I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Abstract: Although a previously developed bump-hole approach has proven powerful in generating specific inhibitors for mapping functions of protein kinases, its application is limited by the intolerance of the large-to-small mutation by certain kinases and the inability to control two kinases separately in the same cells. Herein, we describe the development of an alternative chemical-genetic approach to overcome these limitations. Our approach features the use of an engineered cysteine residue at a particular position as a reactive feature to sensitize a kinase of interest to selective covalent blockade by electrophilic inhibitors and is thus termed Ele-Cys approach. We successfully applied the Ele-Cys approach to identify selective covalent inhibitors of a receptor tyrosine kinase EphB1, and solved cocrystal structures to determine the mode of covalent binding. Importantly, the Ele-Cys and bump-hole approaches afforded orthogonal inhibition of two distinct kinases in the cell, opening the door to their combined use in the study of multi-kinase signaling pathways.
|
May 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Petar O.
Nikiforov
,
Michal
Blaszczyk
,
Sachin
Surade
,
Helena I.
Boshoff
,
Andaleeb
Sajid
,
Vincent
Delorme
,
Nathalie
Deboosere
,
Priscille
Brodin
,
Alain R.
Baulard
,
Clifton E.
Barry 3rd
,
Tom L.
Blundell
,
Chris
Abell
Open Access
Abstract: Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays.
|
Mar 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jennifer
Batson
,
Hamish D.
Toop
,
Clara
Redondo
,
Roya
Babaebi-Jadidi
,
Apirat
Chaikuad
,
Stephen F.
Wearmouth
,
Brian
Gibbons
,
Claire
Allen
,
Cynthia
Tallant
,
Jingxue
Zhang
,
Chunyun
Du
,
Jules
Hancox
,
Tom
Hawtrey
,
Joana
Da Rocha
,
Renate
Griffith
,
Stefan
Knapp
,
David O.
Bates
,
Jonathan C.
Morris
Open Access
Abstract: Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.
|
Feb 2017
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10369]
Open Access
Abstract: Inhibition of protein kinases using ATP-competitive compounds is an important strategy in drug discovery. In contrast, the allosteric regulation of kinases through the disruption of protein–protein interactions has not been widely adopted, despite the potential for selective targeting. Aurora-A kinase regulates mitotic entry and mitotic spindle assembly and is a promising target for anticancer therapy. The microtubule-associated protein TPX2 activates Aurora-A through binding to two sites. Aurora-A recognition is mediated by two motifs within the first 43 residues of TPX2, connected by a flexible linker. To characterize the contributions of these three structural elements, we prepared a series of TPX2 proteomimetics and investigated their binding affinity for Aurora-A using isothermal titration calorimetry. A novel stapled TPX2 peptide was developed that has improved binding affinity for Aurora-A and mimics the function of TPX2 in activating Aurora-A’s autophosphorylation. We conclude that the helical region of TPX2 folds upon binding Aurora-A, and that stabilization of this helix does not compromise Aurora-A activation. This study demonstrates that the preparation of these proteomimetics using modern synthesis methods is feasible and their biochemical evaluation demonstrates the power of proteomimetics as tool compounds for investigating PPIs involving intrinsically disordered regions of proteins.
|
Dec 2016
|
|
I02-Macromolecular Crystallography
|
Dominic I.
James
,
Kate M.
Smith
,
Allan M.
Jordan
,
Emma E.
Fairweather
,
Louise A.
Griffiths
,
Nicola S.
Hamilton
,
James R.
Hitchin
,
Colin P.
Hutton
,
Stuart
Jones
,
Paul
Kelly
,
Alison E.
Mcgonagle
,
Helen
Small
,
Alexandra I. J.
Stowell
,
Julie
Tucker
,
Ian D.
Waddell
,
Bohdan
Waszkowycz
,
Donald J.
Ogilvie
Open Access
Abstract: The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).
|
Nov 2016
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Dominic
Tisi
,
Elisabetta
Chiarparin
,
Emiliano
Tamanini
,
Puja
Pathuri
,
Joseph E.
Coyle
,
Adam
Hold
,
Finn P.
Holding
,
Nader
Amin
,
Agnes C. L.
Martin
,
Sharna J.
Rich
,
Valerio
Berdini
,
Jeff
Yon
,
Paul
Acklam
,
Rosemary
Burke
,
Ludovic
Drouin
,
Jenny E.
Harmer
,
Fiona
Jeganathan
,
Rob
Van Montfort
,
Yvette
Newbatt
,
Marcello
Tortorici
,
Maura
Westlake
,
Amy
Wood
,
Swen
Hoelder
,
Tom D.
Heightman
Abstract: The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.
|
Nov 2016
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Open Access
Abstract: Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a KD = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine–protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C–PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol–1 atom–1). High-resolution crystal structures of the Y220C–ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein–fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells.
|
Jun 2016
|
|
I24-Microfocus Macromolecular Crystallography
|
Ratana
Charoenwattanasatien
,
Salila
Pengthaisong
,
Imogen
Breen
,
Risa
Mutoh
,
Sompong
Sansenya
,
Yanling
Hua
,
Anupong
Tankrathok
,
Liang
Wu
,
Chomphunuch
Songsiriritthigul
,
Hideaki
Tanaka
,
Spencer J.
Williams
,
Gideon
Davies
,
Genji
Kurisu
,
James R. Ketudat
Cairns
Open Access
Abstract: Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of
glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in
spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher
disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum
TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding
and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling
of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.
|
May 2016
|
|
I03-Macromolecular Crystallography
|
Sebastian
Mathea
,
Kamal R.
Abdul Azeez
,
Eidarus
Salah
,
Cynthia
Tallant
,
Finn
Wolfreys
,
Rebecca
Konietzny
,
Roman
Fischer
,
Hua Jane
Lou
,
Paul E.
Brennan
,
Gisela
Schnapp
,
Alexander
Pautsch
,
Benedikt M.
Kessler
,
Benjamin E.
Turk
,
Stefan
Knapp
Abstract: The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here, we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The cocrystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions −1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.
|
Mar 2016
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Xue Zhi
Zhao
,
Steven J.
Smith
,
Daniel
Maskell
,
Mathieu
Metifiot
,
Valerie
Pye
,
Katherine
Fesen
,
Christophe
Marchand
,
Yves
Pommier
,
Peter
Cherepanov
,
Stephen H.
Hughes
,
Terrence R.
Burke
Diamond Proposal Number(s):
[9424]
Open Access
Abstract: HIV integrase (IN) strand transfer inhibitors
(INSTIs) are among the newest anti-AIDS drugs; however,
mutant forms of IN can confer resistance. We developed
noncytotoxic naphthyridine-containing INSTIs that retain low
nanomolar IC50 values against HIV-1 variants harboring all of the
major INSTI-resistant mutations. We found by analyzing crystal
structures of inhibitors bound to the IN from the prototype foamy
virus (PFV) that the most successful inhibitors show striking
mimicry of the bound viral DNA prior to 3′-processing and the
bound host DNA prior to strand transfer. Using this concept of
“bi-substrate mimicry,” we developed a new broadly effective
inhibitor that not only mimics aspects of both the bound target
and viral DNA but also more completely fills the space they would normally occupy. Maximizing shape complementarity and
recapitulating structural components encompassing both of the IN DNA substrates could serve as a guiding principle for the
development of new INSTIs.
|
Feb 2016
|
|
