I03-Macromolecular Crystallography
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Zhanna
Romanyuk
,
Giacomo
Bettin
,
Paul
Brear
,
Sara
Linciano
,
Ylenia
Mazzocato
,
Simone
Bonadies
,
Ilaria
Zanotto
,
Camilla
Mazzucco
,
Alan
Monferone
,
Miguel A.
Soler
,
Gianfranco
Pasut
,
Sara De
Martin
,
Alessandro
Scarso
,
Christian
Heinis
,
Sylvia
Rothenberger
,
Marko
Hyvonen
,
Alessandro
Angelini
Diamond Proposal Number(s):
[33658]
Open Access
Abstract: Macrocyclic peptides (MPs) are valuable molecular formats for drug development, bridging small molecules and larger biologics due to their favorable pharmacological properties. Here, we describe the discovery of low-nanomolar inhibitors of human angiotensin-converting enzyme 2 (hACE2) by quantitatively screening millions of structurally diverse disulfide-cyclized peptide ligands using yeast display technology. The most potent yeast-encoded “one-ring” and “two-ring” MP inhibit hACE2 with Ki values of 1.9 and 1.5 nM, respectively. These inhibitory potencies are comparable to those of other cyclic peptides discovered using well-established in vitro display technologies. Crystal structures of the two MPs in complex with hACE2 reveal the adoption of either a rigid β-hairpin or a cysteine-stabilized α-helix/α-helix motif. Both MPs exhibit binding modes distinct from those of previously reported inhibitors. Thus, yeast display is a valid technology to rapidly generate MPs with desired binding properties for the development of potential therapeutics.
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Mar 2026
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I04-Macromolecular Crystallography
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Ludivine
Moine
,
Wei
Hu
,
Alison
Davis
,
Emanuele
Perola
,
Jian
Guo
,
Kevin
Barvian
,
Yeon Sook
Choi
,
Alexandra
Grassian
,
Joseph L.
Kim
,
Omar K.
Ahmad
,
Thomas A.
Dineen
Open Access
Abstract: Gastrointestinal stromal tumor (GIST) is the most common type of sarcoma of the gastrointestinal tract, with approximately 5000 new cases annually in the USA. Approximately 80% of GIST cases are driven by activating mutations in KIT in exon 9 or 11. Resistance to present therapies like imatinib often arises from secondary KIT mutations, especially V654A (exon 13), which is the most frequent resistance mutation. Tyrosine kinase inhibitors (TKIs) currently approved for GIST can cause dose-limiting side effects due to off-target inhibition of other kinases. Herein, we report the discovery and optimization of BLU-654 (compound 18), a highly potent and kinome-sparing KIT V654A inhibitor. Preclinical efficacy studies demonstrated its prolonged antitumor activity in a KIT V654A cell-derived xenograft mouse model. BLU-654 offers a potent and selective profile suitable for combination therapy for KIT-mutant GIST patients.
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Mar 2026
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I04-Macromolecular Crystallography
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Qiangqiang
Wei
,
Ashley J.
Taylor
,
Nagaraju
Miriyala
,
Mahesh A.
Barmade
,
Zachary O.
Gentry
,
Jordan
Anderson-Daniels
,
Kevin B.
Teuscher
,
Mackenzie M.
Crow
,
Chideraa
Apakama
,
Taylor M.
South
,
Tyson A.
Rietz
,
Kangsa
Amporndanai
,
Jason
Phan
,
John L.
Sensintaffar
,
Mark
Denison
,
Taekyu
Lee
,
Stephen W.
Fesik
Open Access
Abstract: The papain-like protease (PLPro) plays a key role in SARS-CoV-2 replication and represents a promising target for the development of new antiviral therapies. Previous efforts to develop fragment-derived inhibitors of PLPro led to the identification of a novel class of spiro[chromane-2,4′-piperidin]-4-one inhibitors exemplified by lead compound 7. High-resolution covalent cocrystal structures and molecular dynamics simulations were utilized to guide the development of a series of low-nanomolar irreversible PLPro inhibitors, with lead compound 45 demonstrating strong enzymatic inhibition (IC50 = 0.059 μM at T = 60 min) and antiviral activity in A549 cells (EC50 = 2.1 μM at 48 hpi). This novel class of inhibitors represents a promising avenue for the development of therapeutics to overcome the potential of drug-resistant viral strains and future coronavirus outbreaks.
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Feb 2026
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I03-Macromolecular Crystallography
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Chongxun
Ge
,
Dazhi
Feng
,
Song
Shi
,
Xuzhen
Tang
,
Yaqi
Cui
,
Song
Liu
,
Yunyue
Wang
,
Shuangtian
Tang
,
Xinnan
Li
,
Xianqiang
Sun
,
Daopeng
Yuan
,
Jinyi
Xu
,
Hu
He
,
Hong
Yao
Abstract: DNA polymerase theta (Polθ), which mediates microhomology-mediated end joining (MMEJ) in homologous recombination-deficient (HRD) cancers, has recently emerged as a compelling synthetic lethal target. Combining Polθ inhibition with PARP inhibitors represents a promising strategy to overcome PARP inhibitor resistance. Here, we present the discovery of SY-589, a highly potent (ATPase IC50 = 2.29 nM), selective (selectivity index >1800), and orally bioavailable (F = 107%) Polθ helicase inhibitor, which exhibits robust antitumor efficacy in HRD tumors in vitro (CTG IC50 = 2.71 nM). Notably, SY-589 synergized strongly with the PARP inhibitor Olaparib in vitro (Loewe score >20) and in vivo (TGI = 109%), enhancing antitumor effects while permitting reduced Olaparib dosing. Overall, SY-589 is a promising candidate of Polθ inhibitor and has been positioned as a rational combination partner with PARP inhibitors, aiming to overcome PARP inhibitor resistance and mitigate their dose-limiting toxicities.
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Jan 2026
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I04-Macromolecular Crystallography
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Qiangqiang
Wei
,
Ashley J.
Taylor
,
Mahesh Angadrao
Barmade
,
Kevin B.
Teuscher
,
Somanath
Chowdhury
,
Chideraa
Apakama
,
Jordan
Anderson-Daniels
,
Zhu
Yongqing
,
David C.
Schultz
,
Tyson A.
Rietz
,
Taylor M.
South
,
Mackenzie M.
Crow
,
Bin
Zhao
,
Kangsa
Amporndanai
,
John L.
Sensintaffar
,
Jason
Phan
,
Sara
Cherry
,
Mark
Denison
,
Taekyu
Lee
,
Stephen W.
Fesik
Diamond Proposal Number(s):
[42696]
Open Access
Abstract: SARS-CoV-2 papain-like protease (PLPro) plays a key role in viral replication and the host immune response and is a promising target for developing new antiviral treatments. We previously reported a fragment-based screen to identify hits that bind to SARS-CoV-2 PLPro. Here, we describe the discovery of potent PLPro inhibitors by optimizing one of these hits via extensive medicinal chemistry guided by multiple X-ray structures of cocomplexes. Lead compound 46 is shown to bind to the S3 and S4 pockets with nanomolar affinity (0.4 μM) and exhibits robust cellular activity and resistance to mutation. This novel class of PLPro inhibitors can potentially be used as a starting point for the development of inhibitors to combat the emergence of drug-resistant viral strains and future coronavirus outbreaks.
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Jan 2026
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I04-Macromolecular Crystallography
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J. Henry
Blackwell
,
Simon C. C.
Lucas
,
Giovanni
Battocchio
,
Ulf
Börjesson
,
Mark J.
Bostock
,
Erin L.
Braybrooke
,
Tony
Cheung
,
Matthew A.
Cottee
,
Kevin C.
Beaumont
,
Andrea
Gohlke
,
David
Hargreaves
,
Maaike
Van Hoek- Emmelot
,
Vera
Van Hoeven
,
Chimed
Jansen
,
Aarti
Kawatkar
,
Olaf
Kinzel
,
Praveen
Kumar
,
Lea
Kupcova
,
Michael D.
Lainchbury
,
Leonardo
Leon
,
Alexander G.
Milbradt
,
Adeline
Palisse
,
Markus
Schade
,
Kim
Van Rijbroek
,
Claudia
Sacchetto
,
Rick
Schellekens
,
Nancy
Su
,
Hua
Xu
,
Heng
Zhao
,
Yunhua
Chen
,
Shen
Huang
Diamond Proposal Number(s):
[20015]
Abstract: We describe herein the discovery and optimization of a potent and irreversible cellular probe for selective labeling of Bfl-1, a member of the Bcl-2 family. This chemical series demonstrates robust selectivity for Bfl-1 over other related antiapoptotic proteins and exhibits favorable cellular potency as well as promising in vivo pharmacokinetics. Notably, compound 25 achieves a kinact/KI value of 9300 M–1s–1 and elicits caspase activation at submicromolar concentrations in cellular assays. To comprehensively profile proteome-wide selectivity, we performed chemoproteomic analyses on compound 25 alongside our previously reported Bfl-1 inhibitors. This enabled critical insights into potential off-target interactions and facilitated direct comparison of off-target profiles among distinct chemotypes targeting Bfl-1.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36097]
Open Access
Abstract: The tRNA m1G37 methyltransferase (TrmD) is considered essential in various bacteria, including Staphylococcus aureus, a pathogen responsible for a wide range of diseases. Here, we have performed a high-throughput nanomole-scale synthesis campaign (nanoSAR) by late-stage copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC)-functionalizing a library of structurally diverse azides (N = 320) to a pyrrolopyrimidone alkyne. We have identified selective S. aureus TrmD inhibitors with inhibitory activity in the nanomolar to low micromolar range using a direct-to-biology assay read-out. A carbamate-masked guanidine intermediate of the lead structure selectively inhibited S. aureus growth at low micromolar concentrations in cell-based assays, while Gram-negative bacteria and an off-target panel of methyltransferases were not affected. Subsequent cocrystallization resulted in a crystal structure of S. aureus TrmD bound to an inhibitor, providing detailed insights into its binding mode and enabling future structure-guided optimization.
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Dec 2025
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I03-Macromolecular Crystallography
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Hannah L.
Stewart
,
Cinzia
Bordoni
,
Claire E.
Jennings
,
Islam
Al-Khawaldeh
,
Mathew P.
Martin
,
Richard A.
Noble
,
Nicole
Phillips
,
Sara
Pintar
,
Lisa
Prendergast
,
Huw
Thomas
,
Lan-Z.
Wang
,
Jessica E.
Watt
,
Anita
Wittner
,
Agnieszka K.
Bronowska
,
Céline
Cano
,
Martin E. M.
Noble
,
Stephen R.
Wedge
,
Michael J.
Waring
Diamond Proposal Number(s):
[32436, 42280]
Open Access
Abstract: Inhibitors of epidermal growth factor receptor (EGFR) kinase activity are clinically effective treatments for lung cancers driven by activating mutations in EGFR. Resistance to inhibitors develops over time, frequently through further mutations in the kinase domain. On-target resistance to third-generation inhibitor osimertinib, commonly develops through C797S mutation that prevents covalent binding. There is an urgent need for new treatments for osimertinib-resistant EGFR mutants that retain the advantages of the covalent mechanism. Compounds were designed and synthesized to covalently inhibit EGFR through C775, a further cysteine residue we identified in the orthosteric site. Optimisation of the alkynylpyridopyrimidinone scaffold we discovered led to potent compounds that demonstrate inhibition of EGFR phosphorylation and tumor growth in all EGFR mutant cell lines. The covalent C775 mode-of-action was comprehensively established. This work demonstrates that covalent targeting of C775 is a viable mechanism for the treatment of pan-EGFR mutated cancers, particularly those resistant to current therapies.
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Dec 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402, 33658]
Open Access
Abstract: Environment-sensitive fluorescent probes are indispensable tools for studying biological systems and advancing drug discovery. This study reports the development of 4-sulfamoyl-7-aminobenzoxadiazole (SBD)-based fluorescent probes for the allosteric site of the liver isoform of pyruvate kinase (PKL). By integrating SBD moieties into known activator scaffolds, such as mitapivat and diarylsulfonamide (DASA) ligands, probes for indicator displacement assays were designed to quantify ligand interactions in the allosteric site. Compound 4a displayed dose-dependent fluorescence enhancement in response to PKL binding and was used in a competitive binding assay with unlabelled ligands: mitapivat, TEPP-46, DASA-58 and reported activator 21. Structure–activity relationship (SAR) analysis revealed key structural features influencing activity and fluorescence sensitivity. The probes report selectively on the allosteric site ligands as the binding was not affected by natural ligands, such as ADP, fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and phenylalanine. These findings provide a practical framework for detecting allosteric ligand engagement in PKL and expand the repertoire of molecular tools for advancing PKL-targeted therapies.
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Nov 2025
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I02-Macromolecular Crystallography
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Elisabetta
Armani
,
Andrea
Rizzi
,
Daniela
Miglietta
,
Irene
Bassanetti
,
Francesco
Amadei
,
Giandomenico
Brogin
,
Carmelida
Capaldi
,
Fabio
Rancati
,
Chiara
Carnini
,
Sergio
Xanxo Fernandez
,
Maurizio
Civelli
,
Paola
Puccini
,
Marta
Bellini
,
Andrew
Jennings
,
Robert A.
Heald
,
Lilian
Alcaraz
,
Jonathan M.
Sutton
,
Harry
Finch
,
Mary
Fitzgerald
,
Craig
Fox
,
Gino
Villetti
Abstract: The inhibitors of neutrophil elastase (NE) have long attracted interest for the treatment of respiratory diseases. We report the breakthrough of a new potent, selective NE inhibitor with a 24 h duration of action: CHF-6333, is currently undergoing clinical studies for the inhaled treatment of bronchiectasis (BE). The story of the discovery project to identify novel small molecules that inhibit extracellular elastase in the lung with prolonged activity is described. Medicinal chemistry investigation, supported by docking studies, led to N-quaternary compounds with an in vitro profile suitable for inhalatory administration. Compound 15 emerged from in vivo pharmacokinetic and pharmacodynamic studies, also showing safety and no off-target effects in vitro. Salt screening of different counterions, in conjunction with in vivo local irritancy testing, aided in the selection of compound 15-xinafoate (CHF-6333). Efficacy in a lung injury model and no findings in non-GLP toxicity studies promoted CHF-6333 as a clinical candidate.
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Nov 2025
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