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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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[30602]
Open Access
Abstract: Fragment-based drug design offers multiple routes to advance from fragments. One approach is to build structure-activity relationships (SAR) from analogue series in direct-to-biology workflows. Analogues can be prepared by automated chemistry and tested as crude reaction mixtures (CRMs) without purification, but assay noise often leads to hit resynthesis, potentially discarding false negatives and reducing SAR dataset size. High-throughput (HT) X-ray crystallography has the potential to address these issues by resolving hits directly from 100s–1000s of CRMs. However, no systematic analytics exist for extracting SAR models from HT crystallographic evaluation of CRMs. Here, we demonstrate that crystallographic SAR (xSAR) can be extracted from CRMs evaluated via HT X-ray crystallography. We developed a simple rule-based ligand scoring scheme that identifies conserved chemical features associated with crystallographic binding and non-binding. Applied to a crystallographic dataset of 957 fragment elaborations in CRMs targeting PHIP(2), a therapeutically relevant bromodomain, our xSAR model demonstrated effectiveness in two proof-of-concept experiments. First, it recovered 26 missed binders in the initial dataset (false negatives), doubling the hit rate and denoising the dataset. Second, it enabled a prospective virtual screen that identified novel hits with informative chemistries and measurable binding affinities. This work establishes a proof-of-concept that xSAR models can be directly extracted from large-scale crystallographic readouts of CRMs, offering a valuable methodology to build SAR models and accelerate design-make-test iterations without requiring CRM hit resynthesis and confirmation. This invites future work to utilise advanced analytics and modelling techniques to further strengthen purification-agnostic workflows.
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Dec 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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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.
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Feb 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear. To explain the mechanism of pH sensing, we combine analysis of X-ray crystal structures of the KDEL receptor at neutral and acidic pH with advanced computational methods and cell-based assays. We show a critical role for ordered water molecules that allows us to infer a direct connection between protonation in different cellular compartments and the consequent changes in the affinity of the receptor for cargo.
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Jul 2024
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I03-Macromolecular Crystallography
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Yasmin
Bay
,
Raminta
Venskutonytė
,
Stine M.
Frantsen
,
Thor S.
Thorsen
,
Maria
Musgaard
,
Karla
Frydenvang
,
Pierre
Francotte
,
Bernard
Pirotte
,
Philip C.
Biggin
,
Anders S.
Kristensen
,
Thomas
Boesen
,
Darryl S.
Pickering
,
Michael
Gajhede
,
Jette S.
Kastrup
Diamond Proposal Number(s):
[11011]
Abstract: The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1–3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists and positive allosteric modulators of GluK1-3. The half maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 μM kainate was determined to be 26.3 μM for GluK1, 75.4 μM for GluK2 and 639 μM for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 μM and 1.33 μM, respectively, upon co-application of 150 μM BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 μM, in presence of 500 μM BPAM344 and 100 μM kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344 as well as zinc-, sodium- and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790 and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.
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Dec 2023
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I04-Macromolecular Crystallography
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Adam M.
Thomas
,
Marta
Serafini
,
Emma K.
Grant
,
Edward A. J.
Coombs
,
Joseph P.
Bluck
,
Matthias
Schiedel
,
Michael A.
Mcdonough
,
Jessica K.
Reynolds
,
Bernadette
Lee
,
Michael
Platt
,
Vassilena
Sharlandjieva
,
Philip C.
Biggin
,
Fernanda
Duarte
,
Thomas A.
Milne
,
Jacob T.
Bush
,
Stuart J.
Conway
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
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Oct 2023
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I03-Macromolecular Crystallography
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Angelina R.
Sekirnik
,
Jessica K.
Reynolds
,
Larissa
See
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Cynthia
Tallant
,
Bernadette
Lee
,
Katarzyna B.
Leszczynska
,
Rachel L.
Grimley
,
R. Ian
Storer
,
Marta
Malattia
,
Sara
Crespillo
,
Sofia
Caria
,
Stephanie
Duclos
,
Ester M.
Hammond
,
Stefan
Knapp
,
Garrett M.
Morris
,
Fernanda
Duarte
,
Philip C.
Biggin
,
Stuart J.
Conway
Open Access
Abstract: TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18145]
Open Access
Abstract: A novel crystallographic fragment screening data set was generated and used in the SAMPL7 challenge for protein-ligands. The SAMPL challenges prospectively assess the predictive power of methods involved in computer-aided drug design. Application of various methods to fragment molecules are now widely used in the search for new drugs. However, there is little in the way of systematic validation specifically for fragment-based approaches. We have performed a large crystallographic high-throughput fragment screen against the therapeutically relevant second bromodomain of the Pleckstrin-homology domain interacting protein (PHIP2) that revealed 52 different fragments bound across 4 distinct sites, 47 of which were bound to the pharmacologically relevant acetylated lysine (Kac) binding site. These data were used to assess computational screening, binding pose prediction and follow-up enumeration. All submissions performed randomly for screening. Pose prediction success rates (defined as less than 2 Å root mean squared deviation against heavy atom crystal positions) ranged between 0 and 25% and only a very few follow-up compounds were deemed viable candidates from a medicinal-chemistry perspective based on a common molecular descriptors analysis. The tight deadlines imposed during the challenge led to a small number of submissions suggesting that the accuracy of rapidly responsive workflows remains limited. In addition, the application of these methods to reproduce crystallographic fragment data still appears to be very challenging. The results show that there is room for improvement in the development of computational tools particularly when applied to fragment-based drug design.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Petra
Lukacik
,
C. David
Owen
,
Gemma
Harris
,
Jani Reddy
Bolla
,
Sarah
Picaud
,
Irfan
Alibay
,
Joanne E.
Nettleship
,
Louise E.
Bird
,
Raymond
Owens
,
Philip C.
Biggin
,
Panagis
Filippakopoulos
,
Carol V.
Robinson
,
Martin A.
Walsh
Diamond Proposal Number(s):
[4990, 5073, 4988]
Open Access
Abstract: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.
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Oct 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with 10-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL, and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable −4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.
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Jun 2021
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