I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 and BrdT, required a wasteful, late-stage alkylation step and expensive chiral separation. To circumvent these limitations, we developed a route based on stereocontrolled alkylation of an N-Pf protected aspartic acid derivative that was used in a divergent, racemisation-free protocol to yield structurally diverse and enantiopure triazolodiazepines. With this approach, we synthesized bumped thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps and 99% ee without the need for chiral chromatography. Exquisite selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over wild-type bromodomain was established by isothermal titration calorimetry and X-ray crystallography. Our new approach provides unambiguous chemical evidence for the absolute stereochemistry of the active, allele-specific BET inhibitors and a viable route that will open wider access to this compound class.
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Aug 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Storm
Hassell-Hart
,
Andrew
Runcie
,
Tobias
Krojer
,
Jordan
Doyle
,
Ella
Lineham
,
Cory A.
Ocasio
,
Brenno A. D.
Neto
,
Oleg
Fedorov
,
Graham
Marsh
,
Hannah
Maple
,
Robert
Felix
,
Rebecca
Banks
,
Alessio
Ciulli
,
Sarah
Picaud
,
Panagis
Filippakopoulos
,
Frank
Von Delft
,
Paul
Brennan
,
Helen J. S.
Stewart
,
Timothy J.
Chevassut
,
Martin
Walker
,
Carol
Austin
,
Simon
Morley
,
John
Spencer
Diamond Proposal Number(s):
[19301]
Abstract: (+)-JD1, a rationally designed ferrocene analogue of the BET bromodomain (BRD) probe molecule (+)-JQ1, has been synthesized and evaluated in biophysical, cell-based assays as well as in pharmacokinetic studies. It displays nanomolar activity against BRD isoforms, and its cocrystal structure was determined in complex with the first bromodomain of BRD4 and compared with that of (+)-JQ1, a known BRD4 small-molecule probe. At 1 μM concentration, (+)-JD1 was able to inhibit c-Myc, a key driver in cancer and an indirect target of BRD4.
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Dec 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Here, we report the design and synthesis of a first macrocyclic PROTAC by adding a second cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second Brd4 bromodomain validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.
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Nov 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980]
Open Access
Abstract: The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. Here, we report co-crystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.
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Jun 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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William
Farnaby
,
Manfred
Koegl
,
Michael J.
Roy
,
Claire
Whitworth
,
Emelyne
Diers
,
Nicole
Trainor
,
David
Zollman
,
Steffen
Steurer
,
Jale
Karolyi-Oezguer
,
Carina
Riedmueller
,
Teresa
Gmaschitz
,
Johannes
Wachter
,
Christian
Dank
,
Michael
Galant
,
Bernadette
Sharps
,
Klaus
Rumpel
,
Elisabeth
Traxler
,
Thomas
Gerstberger
,
Renate
Schnitzer
,
Oliver
Petermann
,
Peter
Greb
,
Harald
Weinstabl
,
Gerd
Bader
,
Andreas
Zoephel
,
Alexander
Weiss-Puxbaum
,
Katharina
Ehrenhöfer-Wölfer
,
Simon
Wöhrle
,
Guido
Boehmelt
,
Joerg
Rinnenthal
,
Heribert
Arnhof
,
Nicola
Wiechens
,
Meng-Ying
Wu
,
Tom
Owen-Hughes
,
Peter
Ettmayer
,
Mark
Pearson
,
Darryl B.
Mcconnell
,
Alessio
Ciulli
Diamond Proposal Number(s):
[14980]
Abstract: Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.
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Jun 2019
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I24-Microfocus Macromolecular Crystallography
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Vittoria
Zoppi
,
Scott J.
Hughes
,
Chiara
Maniaci
,
Andrea
Testa
,
Teresa
Gmaschitz
,
Corinna
Wieshofer
,
Manfred
Koegl
,
Kristin
Riching
,
Danette L.
Daniels
,
Andrea
Spallarossa
,
Alessio
Ciulli
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers, and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown, and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations.
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Dec 2018
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[5841, 7141]
Abstract: Beyond the targeting of E3 ubiquitin ligases to inhibit protein homeostasis, E3 ligase binders can be converted into targeted protein degraders. We sought to identify new binders of the VHL E3 ligase by biophysical fragment-based screening followed by X-ray crystallographic soaking. We identified fragments binding at the ElonginC:Cullin2 interface and a new cryptic pocket in VHL, along with other potential ligandable sites predicted computationally. The elucidated interactions provide starting points for future ligand development.
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Jul 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrea
Testa
,
Xavier
Lucas
,
Guilherme V.
Castro
,
Kwok-ho
Chan
,
Jane E.
Wright
,
Andrew C.
Runcie
,
Morgan S.
Gadd
,
William T. A.
Harrison
,
Eun-jung
Ko
,
Daniel
Fletcher
,
Alessio
Ciulli
Diamond Proposal Number(s):
[10071, 14980]
Abstract: Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e. fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-L-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia inducible factor 1 alpha (HIF-1α) substrate peptides as well as peptidomimetic ligands that form part of PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein deg-radation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organic chemists, chemical biologists and drug discoverers alike.
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Jun 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10071]
Open Access
Abstract: Thioamide substitution influences hydrogen bond and n→π∗ interactions involved in the conformational stability of protein secondary structures and oligopeptides. Hydroxyproline is the key recognition element of small molecules targeting the von Hippel-Lindau (VHL) E3 ligase, which are of interest as probes of hypoxia signaling and ligands for PROTAC conjugation. We hypothesized that VHL ligands could be a privileged model system to evaluate the contribution of these interactions to protein:ligand complex formation. Herein we report the synthesis of VHL ligands bearing thioamide substitutions at the central hydroxyproline moiety, and characterize their binding by fluorescence polarization, isothermal titration calorimetry, X-ray crystallography and molecular modeling. In spite of a conserved binding mode, the substitution pattern had a pronounced impact on the ligand affinities. Together the results underscore the role of hydrogen bond and n→π∗ interactions in fine-tuning hydroxyproline recognition by VHL.
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Mar 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980, 10071]
Abstract: Plant homeodomain (PHD) zinc fingers are histone reader domains that often associate with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic targets.
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Mar 2018
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