VMXi-Versatile Macromolecular Crystallography in situ
|
Abstract: Macromolecular X-ray crystallography is a powerful tool enabling modern structure- based drug design, where structures of protein-ligand complexes provide a basis for rational design decisions to improve the potency of ligands. However, like the protein itself, small molecules within protein crystals are subject to specific radiation damage (SRD) during the collection of X-ray diffraction data, but the effect of SRD on small molecule ligands has not yet been extensively described. This study is following on recently published work1 investigating SRD to halogenated ligands in protein-ligand structures of the therapeutic cancer target B-cell lymphoma 6 protein (BCL6), which found significant cleavage of carbon-halogen (C-X) bonds during X-ray diffraction data collection. The present work is making use of an improved set of BCL6 ligands to gain further understanding of the role of the halogen type and substitution position on the susceptibility of the C-X bond to SRD. Additionally, diffraction data will not only be collected at cryogenic temperature, but also at room temperature at the VMXi beamline at Diamond Light Source to investigate the impact of data collection temperature on SRD to small molecule ligands.
|
Jun 2025
|
|
I22-Small angle scattering & Diffraction
|
Oleksii
Avdieiev
,
Sergey A.
Denisov
,
Ashkan
Ajeer
,
Lois
Adams
,
Charlene
Greenwood
,
Heather
Nesbitt
,
Keith
Thomas
,
Keith
Rogers
,
Olga
Solovyeva
,
Lev
Mourokh
,
Pavel
Lazarev
Open Access
Abstract: Structural biomarkers determined by X-ray scattering of the tissues can complement conventional diagnostics and provide a pathway for early detection of diseases. In the present study, mouse models were utilized to observe the progression of prostate cancer. We induced cancer in the left lobe of the mouse prostate, whilst the right lobe was left uninoculated. The mice were sacrificed at increasing systematic time points, and lobe samples were subsequently analyzed using X-ray scattering. Control samples were also collected from healthy mice sacrificed at the same time points. This investigation revealed that the ratio between the X-ray scattering peaks associated with the lipids and water can serve as a structural biomarker of cancer, and this biomarker develops as the tumor advances. The obtained cancer trajectory can serve as a baseline for the determination of the disease stage, and the biomarker movement along the trajectory can be evidence of the healing or disease progression.
|
Jun 2025
|
|
B21-High Throughput SAXS
|
Alexander
Leithner
,
Oskar
Staufer
,
Tanmay
Mitra
,
Falk
Liberta
,
Salvatore
Valvo
,
Mikhail
Kutuzov
,
Hannah
Dada
,
Jacob
Spaeth
,
Weijie
Zhou
,
Felix
Schiele
,
Sophia
Reindl
,
Herbert
Nar
,
Stefan
Hoerer
,
Maureen
Crames
,
Stephen
Comeau
,
David
Young
,
Sarah
Low
,
Edward
Jenkins
,
Simon J.
Davis
,
David
Klenerman
,
Andrew
Nixon
,
Noah
Pefaur
,
David
Wyatt
,
Omer
Dushek
,
Srinath
Kasturirangan
,
Michael L.
Dustin
Open Access
Abstract: Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
|
Jun 2025
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[32711]
Open Access
Abstract: The tripartite motif (TRIM) family of RING-type E3 ligases catalyses the formation of many different types of ubiquitin chains, and as such, plays important roles in diverse cellular functions, ranging from immune regulation to cancer signalling pathways. Few ligands have been discovered for TRIM E3 ligases, and these E3s are under-represented in the rapidly expanding field of induced proximity. Here we present the identification of a novel covalent ligand for the PRYSPRY substrate binding domain of TRIM25. We employ covalent fragment screening coupled with high-throughput chemistry direct-to-biology optimisation to efficiently elaborate covalent fragment hits. We demonstrate that our optimised ligand enhances the in vitro auto-ubiquitination activity of TRIM25 and engages TRIM25 in live cells. We also present the X-ray crystal structure of TRIM25 PRYSPRY in complex with this covalent ligand. Finally, we incorporate our optimised ligand into heterobifunctional proximity-inducing compounds and demonstrate the in vitro targeted ubiquitination of a neosubstrate by TRIM25.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Jeffrey A.
Boerth
,
Marianne
Schimpl
,
Simon C. C.
Lucas
,
Jingwen
Zhang
,
Erin L.
Code
,
Kevin J.
Embrey
,
Philip B.
Rawlins
,
Haixia
Wang
,
R. Ian
Storer
,
Paolo
Di Fruscia
,
Jennifer E.
Nelson
,
Alexander G.
Milbradt
,
Ulf
Börjesson
,
Andrea
Gohlke
,
Victoria
Korboukh
,
Ariamala
Gopalsamy
Diamond Proposal Number(s):
[20015]
Abstract: Suppression of oncogenic gene expression is an effective strategy for the treatment of cancer. The SWI/SNF (SWItch/Sucrose Non-Fermentable) complex plays an important role in regulating gene activation or repression, and its dysregulation has been linked to aberrant transcription activity in many types of cancer. Targeting the subunits of this complex, such as SMARCA2, SMARCA4, and PBRM1, which are part of the bromodomain family VIII, has significant therapeutic potential. Herein we report the discovery of pyrimidoindolones as a novel series of bromodomain family VIII binders identified through an NMR-based fragment screen. These binders have been optimized to achieve sub-μM affinity for the family VIII proteins SMARCA2, SMARCA4, and PRBM1, with promising physicochemical properties.
|
May 2025
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Open Access
Abstract: Design and synthesis of two 3-substituted swainsonine derivatives with the aim to improve the potency and selectivity towards Golgi alpha-mannosidase II.
|
May 2025
|
|
I04-Macromolecular Crystallography
|
Syed Feroj
Ahmed
,
Jayanthi
Anand
,
Wei
Zhang
,
Lori
Buetow
,
Loveena
Rishi
,
Louise
Mitchell
,
Jonathan
Bohlen
,
Sergio
Lilla
,
Gary J.
Sibbet
,
Colin
Nixon
,
Amrita
Patel
,
Karolina A.
Majorek
,
Sara
Zanivan
,
Jacinta C.
Bustamante
,
Sachdev S.
Sidhu
,
Karen
Blyth
,
Danny T.
Huang
Diamond Proposal Number(s):
[16258]
Open Access
Abstract: Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase critical for negatively regulating receptor protein tyrosine kinases (RTKs). Deleterious CBL mutants lose E3 activity, but act as adaptors that gain function to cause myeloproliferative neoplasms. Currently, there is no targeted treatment available for patients with CBL mutant-dependent disorders. By combining phage-display technology and structure-based optimization, we discovered CBLock, a nanomolar affinity peptide inhibitor, that binds the substrate-binding site of CBL’s tyrosine kinase binding domain (TKBD). CBLock disrupts the interaction between CBL mutants and RTKs, thereby impairing RTK-mediated priming of adaptor function of CBL mutants and downstream signaling. Notably, CBLock binds TKBD without inducing conformational changes, thereby preserving its ligand-free native conformation. In contrast, when CBL binds RTK substrates, TKBD undergoes a conformational change. Maintaining the native CBL TKBD conformation was crucial for CBLock to inhibit proliferation, induce cell cycle arrest, and promote apoptosis in leukemia cells harboring CBL mutations. In a mouse xenograft model of acute myeloid leukemia (AML), CBLock reduced tumor burden and improved survival rate. Moreover, CBLock inhibited the proliferation of cells derived from patients with CBL mutations. Therefore, inhibiting CBL TKBD in its native state presents a promising therapeutic opportunity in targeting mutant CBL-dependent leukemia.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Zhixiang
Chen
,
Harshil
Dhruv
,
Xuqing
Zhang
,
Rohan Kalyan
Rej
,
Longchuan
Bai
,
Donna
Mceachern
,
Paul
Kirchhoff
,
Rakesh
Nagilla
,
Larry J.
Jolivette
,
Cory T.
Rice
,
Peter
Orth
,
Corey O.
Strickland
,
E. Scott
Priestley
,
Helai P.
Mohammad
,
Meilin
Wang
,
Bo
Wen
,
Duxin
Sun
,
Zhihua
Sui
,
Shaomeng
Wang
Open Access
Abstract: IKZF2 (Helios) is a transcription factor that is selectively expressed by Tregs and is essential for preserving the function and stability of Tregs in the tumor microenvironment (TME), where it suppresses the anti-tumor immune response. Targeted IKZF2 degradation by small molecules represents a promising strategy for the development of a new class of cancer immunotherapy. Herein, we describe the discovery of PVTX-405, a potent, effective, highly selective, and orally efficacious IKZF2 molecular glue degrader. PVTX-405 degrades IKZF2 (DC50 = 0.7 nM and Dmax = 91%) while sparing other CRBN neo-substrates. Degradation of IKZF2 by PVTX-405 increases production of inflammatory cytokine IL-2 and reduces the suppressive activity of Tregs, leading to an increase in Teff cell proliferation. Once-daily oral administration of PVTX-405 as single agent significantly delays the growth of MC38 tumors in a syngeneic tumor model using humanized CRBN mice. PVTX-405 in combination with anti-PD1 or anti-LAG3 significantly increases animal survival compared to anti-PD1 or anti-LAG3 alone. Together, these results demonstrate that PVTX-405 is a promising IKZF2 degrader for clinical development for the treatment of human cancers.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Rohan
Bythell-Douglas
,
Sylvie
Van Twest
,
Lara
Abbouche
,
Elyse
Dunn
,
Rachel J.
Coulthard
,
David C.
Briggs
,
Vincent
Murphy
,
Xinxin
Zhang
,
Winnie
Tan
,
Sarah S
Henrikus
,
Dongming
Qian
,
Yin
Wu
,
Jana
Wolf
,
Laurent
Rigoreau
,
Shabih
Shakeel
,
Kathryn L.
Chapman
,
Neil Q.
Mcdonald
,
Andrew J.
Deans
Open Access
Abstract: FANCM is crucial in genome maintenance, functioning in the Fanconi anemia (FA) pathway, alternative lengthening of telomeres (ALT), and replication fork protection. FANCM recognizes branched DNA structures and promotes their remodeling through ATP-dependent branch migration. The protein has emerged as a promising therapeutic target due to synthetic lethal interactions with BRCA1, SMARCAL1, and RAD52, and in ALT-positive cancers. Here we present crystal structures of FANCM’s N-terminal ATP-dependent translocase domain (2.2 Å) and C-terminal FAAP24-bound region (2.4 Å), both complexed with branched DNA. Through structural analysis, biochemical reconstitution, and cellular studies, we demonstrate that FANCM employs two distinct mechanisms: an ATP-dependent branch migration activity essential for DNA damage survival, and a branched DNA-binding mode that enhances FANCD2-FANCI monoubiquitination through FA core complex interaction. The N-terminal translocase domain specifically recognizes DNA junctions through multiple key elements, while the C-terminal FAAP24-binding domain engages adjacent double-stranded DNA. Our results reveal how FANCM evolved from an ancient DNA repair motor into a sophisticated sensor that couples DNA damage recognition to selective pathway activation, providing a structural framework for developing targeted therapeutics.
|
May 2025
|
|
