I03-Macromolecular Crystallography
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Angelina R.
Sekirnik
,
Jessica K.
Reynolds
,
Larissa
See
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Cynthia
Tallant
,
Bernadette
Lee
,
Katarzyna B.
Leszczynska
,
Rachel L.
Grimley
,
R. Ian
Storer
,
Marta
Malattia
,
Sara
Crespillo
,
Sofia
Caria
,
Stephanie
Duclos
,
Ester M.
Hammond
,
Stefan
Knapp
,
Garrett M.
Morris
,
Fernanda
Duarte
,
Philip C.
Biggin
,
Stuart J.
Conway
Open Access
Abstract: TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.
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Jun 2021
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I03-Macromolecular Crystallography
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Melissa
D'Ascenzio
,
Kathryn M.
Pugh
,
Rebecca
Konietzny
,
Georgina
Berridge
,
Cynthia
Tallant
,
Shaima
Hashem
,
Octovia
Monteiro
,
Jason R.
Thomas
,
Markus
Schirle
,
Stefan
Knapp
,
Brian
Marsden
,
Oleg
Fedorov
,
Chas
Bountra
,
Benedikt M.
Kessler
,
Paul E.
Brennan
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Bromodomain‐containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl‐lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule‐based approach to covalently modify recombinant and endogenous bromodomain‐containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in‐gel visualization and pull‐down of the desired bromodomains.
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Jan 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Epigenetics is of rapidly growing field in drug discovery. Of particular interest is the role of post‐translational modifications to histone and the proteins that read, write, and erase such modifications. The development of inhibitors for reader domains has focused on single domains. One of the major difficulties of designing inhibitors for reader domains, is that with the notable exception of bromodomains, they tend not to possess a well enclosed binding site amenable to small molecule inhibition. As many of the proteins in epigenetic regulation have multiple domains there are opportunities for designing inhibitors that bind at a domain‐domain interface which provide a more suitable interaction pocket. Examination of X‐ray structures of multiple domains involved in recognizing and modifying post‐translational histone marks using the SiteMap algorithm identified potential binding sites at domain‐domain interfaces. For the tandem plant homeodomain‐bromodomain of SP100C, a potential inter‐domain site identified computationally was validated experimentally by the discovery of ligands by X‐ray crystallographic fragment screening.
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Mar 2018
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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William R.
Shadrick
,
Peter J.
Slavish
,
Sergio C.
Chai
,
Brett
Waddell
,
Michele
Connelly
,
Jonathan A.
Low
,
Cynthia
Tallant
,
Brandon M.
Young
,
Nagakumar
Bharatham
,
Stefan
Knapp
,
Vincent A.
Boyd
,
Marie
Morfouace
,
Martine F.
Roussel
,
Taosheng
Chen
,
Richard E.
Lee
,
R.
Kiplin Guy
,
Anang A.
Shelat
,
Philip M.
Potter
Diamond Proposal Number(s):
[10619]
Abstract: Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC50 values comparable to BET inhibitor (BETi) clinical candidates.
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Nov 2017
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I03-Macromolecular Crystallography
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Niall
Igoe
,
Elliott D.
Bayle
,
Cynthia
Tallant
,
Oleg
Fedorov
,
Julia C.
Meier
,
Pavel
Savitsky
,
Catherine
Rogers
,
Yannick
Morias
,
Sarah
Scholze
,
Helen
Boyd
,
Danen
Cunoosamy
,
David M.
Andrews
,
Anne
Cheasty
,
Paul E.
Brennan
,
Susanne
Müller
,
Stefan
Knapp
,
Paul V.
Fish
Abstract: The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.
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Jul 2017
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I02-Macromolecular Crystallography
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Léa
Bouché
,
Clara D.
Christ
,
Stephan
Siegel
,
Amaury E.
Fernández-Montalván
,
Simon J.
Holton
,
Oleg
Fedorov
,
Antonius
Ter Laak
,
Tatsuo
Sugawara
,
Detlef
Stöckigt
,
Cynthia
Tallant
,
James
Bennett
,
Octovia
Monteiro
,
Laura
Díaz-Sáez
,
Paulina
Siejka
,
Julia
Meier
,
Vera
Pütter
,
Jörg
Weiske
,
Susanne
Müller
,
Kilian V. M.
Huber
,
Ingo V.
Hartung
,
Bernard
Haendler
Diamond Proposal Number(s):
[15558, 10619]
Abstract: Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure–activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2– or TAF1–histone H3.3 or H4 interaction assay.
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May 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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.
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Feb 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Niall
Igoe
,
Elliott D.
Bayle
,
Oleg
Fedorov
,
Cynthia
Tallant
,
Pavel
Savitsky
,
Catherine
Rogers
,
Dafydd R.
Owen
,
Gauri
Deb
,
Tim C. P.
Somervaille
,
David M.
Andrews
,
Neil
Jones
,
Anne
Cheasty
,
Hamish
Ryder
,
Paul E.
Brennan
,
Susanne
Müller
,
Stefan
Knapp
,
Paul V.
Fish
Abstract: The BRPF (bromodomain and PHD finger-containing) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Evaluation of the BRPF family as a potential drug target is at an early stage although there is an emerging understanding of a role in acute myeloid leukemia (AML). We report the optimization of fragment hit 5b to 13-d as a biased, potent inhibitor of the BRD of the BRPFs with excellent selectivity over nonclass IV BRD proteins. Evaluation of 13-d in a panel of cancer cell lines showed a selective inhibition of proliferation of a subset of AML lines. Pharmacokinetic studies established that 13-d had properties compatible with oral dosing in mouse models of disease (Fpo 49%). We propose that NI-42 (13-d) is a new chemical probe for the BRPFs suitable for cellular and in vivo studies to explore the fundamental biology of these proteins.
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Jan 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Vassilios
Myrianthopoulos
,
Nicolas
Gaboriaud-Kolar
,
Cynthia
Tallant
,
Michelle-Lynn
Hall
,
Stylianos
Grigoriou
,
Peter Moore
Brownlee
,
Oleg
Fedorov
,
Catherine
Rogers
,
David
Heidenreich
,
Marek
Wanior
,
Nikolaos
Drosos
,
Nikitia
Mexia
,
Pavel
Savitsky
,
Tina
Bagratuni
,
Efstathios
Kastritis
,
Evangelos
Terpos
,
Panagis
Filippakopoulos
,
Susanne
Müller
,
Alexios-Leandros
Skaltsounis
,
Jessica Ann
Downs
,
Stefan
Knapp
,
Emmanuel
Mikros
Open Access
Abstract: Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein–ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).
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Oct 2016
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