C., Pearl, L.H., Workman, P. & Aherne, W.
(2004). High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal biochem,
The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The K-m for ATP determined in the assay was 510 +/- 70 muM. The known HSP90 inhibitors geldanamycin and radicicol gave IC50 values of 4.8 and 0.9 muM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of similar to56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8. (C) 2003 Elsevier Inc. All rights reserved..
(2004). Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2. Nucleic acids research,
(2004). Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. The embo journal,
Millson, S.H., Truman, A.W., Wolfram, F., King, V., Panaretou, B., Prodromou, C., Pearl, L.H. & Piper, P.W.
(2004). Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach. Cell stress & chaperones,
Hinks, J.A., Roe, M., Ho, J.C., Watts, F.Z., Phelan, J., McAllister, M. & Pearl, L.H.
(2003). Expression, purification and preliminary X-ray analysis of the BRCT domain from Rhp9/Crb2. Acta crystallographica section d biological crystallography,
Prodromou, C. & Pearl, L.
(2003). Structure and Functional Relationships of Hsp90. Current cancer drug targets,
Piper, P.W., Panaretou, B., Millson, S.H., Trumana, A., Mollapour, M., Pearl, L.H. & Prodromou, C.
(2003). Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90β, a property that can be exploited in screens for new Hsp90 chaperone inhibitors. Gene,
(2003). Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the -clamp. The embo journal,
Meyer, P., Prodromou, C., Hu, B., Vaughan, C., Roe, S.M., Panaretou, B., Piper, P.W. & Pearl, L.H.
(2003). Structural and Functional Analysis of the Middle Segment of Hsp90: Implications for ATP Hydrolysis and Client Protein and Cochaperone Interactions. Molecular cell,
(2003). Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. The embo journal,
(2003). Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs. The embo journal,
Wibley, J.E., Waters, T.R., Haushalter, K., Verdine, G.L. & Pearl, L.H.
(2003). Structure and Specificity of the Vertebrate Anti-Mutator Uracil-DNA Glycosylase SMUG1. Molecular cell,
Zhang, X., Mark Roe, S., Hou, Y., Bartlam, M., Rao, Z., Pearl, L.H. & Danpure, C.J.
(2003). Crystal Structure of Alanine:Glyoxylate Aminotransferase and the Relationship Between Genotype and Enzymatic Phenotype in Primary Hyperoxaluria Type 1. Journal of molecular biology,
Kumar, S., Nagl, S., Kalsi, J.K., Ravirajan, C.T., Athwal, D., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2003). Anti-cardiolipin/β-2 glycoprotein activities co-exist on human anti-DNA antibody light chains. Molecular immunology,
Meijer, L., Skaltsounis, A.-., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X.P., Vonica, C.A., Brivanlou, A., Dajani, R., et al.
(2003). GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins. Chemistry & biology,
(2003). Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix. Nucleic acids research,
Ryves, W.J., Dajani, R., Pearl, L. & Harwood, A.J.
(2002). Glycogen Synthase Kinase-3 Inhibition by Lithium and Beryllium Suggests the Presence of Two Magnesium Binding Sites. Biochemical and biophysical research communications,
Bjørås, M., Seeberg, E., Luna, L., Pearl, L.H. & Barrett, T.E.
(2002). Reciprocal “flipping” underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase. Journal of molecular biology,
Pappenberger, G., Wilsher, J.A., Mark Roe, S., Counsell, D.J., Willison, K.R. & Pearl, L.H.
(2002). Crystal Structure of the CCTγ Apical Domain: Implications for Substrate Binding to the Eukaryotic Cytosolic Chaperonin. Journal of molecular biology,
Pickles, L.M., Roe, S.M., Hemingway, E.J., Stifani, S. & Pearl, L.H.
(2002). Crystal Structure of the C-Terminal WD40 Repeat Domain of the Human Groucho/TLE1 Transcriptional Corepressor. Structure,
Siligardi, G., Panaretou, B., Meyer, P., Singh, S., Woolfson, D.N., Piper, P.W., Pearl, L.H. & Prodromou, C.
(2002). Regulation of Hsp90 ATPase Activity by the Co-chaperone Cdc37p/p50cdc37. Journal of biological chemistry,
Hinks, J.A., Evans, M.C., de Miguel, Y., Sartori, A.A., Jiricny, J. & Pearl, L.H.
(2002). An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases. Journal of biological chemistry,
Pearl, L.H. & Barford, D.
(2002). Regulation of protein kinases in insulin, growth factor and Wnt signalling. Curr opin struc biol,
Protein kinase cascades provide the regulatory mechanisms for many of the essential processes in eukaryotic cells. Recent structural and biochemical work has revealed the basis of phosphorylation regulation of three consecutive protein kinases - phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB)/Akt and glycogen synthase kinase 3beta (GSK3beta) which transduce signals generated by insulin and/or growth factors binding to cell surface receptors. PDK1 and PKB are both AGC family kinases. Whereas PKB is positively regulated via its phosphorylated C-terminal hydrophobic motif, the activity and specificity of PDK1 are determined by equivalent hydrophobic motifs of substrate AGC kinases. In a contrasting mechanism, GSK3beta is negatively regulated by competitive autoinhibition by its phosphorylated N terminus. GSK3beta also functions in the developmental Wnt signalling pathway, but without cross-talk with the PDK1-PKB/Akt pathway. Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways..
Panaretou, B., Siligardi, G., Meyer, P., Maloney, A., Sullivan, J.K., Singh, S., Millson, S.H., Clarke, P.A., Naaby-Hansen, S., Stein, R., et al.
(2002). Activation of the ATPase activity of Hsp90 by the stress-regulated cochaperone Aha1. Mol cell,
Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle..
Kumar, S., Kalsi, J.K., Ravirajan, C.T., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2002). Molecular expression systems for anti-DNA antibodies—2. Lupus,
Fogg, M.J., Pearl, L.H. & Connolly, B.A.
(2002). Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature structural biology,
Fraser, E., Young, N., Dajani, R., Franca-Koh, J., Ryves, J., Williams, R.S., Yeo, M., Webster, M.T., Richardson, C., Smalley, M.J., et al.
(2002). Identification of the Axin and Frat binding region of glycogen synthase kinase-3. J biol chem,
Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression..
Salek, R.M., Williams, M.A., Prodromou, C., Pearl, L.H. & Ladbury, J.E.
(2002). . Journal of biomolecular nmr,
Harris, R., Maman, J.D., Hinks, J.A., Sankar, A., Pearl, L.H. & Driscoll, P.C.
(2002). . Journal of biomolecular nmr,
Zhang, X., Roe, S.M., Pearl, L.H. & Danpure, C.J.
(2001). Crystallization and preliminary crystallographic analysis of human alanine:glyoxylate aminotransferase and its polymorphic variants. Acta crystallographica section d biological crystallography,
(2001). Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2. The embo journal,
Kumar, S., Kalsi, J., Latchman, D.S., Pearl, L.H. & Isenberg, D.A.
(2001). Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity. Journal of molecular biology,
Dajani, R., Fraser, E., Roe, S.M., Young, N., Good, V., Dale, T.C. & Pearl, L.H.
(2001). Crystal Structure of Glycogen Synthase Kinase 3β. Cell,
TAN, T.H., EDGERTON, S.A., KUMARI, R., McALISTER, M.S., ROWE, S.M., NAGL, S., PEARL, L.H., SELKIRK, M.E., BIANCO, A.E., TOTTY, N.F., et al.
(2001). Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis. Biochemical journal,
(2000). Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus. Nucleic acids research,
O'Hara, B.P., Wilson, S.A., Lee, A.W., Roe, S.M., Siligardi, G., Drew, R.E. & Pearl, L.H.
(2000). Structural adaptation to selective pressure for altered ligand specificity in the Pseudomonas aeruginosa amide receptor, AmiC. Protein engineering, design and selection,
Pearl, L.H. & Prodromou, C.
(2000). Structure and in vivo function of Hsp90. Current opinion in structural biology,
(2000). Structure and function in the uracil-DNA glycosylase superfamily. Mutation research/dna repair,
Kumar, S., Kalsi, J., Ravirajan, C.T., Rahman, A., Athwal, D., Latchman, D.S., Isenberg, D.A. & Pearl, L.H.
(2000). Molecular Cloning and Expression of the Fabs of Human Autoantibodies inEscherichia coli. Journal of biological chemistry,
Rösler, A., Panayotou, G., Hornby, D.P., Barlow, T., Brown, T., Pearl, L.H., Savva, R. & Blackburn, G.M.
(2000). The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues. Nucleosides, nucleotides and nucleic acids,
(2000). The ATPase cycle of Hsp90 drives a molecular clamp' via transient dimerization of the N-terminal domains. The embo journal,
Norman, R.A., Poh, C.L., Pearl, L.H., O'Hara, B.P. & Drew, R.E.
(2000). Steric Hindrance Regulation of thePseudomonas aeruginosaAmidase Operon. Journal of biological chemistry,
(1999). Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex. The embo journal,
Panaretou, B., Pearl, L., Piper, P.W., Prodromou, C., Johal, J. & Sinclair, K.
(1999). The Hsp90 of Candida albicans can confer Hsp90 functions in Saccharomyces cerevisiae: a potential model for the processes that generate immunogenic fragments of this molecular chaperone in C albicans infections. Microbiology,
(1999). Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. The embo journal,
Roe, S.M., Prodromou, C., O'Brien, R., Ladbury, J.E., Piper, P.W. & Pearl, L.H.
(1999). Structural Basis for Inhibition of the Hsp90 Molecular Chaperone by the Antitumor Antibiotics Radicicol and Geldanamycin. Journal of medicinal chemistry,
(1999). Crystal structure of a thwarted mismatch glycosylase DNA repair complex. The embo journal,
Greagg, M.A., Fogg, M.J., Panayotou, G., Evans, S.J., Connolly, B.A. & Pearl, L.H.
(1999). A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. Proceedings of the national academy of sciences,
Barrett, T.E., Savva, R., Panayotou, G., Barlow, T., Brown, T., Jiricny, J. & Pearl, L.H.
(1998). Crystal Structure of a G:T/U Mismatch-Specific DNA Glycosylase. Cell,
Panayotou, G., Brown, T., Barlow, T., Pearl, L.H. & Savva, R.
(1998). Direct Measurement of the Substrate Preference of Uracil-DNA Glycosylase. Journal of biological chemistry,
Barrett, T.E., Savva, R., Barlow, T., Brown, T., Jiricny, J. & Pearl, L.H.
(1998). Structure of a DNA base-excision product resembling a cisplatin inter-strand adduct. Nature structural & molecular biology,
Roe, S.M., Barlow, T., Brown, T., Oram, M., Keeley, A., Tsaneva, I.R. & Pearl, L.H.
(1998). Crystal Structure of an Octameric RuvA–Holliday Junction Complex. Molecular cell,
(1998). ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone invivo. The embo journal,
Prodromou, C., Roe, S.M., Piper, P.W. & Pearl, L.H.
(1997). A molecular clamp in the crystal structure of the N-terminal domain of the yeast Hsp90 chaperone. Nature structural biology,
Chamberlain, D., O'Hara, B.P., Wilson, S.A., Pearl, L.H. & Perkins, S.J.
(1997). Oligomerization of the Amide Sensor Protein AmiC by X-ray and Neutron Scattering and Molecular Modeling. Biochemistry,
Prodromou, C., Roe, S.M., O'Brien, R., Ladbury, J.E., Piper, P.W. & Pearl, L.H.
(1997). Identification and Structural Characterization of the ATP/ADP-Binding Site in the Hsp90 Molecular Chaperone. Cell,
Aguilar, C.F., Sanderson, I., Moracci, M., Ciaramella, M., Nucci, R., Rossi, M. & Pearl, L.H.
(1997). Crystal structure of the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability. Journal of molecular biology,
Wheeler, V.C., Prodromou, C., Pearl, L.H., Williamson, R. & Coutelle, C.
(1996). Synthesis of a modified gene encoding human ornithine transcarbamylase for expression in mammalian mitochondrial and universal translation systems: a novel approach towards correction of a genetic defect. Gene,
Jones, D.T., Moody, C.M., Uppenbrink, J., Viles, J.H., Doyle, P.M., Harris, C.J., Pearl, L.H., Sadler, P.J. & Thornton, J.M.
(1996). Towards meeting the paracelsus challenge: The design, synthesis, and characterization of paracelsin-43, an α-helical protein with over 50% sequence identity to an all-β protein. Proteins: structure, function, and genetics,
Pearl, L.H. & Savva, R.
(1996). The problem with pyrimidines. Nature structural & molecular biology,
Prodromou, C., Piper, P.W. & Pearl, L.H.
(1996). Expression and crystallization of the yeast Hsp82 chaperone, and preliminary x-ray diffraction studies of the amino-terminal domain. Proteins: structure, function, and genetics,
Koulis, A., Cowan, D.A., Pearl, L.H. & Savva, R.
(1996). Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms. Fems microbiology letters,
Wilson, S.A., Wachira, S.J., Norman, R.A., Pearl, L.H. & Drew, R.E.
(1996). Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon. The embo journal,
Danson, M.J., Hough, D.W., Russell, R.J., Taylor, G.L. & Pearl, L.
(1996). Enzyme thermostability and thermoactivity. "protein engineering, design and selection",
Savva, R., McAuley-Hecht, K., Brown, T. & Pearl, L.
(1995). The structural basis of specific base-excision repair by uracil–DNA glycosylase. Nature,
Savva, R. & Pearl, L.H.
(1995). Cloning and expression of the uracil-DNA glycosylase inhibitor (UGI) from bacteriophage PBS-1 and crystallization of a uracil-DNA glycosylase-UGI complex. Proteins: structure, function, and genetics,
Savva, R. & Pearl, L.H.
(1995). Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase–uracil glycosylase inhibitor protein complex. Nature structural & molecular biology,
Pearl, L.H. & Savva, R.
(1995). DNA repair in three dimensions. Trends in biochemical sciences,
O'Hara, B.P., Hemmings, A.M., Buttle, D.J. & Pearl, L.H.
(1995). Crystal Structure of Glycyl Endopeptidase from Carica papaya: A Cysteine Endopeptidase of Unusual Substrate Specificity. Biochemistry,
Wilson, S.A., Williams, R.J., Pearl, L.H. & Drew, R.E.
(1995). Identification of Two New Genes in thePseudomonasaeruginosaAmidase Operon, Encoding an ATPase (AmiB) and a Putative Integral Membrane Protein (AmiS). Journal of biological chemistry,
Pearl, L., O'Hara, B., Drew, R. & Wilson, S.
(1994). Crystal structure of AmiC: the controller of transcription antitermination in the amidase operon of Pseudomonas aeruginosa. The embo journal,
Pearl, L.H., Hemmings, A.M., Nucci, R. & Rossi, M.
(1993). Crystallization and Preliminary X-ray Analysis of the β-Galactosidase from the Extreme Thermophilic Archaebacterium Sulfolobus solfataricus. Journal of molecular biology,
Pearl, L.H., Demasi, D., Hemmings, A.M., Sica, F., Mazzarella, L., Raia, C.A., D' Auria, S. & Rossi, M.
(1993). Crystallization and Preliminary X-ray Analysis of an NAD+ -dependent Alcohol Dehydrogenase from the Extreme Thermophilic Archaebacterium Sulfolobus solfataricus. Journal of molecular biology,
(1993). Similarity of active-site structures. Nature,
Swindells, M.B., Orengo, C.A., Jones, D.T., Pearl, L.H. & Thornton, J.M.
(1993). Recurrence of a binding motif?. Nature,
Wilson, S.A., Wachira, S.J., Drew, R.E., Jones, D. & Pearl, L.H.
(1993). Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein. The embo journal,
Savva, R. & Pearl, L.H.
(1993). Crystallization and Preliminary X-ray Analysis of the Uracil-DNA Glycosylase DNA Repair Enzyme from Herpes Simplex Virus Type 1. Journal of molecular biology,
RAWLINGS, N.D., PEARL, L.H. & BUTTLE, D.J.
(1992). The BaculovirusAutographa californicaNuclear Polyhedrosis Virus Genome Includes a Papain-Like Sequence. Biological chemistry hoppe-seyler,
Prodromou, C. & Pearl, L.H.
(1992). Recursive PCR: a novel technique for total gene synthesis. "protein engineering, design and selection",
Cruzeiro-Hansson, L., Swann, P.F., Pearl, L. & Goodfellow, J.M.
(1992). Molecular dynamics of alkylated DNA. Carcinogenesis,
Wilson, S.A., Chayen, N.E., Hemmings, A.M., Drew, R.E. & Pearl, L.H.
(1991). Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa. Journal of molecular biology,
Blundell, T.L., Jenkins, J.A., Sewell, B.T., Pearl, L.H., Cooper, J.B., Tickle, I.J., Veerapandian, B. & Wood, S.P.
(1990). X-ray analyses of aspartic proteinases. Journal of molecular biology,
Moelling, K., Schulze, T., Knoop, M.-., Kay, J., Jupp, R., Nicolaou, G. & Pearl, L.H.
(1990). In vitro inhibition of HIV-1 proteinase by cerulenin. Febs letters,
Buttle, D.J., Ritonja, A., Pearl, L.H., Turk, V. & Barrett, A.J.
(1990). Selective cleavage of glycyl bonds by papaya proteinase IV. Febs letters,
Blundell, T. & Pearl, L.
(1989). A second front against AIDS. Nature,
McKeating, J.A., Gow, J., Goudsmit, J., Pearl, L.H., Mulder, C. & Weiss, R.A.
(1989). Characterization of HIV-1 neutralization escape mutants. Aids,
Neidle, S., Pearl, L.H., Herzyk, P. & Berman, H.M.
(1988). A molecular model for proflavine--DNA intercalation. Nucleic acids research,
Neidle, S., Pearl, L.H. & Skelly, J.V.
(1987). DNA structure and perturbation by drug binding. Biochemical journal,
Foundling, S.I., Cooper, J., Watson, F.E., Cleasby, A., Pearl, L.H., Sibanda, B.L., Hemmings, A., Wood, S.P., Blundell, T.L., Valler, M.J., et al.
(1987). High resolution X-ray analyses of renin inhibitor-aspartic proteinase complexes. Nature,
(1987). The catalytic mechanism of aspartic proteinases. Febs letters,
PEAR, L.H. & TAYLO, W.R.
(1987). Sequence specificity of retroviral proteases. Nature,
Pearl, L.H. & Taylor, W.R.
(1987). A structural model for the retroviral proteases. Nature,
Ford, K.G., Pearl, L.H. & Neidle, S.
(1987). Molecular modelling of the interactions of tetra-(4-N-methyIpyridyI) porphin with TA and CG sites on DNA. Nucleic acids research,
Pearl, L.H., Skelly, J.V., Hudson, B.D. & Neidle, S.
(1987). The crystal structure of the DNA-binding drug berenil: molecular modelling studies of berenil-DNA complexes. Nucleic acids research,
Pearl, L.H. & Neidle, S.
(1986). Origins of stereospecificity in DNA damage by anti
]pyrene diol-epoxides. Febs letters,
HEMMINGS, A.M., FOUNDLING, S.I., SIBANDA, B.L., WOOD, S.P., PEARL, L.H. & BLUNDELL, T.O.
(1985). Energy calculations on aspartic proteinases: human renin, endothiapepsin and its complex with an angiotensinogen fragment analogue, H-142. Biochemical society transactions,
Pearl, L. & Blundell, T.
(1984). The active site of aspartic proteinases. Febs letters,
Blundell, T., Sibanda, B.L. & Pearl, L.
(1983). Three-dimensional structure, specificity and catalytic mechanism of renin. Nature,
Polier, S., Samant, R.S., Clarke, P.A., Workman, P., Prodromou, C. & Pearl, L.H.
ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system. Nat chem biol.,
Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation..
Elliott, R.J., Jarvis, A., Rajasekaran, M.B., Menon, M., Bowers, L., Boffey, R., Bayford, M., Firth-Clark, S., Key, R., Aqil, R., et al.
Design and discovery of 3-aryl-5-substituted-isoquinolin-1-ones as potent tankyrase inhibitors. Medchemcomm,
The tankyrase proteins (TNKS, TNKS2) are attractive anti-cancer drug targets, particularly as inhibition of their catalytic activity has been shown to antagonise oncogenic WNT signalling.
Peters, N.E., Ferguson, B.J., Mazzon, M., Fahy, A.S., Krysztofinska, E., Arribas-Bosacoma, R., Pearl, L.H., Ren, H. & Smith, G.L.
A Mechanism for the Inhibition of DNA-PK-Mediated DNA Sensing by a Virus. Plos pathogens,
Piper, P.W., Millson, S.H., Mollapour, M., Panaretou, B., Siligardi, G., Pearl, L.H. & Prodromou, C.
Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast. European journal of biochemistry,
Hawkins, T.A., Haramis, A.-., Etard, C., Prodromou, C., Vaughan, C.K., Ashworth, R., Ray, S., Behra, M., Holder, N., Talbot, W.S., et al.
The ATPase-dependent chaperoning activity of Hsp90a regulates thick filament formation and integration during skeletal muscle myofibrillogenesis. Development,
Bigot, N., Day, M., Baldock, R.A., Watts, F.Z., Oliver, A.W. & Pearl, L.H.
Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint. Elife,
Coordination of the cellular response to DNA damage is organised by multi-domain ‘scaffold’ proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint..
Aherne, W., Maloney, A., Prodromou, C., Rowlands, M.G., Hardcastle, A., Boxall, K., Clarke, P., Walton, M.I. & Pearl, L.
Assays for HSP90 and Inhibitors. ,
Pearl, L.H., Bigot, N., Day, M., Baldock, R.A., Watts, F.Z. & Oliver, A.
Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint. ,
Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint..
Day, M., Rappas, M., Ptasińska, K., Boos, D., Oliver, A.W. & Pearl, L.H.
N-terminal BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands. ,
TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which have the capacity to bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 in complex with diverse phospho-ligands – which include human RAD9 and Treslin, as well as S.pombe Crb2 and Sld3 – to define the key determinants of BRCT domain specificity. We use this information to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins..