VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[28534, 34263]
Open Access
Abstract: Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme–nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440, 37593]
Open Access
Abstract: Angiotensin I-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase involved in blood pressure regulation through proteolysis of angiotensin I (Ang-I) into the potent vasoconstrictor, angiotensin II (Ang-II). Inhibition of ACE is therefore used for the treatment of hypertension, heart failure, myocardial infarction, stroke and chronic kidney disease. Current ACE inhibitors (ACEi) bind both the N- and C-catalytic domains of ACE (referred to as nACE and cACE), and this has been linked to the occurrence of side effects due to the wide substrate specificity of ACE. The development of domain selective ACEi with reduced side effects is therefore key for improved therapeutic intervention. Understanding how current ACEi bind nACE and cACE, and their differences in domain selectivity should aid structure-based development of more selective ACEi by identifying different chemical groups that increase or decrease selectivity. We present the kinetic and structural characterisation of nACE and cACE with three thiolate ACEi, captopril (Ki, nACE = 2.53 nm and cACE = 2.04 nm), rentiapril (monomer Ki, nACE = 2.22 nm and cACE = 6.77 nm) and zofenoprilat (Ki, nACE = 2.86 nm and cACE = 0.61 nm). Detailed structural analysis indicated that the S2′ subsite likely contributes to the variation in domain selectivity observed for rentiapril and zofenoprilat due to differences in hydrophobicity and displacement of water molecules that contribute to ACE's hydration shell. Interestingly, in the cACE crystal structure, rentiapril bound as a dimer, and kinetic data revealed that both the monomeric and dimeric (dimer Ki, nACE = 15.11 nm and cACE = 36.38 nm) forms of rentiapril inhibit ACE with nanomolar affinity.
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Feb 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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402]
Open Access
Abstract: Despite the continual emergence of SARS-CoV-2 variants and increasing diversity within the receptor binding domain (RBD), some antibody responses that are directed to conserved regions can display cross-reactivity against variants. We previously isolated an RBD-directed monoclonal antibody (084-7D) from a Beta-infected donor that neutralized Beta and emerging Omicron variants. Here, we solved a high-resolution crystal structure of the 084-7D Fab in complex with the Beta RBD. These data revealed an epitope overlapping both the ACE2 binding site and those of other class 1 antibodies. Furthermore, the epitope includes highly conserved residues, Q409, D420, and Y489, that are present in recent Omicron variants. The N417 residue that emerged with Beta and has since persisted is tolerated within the epitope of 084-7D, explaining the preferential neutralization of contemporaneous N417-containing variants. These structural data defined the mechanism for cross-reactivity of a Beta-elicited neutralizing antibody, potentially informing the design of future broadly reactive SARS-CoV-2 therapeutics.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20015]
Open Access
Abstract: The c-MET kinase is a driver of many cancers, and as such, there are a number of small molecule inhibitors of this kinase approved for clinical use. In this Microperspective, we provide a structural overview of the molecular basis by which these drugs inhibit c-MET, focusing on key features contributing to activity, selectivity, and drug resistance. Where necessary, relevant crystal structures not publicly available were determined and are discussed here alongside existing structural data.
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Feb 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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I03-Macromolecular Crystallography
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Markella
Konstantinidou
,
Holly R.
Vickery
,
Marloes A. M.
Pennings
,
Johanna M.
Virta
,
Shu Yue
Luo
,
Emira J.
Visser
,
Sean D.
Bannier
,
Mrudula
Srikanth
,
Sabine Z.
Cismoski
,
Lucy C.
Young
,
Maxime C. M.
Van Den Oetelaar
,
Frank
Mccormick
,
Christian
Ottmann
,
Luc
Brunsveld
,
Michelle R.
Arkin
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Molecular glues, compounds that bind cooperatively at protein–protein interfaces (PPIs), are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional “undruggable” targets. Here, we focus on a native regulatory PPI between the scaffolding protein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway. Extensive drug discovery efforts have focused on the MAPK pathway due to its central role in oncology and developmental disorders (RASopathies). However, the modulation of its protein complexes is underexplored. C-RAF activity is regulated on multiple levels including dimerization, phosphorylation, and complex formation with 14-3-3, which prevents C-RAF activation by binding to a C-RAF sequence centered on phospho-serine 259. We used a fragment-merging approach to design molecular glues that bound to the composite surface of this 14-3-3/C-RAFpS259 complex. Molecular glues stabilized the inhibitory complex up to 300-fold; their glue-based mechanism of action was confirmed by crystallography and biophysical studies. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Cellular assays demonstrated on-target engagement, enhanced phosphorylation levels of C-RAFpS259, and reduced levels of RAF dimerization and ERK phosphorylation. Overall, this approach enabled chemical biology studies for a C-RAF site that was intrinsically disordered prior to 14-3-3 binding and had not been targeted previously. These molecular glues will be useful chemical probes and starting points for drug discovery efforts to modulate native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.
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Jan 2026
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Open Access
Abstract: Fragment-based Drug Discovery (FBDD) is a proven methodology for the discovery of new therapeutics. After the identification of small molecular fragments, subsequent steps are guided by the “Design, Make, Test” (DMT) cycle. During the “Design” phase, chemical modifications are proposed that generate Structure-Activity Relationship information, improve interaction profiles and physicochemical properties. In the “Make” phase, designs are synthesised into viable compounds, with an emphasis on feasibility, scalability and the incorporation of novel chemistries enabling broad chemical space sampling. Finally, the “Test” phase evaluates these compounds through a series of assays, identifying binders and enabling Structure-Activity Relationship models that guide subsequent designs. Within DMT cycles, fragment progression – the process of converting initial hits into more potent follow-up lead compounds – is an essential component, but has many challenges associated with it. Here, we review such challenges along with recent developments designed to mitigate them.
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Jan 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[37115]
Open Access
Abstract: α7 nicotinic receptors are neurotransmitter-gated ion channels involved in neurological and inflammatory diseases. Ligands acting on its neurotransmitter binding site and on the channel domain of α7 have been extensively developed, yielding a wide range of orthosteric effectors and allosteric positive modulators. Here, we present the functional and structural characterization of two camelid antibody fragments, or nanobodies, F1 and E6, that inhibit α7 activity by acting as negative allosteric modulators, an underrepresented class of ligands. Cryo-EM structures of the nanobodies in complex with α7 show that both nanobodies form a pentameric bundle at the apex of the receptor, each nanobody interacting through a conserved set of residues at α7 subunit interfaces. Electrophysiological experiments suggest that E6 inhibits the activity of α7 by stabilizing its resting conformation, and that internanobodies interactions are key to its activity. Those two nanobodies expand the toolbox for human α7 modulation, opening new possibilities for its pharmacological control with far reaching potentialities in clinics.
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Jan 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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