B23-Circular Dichroism
|
Xi
Lei
,
Sibo
Peng
,
Nijas
Aliu
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Tiberiu-Marius
Gianga
,
Xin
Jin
,
Sabrina
Ruggiero
,
Shilpa
Chokshi
,
Luca
Urbani
,
Bernhard F.
Gibbs
,
Elena
Klenova
,
Gurprit S.
Lall
,
Gabriela
Dekany
,
Dietmar
Cholewa
,
Steffen M.
Berger
,
Maryam
Abooali
,
Inna M.
Yasinska
,
Song
Xu
,
Elizaveta
Fasler-Kan
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[36941, 38189]
Abstract: Human cancer cells and malignant tumours operate immune evasion machinery, which consists of a set of cross-linked biochemical pathways that suppress the activities of immune cells, such as cytotoxic T cells and natural killer cells. Recent evidence demonstrated that the immune evasion machinery operated by human cancer cells is self-sustaining. However, the mechanisms underlying self-regulation of cancer immune evasion networks remain to be understood. Here we report for the very first time that the cell surface receptor CD44 acts as an enhancer of immune evasion activity in human cancer cells rather than an immune checkpoint per se. Furthermore, upon interaction with galectin-9, CD44 was found to alter cell membrane potential and induce the secretion of TGF-β. Secreted TGF-β displays autocrine and paracrine activities following TGF-β receptor (TGFBR) engagement, which was shown to then activate the Smad3 transcriptional pathway. CD44 complexed to galectin-9 allosterically enhances activity of the TGFBR complex and this was shown to substantially amplify the expression of immune checkpoint proteins crucially associated with suppressing the anti-cancer activities of cytotoxic lymphoid cells. We also demonstrated that this is a fundamental pathway used by human embryos to prevent immune rejection and by innate immune cells to facilitate anti-bacterial innate immune response. Cancer cells and malignant tumours hijack and successfully adapt this fundamental pathway to escape immune surveillance and suppress anti-cancer immune reactions. As such, this autocrine loop allows for the self-regulation of immune evasion networks operated by cancer cells and can be considered as a promising target for highly efficient immunotherapy of cancer.
|
Nov 2026
|
|
I03-Macromolecular Crystallography
|
Rob F.
Lammers
,
Qin
Su
,
Laura
Mazo
,
Wendy A.
Offen
,
Roelof
Ottenhoff
,
Mats J.
Bulterman
,
Martijn
Van Der Lienden
,
Maria J.
Ferraz
,
Florian
Küllmer
,
Zahid
Ilhan
,
Hans
Van Den Elst
,
Ingeborg
Van Der Made
,
Cindy P. A. A.
Van Roomen
,
Anne
De Jongh
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Rolf G.
Boot
,
Noam
Zelcer
,
Gideon J.
Davies
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
,
Marta
Artola
Diamond Proposal Number(s):
[39189]
Open Access
Abstract: Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.
|
Sep 2026
|
|
I04-Macromolecular Crystallography
|
Michael H. N.
Meulendijks
,
Yun
Li
,
Frederik J.
Steiner
,
Rasmus
Klintrot
,
Clemens
Schulte
,
Seline
Roose
,
Alejandro
Yeste‐vázquez
,
Hans M.
Maric
,
Sven
Hennig
,
Felix M.
Paulussen
,
Ioana M.
Ilie
,
Tom N.
Grossmann
Diamond Proposal Number(s):
[34465]
Open Access
Abstract: Peptide-based inhibitors are promising tools to target challenging protein interfaces, yet improving their affinity and drug-like properties is still a major hurdle. This holds particularly true for β-sheet motifs with only limited strategies available to robustly stabilize their conformation. Previously, we reported linear β-hairpin peptide l12 with micromolar affinity for β-catenin, a central effector of aberrant Wnt signaling in cancer. Here, we show that a single N4V substitution markedly enhances binding, despite not directly contacting the protein surface as evidenced by obtained X-ray structures and molecular dynamics simulations. Notably, the substitution combines additively with head-to-tail cyclization yielding a peptide with strongly increased affinity. This mechanistic study presents structural and computational analyses revealing that the affinity improvements arise from solution-state preorganization and β-hairpin stabilization mediated by an altered intramolecular hydrogen bond network. Our findings highlight the critical role of conformational preorganization in peptide-protein recognition, demonstrating that optimizing solution-state properties can unlock substantial affinity gains that are not obvious from crystal structures alone.
|
Sep 2026
|
|
I04-Macromolecular Crystallography
|
Juan
Del Pozo
,
Elisabeth
Hennessy
,
Patrick
Schoepf
,
Kazuaki
Shibata
,
Kei
Akemoto
,
Naomi
Abe
,
Christopher
Agnew
,
Hiroki
Asakura
,
David J.
Bennett
,
Samantha A.
Burgess
,
Xiaomei
Chai
,
Gianni
Chessari
,
Philip J.
Day
,
Ruchia
Duggal
,
George M.
Giambasu
,
Christopher C. F.
Hamlett
,
Yuan
Jiang
,
Taiki
Kida
,
Toshiharu
Komori
,
Hitomi
Kondo
,
Brian M.
Lacey
,
Todd W.
Mayhood
,
Risako
Miura
,
Shinji
Mizuarai
,
Erik V.
Munsell
,
Marc
O’reilly
,
Barbara
Rath
,
David C.
Rees
,
Michael C.
Ryan
,
Takeshi
Sagara
,
Toshihiro
Sakamoto
,
Jordan
De Jesus Silva
,
Nicolas
Solban
,
Xuelei Sherry
Song
,
Tetsuya
Sugimoto
,
Miki
Terasaka
,
David G.
Twigg
,
Hiroyuki
Ueno
,
Kentaro
Wakayama
,
Zangwei
Xu
,
Tomohiro
Yamamoto
,
Alexander
Stoeck
,
Yongxin
Han
Diamond Proposal Number(s):
[27057]
Abstract: Current KRASG12C inhibitors mainly target the inactive GDP-bound state, but clinical efficacy is limited, and resistance mechanisms have been identified. We disclose novel covalent inhibitors that selectively target the active GTP-bound KRASG12C. Starting from a high-throughput screen, we identified a keto-indolizine scaffold featuring an aniline-derived acrylamide warhead that covalently binds Cys12 in the switch-II pocket of active KRASG12C. X-ray crystallography revealed key interactions of acrylamide with GMPPCP′s γ-phosphate, stabilizing the GTP state. We present medicinal chemistry efforts leading to the discovery of compound 17, which shows favorable oral bioavailability in preclinical species, exhibiting prolonged disruption of the KRAS-cRAF interaction and downstream ERK phosphorylation in vivo. In a KRASG12C-driven pancreatic cancer xenograft model (MIA PaCa-2), orally administered compound 17 achieved complete tumor regression for up to 45 days with good tolerability. This study establishes a novel class of KRASG12C inhibitors targeting the active GTP-bound state, which may have the potential to overcome some resistance mechanisms.
|
Sep 2026
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[34438, 31440, 37593]
Open Access
Abstract: In targeted protein degradation (TPD), specific subcellular proteins are removed by routing them to the ubiquitin-proteasome, autophagy, or lysosome machinery. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic heterobifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging, and the number of currently addressable ubiquitin E3 ligases is limited. Here, we design a heterobifunctional de novo protein to trigger the degradation of a common cancer target, resulting in a desired phenotypic output. First, we developed a highly stable and adaptable helix–turn–helix scaffold for presenting multiple binding sites. Next, we use computational protein design to incorporate and embellish hot-spot-binding sites to target the antiapoptotic mediators BCL-xL and MCL-1. We show a 75% success rate for creating submicromolar binders against these targets. Crystal structures of the complexes confirmed the designed binding poses. Then, we designed short linear motifs (SLiMs) into the loop of the scaffold to recruit KLHL20 and the ubiquitin ligase machinery. These designs have low micromolar affinity for KLHL20 comparable to that of the natural SLiMs. Moreover, the bifunctionalized proteins degrade BCL-xL in cells, leading to apoptosis.
|
Aug 2026
|
|
I04-Macromolecular Crystallography
|
Joel L.
Syphers
,
Josephine A.
Wright
,
Adarsh
Kumar
,
Nikos
To
,
Lewis
Elson
,
Andreas
Kramer
,
Susanne
Müller
,
Viktoria
Morasch
,
Aeson
Chang
,
Savannah
Young
,
Erica K.
Sloan
,
Rebekah
De Nys
,
Tharindie N.
Silva
,
Laura
Vrbanac
,
Kate R.
Barratt
,
Julia
Leeflang
,
Sadia T.
Hasan
,
Robert W.
Gable
,
Stefan
Knapp
,
Daniel L
Worthley
,
Siddhartha
Mukherjee
,
Kieran
Stockton
,
Susan L.
Woods
,
Daniel L.
Priebbenow
,
Jonathan B.
Baell
Open Access
Abstract: Macrocyclization can enhance the selectivity of acyclic compounds toward structurally similar biological targets such as kinases. WEE1 regulates cellular homeostasis and is a promising target in oncology. The clinical candidate AZD1775 (1) failed to progress past Phase II trials because of patient tolerability issues, likely due to off-target inhibition of polo-like kinase 1 (PLK1). Herein, a computer-aided drug design approach was conducted to develop a macrocycle based on the 1-WEE1 X-ray cocrystal structure. Significantly enhanced WEE1 inhibitory selectivity over PLK1 was determined for leading macrocycle 2, which also demonstrated broader kinome-wide selectivity. Patient-derived organoids from colorectal cancer (CRC) peritoneal and liver metastases, treated with 2, demonstrated comparably strong or enhanced anticancer efficacy compared to that of 1. Against patient-matched normal colon vs primary CRC organoids, 2 potently and selectively treated CRC, as well as enhanced DNA damage compared to 1. Finally, the X-ray cocrystal structure of 2 bound to WEE1 validated its computationally predicted bioactive binding mode.
|
Aug 2026
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[34035]
Open Access
Abstract: The rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (RAF/MEK/ERK) signaling cascade regulates cell proliferation and differentiation and is frequently dysregulated in cancer. Approximately 90% of RAF-mutant cancers harbor mutations in B-type rapidly accelerated fibrosarcoma (BRAF). Its proto-oncogenicity is attributed to a four-residue N-terminal acidic (NtA) motif. Although a long-standing model proposes that the NtA promotes activating asymmetric RAF dimerization, the model lacks structural support. Here, we present structures of NtA-mediated asymmetric BRAF dimers bound to their substrate MEK1. Cellular and biochemical data show that the NtA is not required for KRAS-mediated BRAF recruitment to the plasma membrane but is required for the fully catalytically active state. The structure capturing BRAF in a post-catalytic state bound to Ser222-phosphorylated MEK1 further supports this model. The combination of structural and cellular data corroborates the model of NtA-driven asymmetry in BRAF activation and resolves a long-standing disconnect between RAF cancer genetics and structural biology.
|
Aug 2026
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[34035]
Open Access
Abstract: The discovery of tubulysins sparked considerable interest due to their high cytotoxic activity against multidrug resistant tumors. Total synthesis of these complex natural products—peptidic metabolites from myxobacteria Archangium gephyra and Angiococcus disciformis—demonstrated the power of organic chemistry and paved the way for the development of simpler, more stable, and selective derivatives. Despite these significant achievements, tubulysins are not used as stand-alone drugs due to their toxic side effects. In this study, we outline the design, synthesis, and evaluation of photoswitchable tubulysin analogues, which could be starting points for development of photopharmacological therapies aimed at addressing the toxicity challenges associated with tubulysins. The cytotoxic activity of one of the key analogues was shown to be light-controllable. For the first time, we provide a comparative analysis of the crystal structures of both photoisomers of a diarylethene-containing compound complexed with target proteins. This comparison enables a mechanistic explanation for the experimentally observed differences in the target binding efficiency and respective activity between the two photoisomers of the photoswitchable tubulysin analogue.
|
Aug 2026
|
|
I03-Macromolecular Crystallography
|
Athina
Zerva
,
Nicolai D.
Raig
,
Zaile
Zhuang
,
Andreas
Kramer
,
Johannes
Dopfer
,
Riley K.
Togashi
,
Martin Peter
Schwalm
,
Lewis
Elson
,
Julia M.
Frischkorn
,
Benedict-Tilman
Berger
,
Susanne
Müller
,
James K.
Chen
,
Stefan
Knapp
,
Thomas
Hanke
Diamond Proposal Number(s):
[44264]
Open Access
Abstract: Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest its involvement in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. These investigations are currently hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50: 11 nM; cellular EC50: 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.
|
Aug 2026
|
|
B21-High Throughput SAXS
|
Open Access
Abstract: Semaglutide is a glucagon-like peptide-1 receptor agonist widely used for the treatment of type 2 diabetes and obesity. Despite its clinical efficacy, oral administration remains challenging because of its limited gastrointestinal stability, poor epithelial permeability, and low affinity for lipid-based delivery systems. In the present study, a combined hydrophobic ion pairing (HIP) and solid lipid nanoparticle (SLN) approach was explored to improve semaglutide incorporation and delivery-related properties. Semaglutide was complexed with the cationic lipid DOTAP at different molar ratios (1:0–1:18) and subsequently incorporated into cetyl palmitate-based SLNs produced by microfluidic mixing using a herringbone device. The resulting formulations were characterized in terms of particle size, ζ-potential, encapsulation efficiency, morphology, solid-state organization, colloidal stability, release behavior, mucus interaction, cytocompatibility, and epithelial permeability. Among the various formulations prepared, the one prepared with a molar ratio semaglutide: DOTAP of 1:18 and a peptide concentration of 10% (w/w) (F10) showed the best results, combining particle sizes of less than 300 nm with almost complete encapsulation efficiency and a highly positive ζ-potential. FTIR, DSC, TGA and SAXS analyses confirmed the correct formation of the complex and its incorporation into the lipid matrix. The F10 formulation demonstrated good stability under simulated gastrointestinal conditions and a sustained-release profile. The formulation also exhibited strong interactions with mucus, whilst retaining the ability to diffuse through the mucin network. Cytocompatibility studies demonstrated acceptable cell viability at relevant concentrations, whilst permeability experiments through Caco-2 monolayers revealed an approximately 6-fold increase in apparent permeability compared to free semaglutide. Therefore, these findings indicate that the combination of DOTAP-mediated hydrophobic ion pairing and microfluidic-assisted SLN production represents a potentially promising strategy for improving semaglutide encapsulation, gastrointestinal stability, and epithelial transport, while maintaining a favorable balance between mucus interaction and mucodiffusion.
|
Aug 2026
|
|
