I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[36097]
Open Access
Abstract: The cavity-creating p53 cancer mutation Y220C, which accounts for an estimated 125,000 new cancer cases per year, serves as an excellent paradigm for the development of mutant p53 reactivators. Several molecules that reactivate this thermolabile cancer mutant by targeting the mutation-induced crevice have been developed, and one of them, rezatapopt, is currently in clinical trials. The less frequently occurring Y220N and Y220S mutations are even more destabilizing than Y220C but create a similar surface crevice, raising the question of whether cancer patients with these mutations might also benefit from rezatapopt treatment. Here, we show that rezatapopt also binds to the Y220N and Y220S mutants, with nanomolar affinity, resulting in a full recovery of wild-type-like stability for the latter. High-resolution crystal structures of all three mutants bound to rezatapopt revealed a conserved binding mode, highlighting key interactions, including multipolar interactions of a fluorine substituent at a chiral center with the protein backbone. Consistent with the biophysical and structural data, rezatapopt reactivated p53 signaling in both Y220C and Y220S mutant cells by restoring the folded conformation and transcriptional activity, leading to anti-proliferative effects and apoptosis, albeit requiring higher compound concentrations in Y220S cells. The Y220N mutant, despite exhibiting high-nanomolar affinity for rezatapopt and substantial stabilization, did not show noticeable effects in cells at the concentrations tested, as rezatapopt binding resulted in only partial compensation for the mutation-induced loss of stability, for which we provide a structural explanation. Our data suggest that the development of clinical pan-Y220C/N/S reactivators, which could benefit an additional 10,000 patients per year, is challenging but not impossible.
|
Feb 2026
|
|
I03-Macromolecular Crystallography
|
Anthony K.
Edmonds
,
Dimitrios-Ilias
Balourdas
,
Graham P.
Marsh
,
Robert
Felix
,
Bradley
Brasher
,
Jeff
Cooper
,
Cari
Graber-Feesl
,
Madhu
Kollareddy
,
Karim
Malik
,
Helen
Stewart
,
Timothy J. T.
Chevassut
,
Ella
Lineham
,
Simon
Morley
,
Oleg
Fedorov
,
James
Bennett
,
Mohan B.
Rajasekaran
,
Samuel
Ojeda
,
Drew A.
Harrison
,
Christopher J.
Ott
,
Andreas C.
Joerger
,
Hannah J.
Maple
,
John
Spencer
Open Access
Abstract: Degraders with dual activity against BRD4 and CBP/EP300 were designed. A structure-guided design approach was taken to assess and test potential exit vectors on the dual BRD4 and CBP/EP300 inhibitor, ISOX-DUAL. Candidate degrader panels revealed that VHL-recruiting moieties could mediate dose-responsive ubiquitination of BRD4. A panel of CRBN-recruiting thalidomide-based degraders was unable to induce ubiquitination or degradation of target proteins. High-resolution protein cocrystal structures revealed an unexpected interaction between the thalidomide moiety and Trp81 on the first bromodomain of BRD4. The inability to form a ternary complex provides a potential rationale for the lack of degrader activity with these compounds, some of which have remarkable affinities close to those of (+)-JQ1, as low as 65 nM in a biochemical assay, vs 1.5 μM for their POI ligand, ISOX-DUAL. Such a “degrader collapse” may represent an under-reported mechanism by which some putative degrader molecules are inactive with respect to target protein degradation.
|
Apr 2025
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Harold
Grosjean
,
Anthony
Aimon
,
Storm
Hassell-Hart
,
Warren
Thompson
,
Lizbe
Koekemoer
,
James
Bennett
,
Anthony
Bradley
,
Cameron
Anderson
,
Conor
Wild
,
William J.
Bradshaw
,
Edward A.
Fitzgerald
,
Tobias
Krojer
,
Oleg
Fedorov
,
Philip C.
Biggin
,
John
Spencer
,
Frank
Von Delft
Diamond Proposal Number(s):
[19301]
Abstract: Fragment approaches are long-established in target-based ligand discovery yet their full transformative potential lies dormant, because progressing hits to potency remains underserved by methodological work. The only credible progression paradigm is multiple cycles of costly conventional design-make-test-analyse (DMTA) medicinal chemistry, necessitating picking winners early and discarding others. It is effective to cheaply parallelize large numbers of non-uniform multi-step reactions, because, even without compound purification, a high-quality readout of binding is available, viz. crystallography. This can detect low-level binding of slightly active compounds, which the targeted binding site extracts directly from crude reaction mixtures (CRMs). In this proof-of-concept study, we expand a fragment hit from a crystal-based screen of the bromodomain PHIP2, using array synthesis on low-cost robotics to implement 6 independent multi-step reaction routes of up to 5 steps, attempting the synthesis of 1876 diverse expansions, designs entirely driven by synthetic tractability. The expected product was present in 1108 (59%) CRMs, detected by automated mass spectrometry, 22 individual products were resolved in crystal structures of CRMs added to crystals, providing an initial SAR map, pose stability in 19 and instability in 3 products and resolved stereochemical preference. One compound showed biochemical potency (IC50=34 μM) and affinity (Kd=50 μM) after resynthesis.
|
Feb 2025
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[26617]
Open Access
Abstract: Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer’s disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-β (Aβ) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.
|
Jun 2023
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Open Access
Abstract: A library of thiazoles and selenothiazoles were synthesized via Ir-catalyzed ylide insertion chemistry. This process is a functional group, particularly heterocycle-substituent tolerant. This was applied to the synthesis of fanetizole, an anti-inflammatory drug, and a thiazole-containing drug fragment that binds to the peptidyl-tRNA hydrolase (Pth) in Neisseria gonorrheae bacteria.
|
Nov 2022
|
|
I03-Macromolecular Crystallography
|
Arathy
Jose
,
Daniel
Guest
,
Remi
Legay
,
Graham J.
Tizzard
,
Simon
Coles
,
Mariliza
Derveni
,
Edward
Wright
,
Lester
Marrison
,
Alpha A.
Lee
,
Aaron
Morris
,
Matt
Robinson
,
Frank
Von Delft
,
Daren
Fearon
,
Lizbe
Koekemoer
,
Tetiana
Matviuk
,
Anthony
Aimon
,
Christopher J.
Schofield
,
Tika R.
Malla
,
Nir
London
,
Barnaby W.
Greenland
,
Mark C.
Bagley
,
John
Spencer
Diamond Proposal Number(s):
[19301]
Open Access
Abstract: The pentafluorosulfanyl (-SF5) functional group is of increasing interest as a bioisostere in medicinal chemistry. A library of SF5-containing compounds, including amide, isoxazole, and oxindole derivatives, was synthesised using a range of solution-based and solventless methods, including microwave and ball-mill techniques. The library was tested against targets including human dihydroorotate dehydrogenase (HDHODH). A subsequent focused approach led to synthesis of analogues of the clinically used disease modifying anti-rheumatic drugs (DMARDs), Teriflunomide and Leflunomide, considered for potential COVID-19 use, where SF5 bioisostere deployment led to improved inhibition of HDHODH compared with the parent drugs. The results demonstrate the utility of the SF5 group in medicinal chemistry.
|
Feb 2022
|
|
I03-Macromolecular Crystallography
|
John
Spencer
,
Storm
Hassell
,
Sarah
Picaud
,
Ralph
Lengacher
,
Joshua
Csuker
,
Regis
Millet
,
Gilles
Gasser
,
Roger
Alberto
,
Hannah
Maple
,
Robert
Felix
,
Zbigniew
Leśnikowski
,
Helen
Stewart
,
Timothy
Chevassut
,
Simon
Morley
,
Panagis
Filippakopoulos
Diamond Proposal Number(s):
[19301]
Abstract: A series of bulky organometallic and organic analogues of the bromodomain (BRD) inhibitor (+)‐JQ1 have been prepared. The most potent, N‐[(adamantan‐1‐yl)methyl]‐2‐[(9S)‐7‐(4‐chlorophenyl)‐4,5,13‐trimethyl‐3‐thia‐1,8,11,12‐tetraazatricyclo[8.3.0.02,6]trideca‐2(6),4,7,10,12‐pentaen‐9‐yl]acetamide, 2e , showed excellent potency with an K D = ca. 130 nM vs BRD4(1) and a ca. 2‐fold selectivity over BRD4(2) (K D = ca. 260 nM). Its binding to the first bromodomain of BRD4 was determined by a protein cocrystal structure.
|
Jan 2021
|
|
I24-Microfocus Macromolecular Crystallography
|
Angeliki
Ditsiou
,
Chiara
Cilibrasi
,
Nikiana
Simigdala
,
Athanasios
Papakyriakou
,
Leanne
Milton-Harris
,
Viviana
Vella
,
Joanne E.
Nettleship
,
Jae Ho
Lo
,
Shivani
Soni
,
Goar
Smbatyan
,
Panagiota
Ntavelou
,
Teresa
Gagliano
,
Maria Chiara
Iachini
,
Sahir
Khurshid
,
Thomas
Simon
,
Lihong
Zhou
,
Storm
Hassell-Hart
,
Philip
Carter
,
Laurence H.
Pearl
,
Robin L.
Owen
,
Raymond J.
Owens
,
S. Mark
Roe
,
Naomi E.
Chayen
,
Heinz-Josef
Lenz
,
John
Spencer
,
Chrisostomos
Prodromou
,
Apostolos
Klinakis
,
Justin
Stebbing
,
Georgios
Giamas
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.
|
Nov 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Raysa
Khan Tareque
,
Storm
Hassell-Hart
,
Tobias
Krojer
,
Anthony
Bradley
,
Srikannathasan
Velupillai
,
Romain
Talon
,
Michael
Fairhead
,
Iain J.
Day
,
Kamlesh
Bala
,
Robert
Felix
,
Paul D.
Kemmitt
,
Paul
Brennan
,
Frank
Von Delft
,
Laura
Diaz Saez
,
Kilian
Huber
,
John
Spencer
Diamond Proposal Number(s):
[18145]
Abstract: Combined photochemical arylation, “nuisance effect” (SNAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium‐throughput X‐ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho‐arylation of 2‐phenylpyridine gave five products resulting from mono‐ and bisarylations combined with SNAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.
|
Aug 2020
|
|
I03-Macromolecular Crystallography
|
Open Access
Abstract: We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.
|
Jan 2020
|
|
