Darby, J.F.
Vidler, L.R.
Simpson, P.J.
Al-Lazikani, B.
Matthews, S.J.
Sharp, S.Y.
Pearl, L.H.
Hoelder, S.
Workman, P.
(2020). Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches. Scientific reports,
Vol.10
(1),
pp. 16000-?.
show abstract
Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic 'client' proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native 'carboxylate clamp' interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1 H- 15 N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors..
Polo, L.M.
Xu, Y.
Hornyak, P.
Garces, F.
Zeng, Z.
Hailstone, R.
Matthews, S.J.
Caldecott, K.W.
Oliver, A.W.
Pearl, L.H.
(2019). Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1. Cell reports,
Vol.26
(3),
pp. 573-581.e5.
Leimbacher, P.-.
Jones, S.E.
Shorrocks, A.-.
de Marco Zompit, M.
Day, M.
Blaauwendraad, J.
Bundschuh, D.
Bonham, S.
Fischer, R.
Fink, D.
Kessler, B.M.
Oliver, A.W.
Pearl, L.H.
Blackford, A.N.
Stucki, M.
(2019). MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis. Molecular cell,
Vol.74
(3),
pp. 571-583.e8.
show abstract
In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division..
Bigot, N.
Day, M.
Baldock, R.A.
Watts, F.Z.
Oliver, A.W.
Pearl, L.H.
(2019). Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint. Elife,
Vol.8.
show abstract
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.
Ptasinska, K.
Boos, D.
Oliver, A.W.
Pearl, L.H.
(2018). BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands. Elife,
Vol.7.
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.
Pearl, L.H.
Llorca, O.
(2018). RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex. Nature communications,
Vol.9
(1),
pp. 1501-?.
show abstract
The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins..
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.
Oliver, A.W.
(2017). Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nature communications,
Vol.8
(1).
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,
Vol.19
(12),
pp. 2515-2528.
Jeggo, P.A.
Pearl, L.H.
Carr, A.M.
(2016). DNA repair, genome stability and cancer: a historical perspective. Nature reviews cancer,
Vol.16
(1),
pp. 35-42.
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.
Pearl, L.H.
Neely, R.
Keep, N.H.
Watts, F.Z.
Morris, J.R.
(2016). Human BRCA1–BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nature structural & molecular biology,
Vol.23
(7),
pp. 647-655.
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,
Vol.12
(8),
pp. 628-635.
Pearl, L.H.
(2016). Review: The HSP90 molecular chaperone—an enigmatic ATPase. Biopolymers,
Vol.105
(8),
pp. 594-607.
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,
Vol.7,
pp. 11242-?.
show abstract
The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ..
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.
Matthews, S.J.
Pearl, L.H.
Caldecott, K.W.
(2016). PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2). Nature communications,
Vol.7,
pp. 12404-?.
show abstract
PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break..
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,
Vol.34
(1),
pp. 15-26.
show abstract
The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus-truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wild-type CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wild-type CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37's client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37..
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,
Vol.71
(5),
pp. 1197-1206.
show abstract
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..
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,
Vol.15
(3),
pp. 166-180.
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-BRCT2 Domain with γH2AX. Cell reports,
Vol.13
(10),
pp. 2081-2089.
show abstract
53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications--H4K20me2 and H2AK13/K15ub--downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1..
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.
Atack, J.R.
(2014). Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Analytical biochemistry,
Vol.454,
pp. 17-22.
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,
Vol.22
(6),
pp. 805-818.
Qu, M.
Rappas, M.
Wardlaw, C.P.
Garcia, V.
Ren, J.-.
Day, M.
Carr, A.M.
Oliver, A.W.
Du, L.-.
Pearl, L.H.
(2013). Phosphorylation-Dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1. Molecular cell,
Vol.51
(6),
pp. 723-736.
Fugel, W.
Oberholzer, A.E.
Gschloessl, B.
Dzikowski, R.
Pressburger, N.
Preu, L.
Pearl, L.H.
Baratte, B.
Ratin, M.
Okun, I.
Doerig, C.
Kruggel, S.
Lemcke, T.
Meijer, L.
Kunick, C.
(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,
Vol.56
(1),
pp. 264-275.
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,
Vol.41
(19),
pp. 9168-9182.
Meier, C.
Brookings, D.C.
Ceska, T.A.
Doyle, C.
Gong, H.
McMillan, D.
Saville, G.P.
Mushtaq, A.
Knight, D.
Reich, S.
Pearl, L.H.
Powell, K.A.
Savva, R.
Allen, R.A.
(2012). Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting. Journal of structural biology,
Vol.177
(2),
pp. 329-334.
Spagnolo, L.
Barbeau, J.
Curtin, N.J.
Morris, E.P.
Pearl, L.H.
(2012). Visualization of a DNA-PK/PARP1 complex. Nucleic acids research,
Vol.40
(9),
pp. 4168-4177.
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,
Vol.19
(7),
pp. 685-692.
Tahtouh, T.
Elkins, J.M.
Filippakopoulos, P.
Soundararajan, M.
Burgy, G.
Durieu, E.
Cochet, C.
Schmid, R.S.
Lo, D.C.
Delhommel, F.
Oberholzer, A.E.
Pearl, L.H.
Carreaux, F.
Bazureau, J.-.
Knapp, S.
Meijer, L.
(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,
Vol.55
(21),
pp. 9312-9330.
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,
Vol.39
(1),
pp. 313-324.
Cuomo, M.E.
Platt, G.M.
Pearl, L.H.
Mittnacht, S.
(2011). Cyclin-Cyclin-dependent Kinase Regulatory Response Is Linked to Substrate Recognition. Journal of biological chemistry,
Vol.286
(11),
pp. 9713-9725.
Mollapour, M.
Tsutsumi, S.
Truman, A.W.
Xu, W.
Vaughan, C.K.
Beebe, K.
Konstantinova, A.
Vourganti, S.
Panaretou, B.
Piper, P.W.
Trepel, J.B.
Prodromou, C.
Pearl, L.H.
Neckers, L.
(2011). Threonine 22 Phosphorylation Attenuates Hsp90 Interaction with Cochaperones and Affects Its Chaperone Activity. Molecular cell,
Vol.41
(6),
pp. 672-681.
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,
Vol.3
(3),
pp. 271-282.
show abstract
Background: Successful structural investigations of protein–protein interactions can be facilitated by studying only the core interacting regions of the constituent proteins. However, attempting the discovery of stable core complexes using informed trial-and-error approaches can prove time and resource intensive. Methods: We describe a valuable extension of combinatorial domain hunting (CDH), a technology for the timely elucidation of soluble protein truncations. The new method, CDH2, enables empirical discovery of stable protein–protein core complexes. CDH2 is demonstrated ab initio using a previously well-characterized Hsp90/Cdc37 complex. Results: Without using a priori information, we demonstrate the isolation of stable protein–protein complexes, suitable for further analyses. Discussion: This resource-efficient process can provide protein complexes for screening of compounds designed to modulate protein–protein interactions, thus facilitating novel drug discovery. .
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 research,
Vol.39
(13),
pp. 5757-5767.
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,
Vol.286
(29),
pp. 26220-26227.
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,
Vol.21
(13),
pp. 1152-1157.
Ali, M.M.
Collins, I.
Bagratuni, T.
Davenport, E.L.
Nowak, P.R.
Silva-Santisteban, M.C.
Hardcastle, A.
McAndrews, C.
Rowlands, M.G.
Morgan, G.J.
Aherne, W.
Collins, I.
Davies, F.E.
Pearl, L.H.
(2011). Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response. Embo j,
Vol.30
(5),
pp. 894-905.
show abstract
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 Ire1α 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 Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α 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..
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 theStreptomyces hygroscopicusHtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90. The faseb journal,
Vol.25
(11),
pp. 3828-3837.
Garces, F.
Pearl, L.H.
Oliver, A.W.
(2011). The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase. Molecular cell,
Vol.44
(3),
pp. 385-396.
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.
Garrett, M.D.
(2011). CCT241533 is a potent and selective inhibitor of CHK2 that potentiates the cytotoxicity of PARP inhibitors. Cancer res,
Vol.71
(2),
pp. 463-472.
show abstract
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 IC(50) of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 μmol/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..
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.
Workman, P.
Moody, C.J.
(2011). Targeting the Hsp90 Molecular Chaperone with Novel Macrolactams Synthesis, Structural, Binding, and Cellular Studies. Acs chemical biology,
Vol.6
(12),
pp. 1339-1347.
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.
Raynham, T.
Aherne, G.W.
Pearl, L.H.
Oliver, A.W.
Garrett, M.D.
Collins, I.
(2011). Structure-based design of potent and selective 2-(quinazolin-2-yl)phenol inhibitors of checkpoint kinase 2. J med chem,
Vol.54
(2),
pp. 580-590.
show abstract
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 (IC(50) 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.
Colombo, G.
Blagg, B.S.
Panaretou, B.
Stetler-Stevenson, W.G.
Trepel, J.B.
Piper, P.W.
Prodromou, C.
Pearl, L.H.
Neckers, L.
(2010). Swe1Wee1-Dependent Tyrosine Phosphorylation of Hsp90 Regulates Distinct Facets of Chaperone Function. Molecular cell,
Vol.37
(3),
pp. 333-343.
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 TranscreenerTM ADP assay kit. J biomol screen,
Vol.15
(3),
pp. 279-286.
show abstract
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 ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). 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 reagents and compared well to that determined using other assay formats..
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.
(2010). An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity. Dna repair,
Vol.9
(9),
pp. 1003-1010.
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,
Vol.39
(2),
pp. 269-281.
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.
(2010). Inhibition of Hsp90 with Resorcylic Acid Macrolactones: Synthesis and Binding Studies. Chemistry - a european journal,
Vol.16
(34),
pp. 10366-10372.
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.
Wynne Aherne, G.
Garrett, M.D.
Collins, I.
(2010). Identification and characterisation of 2-aminopyridine inhibitors of checkpoint kinase 2. Bioorg med chem,
Vol.18
(2),
pp. 707-718.
show abstract
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..
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 research,
Vol.37
(Database issue),
pp. D824-D831.
show abstract
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,
Vol.276
(1),
pp. 199-209.
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.
Gierschik, P.
Pearl, L.H.
Katan, M.
(2009). Structural Insights into Formation of an Active Signaling Complex between Rac and Phospholipase C Gamma 2. Molecular cell,
Vol.34
(2),
pp. 223-233.
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,
Vol.37
(5),
pp. 1701-1712.
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,
Vol.34
(6),
pp. 735-745.
Oliver, A.W.
Swift, S.
Lord, C.J.
Ashworth, A.
Pearl, L.H.
(2009). Structural basis for recruitment of BRCA2 by PALB2. Embo reports,
Vol.10
(9),
pp. 990-996.
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,
Vol.392
(4),
pp. 923-936.
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,
Vol.8
(12),
pp. 1380-1389.
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,
Vol.4
(4),
pp. 289-297.
show abstract
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 low 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..
Kilkenny, M.L.
Doré, 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,
Vol.22
(15),
pp. 2034-2047.
show abstract
Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor2 and BRCT2 domains. Crb2-Tudor2 domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT2 domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT2 domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with γ-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect..
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.
Zhang, M.-.
(2008). Molecular Characterization of Macbecin as an Hsp90 Inhibitor. Journal of medicinal chemistry,
Vol.51
(9),
pp. 2853-2857.
Pearl, L.H.
Prodromou, C.
Workman, P.
(2008). The Hsp90 molecular chaperone: an open and shut case for treatment. Biochemical journal,
Vol.410
(3),
pp. 439-453.
show abstract
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.
de Haven Brandon, A.
Gowan, S.
Boxall, F.
Aherne, W.
Rowlands, M.
Hayes, A.
Martins, V.
Urban, F.
Boxall, K.
Prodromou, C.
Pearl, L.
James, K.
Matthews, T.P.
Cheung, K.M.
Kalusa, A.
Jones, K.
McDonald, E.
Barril, X.
Brough, P.A.
Cansfield, J.E.
Dymock, B.
Drysdale, M.J.
Finch, H.
Howes, R.
Hubbard, R.E.
Surgenor, A.
Webb, P.
Wood, M.
Wright, L.
Workman, P.
(2008). NVP-AUY922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis. Cancer res,
Vol.68
(8),
pp. 2850-2860.
show abstract
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-1alpha, 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,
Vol.413
(2),
pp. 261-268.
show abstract
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..
Hawkins, T.A.
Haramis, A.-.
Etard, C.
Prodromou, C.
Vaughan, C.K.
Ashworth, R.
Ray, S.
Behra, M.
Holder, N.
Talbot, W.S.
Pearl, L.H.
Strähle, U.
Wilson, S.W.
(2008). The ATPase-dependent chaperoning activity of Hsp90a regulates thick filament formation and integration during skeletal muscle myofibrillogenesis. Development,
Vol.135
(6),
pp. 1147-1156.
show abstract
The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish slothu45 mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly..
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,
Vol.27
(4),
pp. 704-714.
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,
Vol.27
(20),
pp. 2789-2798.
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.
Dymock, B.
Eccles, S.A.
Finch, H.
Fink, A.
Hayes, A.
Howes, R.
Hubbard, R.E.
James, K.
Jordan, A.M.
Lockie, A.
Martins, V.
Massey, A.
Matthews, T.P.
McDonald, E.
Northfield, C.J.
Pearl, L.H.
Prodromou, C.
Ray, S.
Raynaud, F.I.
Roughley, S.D.
Sharp, S.Y.
Surgenor, A.
Walmsley, D.L.
Webb, P.
Wood, M.
Workman, P.
Wright, L.
(2008). 4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer. J med chem,
Vol.51
(2),
pp. 196-218.
show abstract
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 GI50 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 approximately 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.
Foster, T.A.
Sheridan, R.M.
Gregory, M.A.
Roe, S.M.
Prodromou, C.
Pearl, L.
Boyd, S.M.
Wilkinson, B.
Martin, C.J.
(2008). Optimizing Natural Products by Biosynthetic Engineering: Discovery of Nonquinone Hsp90 Inhibitors. Journal of medicinal chemistry,
Vol.51
(18),
pp. 5494-5497.
Vaughan, C.K.
Mollapour, M.
Smith, J.R.
Truman, A.
Hu, B.
Good, V.M.
Panaretou, B.
Neckers, L.
Clarke, P.A.
Workman, P.
Piper, P.W.
Prodromou, C.
Pearl, L.H.
(2008). Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol cell,
Vol.31
(6),
pp. 886-895.
show abstract
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 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 pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 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.
Patterson, L.
Matthews, T.P.
Cheung, K.M.
Ball, K.
Hayes, A.
Raynaud, F.
Marais, R.
Pearl, L.
Eccles, S.
Aherne, W.
McDonald, E.
Workman, P.
(2007). In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors. Cancer res,
Vol.67
(5),
pp. 2206-2216.
show abstract
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 HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-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 mumol/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,
Vol.8
(1),
pp. 56-62.
Sharp, S.Y.
Prodromou, C.
Boxall, K.
Powers, M.V.
Holmes, J.L.
Box, G.
Matthews, T.P.
Cheung, K.M.
Kalusa, A.
James, K.
Hayes, A.
Hardcastle, A.
Dymock, B.
Brough, P.A.
Barril, X.
Cansfield, J.E.
Wright, L.
Surgenor, A.
Foloppe, N.
Hubbard, R.E.
Aherne, W.
Pearl, L.
Jones, K.
McDonald, E.
Raynaud, F.
Eccles, S.
Drysdale, M.
Workman, P.
(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,
Vol.6
(4),
pp. 1198-1211.
show abstract
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 IC(50) 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,
Vol.32
(8),
pp. 351-356.
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. Molecular cell,
Vol.28
(3),
pp. 371-385.
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,
Vol.26
(20),
pp. 4402-4412.
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.
Gierschik, P.
Pearl, L.H.
Driscoll, P.C.
Katan, M.
(2006). Structural and Mechanistic Insights into Ras Association Domains of Phospholipase C Epsilon. Molecular cell,
Vol.21
(4),
pp. 495-507.
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,
Vol.22
(4),
pp. 511-519.
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,
Vol.22
(5),
pp. 645-655.
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,
Vol.34
(16),
pp. 4515-4526.
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,
Vol.23
(5),
pp. 697-707.
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.
Driscoll, P.C.
Savva, R.
(2006). Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications. Protein science,
Vol.15
(10),
pp. 2356-2365.
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.
(2006). Inhibition of Hsp90 with synthetic macrolactones: synthesis and structural and biological evaluation of ring and conformational analogs of radicicol. Chem biol,
Vol.13
(11),
pp. 1203-1215.
show abstract
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 adopt the 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 Yeast
Saccharomyces 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,
Vol.5
(11),
pp. 1914-1924.
show abstract
ABSTRACT
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 the
slt2
Δ
Saccharomyces cerevisiae
mutant 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 the
slt2
Δ 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. The embo journal,
Vol.25
(13),
pp. 3179-3190.
Ioannou, Y.
Giles, I.
Lambrianides, A.
Richardson, C.
Pearl, L.H.
Latchman, D.S.
Isenberg, D.A.
Rahman, A.
(2006). A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli. Bmc biotechnology,
Vol.6
(1).
show abstract
Abstract
Background
The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β2GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL.
Methods
Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter.
Results
Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS.
Conclusion
By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of β2GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production.
.
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,
Vol.440
(7087),
pp. 1013-1017.
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,
Vol.42
(1),
pp. 39-48.
Pearl, L.H.
(2005). Hsp90 and Cdc37 – a chaperone cancer conspiracy. Current opinion in genetics & development,
Vol.15
(1),
pp. 55-61.
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,
Vol.13
(2),
pp. 243-255.
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,
Vol.114
(3),
pp. 418-427.
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,
Vol.55
(6),
pp. 1735-1750.
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,
Vol.4
(5),
pp. 849-860.
show abstract
ABSTRACT
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 the
Saccharomyces cerevisiae
proteome 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 (T22I
hsp82
) 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.
.
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. The biochemical journal,
Vol.389
(Pt 3),
pp. 657-664.
show abstract
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/SNFbeta). We present in this paper the crystal structure of the proximal BAH domain from chicken polybromo (BAH1), at a resolution of 1.6 A (1 A=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein-protein interactions..
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,
Vol.20
(4),
pp. 525-538.
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.
Aherne, G.W.
McDonald, E.
Workman, P.
(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,
Vol.15
(14),
pp. 3338-3343.
show abstract
High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) 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..
Polychronopoulos, P.
Magiatis, P.
Skaltsounis, A.-.
Myrianthopoulos, V.
Mikros, E.
Tarricone, A.
Musacchio, A.
Roe, S.M.
Pearl, L.
Leost, M.
Greengard, P.
Meijer, L.
(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,
Vol.47
(4),
pp. 935-946.
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,
Vol.335
(2),
pp. 573-582.
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,
Vol.116
(1),
pp. 87-98.
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,
Vol.41
(5),
pp. 495-510.
Meyer, P.
Prodromou, C.
Liao, C.
Hu, B.
Mark Roe, S.
Vaughan, C.K.
Vlasic, I.
Panaretou, B.
Piper, P.W.
Pearl, L.H.
(2004). Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery. The embo journal,
Vol.23
(3),
pp. 511-519.
Llorca, O.
Pearl, L.H.
(2004). Electron microscopy studies on DNA recognition by DNA-PK. Micron,
Vol.35
(8),
pp. 625-633.
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,
Vol.279
(50),
pp. 51989-51998.
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,
Vol.327
(2),
pp. 176-183.
show abstract
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 Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM 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 approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8..
Oliver, A.W.
(2004). Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2. Nucleic acids research,
Vol.32
(2),
pp. 456-464.
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,
Vol.9
(4),
pp. 359-359.
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,
Vol.59
(7),
pp. 1230-1233.
Prodromou, C.
Pearl, L.
(2003). Structure and Functional Relationships of Hsp90. Current cancer drug targets,
Vol.3
(5),
pp. 301-323.
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,
Vol.302
(1-2),
pp. 165-170.
Bunting, K.A.
(2003). Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the -clamp. The embo journal,
Vol.22
(21),
pp. 5883-5892.
Dajani, R.
(2003). Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. The embo journal,
Vol.22
(3),
pp. 494-501.
Boskovic, J.
(2003). Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs. The embo journal,
Vol.22
(21),
pp. 5875-5882.
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,
Vol.11
(3),
pp. 647-658.
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,
Vol.11
(6),
pp. 1647-1659.
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,
Vol.331
(3),
pp. 643-652.
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,
Vol.40
(8),
pp. 517-530.
Meijer, L.
Skaltsounis, A.-.
Magiatis, P.
Polychronopoulos, P.
Knockaert, M.
Leost, M.
Ryan, X.P.
Vonica, C.A.
Brivanlou, A.
Dajani, R.
Crovace, C.
Tarricone, C.
Musacchio, A.
Roe, S.M.
Pearl, L.
Greengard, P.
(2003). GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins. Chemistry & biology,
Vol.10
(12),
pp. 1255-1266.
Bunting, K.A.
(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,
Vol.31
(6),
pp. 1633-1639.
Pearl, L.
(2002). Regulation of protein kinases in insulin, growth factor and Wnt signalling. Current opinion in structural biology,
Vol.12
(6),
pp. 761-767.
Fraser, E.
Young, N.
Dajani, R.
Franca-Koh, J.
Ryves, J.
Williams, R.S.
Yeo, M.
Webster, M.-.
Richardson, C.
Smalley, M.J.
Pearl, L.H.
Harwood, A.
Dale, T.C.
(2002). Identification of the Axin and Frat Binding Region of Glycogen Synthase Kinase-3. Journal of biological chemistry,
Vol.277
(3),
pp. 2176-2185.
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,
Vol.290
(3),
pp. 967-972.
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,
Vol.317
(2),
pp. 171-177.
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,
Vol.277
(19),
pp. 16936-16940.
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,
Vol.318
(5),
pp. 1367-1379.
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/p50. Journal of biological chemistry,
Vol.277
(23),
pp. 20151-20159.
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,
Vol.10
(6),
pp. 751-761.
Panaretou, B.
Siligardi, G.
Meyer, P.
Maloney, A.
Sullivan, J.K.
Singh, S.
Millson, S.H.
Clarke, P.A.
Naaby-Hansen, S.
Stein, R.
Cramer, R.
Mollapour, M.
Workman, P.
Piper, P.W.
Pearl, L.H.
Prodromou, C.
(2002). Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. Mol cell,
Vol.10
(6),
pp. 1307-1318.
show abstract
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..
Fogg, M.J.
Pearl, L.H.
Connolly, B.A.
(2002). Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature structural biology,
Vol.9
(12),
pp. 922-927.
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,
Vol.11
(12),
pp. 833-842.
show abstract
Antibodies to double-stranded DNA are the best-known serological markers of systemic lupus erythematosus, and are closely associated with its renal pathogenesis. How these antibodies recognize DNA is not fully understood.An understandingof the relationship between the functional attributes of an antibody with the three-dimensional structure of its antigen-combining site would allow an insight into the rules that dictate auto-antibody–nucleic acid interaction and consequent pathogenicity of the autoantibody. Data from such studies could assist the development of novel drugs as an approach to specific therapies that can inhibit or disrupt protein–nucleic acid interactions. A full understanding of the binding specificities can be achieved only by experimental determination of detailed three-dimensional structure of these antibodies alone, and of their complexes with specific DNA antigens. A prerequisite of such a study is the ability to produce multimilligram quantities of the antibody protein. However, these antibodies are particularly difficult to express, probably due to their DNA-binding activity. This review attempts to focus on the recent developmentson the over-expressionof anti-DNA antibody fragments in heterologous cell expression systems and their purification to homogeneity that would in turn allow their structural studies via crystallization. .
Harris, R.
Maman, J.D.
Hinks, J.A.
Sankar, A.
Pearl, L.H.
Driscoll, P.C.
(2002). . Journal of biomolecular nmr,
Vol.22
(4),
pp. 373-374.
Salek, R.M.
Williams, M.A.
Prodromou, C.
Pearl, L.H.
Ladbury, J.E.
(2002). . Journal of biomolecular nmr,
Vol.23
(4),
pp. 327-328.
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,
Vol.57
(12),
pp. 1936-1937.
Ceschini, S.
(2001). Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2. The embo journal,
Vol.20
(23),
pp. 6601-6611.
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,
Vol.308
(3),
pp. 527-539.
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,
Vol.105
(6),
pp. 721-732.
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.
ENGWERDA, C.
GRAY, C.A.
J. MEYER, D.
(2001). Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis. Biochemical journal,
Vol.357
(2),
pp. 373-373.
Evans, S.J.
(2000). Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus. Nucleic acids research,
Vol.28
(5),
pp. 1059-1066.
Pearl, L.H.
Prodromou, C.
(2000). Structure and in vivo function of Hsp90. Current opinion in structural biology,
Vol.10
(1),
pp. 46-51.
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,
Vol.13
(2),
pp. 129-132.
Pearl, L.H.
(2000). Structure and function in the uracil-DNA glycosylase superfamily. Mutation research/dna repair,
Vol.460
(3-4),
pp. 165-181.
Norman, R.A.
Poh, C.L.
Pearl, L.H.
O'Hara, B.P.
Drew, R.E.
(2000). Steric Hindrance Regulation of the Pseudomonas aeruginosa Amidase Operon. Journal of biological chemistry,
Vol.275
(39),
pp. 30660-30667.
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,
Vol.19
(10-12),
pp. 1505-1516.
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 in Escherichia coli. Journal of biological chemistry,
Vol.275
(45),
pp. 35129-35136.
Prodromou, C.
(2000). The ATPase cycle of Hsp90 drives a molecular clamp' via transient dimerization of the N-terminal domains. The embo journal,
Vol.19
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pp. 4383-4392.
O'Hara, B.P.
(1999). Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex. The embo journal,
Vol.18
(19),
pp. 5175-5186.
Barrett, T.E.
(1999). Crystal structure of a thwarted mismatch glycosylase DNA repair complex. The embo journal,
Vol.18
(23),
pp. 6599-6609.
Panaretou, B.
Sinclair, K.
Prodromou, C.
Johal, J.
Pearl, L.
Piper, P.W.
(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,
Vol.145
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pp. 3455-3463.
Prodromou, C.
(1999). Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones. The embo journal,
Vol.18
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pp. 754-762.
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,
Vol.42
(2),
pp. 260-266.
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,
Vol.96
(16),
pp. 9045-9050.
show abstract
Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⋅C → A⋅U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and
Pfu
) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.
.
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,
Vol.92
(1),
pp. 117-129.
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,
Vol.273
(1),
pp. 45-50.
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,
Vol.5
(8),
pp. 697-701.
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,
Vol.2
(3),
pp. 361-372.
Panaretou, B.
(1998). ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone invivo. The embo journal,
Vol.17
(16),
pp. 4829-4836.
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,
Vol.36
(26),
pp. 8020-8029.
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,
Vol.4
(6),
pp. 477-482.
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,
Vol.90
(1),
pp. 65-75.
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,
Vol.271
(5),
pp. 789-802.
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,
Vol.169
(2),
pp. 251-255.
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,
Vol.24
(4),
pp. 502-513.
Pearl, L.H.
Savva, R.
(1996). The problem with pyrimidines. Nature structural & molecular biology,
Vol.3
(6),
pp. 485-487.
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,
Vol.25
(4),
pp. 517-522.
Koulis, A.
Cowan, D.A.
Pearl, L.H.
Savva, R.
(1996). Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms. Fems microbiology letters,
Vol.143
(2-3),
pp. 267-271.
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,
Vol.15
(21),
pp. 5907-5916.
Danson, M.J.
Hough, D.W.
Russell, R.J.
Taylor, G.L.
Pearl, L.
(1996). Enzyme thermostability and thermoactivity. "protein engineering, design and selection",
Vol.9
(8),
pp. 629-630.
Savva, R.
McAuley-Hecht, K.
Brown, T.
Pearl, L.
(1995). The structural basis of specific base-excision repair by uracil–DNA glycosylase. Nature,
Vol.373
(6514),
pp. 487-493.
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,
Vol.22
(3),
pp. 287-289.
Wilson, S.A.
Williams, R.J.
Pearl, L.H.
Drew, R.E.
(1995). Identification of Two New Genes in the Pseudomonasaeruginosa Amidase Operon, Encoding an ATPase (AmiB) and a Putative Integral Membrane Protein (AmiS). Journal of biological chemistry,
Vol.270
(32),
pp. 18818-18824.
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,
Vol.2
(9),
pp. 752-757.
Pearl, L.H.
Savva, R.
(1995). DNA repair in three dimensions. Trends in biochemical sciences,
Vol.20
(10),
pp. 421-426.
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,
Vol.34
(40),
pp. 13190-13195.
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,
Vol.13
(24),
pp. 5810-5817.
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,
Vol.229
(2),
pp. 561-563.
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,
Vol.229
(3),
pp. 782-784.
Pearl, L.
(1993). Similarity of active-site structures. Nature,
Vol.362
(6415),
pp. 24-24.
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,
Vol.12
(9),
pp. 3637-3642.
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,
Vol.234
(3),
pp. 910-912.
Swindells, M.B.
Orengo, C.A.
Jones, D.T.
Pearl, L.H.
Thornton, J.M.
(1993). Recurrence of a binding motif?. Nature,
Vol.362
(6418),
pp. 299-299.
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,
Vol.373
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pp. 1211-1216.
Prodromou, C.
Pearl, L.H.
(1992). Recursive PCR: a novel technique for total gene synthesis. "protein engineering, design and selection",
Vol.5
(8),
pp. 827-829.
Cruzeiro-Hansson, L.
Swann, P.F.
Pearl, L.
Goodfellow, J.M.
(1992). Molecular dynamics of alkylated DNA. Carcinogenesis,
Vol.13
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pp. 2067-2073.
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,
Vol.222
(4),
pp. 869-871.
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,
Vol.211
(4),
pp. 919-941.
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,
Vol.261
(2),
pp. 373-377.
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,
Vol.260
(2),
pp. 195-197.
Blundell, T.
Pearl, L.
(1989). A second front against AIDS. Nature,
Vol.337
(6208),
pp. 596-597.
McKeating, J.A.
Gow, J.
Goudsmit, J.
Pearl, L.H.
Mulder, C.
Weiss, R.A.
(1989). Characterization of HIV-1 neutralization escape mutants. Aids,
Vol.3
(12),
pp. 777-784.
Neidle, S.
Pearl, L.H.
Herzyk, P.
Berman, H.M.
(1988). A molecular model for proflavine--DNA intercalation. Nucleic acids research,
Vol.16
(18),
pp. 8999-9016.
Neidle, S.
Pearl, L.H.
Skelly, J.V.
(1987). DNA structure and perturbation by drug binding. Biochemical journal,
Vol.243
(1),
pp. 1-13.
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.
Norey, C.G.
Kay, J.
Boger, J.
Dunn, B.M.
Leckie, B.J.
Jone, D.M.
Atrash, B.
Hallett, A.
Szelke, M.
(1987). High resolution X-ray analyses of renin inhibitor-aspartic proteinase complexes. Nature,
Vol.327
(6120),
pp. 349-352.
Pearl, L.H.
(1987). The catalytic mechanism of aspartic proteinases. Febs letters,
Vol.214
(1),
pp. 8-12.
PEAR, L.H.
TAYLO, W.R.
(1987). Sequence specificity of retroviral proteases. Nature,
Vol.328
(6130),
pp. 482-482.
Pearl, L.H.
Taylor, W.R.
(1987). A structural model for the retroviral proteases. Nature,
Vol.329
(6137),
pp. 351-354.
Foundling, S.I.
Cooper, J.
Watson, F.E.
Pearl, L.H.
Hemmings, A.
Wood, S.P.
Blundell, T.
Hallett, A.
Jones, D.M.
Sueiras, J.
Atrash, B.
Szelke, M.
(1987). Crystallographic Studies of Reduced Bond Inhibitors Complexed with an Aspartic Proteinase. Journal of cardiovascular pharmacology,
Vol.10,
pp. 59-68.
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,
Vol.15
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pp. 6553-6562.
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,
Vol.15
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pp. 3469-3478.
Pearl, L.H.
Neidle, S.
(1986). Origins of stereospecificity in DNA damage by anti
-benzo[a
]pyrene diol-epoxides. Febs letters,
Vol.209
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pp. 269-276.
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,
Vol.13
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pp. 1036-1041.
Pearl, L.
Blundell, T.
(1984). The active site of aspartic proteinases. Febs letters,
Vol.174
(1),
pp. 96-101.
Blundell, T.
Sibanda, B.L.
Pearl, L.
(1983). Three-dimensional structure, specificity and catalytic mechanism of renin. Nature,
Vol.304
(5923),
pp. 273-275.
Dekker, C.
Roe, S.M.
McCormack, E.A.
Beuron, F.
Pearl, L.H.
Willison, K.R.
The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. The embo journal,
Vol.30
(15),
pp. 3078-3090.
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.,
Vol.9,
pp. 307-312.
show abstract
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..
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.
Endicott, J.A.
Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System. Cell reports,
Vol.21
(5),
pp. 1386-1398.
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. ,
,
pp. 149-162.
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,
Vol.9
(10),
pp. e1003649-e1003649.
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,
Vol.270
(23),
pp. 4689-4695.
Elliott, R.J.
Jarvis, A.
Rajasekaran, M.B.
Menon, M.
Bowers, L.
Boffey, R.
Bayford, M.
Firth-Clark, S.
Key, R.
Aqil, R.
Kirton, S.B.
Niculescu-Duvaz, D.
Fish, L.
Lopes, F.
McLeary, R.
Trindade, I.
Vendrell, E.
Munkonge, F.
Porter, R.
Perrior, T.
Springer, C.
Oliver, A.W.
Pearl, L.H.
Ashworth, A.
Lord, C.J.
Design and discovery of 3-aryl-5-substituted-isoquinolin-1-ones as potent tankyrase inhibitors. Medchemcomm,
Vol.6
(9),
pp. 1687-1692.
show abstract
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.
.
Chen, X.
Ali, Y.I.
Fisher, C.E.
Arribas-Bosacoma, R.
Rajasekaran, M.B.
Williams, G.
Walker, S.
Booth, J.R.
Hudson, J.J.
Roe, S.M.
Pearl, L.H.
Ward, S.E.
Pearl, F.M.
Oliver, A.W.
Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein. Elife,
Vol.10.
show abstract
BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM..
Day, M.
Oliver, A.W.
Pearl, L.H.
Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1. Dna repair,
Vol.108,
pp. 103232-?.
show abstract
The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 - a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage..