Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

Article abstract of DOI:10.1039/c9cc08362f

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-Target modification with blood thiols (e.g., glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker.

Full text of DOI:10.1039/c9cc08362f

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Use of pyridazinediones as extracellular cleavable
linkers through reversible cysteine conjugation†
Cite this: Chem. Commun., 2019,
55, 14829
a
Calise Bahou, ‡a Richard J. Spears, ‡a Abil E. Aliev,a Antoine Maruani,
a
a
a
James R. Baker *a and
Received 25th October 2019,
Accepted 15th November 2019
Marcos Fernandez,
Vijay Chudasama
Faiza Javaid,
Peter A. Szijj,
ab
*
DOI: 10.1039/c9cc08362f
rsc.li/chemcomm
Herein we report a retro-Michael deconjugation pathway of thiol– require extracellular cleavable linkers in order to operate
pyridazinedione linked protein bioconjugates to provide a novel efficiently.11 The two most common types of extracellular cleavable
cleavable linker technology. We demonstrate that the novel pyri- linkers are disulfide based linkers12 and dipeptide valine–citrulline
dazinedione linker does not suffer from off-target modification (Val–Cit) linkers.13,14 Disulfide linkers release cargo through dis-
with blood thiols (e.g., glutathione, human serum albumin (HSA)), ulfide exchange in environments containing high concentrations
which is in sharp contrast to an analogous maleimide linker.
of thiols.12 The Val–Cit linker undergoes enzymatic cleavage by
cathepsin B, a cysteine protease that is typically overexpressed in
Site-selective modification of proteins lies at the forefront of con- cancers;11,14 although this protease is usually found in the lysosome
temporary approaches to problem solving in chemical biology.1,2 it has also displayed extracellular activity in cancers.11 These linkers
Construction of these protein bioconjugates frequently uses cysteine are not without their disadvantages however; exposed disulfide
as a handle for bioconjugation,3,4 owing to its favourable properties linkers are susceptible to reduction before the ADC reaches its
(high nucleophilicity of the thiol side chain at neutral pH (pKa E 8), target.10 Whilst hindered disulfides have (to some extent)
low natural abundance (o2%) and ease of incorporation via site- addressed the issue of stability of disulfide linkers in blood they
directed mutagenesis).5 In particular, cysteine modification features are, to the best of our knowledge, not viable for application as
heavily in the synthesis of antibody–drug conjugates (ADCs), a class extracellular cleavable linkers, i.e., they only cleave post-inter-
of therapeutics that combines the selective targeting capabilities of nalisation into an intracellular environment.15,16 Val–Cit linkers
an antibody with the potent toxicity of small molecule drugs.6 are also susceptible to other protease-related activity, which can
Currently, five ADCs are FDA-approved, including Adcetrist and hamper their stability in blood plasma.10 An alternative system
Polivyt whose payloads are linked directly via cysteine bio- explored within the context of cleavable linkers is that of
conjugation.7,8 Mechanistically, ADCs operate via either internali- maleimide–thiol linkages (Fig. 1a),17,18 which can cleave via a
sing or non-internalising pathways depending on whether endo- retro-Michael pathway and the released maleimide can be trapped
cytosis is initiated following antigen binding,9 with toxic cargo by other abundant thiols in the tumour environment. However, the
release dependent on whether a ‘‘non-cleavable linker’’ or ‘‘cleavable rapid kinetics of maleimide–thiol conjugation makes any released
linker’’ is used when constructing the ADC.10 For non-cleavable maleimide, before the maleimide–thiol conjugate reaches its
linkers, drug release occurs following lysosomal degradation of the desired destination, highly susceptible to off-target thiol modifica-
internalised ADC, whereas for cleavable linkers drug release is tion by other biomolecules in vivo (e.g., HSA),19 thus limiting their
facilitated through a stimulus pre- and/or post-internalisation, e.g., use as cleavable systems in a biological context. Furthermore,
triggered by a change in cancer microenvironment conditions.
maleimide–thiol linkages can undergo competitive hydrolysis;
Historically, many ADCs relied on internalising targets to enable terminating the retro-Michael deconjugation pathway and hinder-
drug release, however, in recent years there has been increasing ing overall cleavage and ultimately cargo release.
interest in non-internalising ADCs. These non-internalising ADCs
Over the past decade, we have reported on the use of bromo-
pyridazinediones (BrPDs) as tools for efficient cysteine bioconjuga-
tion, particularly in the synthesis of dually functionalised conjugates,
nanoparticle constructs, and therapeutically relevant biomolecules
such as ADCs.20–22 Unlike maleimides, conjugates synthesised using
BrPDs do not undergo ring hydrolysis, and the unsaturated ring
scaffold is otherwise stable under aqueous conditions.23 We were
intrigued, however, as to whether bioconjugates containing
a Department of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail: v.chudasama@ucl.ac.uk, j.r.baker@ucl.ac.uk
b Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy,
Universidade de Lisboa, 1649-004 Lisbon, Portugal
† Electronic supplementary information (ESI) available: [DETAILS]. See DOI:
10.1039/c9cc08362f
‡ These authors contributed equally to this article.
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Fig. 1 (a) Competing reactions of cysteine–maleimide conjugates including
reversible retro-Michael deconjugation and irreversible hydrolysis of the succi-
nimide motif. (b) Proposed reaction for cysteine–pyridazinedione conjugates
involving retro-Michael deconjugation only.
a saturated pyridazinedione scaffold (synthesised as previously
described)23 could proceed down a similar reversible retro-Michael
deconjugation pathway displayed by classical maleimides and other
Michael acceptors, e.g., cyanoacrylates.24 In the context of a novel
cleavable linker, we hypothesised that if Michael addition is
observed with PDs the lack of ring hydrolysis would avoid termina-
tion of the retro-Michael pathway as seen in maleimide conjugates.
Moreover, a different reactivity profile for conjugation and retro-
Michael on PDs (compared to other acceptors) could also provide
novel opportunities, e.g., a dynamic system with a slow release of
PDs that do not react with blood thiols would be a useful addition to
the extracellular non-internalising ADC linker toolbox.
Fig. 2 (a) Structure of Boc-Cys(PD)-OMe 9. (b) Structural representation
of pseudoaxial conformation of Boc-Cys(PD)-OMe 9. (c) NMR analysis
showing slow release of PD from Boc-Cys(PD)-OMe 9 to form diethyl PD
11 and Boc-Cys-OMe 10. (d) Schematic of TNB 13 release from reaction
with 9 and DTNB 12 with graph depicting calculated % deconjugation.
Our initial assessment of PDs as a platform for reversible
cysteine modification began with the synthesis of small molecule was incubated with DTNB (Ellman’s reagent) 12, andanticipatedTNB
Boc-Cys(PD)-OMe 9 (from Boc-Cys-OMe 10 and PD 11) to act as a 13 release (from reaction of liberated Boc-Cys-OMe 10 with DTNB 12)
model system for our studies (Fig. 2a). We noted that, compared to was monitored by UV-Vis absorption at 412 nm at 24 h intervals over
the 5-membered ring of the thioether succinimide motif obtained the course of 96 h (Fig. 2d). Over this time course, we noted a 36%
from classical maleimide–thiol conjugates, PD–thiol conjugates increase in the amount of deconjugated species determined from
comprise a 6-membered ring that may allow for more conforma- absorbance at 412 nm when DTNB 12 was incubated with conjugate 9,
tional freedom along the –CH2–CH(SR)– backbone of the ring, compared to a control experiment (Fig. 2d, see ESI† for details). These
giving rise to two possible ‘‘pseudoaxial’’ and ‘‘pseudoequatorial’’ experiments confirmed the presence of a retro-Michael deconjugation
conformers. Using molecular mechanics calculations of Boc- pathway being effective on a thiolated PD.
Cys(PD)-OMe 9, applying the MMX force field25 to find the opti-
Next, we turned our attention towards the reversibility of PDs
mised geometries of the potential pseudoaxial/pseudoequatorial within the context of protein bioconjugates, utilising a mutant green
conformers, we experimentally determined conformer ratio in a fluorescent protein containing a solvent-accessible free cysteine as a
phosphate buffer (PB) pH 7.4 : CD3CN (7 : 3) solvent system at 37 1C model system (GFPS147C 14, expressed as described previously).28
by 1H NMR (see ESI†).26,27 We found that the PD ring of Boc- Additionally, we also used a therapeutically relevant antibody Fab 15
Cys(PD)-OMe 9 adopts both conformers, but with the pseudoaxial (Fab, produced through digestion of the corresponding full anti-
conformer, which we assume is more likely to be prone to eventual body). Following reduction with tris(2-carboxyethyl)phosphine
elimination, dominating the equilibrium (3 : 1 ratio of pseudoaxial : (TCEP, see ESI,† the GFP single cysteine formed a dimer sponta-
pseudoequatorial) (Fig. 2b). With small molecule Boc-Cys(PD)-OMe neously in storage so reduction was required) GFPS147C 14 and an
9 in-hand, we next monitored the potential deconjugation of the PD antibody Fab 15 were fully modified with model PD 11 as judged by
by 1H NMR at 24 h intervals over the course of 96 h. New peaks in LC-MS to give GFP–PD 16 and Fab–PD 17 respectively (Fig. 3a-ii,
the 1H NMR corresponding to deconjugated PD 11 emerged, with Fab–PD 17 retained binding by ELISA – see ESI†). As a comparative
integrations of the newly appearing PD 11 CH protons suggesting model, GFPS147C 14 and Fab fragment 15 were also modified with a
gradual PD liberation from the conjugate over time (Fig. 2c). slowly hydrolysing N-methyl maleimide 23 to give GFP–maleimide
To further confirm that deconjugation was taking place, we next 18 and Fab–maleimide 19 conjugates (Fig. 3a-iii).29 These bioconju-
performed an Ellman’s test to establish the presence of free thiol gates were then subjected to a seven-day incubation period in buffer
generated from deconjugation of conjugate 9. Boc-Cys(PD)-OMe 9 representing physiological conditions (pH 7.4, 37 1C, no EDTA), and
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Fig. 3 (a) Mass spectrometry data showing (i) GFPS147C 14 and model Fab fragment 15; (ii) GFP–PD species 16 and Fab–PD species 17; (iii) GFP-maleimide species
18 and Fab-maleimide species 19; (b) mass spectrometry analysis of (i) incubation of GFP conjugates 16 and 18 (50 mM) in PBS pH 7.4 for 7 days at 37 1C; (ii) incubation
of Fab conjugates 17 and 19 (20 mM) in PBS pH 7.4 for 7 days at 37 1C. (c) Kinetic analysis of reaction between GFP conjugates (16 and 18) and DTNB 12 in PBS pH 7.4
for 12 h at 37 1C to form TNB 13 that was monitored by UV absorbance at A412. Plots show changing [TNB] and ln[GFP conjugate] vs. time.
deconjugation/ring hydrolysis of the conjugates was monitored by deconjugation seen by LC-MS, we can infer that the difference in
LC-MS analysis (Fig. 3b).
equilibrium shift between maleimides and PDs is likely a consequence
Significant release of the PD species from both GFP–PD 16 and of the vastly different rates of Michael addition presented by these two
Fab–PD 17 was noted, yielding GFPS147C 14 and reoxidised Fab 15 motifs, i.e., maleimide and PD scaffolds are releasing at a similar rate
respectively with no conjugates bearing ring hydrolysis observed. but maleimides immediately react with the released cysteine.
In sharp contrast, GFP–maleimide 18 and Fab–maleimide 19
In view of the relatively slow release of PD from the protein
afforded only the succinamic acid species 21 under the same conjugates, we thought that it could be exploited as a novel extra-
conditions; succinamic acids have previously been shown to be cellular cleavable linker for use in protein conjugates where the
stable to retro-Michael deconjugation.30 GFP and Fab conjugates tumour targeting protein carrier is non-internalising (e.g., an alter-
16–19 were also subjected to incubation with a model monoBr PD native to conventional non-internalising ADC cleavable linkers such
species, which would theoretically form an irreversible unsaturated as disulfide and Val–Cit as discussed in the introduction). Whilst
GFP–PD species/Fab–PD species that would act as a ‘‘trap’’ for any maleimide conjugates have been explored in this context (i.e., as a
available cysteine liberated from a retro-Michael pathway (see ESI†). reversible cleavable linker),31 the PD conjugate does not undergo
For the GFP–PD 16 and Fab–PD 17, a newly formed unsaturated hydrolysis and the low reactivity of unsaturated PDs with protein
GFP–PD conjugate and Fab–PD conjugate was obtained, with LC-MS thiols would ideally result in any prematurely released cargo (if any)
analysis indicating near-full release of the original PD cargo (see being rapidly cleared from the body as a small molecule.
ESI†). In contrast, incubation of monoBr PD with GFP–maleimide 18
To this end, Fab–PD conjugate 17 was incubated with common
and Fab–maleimide 19 showed minor formation of unsaturated blood thiols glutathione (5 mM, 4 h) and HSA 5 (20 mM, 48 h).
GFP–PD species and Fab–PD species with a significant amount of An antibody Fab fragment was seen as a relevant model for these
maleimide hydrolysis observed (see ESI†). These results confirm that studies as various antibodies and antibody fragments have found
both Michael acceptors (nonBr PDs and maleimides) exist in an application as non-internalising ADCs.32,33 To our delight, no transfer
equilibrium between conjugation and retro-Michael deconjugation. of PD to HSA 5 was observed (Fig. 4a) and addition of excess
It also highlights the competing hydrolysis pathway when using male- glutathione did not accelerate PD release (see ESI†). In sharp contrast,
imide conjugates in this context. We next investigated the compara- when Fab–maleimide conjugate 19 was subjected to HSA incubation
tive rates of retro-Michael deconjugation PDs and maleimides. GFP conditions, significant amounts of HSA–maleimide species 24 was
conjugates 16 and 18 were incubated with excess DTNB 12 at 37 1C, observed; confirming previous reports of blood instability of male-
with any liberated cysteine anticipated to react quantitatively to form imide conjugates (Fig. 4b).19 Furthermore, incubation of each of HSA 5
TNB 13, giving a characteristic absorbance at 412 nm which could be (50 mM) and glutathione (500 mM) with diethyl PD 11 (2.5 mM and
used to determine PD/maleimide deconjugation (Fig. 3c and ESI†). 500 mM, respectively) for 24 h at pH 7.4 to appraise whether any
Approximate rate constants for the pseudo first order retro-Michael reaction occurred, even under these forcing conditions, led to no
deconjugation reactions were found to be 2.34 Â 10À5 sÀ1 for the observed reaction in either case (see ESI†). Finally, we wanted to assess
GFP–maleimide species 18 and 2.29 Â 10À5 sÀ1 for the GFP–PD the applicability of the PD conjugate as an extracellular cleavable linker,
species 16, suggesting no significant difference between the rate of in a partially acidic environment such as that of a tumour (ca. pH 6.5).34
deconjugation. By comparing retro-Michael deconjugation rates with Fab–PD species 17 was incubated under buffered conditions at pH 6.5
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maleimide–thiol conjugates are used as degradable systems (e.g.,
synthesis of hydrogels).35
We gratefully acknowledge the EPSRC (CASE Award with LifeArc,
173621) for funding C. B., and the Leverhulme Trust (RPG-2017-288,
176274) for funding R. J. S. A. M. would like to acknowledge the
Ramsay Memorial trust for provision of a Ramsay Fellowship.
F. J. and P. S. would like to thank the Wellcome Trust for funding.
Conflicts of interest
There are no conflicts to declare.
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14832 | Chem. Commun., 2019, 55, 14829--14832
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