Discovery and optimisation of a covalent ligand for TRIM25 and its application to targeted protein ubiquitination

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.

Full text

Discovery and optimisation of a covalent ligand for
TRIM25 and its application to targeted protein
ubiquitination†
Katherine A. McPhie,
a
Diego Esposito,
a
Jonathan Pettinger,
b
Daniel Norman,
c
Thilo Werner,
d
Toby Mathieson,
d
Jacob T. Bush
b
and Katrin Rittinger *
a
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.
Introduction
E3 ubiquitin ligases form a diverse family of proteins that
mediate ubiquitination, a critical post-translational modica-
tion involved in regulating the majority of cellular processes,
including protein degradation, cell signalling and DNA damage
response.
1,2
Ubiquitination occurs via an ATP-dependent
cascade involving three enzymes: an E1-activating enzyme, an
E2-conjugating enzyme, and an E3 ligase enzyme. The initial
ubiquitin modication can be further extended into structurally
diverse polyubiquitin chains, linked via one of ubiquitin's seven
lysine (Lys) residues, or N-terminal methionine (Met).
3
The >600
human E3 ligases provide substrate specicity, and oen work
in synergy with specic E2-conjugating enzymes (of which there
are ∼40) to regulate ubiquitin chain architecture and topology.
4
Induced proximity modalities that exploit the role of ubiq-
uitination in proteasomal degradation have emerged as powerful
tools and therapeutic strategies. Heterobifunctional molecules,
called proteolysis targeting chimeras (PROTACs), are used to
redirect E3 ligases to modify disease-causing proteins with Lys48-
linked ubiquitin chains, thus inducing their proteasomal degra-
dation.
5
Targetedprotein degradation (TPD) via non-proteasomal
pathways, such as lysosomal degradation and autophagy, has also
been demonstrated,
6–8
but never through direct engagement of an
E3 ligase that activates these pathways (via Lys11- or Lys63-linked
ubiquitin chains). Identifying ligands for these Lys11- or Lys63-
specic E3 ligases could enable alternative non-proteasomal
degradation strategies. In recent years, the eld of induced
proximity has expanded beyond TPD to hijack alternative
enzymes such as deubiquitinating enzymes and phosphatases.
9,10
Despite the diverse roles of E3 ligases in regulating the majority of
cellular processes, only a small proportion of the >600 E3 ligases
have been liganded and repurposed for induced proximity
modalities.
11,12
The discovery of new ligands for E3 ligases,
particularly those with non-degradative ubiquitination activity,
presents a promising strategy to activate alternative cellular
outcomes for disease-related proteins.
13
We sought to assess whether the E3 ligase TRIM25, reported
to catalyse the formation of both Lys48- and Lys63-linked
ubiquitin chains,
14
could be liganded and repurposed for tar-
geted protein ubiquitination. TRIM25 is a member of the TRIM
family of RING-type E3 ligases and comprises a canonical N-
terminal tripartite motif (TRIM) and a variable C-terminal
PRYSPRY substrate binding domain (Fig. 1A).
15
As the
majority of current proximity-inducing small molecules for E3s
recruit substrates to the physiological substrate binding
component (e.g. the substrate adaptor of Cullin RING E3s),
11
we
focused our efforts towards liganding the PRYSPRY substrate
binding domain of TRIM25. TRIM25 has been reported to
a
Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute, 1
Midland Road, London, NW1 1AT, UK. E-mail: katrin.rittinger@crick.ac.uk
b
Crick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, Stevenage, Hertfordshire,
SG1 2NY, UK
c
Chemical Biology, GSK, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK
d
Cellzome GmbH, a GSK Company, Meyerhofstrasse 1, Heidelberg, 69117, Germany
†Electronic supplementary information (ESI) available. See DOI:
https://doi.org/10.1039/d5sc01540e
Cite this: Chem. Sci.,2025,16,10432
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Received 26th February 2025
Accepted 30th April 2025
DOI: 10.1039/d5sc01540e
rsc.li/chemical-science
10432 |Chem. Sci.,2025,16,10432–10443 © 2025 The Author(s). Published by the Royal Society of Chemistry
Chemical
Science
EDGE ARTICLE

ubiquitinate a number of different substrates, possibly in some
cases mediated through RNA binding,
16
including RIG-I,
15,17,18
DDX3X
19
and ZAP,
20–22
with diverse roles in immune regulation,
cancer signalling pathways and antiviral activity.
23–26
As such,
TRIM25 is a promising candidate for redirection to a variety of
neosubstrates, and the development of novel chemical tools
Fig. 1 Covalent fragment screening against recombinant TRIM25 PRYSPRY. (A) Cartoon representation of the TRIM25 protein with domain
boundaries: RING domain (blue), B-box domain (purple), coiled coil domain (yellow), and PRYSPRY domain (green); (B) schematic of irreversible
covalent binding equilibrium kinetics and intact protein LCMS assay. Created in BioRender. McPhie, K. (2025) https://BioRender.com/e02g172;
(C) summary of covalent fragment screening by intact protein LCMS. % labelling of 221 chloroacetamides (50 mM) against TRIM25 PRYSPRY (0.25
mM) at 4 °C for 24 h. The green line represents the mean + 2SD; (D) chemical structures of hit fragments 1–3for TRIM25 PRYSPRY selected for
HTC-D2B progression. Chemical structures of hit fragments 4–8are shown in Fig. S1A, ESI;†(E) k
obs
(h
−1
) plotted against concentration (mM),
fitted using a straight line fit. Data are presented as n=2, mean ±SE of fit; (F) table of % labelling, k
inact
/K
I
(M
−1
s
−1
) values, and t
1/2
(h) (in the
presence of 4 mM GSH) for fragments 1–3. Kinetic characterisation for hit fragments 4–8is shown in Fig. S3, ESI.†
© 2025 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2025,16,10432–10443 | 10433
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that target TRIM25 could help to deconvolute its many
proposed functions.
Within the TRIM family, only one other protein, TRIM21, has
been repurposed for targeted protein ubiquitination and degra-
dation. The TPD strategy called ‘Trim-Away’harnesses TRIM21's
innate ability to cluster around antigens through binding to the
Fc receptor of antibodies.
27,28
More recently, small molecule
binders of TRIM21, functioning as both molecular glues and
components of heterobifunctional PROTACs, have been demon-
strated to selectively degrade multimeric protein complexes.
29
Additionally, small molecule ligands for the PRYSPRY domains of
two other PRYSPRY-containing TRIM E3 ligases, TRIM7 and
TRIM58, have been described,
30,31
providing rst evidence for the
ligandability of the PRYSPRY substrate binding domain.
To identify ligands for the TRIM25 PRYSPRY domain, we
employed a covalent fragment-based discovery approach, using
intact protein liquid chromatography mass spectrometry (LCMS)
to screen for covalent binders (Fig. 1B). Covalent fragments
comprise a reversible molecular recognition motif (typically <300
Da), and an electrophilic ‘warhead’for covalent modication to
proximal nucleophilic residues, such as cysteine (Cys). This
covalent functionality overcomes limitations associated with the
modest reversible affinity of fragments and improves the ease
and sensitivity of hit detection.
32
Moreover, covalent ligands
enable the targeting of traditionally ‘undruggable’shallow
protein surfaces, oen found in scaffold proteins or mediators of
protein–protein interactions (PPIs), such as the PRYSPRY
domain.
33
With a well-tuned electrophile, covalent ligands offer
high potency and selectivity driven by increased occupancy of the
irreversible modication. Furthermore, covalent ligands have
successfully been applied to induced proximity modalities, as
demonstrated for several members of the CRL (Cullin-RING
ligase) and RING-type E3 families, including DCAF1, DCAF16,
RNF114, and FEM1B.
34–37
While most heterobifunctional mole-
cules employ reversible ligands to facilitate a catalytic mecha-
nism of action, there is an increasing use of covalent ligands
targeting the E3 ligase component.
38–41
This can be advanta-
geous, enabling potent degradation even at low ligand occu-
pancy, as well as improved physicochemical properties of
traditionally large (>500 Da) heterobifunctional molecules, oen
due to the typically smaller size of covalent ligands.
42,43
In this study, we coupled covalent fragment screening with
our recently reported high-throughput chemistry direct-to-
biology (HTC-D2B) chloroacetamide fragment elaboration
platform.
44
HTC-D2B enabled efficient fragment optimisation
and facilitated the rapid selection of optimised covalent binders
of TRIM25 PRYSPRY for resynthesis and downstream valida-
tion. Optimised binders were characterised and incorporated
into heterobifunctional molecules capable of repurposing
TRIM25 to ubiquitinate a neosubstrate in vitro.
Results and discussion
Covalent fragment screening identies binders of the TRIM25
PRYSPRY domain
To explore the ligandability of TRIM25 PRYSPRY, we employed
a binding site-agnostic covalent fragment screening approach.
Intact protein liquid chromatography mass spectrometry
(LCMS) was used to screen a library of 221 cysteine-reactive
chloroacetamide fragments
44,45
(ESI Data S1†)at50mM
against the recombinantly expressed TRIM25 PRYSPRY domain
(0.25 mM), with incubation at 4 °C for 24 hours (Fig. 1C).
Percentage labelling by each covalent fragment was assessed, by
comparing the relative intensities of apo protein and protein–
fragment complexes. Fragments were selected as hits if the
percentage labelling was greater than 33.9% (mean of labelling
across the whole library + 2SD). We identied eight fragment
hits (1–8, Fig. 1D and S1A, ESI†) that surpassed this threshold,
representing a 3.6% hit rate. All hits were observed to modify
the TRIM25 PRYSPRY domain with a majority single labelling
event (Fig. S1B, ESI†). We counter-screened against the
PRYSPRY domain of a related TRIM protein, TRIM21, which has
received signicant attention within the induced proximity eld
(Fig. S2, ESI†).
27,29
No signicant labelling of TRIM21 PRYSPRY
for any fragment within the chloroacetamide fragment library
was observed, despite the presence of solvent-accessible Cys
residues in both TRIM21 and TRIM25 PRYSPRY domains.
To validate covalent binding of fragment hits 1–8and to
elucidate the rate of covalent labelling, kinetic characterisation
was performed. A 10-point dilution series of each fragment hit
was incubated with TRIM25 PRYSPRY (0.5 mM) at 4 °C and
sampled at eight different timepoints across 24 hours (Fig. S3A,
ESI†). The pseudo-rst order rate constant (k
obs
) was plotted for
each concentration (Fig. 1E and S3B, ESI†), and from the
gradient of the tted straight line, the second-order rate
constant, k
inact
/K
I
, for each fragment was determined (Fig. S3C,
ESI†). The three fragments with the highest k
inact
/K
I
values,
fragments 1,2and 3(Fig. 1F), were selected for elaboration
using high-throughput chemistry direct-to-biology (HTC-D2B)
optimisation approaches.
The intrinsic reactivity of chloroacetamide fragment hits 1
and 2was also assessed using an LCMS-based glutathione
(GSH) assay to measure compound half-life (t
1/2
). Fragment 3
does not contain a UV-active chromophore, so it was incom-
patible with the LCMS-based reactivity assay. In the presence of
4 mM GSH, t
1/2
values of 1.7 h and 1.1 h for 1and 2, respectively,
were calculated and used as a benchmark for further covalent
optimisation (Fig. 1F and S3D, ESI†). These half-life values fall
within the expected range for covalent inhibitors,
44
suggesting 1
and 2are good starting points for further optimisation.
HTC-D2B optimisation enables efficient discovery of potent
covalent ligands that enhance TRIM25 auto-ubiquitination
activity
To optimise fragments 1–3, we employed a high-throughput
chemistry direct-to-biology (HTC-D2B) strategy (Fig. S4A,
ESI†). We have previously described the 384-well plate-based
HTC-D2B platform,
44,45
where a library of elaborated parent
amines is designed based on structural similarity to the hit
fragment. A single-step amide coupling reaction is performed to
install the reactive chloroacetamide electrophile (Fig. 2A), and
the resulting elaborated chloroacetamides are screened without
purication, by intact protein LCMS. The speed and minimal
10434 |Chem. Sci.,2025,16,10432–10443 © 2025 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

handling between synthesis and biological screening enable
rapid exploration of chemical space to identify improved
binders, which can then be puried for conrmation and
accurate potency determination.
Three separate HTC-D2B hit expansion libraries were
designed based on fragments 1–3. For each fragment,
a Tanimoto-based similarity search
46
around the parent amine
(1a–3a, Fig. S4A, ESI†) was performed, ltering for readily
available amines with a molecular weight between 110 and
350 Da. This resulted in a curated library of 83 parent amines
based on fragment 1(HTC-D2B plate 1), 212 parent amines
based on fragment 2(HTC-D2B plate 2), and 186 parent amines
Fig. 2 High-throughput chemistry direct-to-biology (HTC-D2B) optimisation of fragments 1–3. (A) HTC reaction scheme;
44
(B) pie charts of
HTC conversion analysed by LCMS across HTC-D2B plate 1 (83 compounds, based on 1), HTC-D2B plate 2 (186 compounds, based on 2), and
HTC-D2B plate 3 (212 compounds, based on 3). % AUC (area under the curve) represents AUC for the product-containing peak relative to the
starting material-containing peak, selected on identification of the expected m/z;
47
(C) comparison of % labelling of TRIM25 PRYSPRY (0.5 mM) by
the original fragment library (50 mM) and each HTC-D2B library (50 mM and 5 mM) at 4 °C for 24 h. Black lines represent mean % labelling.
Fragment 1was not included in HTC-D2B plate 1, so a close analogue is highlighted instead (9, structure shown in Fig. S4B, ESI†); (D) chemical
structures of optimised chloroacetamides selected for progression; (E) k
obs
(h
−1
) plotted against concentration (mM), fitted using a straight line fit.
Data are presented as n=3; (F) table of % labelling, k
inact
/K
I
(M
−1
s
−1
) values (mean ±SD, n=3), and t
1/2
(h) (in the presence of 4 mM GSH) for
compounds 10,11 and 12.
© 2025 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2025,16,10432–10443 | 10435
Edge Article Chemical Science

based on fragment 3(HTC-D2B plate 3). Parent amine 1a was
not available for inclusion in HTC-D2B plate 1, so a matched
molecular pair analogue was included instead (9, structure
shown in Fig. S4B, ESI†). For all three libraries, installation of
the chloroacetamide electrophile by HTC proceeded with
satisfactory chemical conversion (Fig. 2B). Following a hydrox-
ylamine quench, the three libraries (50 mM, 481 chlor-
oacetamides total) were incubated with TRIM25 PRYSPRY (0.5
mM) at 4 °C for 24 hours and screened by intact protein LCMS. A
signicant improvement in labelling was observed across the
libraries (Fig. 2C). To triage the best hits, the libraries were also
screened at 5 mM (Fig. 2C). Synthetic tractability and percentage
labelling were evaluated for selecting optimised HTC-D2B
compounds for progression. Additionally, compounds with
low chemical conversion but high protein labelling at 5 mM were
considered particularly efficient labellers and were prioritised
over compounds with high chemical conversion and compa-
rable protein labelling. Consequently, three optimised HTC-
D2B compounds 10–12 were selected for resynthesis and puri-
cation (Fig. 2D and S4C–E, ESI†).
Optimised hits 10–12 were resynthesised and puried for
further characterisation (Scheme S1–S3, ESI†). As before, the
rate of labelling (k
inact
/K
I
) was measured (Fig. S5A, ESI†). The
k
inact
/K
I
values of compounds 10 and 11 were calculated as 1.33
±0.14 M
−1
s
−1
and 2.34 ±0.19 M
−1
s
−1
, a 6-fold improvement
over hit fragment 1and an 18-fold improvement over hit frag-
ment 3, respectively (Fig. 2E and F). Unfortunately, puried
compound 12 did not reproduce the labelling observed in HTC-
D2B screening (Fig. S4E, ESI†), and did not show a notable
improvement in kinetics. Therefore, 12 was not progressed any
further.
Additionally, compound 11, a mixture of diastereomers, was
further puried into its component diastereomers (11-1,11-2,
11-3 and 11-4, Fig. S5B, ESI†). Although we were unable to
assign absolute stereochemistry, we observed a clear difference
in k
inact
/K
I
values between enantiomers 11-1 and 11-2, and
enantiomers 11-3 and 11-4, suggesting a stereoselective prefer-
ence for TRIM25 PRYSPRY (Fig. S5C–E, ESI†).
To ensure that the observed increase in k
inact
/K
I
for
compounds 10 and 11 was driven by an increase in the potency
of reversible molecular recognition, rather than an increase in
the intrinsic reactivity of the chloroacetamide electrophile, t
1/2
values for 10 and 11 were also obtained. As before, the rate of
covalent modication with GSH was measured using an LCMS-
based assay. Compounds 10 and 11 had a calculated half-life of
1.9 h in the presence of 4 mM GSH, conrming that the HTC-
D2B campaign had not increased the reactivity of the chlor-
oacetamide electrophile (Fig. 2F and S3D, ESI†). Moreover, the
measured t
1/2
values of 10 and 11 were comparable to that of the
approved cysteine-reactive covalent cancer therapeutic, osi-
mertinib (t
1/2
=1.3 h),
44,48
demonstrating the value of
chloroacetamide-based tool molecules.
To test if covalent binding of compounds 10 and 11 affected
TRIM25 catalytic activity, in vitro auto-ubiquitination assays
were performed using recombinant proteins to reconstitute the
ubiquitin system (Fig. S6A, ESI†). Full-length recombinant
TRIM25 was pre-treated with either DMSO, 10 or 11 for 16 hours
at 4 °C, prior to performing the auto-ubiquitination assay.
Interestingly, the rate of TRIM25 auto-ubiquitination was
signicantly increased by compounds 10 and 11 (Fig. 3A, S6B
and C, ESI†). We speculate that the compounds may be acting
as molecular glues to cluster TRIM25 molecules and stabilise
a higher-order oligomeric state of TRIM25, which is known to be
required for TRIM E3 activity.
49,50
Alternatively, the compounds
may be inducing the stabilisation of a particular conformation
where a greater number of Lys residues are accessible for
ubiquitination.
TRIM25 is engaged by compound 10 in live cells and the
protein–ligand complex crystal structure gives insight into
binding mode
We next explored the selectivity of 10 and 11, as well as their
target engagement in a cellular context. First, compounds 10
and 11 were screened against a panel of recombinant ubiquitin
system proteins by intact protein LCMS. The recombinant
proteins selected were three E2 enzymes (UBCH5B, UBC13
K92A
,
and UEV1A), two other TRIM protein substrate binding
domains (TRIM21 PRYSPRY and TRIM2 NHL), and the catalytic
OTU domains of three deubiquitinating enzymes containing
active site cysteines (OTUD4, OTUD7B and ZRANB1). Pleasingly,
10 and 11 were observed to selectively label TRIM25 PRYSPRY,
with minimal to no labelling of other ubiquitin system proteins
(Fig. 3B). Unfortunately, the E1 enzyme and full-length TRIM25
could not be assessed in this panel as both proteins were too
large for accurate deconvolution of intact protein mass spectra.
Instead, an E1∼Ub loading assay was performed under
reducing and non-reducing conditions
51
to assess whether
ubiquitin loading was inhibited by compounds 10 and 11.
E1∼Ub loading was not inhibited, indicating that 10 and 11 did
not covalently modify the E1 active site Cys (Fig. S6D, ESI†).
The selectivity and cellular interaction proles of 10 and 11
were further characterised by chemoproteomics in live THP-1
cells. We employed an established competitive chemo-
proteomics workow, using an iodoacetamide desthiobiotin
(IA-DTB) probe,
52,53
to assess compound selectivity (at 50 mM
and 10 mM) and identify the site of modication on TRIM25.
Comparison of IA-DTB labelled peptide intensities between
compound-treated samples and DMSO controls enables
compound engagement to be determined, reported as a fold
change (FC) ratio. Compound 10 engaged TRIM25 in cells at the
PRYSPRY domain at 50 mM, and we identied the peptide
FTYC
498
SQVLGLHC
506
YK as the site of covalent modication.
Although we detected two peptides for this sequence modied
by the IA-DTB probe, we were unable to determine which of the
two cysteine residues (Cys498 or Cys506) was modied from this
experiment (Fig. 3C). At 50 mM, 50 other protein targets were
also competed by compound 10, including VCPIP (a deubiqui-
tinating enzyme) and DNMT1 (a DNA methyltransferase) among
the most signicant off-targets. Although 10 was not observed to
be highly selective across the proteome and remains an early-
stage covalent lead compound, TRIM25 was the only E3 ligase
identied to be labelled by 10 in cells. Given the compound's
size (370 Da) and minimal molecular recognition features
10436 |Chem. Sci.,2025,16,10432–10443 © 2025 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

Fig. 3 Biochemical characterisation, target engagement and crystal structure of compound 10. (A) Auto-ubiquitination time course assays with
TRIM25 pre-treated with either DMSO or compound 10 or 11. TRIM25 (4 mM) was incubated with compounds (50 mM) or DMSO (1%) for 16 h at 4 °
C, before addition of E1 (0.2 mM), UBCH5B (2 mM), Ub (50 mM), and Ub
ATTO
(1 mM). The assay was initiated by the addition of ATP and performed for
60 min at 30 °C. Time point 0 was taken before the addition of ATP. The samples were analysed by SDS-PAGE, with Coomassie staining and
scanning at 700 nm wavelength (for ATTO emission). Coomassie gel is shown in Fig. S6B, ESI;†(B) Heatmap of % labelling of purified chlor-
oacetamides 10 and 11 (50 mM) against a panel of recombinant Ub system proteins (10 mM) with incubation at 4 °C for 24 h, and analysis by intact
protein LCMS; (C) cellular target identification for 10 (50 mM, left, and 10 mM, right, 4 h incubation at 37 °C in live THP-1 cells) using an
iodoacetamide desthiobiotin (IA-DTB) probe-based competitive profiling approach. Target engagement is considered significant if FC #0.25; p-
value #0.01 (top left corner, outlined by dotted lines). Significantly competed sites Cys498 and Cys506 on the TRIM25 peptide,
FTYC
498
SQVLGLHC
506
YK, are highlighted in teal. The top five significantly competed off-target Cys sites are highlighted in grey. Volcano plots for
compound 11 are shown in Fig. S6E, ESI;†(D) labelling of 10 (50 mM) using recombinant TRIM25 PRYSPRY cysteine mutants (10 mM) by intact
protein LCMS, with incubation at 4 °C for 24 h; (E) X-ray crystal structure of 10 (teal) bound to TRIM25 PRYSPRY (pale green) with protein–
fragment contacts displayed (dashed, yellow), PDB 9I0T; (F) X-ray crystal structure of 10 (teal) bound to TRIM25 PRYSPRY (pale green) with
protein–fragment contacts displayed (dashed, yellow) and 2Fo-Fc density maps displayed at s-level 1.0 (grey mesh); (G) X-ray crystal structure of
10 (teal) bound to TRIM25 PRYSPRY (pale green) with the protein surface displayed and an exit vector for heterobifunctional compound design
indicated by a blue arrow.
© 2025 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2025,16,10432–10443 | 10437
Edge Article Chemical Science

(hydrophobic biphenyl ring system), 10 was considered a good
starting point for further tool compound development. In
contrast, compound 11 was not observed to engage TRIM25 or
many other proteins in cells, despite having a similar k
inact
/K
I
and GSH reactivity, suggesting poor cell permeability. There-
fore, it was not progressed any further (Fig. S6E, ESI†).
To conrm the exact site of covalent modication, we per-
formed mutagenesis on Cys498 and Cys506 of TRIM25
PRYSPRY, followed by incubation of recombinant mutants (10
mM) with compound 10 (50 mM) at 4 °C for 24 hours. Analysis by
intact protein LCMS revealed Cys498 as the site of covalent
modication by compound 10 (Fig. 3D). Multiple sequence
alignment of all PRYSPRY-containing TRIM proteins showed
that Cys498 is not conserved across the family (Fig. S7A, ESI†).
Thus, the covalent targeting of Cys498 by compound 10 reveals
a unique binding mode for TRIM25.
Next, structural characterisation of the protein–ligand
complex provided insight into the binding mode of compound
10 and helped guide further compound elaboration. We solved
the X-ray crystal structure of TRIM25 PRYSPRY in complex with
compound 10 (PDB 9I0T, Table S1, ESI†), which showed 10
covalently bound to Cys498 within a network of hydrophobic
aromatic residues. The biphenyl ring system of 10 forms p-
stacking interactions with the aromatic side chains of Phe523,
Phe554, Trp616, and Phe618 (Fig. 3E). Furthermore, the
carbonyl of the Cys-linked acetamide bond of 10 acts as an H-
bond acceptor for both the side chain hydroxyl and main
chain –NH of Ser499, and the side chain indole amine of
Trp616. The carbonyl of the diazepane amide linker also forms
an H-bond interaction with the terminal guanidino group of
Arg541 (Fig. 3F). Compared to the apo-TRIM25 PRYSPRY
structure (PDB 6FLM),
15
no signicant conformational changes
within the binding site occurred (Fig. S7B, ESI†). Additionally,
we used ligand-based
1
H-NMR to further validate the binding
pose of compound 10, and conrmed its binding mode in
solution (Fig. S7C, ESI†). Interestingly, structural overlay of
TRIM25 PRYSPRY-compound 10 with the PRYSPRY domains of
TRIM7, TRIM21 and TRIM58, for which small molecule ligands
have been reported,
29–31
showed that the ligands all bind at the
same site on the PRYSPRY surface, which has also been sug-
gested to be a substrate binding interface
31
(Fig. S7D, ESI†). This
may present an opportunity to target future PRYSPRY ligand
discovery campaigns to this particular privileged ligand- and
substrate-binding site.
Heterobifunctional proximity-inducers enable in vitro
targeted protein ubiquitination of a neosubstrate by TRIM25
Using our TRIM25 PRYSPRY-compound 10 complex structure,
we identied potential exit vectors for incorporation of 10 into
proximity-inducing heterobifunctional molecules. The meta
position on the pendant phenyl ring was determined to be both
solvent accessible and synthetically tractable (Fig. 3G). Three
heterobifunctional compounds (HB1,HB2, and HB3) were
designed and synthesised with varying PEG linker lengths,
incorporating 10, and the well-characterised BRD4 ligand, JQ1
(Fig. 4A and Scheme S4, ESI†).
54
Compounds HB1–HB3 (50 mM)
were incubated with the TRIM25 PRYSPRY domain (10 mM) at 4
°C for 20 hours, and labelling was analysed by intact protein
LCMS (49%, 75% and 87%, respectively, Fig. S8A, ESI†).
Although labelling was reduced compared to compound 10
alone (94%), the labelling observed conrmed that an exit
vector at the meta position of the pendant phenyl ring was
tolerated.
Next, we assessed in vitro ternary complex formation by
recombinant protein pull-down, using isolated TRIM25
PRYSPRY and BRD4 BD2 domains. Ternary complex formation
was observed for all three linker lengths (Fig. S8B, ESI†).
However, an intermediate wash step aer overnight incubation
of the TRIM25 PRYSPRY domain (2 mM) with compounds HB1,
HB2 or HB3 (50 mM) was required to remove excess unreacted
heterobifunctional compound. This suggests the presence of
a large hook effect, where, at the concentrations required for
covalent labelling and without the intermediate wash step,
binary complex interactions outcompeted ternary complex
formation. This ternary complex formation, when carried out
with the intermediate wash step, was also reproduced using full
length TRIM25 and BRD4 proteins (Fig. S8C, ESI†). These long
incubation times and the intermediate wash step indicate that
compounds HB1–HB3 would require further optimisation for
use in cellular assays. Incorporating a more potent binder of
TRIM25 than compound 10 would reduce incubation times and
likely remove the necessity for the intermediate wash step.
Additionally, we performed SPR to obtain the affinity of
BRD4 BD2 for TRIM25 PRYSPRY pre-labelled with HB1–HB3.In
the absence of a heterobifunctional compound, BRD4 BD2
demonstrated no affinity for TRIM25 PRYSPRY. Upon pre-
labelling TRIM25 PRYSPRY with HB1–HB3, we observed
strong binding of BRD4 BD2, with very little difference between
each compound (K
D
=15 ±1.9 nM, 20 ±2.1 nM, and 22.5 ±
3.6 nM, respectively) (Fig. S8D, ESI†). Although ternary complex
formation is a critical component of induced proximity phar-
macology, incorporation of higher affinity ligands into hetero-
bifunctional molecule design does not correlate directly with
improved ubiquitination. To maintain optimal processivity,
a balance between longevity of ternary complex association and
rate of dissociation must be obtained, as well as optimal ternary
complex conformation.
55
Thus, we turned to structural biology
to examine the ternary complex structure.
Unfortunately, our attempts to crystallise the ternary
complex between TRIM25 PRYSPRY, BRD4 BD2 and any of the
three heterobifunctional molecules were unsuccessful. Instead,
we used small-angle X-ray scattering (SAXS) to gain structural
information and assess the exibility of the TRIM25 PRYPSRY-
HB2-BRD4 BD2 ternary complex (Table S2, ESI†). SAXS analysis
revealed that both TRIM25 PRYSPRY and BRD4 BD2 are
monomeric in solution (Fig. S9A–D, ESI†). Upon ternary
complex formation, we observed an elongated molecular shape,
characterised by an expansion in both maximum particle
dimension (D
max
) and radius of gyration (R
g
), without signi-
cant changes in the cross-section radius of gyration (R
c
)
(Fig. S9E–G, ESI†). Flexibility analysis and DAMMIF ab initio
modelling of the ternary complex supported this, and revealed
aexible, elongated envelope exceeding individual domain
10438 |Chem. Sci.,2025,16,10432–10443 © 2025 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

dimensions (Fig. 4B). This suggests that compound HB2 acts as
a dynamic linker between TRIM25 PRYSPRY and BRD4 BD2
domains, with PEG groups providing rotational exibility that
can separate domains by up to ∼15 Å, precluding stable direct
interdomain interactions in solution. Thus, our SAXS analysis
reveals a ternary complex characterised by signicant inter-
domain exibility.
Finally, the targeted ubiquitination of BRD4 by TRIM25 was
investigated, using the in vitro ubiquitination assay described
earlier. Full-length TRIM25 (4 mM) was pre-labelled with either
DMSO or compound HB1,HB2 or HB3 (50 mM) for 20 hours at 4
°C, and excess compound was removed with an intermediate
wash step, and the TRIM25-compound complex was used as the
E3 component in the in vitro ubiquitination assay. We observed
heterobifunctional compound-induced ubiquitination of BRD4
(Fig. 4C and S8E, ESI†) with all three linker lengths. To conrm
that this ubiquitination was induced by the formation of
a ternary complex, we also performed this experiment using
compound 10 and HB1a (JQ1-2PEG) alone, and no targeted
ubiquitination of BRD4 was observed. Thus, although our
Fig. 4 Design, SAXS and biochemical characterisation of heterobifunctional compounds for targeted protein ubiquitination of BRD4. (A)
Chemical structures of heterobifunctional BRD4-recruiting compounds HB1,HB2 and HB3; (B) best DAMMIF ab initio molecular envelope for the
TRIM25 PRYSPRY-HB2-BRD4 BD2 complex, with cartoon representations of BRD4 BD2-JQ1 (pale pink, PDB 3ONI),
54
TRIM25 PRYSPRY-
compound 10 (pale green, PDB 9I0T), and compound HB2 (orange); (C) In vitro targeted protein ubiquitination time course assay for BRD4, using
His
10
-TRIM25 pre-treated with either DMSO or compounds. His
10
-TRIM25 (4 mM) was incubated with compounds (50 mM) or DMSO (1%) for 20 h
at 4 °C. Excess unreacted compound was removed prior to the addition of E1 (0.5 mM), UBCH5B (2 mM), Ub (50 mM), Ub
ATTO
(1 mM), and BRD4 (4
mM, no tags). Ubiquitination was initiated by the addition of ATP and performed for 60 min at 30 °C. The samples were analysed by SDS-PAGE,
western blotting with a-BRD4, and scanning at 700 nm wavelength (for ATTO emission). Coomassie staining is shown in Fig. S8E, ESI.†
© 2025 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2025,16,10432–10443 | 10439
Edge Article Chemical Science

biochemical, SPR, and SAXS data suggest a lack of positive
cooperativity, we still observe proximity-induced targeted
ubiquitination of BRD4 by TRIM25. Taken together, our results
demonstrate that TRIM25, a previously unexplored E3 ligase in
the induced proximity eld, can be repurposed to ubiquitinate
a neosubstrate in vitro.
Conclusions
The discovery of small molecules targeting previously unli-
ganded E3 ligases is needed to expand the repertoire of enzymes
that can be repurposed for induced proximity modalities. In
this work, we identied a novel covalent ligand for the E3 ligase,
TRIM25, and demonstrated that it can be incorporated into
heterobifunctional molecules capable of recruiting TRIM25 for
proximity-induced in vitro ubiquitination of a neosubstrate.
Using covalent fragment screening coupled with a high-
throughput chemistry direct-to-biology strategy to expedite the
process of fragment optimisation, we identied two chlor-
oacetamide ligands (compounds 10 and 11) that bind to the
PRYSPRY domain of TRIM25. Further characterisation eluci-
dated that these ligands enhance the auto-ubiquitination of
TRIM25 in vitro, and selectively label the PRYSPRY domain of
TRIM25 (at Cys498) over other ubiquitin system proteins in vitro
and, for 10, in live THP-1 cells. A crystal structure of the TRIM25
PRYSPRY-compound 10 complex was solved at 1.8 Å resolution,
which, to our knowledge, represents the rst published struc-
ture of TRIM25 in complex with a small molecule. Structure-
informed design guided the incorporation of compound 10
into proximity-inducing heterobifunctional molecules. We
demonstrated heterobifunctional compound-induced ternary
complex formation and targeted in vitro ubiquitination of BRD4
by TRIM25.
Further cellular studies to investigate the effects of neo-
substrate recruitment and ubiquitination by TRIM25 will rst
require optimisation of compound 10. The current requirement
for long incubation periods and removal of excess compound
with HB1–HB3 in vitro precludes the use of these compounds in
cellular assays. Structure-based medicinal chemistry campaigns
to improve the potency and selectivity of 10 will likely reduce the
large hook effect observed with heterobifunctional compounds
HB1–HB3, enabling induced ubiquitination at a range of
concentrations, without an intermediate excess compound
removal step. Investigating the function of optimised hetero-
bifunctional molecules in a cellular context will help to uncover
whether TRIM25 acts as a canonical degrader ligase or mediates
a more complex signalling pathway. Orthogonally, optimised
ligands could be further studied in a cellular context, either as
inhibitors of reported TRIM25 substrates or as molecular glues
to identify novel neosubstrates of TRIM25.
Overall, we have developed small molecules for TRIM25 and
expanded the repertoire of liganded E3 ligases. Our results
highlight the power in using covalent approaches for targeting
shallow protein–protein interaction surfaces, and demonstrate
that another E3 ligase within the TRIM family can be repur-
posed to direct ubiquitination towards a neosubstrate. As such,
targeted ligand discovery for the recruitment of TRIM family
proteins presents an attractive strategy for future induced
proximity modalities.
Data availability
The X-ray crystal structure reported in this study has been
deposited in the Protein Data Bank under accession code 9I0T.
Additional information is also available in the attached Table
S1, ESI.†The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium (https://
proteomecentral.proteomexchange.org)via the PRIDE partner
repository
56
with the dataset identier PXD061182. Additional
information is also available in the attached ESI Data S2.†
Author contributions
KAM wrote the manuscript and performed protein biochem-
istry, intact protein LCMS, HTC-D2B synthesis and screening, X-
ray crystallography, synthetic chemistry, and biochemical and
biophysical assays. DE performed crystallographic data pro-
cessing and SAXS analysis. JP performed the initial covalent
fragment screening experiment and carried out library
management. DN, TW and TM performed chemoproteomics
experiments and analysis. JP, JB and KR contributed to data
analysis and discussion. JB and KR jointly supervised the study.
All authors read and edited the nal manuscript.
Conflicts of interest
The authors declare no competing interests.
Acknowledgements
The authors would like to thank Harry Wilders and Sam Rowe
(GSK) for HTC-D2B platform development; Emma Grant (GSK)
for reactive fragment platform maintenance; David House
(GSK) for discussions and continued support; Andy Purkiss
(Structural Biology STP) for crystallographic data collection; the
staffof Diamond Light Source, Oxford, UK for synchrotron
access (I04 beamline); Melanie Jundt, Kerstin Kammerer and
Michael Steidel (Cellzome) for chemoproteomics support;
Laura Masino and Evangelos Christodoulou (Structural Biology
STP) for SPR and large-scale protein expression support; Sarah
Maslen (Proteomics STP) for intact protein LCMS support; Jes-
sica Waters for HRMS sample processing; Roger George
(Structural Biology STP) and Karen Chau for giing recombi-
nant proteins; Jasmine Green (GSK) for chiral purication; and
George Biggs and Jane Dudley-Fraser for chemoproteomics and
cell biology guidance. This work was supported by the Francis
Crick Institute, which receives its core funding from Cancer
Research United Kingdom (CC 2075), the United Kingdom
Medical Research Council (CC 2075), and the Wellcome Trust
(CC 2075) and by the Engineering and Physical Sciences
Research Council, EP/V038028/1. For the purpose of open
access, the author has applied a CC BY public copyright license
to any author-accepted manuscript version arising from this
submission. Fig. 1B, S4A and S6A, ESI†were made using https://
10440 |Chem. Sci.,2025,16,10432–10443 © 2025 The Author(s). Published by the Royal Society of Chemistry
Chemical Science Edge Article

www.biorender.com under an institutional license belonging to
the Francis Crick Institute.
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