I03-Macromolecular Crystallography
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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.
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Feb 2009
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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James E. H.
Day
,
Swee Y.
Sharp
,
Martin G.
Rowlands
,
Wynne
Aherne
,
Angela
Hayes
,
Florence I.
Raynaud
,
William
Lewis
,
S. Mark
Roe
,
Chrisostomos
Prodromou
,
Laurence H.
Pearl
,
Paul
Workman
,
Christopher J.
Moody
Diamond Proposal Number(s):
[6385]
Abstract: A series of resorcylic acid macrolactams, nitrogen analogues of the naturally occurring macrolactone radicicol, have been prepared by chemical synthesis and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves, as key steps, ring opening of an isocoumarin intermediate, followed by a ring- closing metathesis reaction to form the macrocycle. Subsequent manipulation of the ester group into a range of amides allows access to a range of new macrolactams following deprotection of the two phenolic groups. These new resorcylic acid lactams exhibit metabolic stability greater than that of related lactone counterparts, while co-crystallization of three macrolactams with the N-terminal domain ATP site of Hsp90 conforms that they bind in a similar way to the natural product radicicol and to our previous synthetic lactone analogues. Interestingly, however, in the case of the N- benzylamide, additional binding to a hydrophobic pocket of the protein was observed. In biological assays, the new macrocyclic lactams exhibit a biological pro\ufb01le equivalent or superior to that of the related lactones and show the established molecular signature of Hsp90 inhibitors in human colon cancer cells.
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Dec 2011
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: CDK9 is the kinase of positive transcription elongation factor b and facilitates the transition of paused RNA polymerase II to processive transcription elongation. CDK9 is a validated target for the treatment of cancer, cardiac hypertrophy, and human immunodeficiency virus. Here we analyze different CDK9/cyclin T variants to identify a form of the complex amenable to use in inhibitor design. To demonstrate the utility of this system, we have determined the crystal structures of CDK9/cyclin T and CDK2/cyclin A bound to the CDK9-specific inhibitor CAN508. Comparison of the structures reveals CDK9-specific conformational changes and identifies a CDK9-specific hydrophobic pocket, adjacent to the áC-helix. By comparison with a previously published structure of CDK9/cyclin T/human immunodeficiency virus TAT we find that the CDK9 áC-helix has a degree of conformational variability that has the potential to be exploited for inhibitor design.
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May 2012
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Magnus
Alphey
,
Lisa
Pirrie
,
Leah S.
Torrie
,
Wassila Abdelli
Boulkeroua
,
Mary
Gardiner
,
Aurijit
Sarkar
,
Marko
Maringer
,
Wulf
Oehlmann
,
Ruth
Brenk
,
Michael S.
Scherman
,
Michael
Mcneil
,
Martin
Rejzek
,
Robert A.
Field
,
Mahavir
Singh
,
David
Gray
,
Nicholas J.
Westwood
,
James H.
Naismith
Open Access
Abstract: Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.
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Nov 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Mathieu
Métifiot
,
Kasthuraiah
Maddali
,
Barry C.
Johnson
,
Stephen
Hare
,
Steven J.
Smith
,
Xue Zhi
Zhao
,
Christophe
Marchand
,
Terrence R.
Burke
,
Stephen H.
Hughes
,
Peter
Cherepanov
,
Yves
Pommier
Abstract: On the basis of a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new molecule, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency
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Dec 2012
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I02-Macromolecular Crystallography
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Rasheduzzaman
Chowdhury
,
José Ignacio
Candela-Lena
,
Mun Chiang
Chan
,
David Jeremy
Greenald
,
Kar Kheng
Yeoh
,
Ya-Min
Tian
,
Michael A.
Mcdonough
,
Anthony
Tumber
,
Nathan R.
Rose
,
Ana
Conejo-Garcia
,
Marina
Demetriades
,
Sinnakaruppan
Mathavan
,
Akane
Kawamura
,
Myung Kyu
Lee
,
Freek
Van Eeden
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Abstract: The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on thefunctional effects of PHD inhibition in cells and in animals.
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May 2013
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I04-Macromolecular Crystallography
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Anders E. G.
Lindgren
,
Tobias
Karlberg
,
Ann-Gerd
Thorsell
,
Mareike
Hesse
,
Sara
Spjut
,
Torun
Ekblad
,
C. David
Andersson
,
Ana Filipa
Pinto
,
Johan
Weigelt
,
Michael O.
Hottiger
,
Anna
Linusson
,
Mikael
Elofsson
,
Herwig
Schüler
Diamond Proposal Number(s):
[6603]
Abstract: Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.
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Jun 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Open Access
Abstract: Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients.
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Jul 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Abstract: Acetolactate decarboxylase catalyzes the conversion of both enantiomers of acetolactate to the (R)-enantiomer of acetoin, via a mechanism that has been shown to involve a prior rearrangement of the non-natural (R)-enantiomer substrate to the natural (S)-enantiomer. In this paper, a series of crystal structures of ALDC complex with designed transition state mimics are reported. These structures, coupled with inhibition studies and site-directed mutagenesis provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation/protonation events. A mechanism for the transformation of each enantiomer of acetolactate is proposed.
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Aug 2013
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8359]
Open Access
Abstract: Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation.
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Aug 2013
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