Impact of Target Warhead and Linkage Vector on Inducing Protein Degradation: Comparison of Bromodomain and Extra-Terminal (BET) Degraders Derived from Triazolodiazepine (JQ1) and Tetrahydroquinoline (I-BET726) BET Inhibitor Scaffolds

Article abstract of DOI:10.1021/acs.jmedchem.6b01912

The design of proteolysis-targeting chimeras (PROTACs) is a powerful small-molecule approach for inducing protein degradation. PROTACs conjugate a target warhead to an E3 ubiquitin ligase ligand via a linker. Here we examined the impact of derivatizing two different BET bromodomain inhibitors, triazolodiazepine JQ1 and the more potent tetrahydroquinoline I-BET726, via distinct exit vectors, using different polyethylene glycol linkers to VHL ligand VH032. Triazolodiazepine PROTACs exhibited positive cooperativities of ternary complex formation and were more potent degraders than tetrahydroquinoline compounds, which showed negative cooperativities instead. Marked dependency on linker length was observed for BET-degrading and cMyc-driven antiproliferative activities in acute myeloid leukemia cell lines. This work exemplifies as a cautionary tale how a more potent inhibitor does not necessarily generate more potent PROTACs and underscores the key roles played by the conjugation. The provided insights and framework for structure-activity relationships of bivalent degraders are anticipated to have wide future applicability.

Full text of DOI:10.1021/acs.jmedchem.6b01912

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Article
Impact of target warhead and linkage vector on inducing
protein degradation: comparison of Bromodomain and Extra-
Terminal (BET) degraders derived from triazolodiazepine (JQ1)
and tetrahydroquinoline (I-BET726) BET inhibitor scaffolds
Kwok-Ho Chan, Michael Zengerle, Andrea Testa, and Alessio Ciulli
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017
Downloaded from http://pubs.acs.org on June 8, 2017
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Impact of target warhead and linkage vector on
inducing protein degradation: comparison of
Bromodomain and ExtraꢀTerminal (BET)
degraders derived from triazolodiazepine (JQ1)
and tetrahydroquinoline (IꢀBET726) BET
inhibitor scaffolds
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*
KwokꢀHo Chan, Michael Zengerle, Andrea Testa, Alessio Ciulli
Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of
Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.
*
Correspondence and requests for materials should be addressed to A.C. Eꢀmail:
a.ciulli@dundee.ac.uk
KEYWORDS: protein degradation, PROTACs, bromodomain, cooperativity, acute myeloid
leukemia
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ABSTRACT
The design of proteolysisꢀtargeting chimeras (PROTACs) is a powerful smallꢀmolecule
approach for inducing protein degradation. PROTACs conjugate a target warhead to an E3
ubiquitin ligase ligand via a linker. Here we examined the impact of derivatizing two
different BET bromodomain inhibitors, triazolodiazepine JQ1 and the more potent
tetrahydroquinoline IꢀBET726, via distinct exit vectors, using different polyethylene glycol
linkers to VHL ligand VH032. Triazolodiazepine PROTACs exhibited positive
cooperativities of ternary complex formation, and were more potent degraders than
tetrahydroquinoline compounds, which showed negative cooperativities instead. Marked
dependency on linker length was observed for BETꢀdegrading and cMycꢀdriven
antiproliferative activities in acute myeloid leukemia cell lines. This work exemplifies as a
cautionary tale how a more potent inhibitor does not necessarily generate more potent
PROTACs, and underscores the key roles played by the conjugation. The provided insights
and framework for structureꢀactivity relationships of bivalent degraders is anticipated to have
wide future applicability.
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INTRODUCTION
Targeted protein degradation by exploiting the ubiquitin proteasome system has
1ꢀ3
recently emerged as a new modality of intervention for medicinal chemistry. One approach
to induce protein degradation is to design heteroꢀbifunctional molecules called proteolysisꢀ
targeting chimeras (also known as PROTACs) which comprise of a ligand binding an E3
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,5
ubiquitin ligase conjugated to a ligand binding the target protein. First introduced by Crews
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and Deshaies in 2001 (ref. ), developments of the technology over the following decade
were in large part hampered by poor drugꢀlikeness of the early generation compounds that
6
,7
typically incorporated peptidic binders for E3 ligases. Recently discovered highꢀaffinity
8
small molecules for the Cullin RING E3 ubiquitin ligases (CRLs), in particular against von
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ꢀ11
HippelꢀLindau (VHL e.g. 1 (VH032), Chart 1)
and cereblon (CRBN e.g. 2
12ꢀ15
(pomalidomide), Chart 1)
greatly contributed to full realization of the technology’s
potential. As a result of these developments, we and others recently reported potent activities
5
,16ꢀ20
18,20ꢀ25
and specificity in cells and in vivo of both VHLꢀbased
and CRBNꢀbased
PROTACs against several targets – including the Bromodomain and ExtraꢀTerminal (BET)
16,19,21,22
proteins Brd2, Brd3 and Brd4.
BET proteins are particularly attractive targets, with a
2
6,27
dozen of BET inhibitors from different scaffolds,
that are in >20 clinical trials against a
variety of diseases, mainly solid and hematological cancers including acute myeloid
2
8,29
leukaemia (AML) and mixed lineage leukemia (MLL)
as well as NUTꢀmidline
30
carcinomas. BETꢀtargeting PROTACs could provide advantageous therapeutic profiles over
19
BET inhibitors. In addition to their therapeutic potential, BETꢀtargeting PROTACs provide
useful chemical tools for posttranslational protein knockdown. The acute, profound and
reversible effect of compounds make it an alternative and advantageous approach to genetic
knockdowns to study the function of BET proteins in physiological and disease cellular state.
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Chart 1. Chemical structures of ligands for VHL (1) and CRBN (2) and BET
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inhibitors 3 (JQ1) and 4 (IꢀBET726) .
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One potential advantage of transforming inhibitors into degraders using the PROTAC
approach is that removal of the entire protein is expected to be mechanistically different from
blockade of a single domain interaction with an inhibitor, and to more closely phenocopy
genetic downregulation. This limitation is exemplified by smallꢀmolecule inhibitors of the
bromodomain of SMARCA2 and SMARCA4, which fail to display the antiproliferative
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phenotype expected based on genetic protein knockdown. A second advantage of ligand
directed protein degradation is the potential to enhance selectivity of target modulation over
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,16
and above the binary target engagement selectivity of the constitutive inhibitor. Selective
targeting of a single BET protein while sparing its paralogs would allow to better decipher
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their individual physiological roles. This is particularly relevant given traditional genetic
techniques have proven challenging, exemplified by the embryonic lethality of BET gene
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knockꢀouts. While selective inhibition of BET bromodomains can be achieved using alleleꢀ
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selective bumpꢀandꢀhole approaches, singleꢀpoint mutations need to be introduced ideally
using isogenic knockꢀins to enable selective target inhibition.
We previously reported VHLꢀtargeting PROTAC compounds 6 (MZ1) and analogue
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(MZ2) (Chart 2, see ref. ) that induced preferential depletion of a single BET member,
Brd4, over Brd2 and Brd3, despite binding the different BET bromodomains with
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6
comparable affinities. Our recent work disclosing the crystal structure of VHLꢀ6ꢀBrd4
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ternary complex, the first crystal structure of a PROTAC bound to both target protein and E3
ligase, showed how PROTAC 6 folds into itself to allow the two proteins to form productive
5
interactions. Our discovery provided structural insights into ligandꢀinduced proteinꢀprotein
interactions driving cooperative and preferential formation of ternary complexes as a basis
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for effective target degradation. This realization has important implications for PROTACs,
as it demonstrates an added layer of target depletion selectivity through PROTACꢀinduced
interactions between the target and the ligase, and supports important roles for the
derivatization mode of the two warhead ligands via the linker. All BETꢀdegrading PROTACs
1
6,19,21,22
reported so far by us and others
are based on the panꢀselective triazolodiazepineꢀ
3
4
based BET inhibitor 3 (Chart 1). However, while this manuscript was under review, a
study has reported active CRBNꢀbased BET degraders based on an azacarbazole containing
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BET inhibitor. To interrogate the impact of using a different, more potent BET inhibitor
than 3, and of exploring a different vector out of the warhead, on the activity and intraꢀBET
selectivity profile of BETꢀtargeting PROTACs, we here report novel VHLꢀrecruiting
PROTACs derived from a highꢀaffinity BET ligand, the tetrahydroquinolineꢀbased BET
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inhibitor 4 (Chart 1).
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RESULTS AND DISCUSSION
3
6
d d
Crystal structures of 4 (K for Brd4 tandem bromodomain = 4 nM; compare to K s
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00 nM for 3, ref. ) bound to BET bromodomains show that the free carboxylic acid of the
BET inhibitor is solvent exposed and is not involved in direct interactions with the protein
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(Figure 1b).
We therefore hypothesized that the carboxylate group could be exploited to
readily conjugate a linker e.g. via amide bond formation, without impairing binding to BET
bromodomains. Superposition of the coꢀcrystal structures of 3 and 4 each bound to the Nꢀ
terminal bromodomain of Brd4 (Figure 1) additionally showed that the benzoic acid group of
4
extends in a different direction from the tertꢀbutyl ester group of 3. We therefore became
interested in exploring the tolerance of the PROTAC approach to different exit vectors from
BET inhibitor scaffolds. Based on this design strategy, 4 was connected to the terminal
10
acetamide group of VHL ligand 1 (ref. ) to obtain PROTACs 8 (MZPꢀ61), 9 (MZPꢀ54), and
10 (MZPꢀ55) which bear a 2ꢀ, 3ꢀ and 4ꢀunit PEG linker, respectively, consistently with 5
2
2
(MZ4), 6 and 7 (Chart 2). Cereblonꢀbased compound 11 (Chart 2 ref. ) was also included
to provide a first direct comparison with VHLꢀbased PROTACs.
Figure 1. Coꢀcrystal structures to guide PROTAC linking design. First bromodomain of
Brd4 with bound (a) 3 (green carbons, PDB code 3MXF ) and (b) 4 (cyan carbons, 4BJX ).
Arrows highlight exit vectors for linking.
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Chart 2. Chemical structures of VHLꢀtargeting PROTACs based on 4 and 3 used in this
study and chemical structure of CRBNꢀtargeting PROTAC 11 (ARVꢀ825).
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To assess BET degradation activities, compounds were first profiled in HeLa cancer
cells because these cells are less susceptible to the cytotoxic effects of BET knockdown or
inhibition (Figure 2 and Fig. S1, see full blots in Fig. S5). Representative PROTACs 10 and
7
(each containing a PEGꢀ4 linker unit) induced marked concentrationꢀdependent knockdown
of BET proteins (Figure 2).
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Figure 2. Protein degradation profile of VHLꢀbased BET degraders. HeLa cells were
treated for 24 h. Protein levels are shown from one representative of two biological
replicates, visualized by immunoblot (a,c) and quantified relative to DMSO control (b,d).
Intensity values were quantified as described in the Methods.
Interestingly, tetrahydroquinolineꢀbased compound 10 showed depletion selectivity for Brd4
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and Brd3 over Brd2, in contrast to 7 that is a Brd4ꢀselective degrader (Figure 2). A similar
pattern of BET proteins degradation was observed with PEGꢀ3 linked compounds 9 and 6
(Fig. S1cꢀd,gꢀh). In contrast, PEGꢀ2 linked PROTACs 5 (Fig. S1eꢀf), and 8 (Fig. S1aꢀb)
showed lower activity over all BET proteins. Similar to tetrahydroquinolineꢀbased
PROTACs, 11 showed some preference for degrading Brd3/4 over Brd2, although all BET
proteins were potently depleted at 100 nM (Fig. S1iꢀj). Interestingly, treatments with
tetrahydroquinolineꢀbased PROTACs 9 and 10, revealed increased levels of BET proteins at
the higher concentration (1ꢀ10 µM, Fig. S1cꢀd and Figure 2aꢀb), thought to be due to the
4
“
hook effect”. Brd2 levels even increased beyond vehicle control level (Fig. S1cꢀd and
Figure 2aꢀb). These effects were largely recapitulated when the degradation assays were
repeated with shorter treatments of 6 h (Fig. S2), suggesting that the observed increase in
protein levels are not due to secondary effects at the longer time point. Control treatments
with the parent BET inhibitors 3 and 4 also led to increased levels of BET proteins (Fig. S1kꢀ
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n). Marked upꢀregulation was seen for Brd2 with inhibitor 4 treatment (Fig. S1kꢀl) and for
Brd4 long isoform with inhibitor 3 (Fig. S1mꢀn). Similar upꢀregulation of Brd4 with 3 were
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observed in Burkitt’s lymphoma cell lines.
Together, the data suggest that
tetrahydroquinoline based PROTACs function more as inhibitors than as degraders at the
higher concentrations. These results underscore the importance to identify suitable
concentrations to dissect effects due to PROTACꢀinduced degradation activity from those
due to inhibitory activity and potential cellular feedback mechanisms, which could
compensate pharmacological activity. Nonetheless, compounds 9 and 10 act as selective
degraders of BRD3/4 within appropriate window of concentration (30ꢀ100 nM).
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The distinctive activity profile of tetrahydroquinolineꢀbased PROTACs prompted us
to compare and contrast thermodynamics of ternary complex formation equilibria for this
class of PROTACs relative to the triazolodiazepineꢀbased series. We applied an isothermal
titration calorimetry (ITC) based assay setꢀup that we recently developed to circumvent
potential hookꢀeffects in ternary complex formation, and that we used to characterize
thermodynamics and cooperativities for binding of 6 to VHL and different BET
5
bromodomains. In our previous work, we showed how 6 forms highly cooperative and stable
complexes between VHL and BET bromodomains, and preferentially with the second
BD2
5
d
bromodomain of Brd4 (Brd4 ). We therefore set out to measure dissociation constants K s
of binary and ternary complexes formed between compounds 5–10, the VHLꢀEloCꢀEloB
BD2
protein (VCB), and Brd4 , and the resulting cooperativities (Table 1, see also Fig. S3). At
the binary level, the bromodomain warhead of the PROTACs 8–10 bound the BET
bromodomain consistently with higher potency that the corresponding bromodomain ligand
warhead within 5–7, while the VHL ligand warhead bound VCB with comparable affinities
across all PROTACs (Table 1). However strikingly, all tetrahydroquinoline based PROTACs
exhibited negative cooperativities of ternary complex formation, meaning that they bound the
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first protein more tightly on their own than in the presence of the second protein (α < 1,
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where α values are defined as ratio between binary and ternary K
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s, Table 1, see Figure 3
for representative binary and ternary titrations of VCB into 10 in the absence and presence of
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bromodomain).
Figure 3. Measuring cooperativities of ternary complex formation by ITC. (a) VCB
BD2
titrated into 10 alone. (b) VCB titrated into Brd4 ꢀ10 binary complex and (c) VCB titrated
BD1
BD2
into Brd2 ꢀ10. VCB binds more strongly to 10 alone (K = 110 nM) than to Brd4 ꢀ10 (Kd
d
BD1
=
180 nM) or Brd2 ꢀ10 (K = 330 nM), highlighting negative cooperativity.
d
Negative cooperativities were confirmed against all six BET bromodomains, as shown for
representative compound 10, with the Brd2 bromodomains showing the lowest α values
(Table S1). This feature was in stark contrast to the triazolodiazepineꢀbased series 5–7,
which all showed positive cooperativities (α values > 1, Table 1). The thermodynamic data
highlight an important feature; that is, cooperativities of PROTACs ternary complex
formation do not follow the binding affinities of the target warheads. Our data exemplifies
how PROTACs made from more potent target warhead ligands can form ternary complexes
less productively. It is interesting that despite being negatively cooperative, compounds 8–10
can still act as effective degraders at low concentration, underscoring the power of the subꢀ
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stoichiometric catalytic activity of PROTACs. The observation of stronger hook effects for
8
–10 compared to 5–7 in the degradation assays is however consistent with their negative
cooperativity, i.e. with them behaving more like inhibitors than degraders at higher
concentration. We previously demonstrated the importance of the ligandꢀinduced proteinꢀ
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protein contacts in dictating the large positive cooperativity of the VHL:6:Brd4 system. It is
therefore likely that the different exit vector from the tetrahydroquinoline warhead forces an
unfavourable relative orientation between the E3 ligase and the bromodomain. Comparing
the different linker lengths within a given series, it was found that PEGꢀ3 linked 6 showed the
highest cooperativity amongst the triazolodiazepineꢀbased series, whereas PEGꢀ2 linked 8
showed the lowest cooperativity amongst the tetrahydroquinolineꢀbased series (Table 1). In
both series overall, short linker proved to be less efficient in forming ternary complex and
inducing protein degradation.
To provide a functional downstream readout of the cellular activity of BET degraders,
we assessed antiꢀproliferative effects of PROTACs in AML MV4;11 (Figure 4aꢀb) and
HL60 (Fig. S4aꢀb), as these are well characterized BETꢀsensitive cell lines (see full blots in
Fig. S6). All compounds showed marked antiꢀproliferative activity in both cell lines.
Although some PROTAC compounds exhibited comparable nanomolar halfꢀmaximal antiꢀ
proliferative concentrations (pEC50s) relative to the constitutive inhibitors alone, the
maximal response to baseline level at the higher concentrations (Emax) of all VHLꢀbased
PROTACs presented exceeded that of the BET inhibitors (see Figure 4aꢀb and Fig. S4aꢀb,
and values tabulated in Table 2). This activity is likely owing to the more profound effect
associated with removing the entire protein compared to blocking an individual binding site,
which leaves other parts and domains of the proteins (e.g. the extraꢀterminal ET domain) still
functional. PEGꢀ3 and PEGꢀ4 based PROTACs proved overall more potent than PEGꢀ2,
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consistent with the trends in degradation activities in HeLa and cooperativities (Figure 4c
and Table 2).
To confirm Brd4 degradation and downstream impact on cMyc levels, we examined
protein levels in the same cellular context following acute pharmacological intervention (4 h
treatments). In each of the two series, PEGꢀ3 and PEGꢀ4 based PROTACs (6 and 7, 9 and 10)
induced superior depletion of both Brd4 and cMyc over their respective PEGꢀ2 analogues 5
and 8 in both cell lines, at two different concentrations (Figure 4d and Fig. S4cꢀe).
PROTACs 6 and 7 and 9 also showed higher depletion of cMyc levels compared to their
inhibitor counterparts, indicating a greater downstream response with more efficient chemical
degraders, while 5 and 8 induce lower cMyc depletion than the corresponding inhibitors 3
and 4, respectively (Fig. 4c). Together, the results confirmed the PEGꢀ2 linker length to be
too short for optimal PROTAC activity.
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Figure 4. Antiꢀproliferative and Mycꢀsuppression activity of BET degraders and
inhibitors. (aꢀb) MV4;11 cells were treated with PROTACs and their corresponding BET
targeting ligands for 48 h prior to quantitation of cell viability. (c) Halfꢀeffective
concentrations of BET degraders and corresponding inhibitors; (d) MV4;11 cells were treated
for 4 h with BET PROTACs or inhibitors (50 nM) or DMSO control. Protein levels are
shown from one representative of two biological replicates.
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Structureꢀactivity relationschips (SARs) are typically quantified by measuring binding
d i
or inhibition constants (K , K ), or inhibitory dose response curves (IC50). Because induced
protein degradation features catalytic depletion of protein levels over time, different
parameters are needed to quantify compounds potency and efficacy. To evaluate SAR in a
quantitative fashion, we evaluted: pDC50 (concentration causing 50% reduction of protein
level relative to vehicle) and Dmax (maximum reduction of protein level relative to vehicle)
for HeLa protein degradation responses; pEC50 (halfꢀmaximal effective concentrations) and
Emax (maximal response to baseline level at the highest concentrations) from cell viability
assays; % reduction of Brd4 and cMyc levels in AML cell lines; and cooperativity (α) of
BD2
ternary complex formation with VCB and Brd4
(values reported in Table 2). To evaluate
the main drivers of the observed antiꢀproliferative effects, we plotted PROTACs pEC50
values from AML cell viability assays relative to other parameters (Figure 4). Strong
correlation was found between pEC in MV4;11 and pDC on the long isoform of Brd4 in
50
50
2
HeLa (r = 0.84, Figure 4a). The antiꢀproliferative activities against AML of BET PROTACs
and their parent inhibitors correlated well with depletion of cellular levels of cMyc in both
2
2
MV4;11 (r = 0.69, Figure 4b) and HL60 (r = 0.62), consistent with AML cells proliferation
3
8
being cMycꢀdriven. Overall, PEGꢀ3 linked 6 and 9 confirmed to be the most effective
amongst the VHLꢀbased PROTACs, with activities comparable to those of CRBNꢀbased
PROTAC 11. Importantly, for
a
given linker length, the trends confirmed
tetrahydroquinolineꢀbased PROTACs to be less effective degraders than the triazolodiazepine
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based, despite the constitutive ligand 4 confirming to be a more potent BET inhibitor than 3
in these cell lines (Figure 4c).
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Figure 5. PROTACs’ SAR correlation plots. AntiꢀAML activities (48 h treatments)
plotted against (a) HeLa degradation of Brd4 long isoform (24 h), and (b) reduction in cMyc
levels in MV4;11 (4 h).
CONCLUSIONS
We describe novel VHLꢀtargeting BET degraders designed based on a highꢀaffinity
tetrahydroquinoline inhibitor, and explore the impact of varying the BETꢀrecruiting scaffold
and the linkage vector on PROTAC ternary complex recognition and cellular activity.
Despite being derivatized from a more potent BET inhibitor, the tetrahydroquinoline based
series showed negative cooperativities of ternary complex formation and proved to be less
effective degraders than the positively cooperative triazolodiazepine series. These results
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exemplify how more potent inhibitors do not necessarily generate more potent PROTACs,
and underscore how the ability to strongly form the ternary complex is critical to the
mechanism of action of bivalent degraders. Sideꢀbyꢀside comparisons demonstrated
remarkable dependency of cellular activity on the linker length, with a trend of PEGꢀ3 >
PEGꢀ4 >> PEGꢀ2 observed for both chemical series, potentially suggesting a “sweetꢀspot”
for optimal linking within a given E3 ligase:target pair. We also show how by changing the
BETꢀrecruiting warhead and linkage vector, the intraꢀBET degradation selectivity profile
could be tuned from Brd4ꢀselective for one series to Brd3/4 selective for another. Further
SAR on either the linker or warhead ligand and exit vector could further increase potency and
selectivity of degrading the different BET proteins. Future work assessing the impact of
varying other parameters, such as the nature of the E3 ligase recruited and the E3 warhead
used is also warranted. More generally, we provide a framework for establishing future
structureꢀactivity relationships of chemical degraders based on measurable in vitro
parameters that we anticipate will prove useful to the burgeoning new field of inducing
protein degradation with small molecules.
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EXPERIMENTAL SECTION
A. Chemistry
All chemicals, unless otherwise stated were commercially available and used without
further purification. Enantiopure (+)ꢀ3 and 4 were purchased from Medchemexpress LLC,
Princeton, USA. (+)ꢀ3 was deprotected to the carboxylic acid form 6HꢀThieno[3,2ꢀ
16
f][1,2,4]triazolo[4,3ꢀa][1,4]diazepineꢀ6ꢀacetic acid, as previously described. 11 was
22
16
synthesized as described previously. 6 and 7 were synthesized as described. Reactions
were magnetically stirred; commercially available anhydrous solvents were used. NMR
spectra were recorded on a Bruker Ascend 400. Chemical shifts are quoted in ppm and
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13
referenced to the residual solvent signals: H δ = 7.26 (CDCl
3
), C δ = 77.16; signal splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad
br). Coupling constants (JHꢀH) are measured in Hz. High Resolution Mass Spectra (HRMS)
(
were recorded on a Bruker microTOF. Low resolution MS and analytical HPLC traces were
recorded on an Agilent Technologies 1200 series HPLC connected to an Agilent
Technologies 6130 quadrupole LCꢀMS, connected to an Agilent diode array detector.
Preparative HPLC was performed on a Gilson Preparative HPLC System with a Waters Xꢀ
Bridge C18 column (100 mm x 19 mm; 5 ꢁm particle size) and a gradient of 5 % to 95 %
acetonitrile in water over 10 min, flow 25 mL/min, with 0.1 % ammonia in the aqueous
phase. The purity of all compounds was analyzed by HPLCꢀMS (ESI) and was > 95%.
General procedure for synthesis of VHL ligand–linker conjugates. The azideꢀ(PEG)n
0
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derivatives of compound 1 were synthesized as previously described.
(2
S,4
R
)ꢀ1ꢀ((
S
)ꢀ2ꢀ(2ꢀ(2ꢀ(2ꢀAzidoethoxy)ethoxy)acetamido)ꢀ3,3ꢀdimethylbutanoyl)ꢀ4ꢀ
hydroxyꢀ
N
ꢀ(4ꢀ(4ꢀmethylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀcarboxamide.
Prepared
accordingly to the general procedure for synthesis of VHL ligand–linker conjugates.
1
Obtained 411 mg, colorless oil, 68% yield. HꢀNMR (CDCl
3
, 400 MHz) δ ppm: 8.68 (s,
1
H), 7.38–7.33 (m, 5H), 7.24 (d, J = 8.5 Hz, 1H), 4.75 (t, J = 7.9 Hz, 1H), 4.59–4.54 (m,
H), 4.48 (d, J = 8.6 Hz, 1H), 4.33 (dd, J = 14.9 Hz, J = 5.2 Hz, 1H), 4.12–4.09 (m, 1H),
2
4.06–3.96 (m, 2H), 3.70–3.66 (m, 6H), 3.61 (dd, J = 11.4 Hz, J = 3.7 Hz, 1H), 3.41–3.38 (m,
13
2
H), 2.89 (br s, 1H), 2.63–2.58 (m, 1H), 2.52 (s, 3H), 2.13–2.08 (m, 1H), 0.95 (s, 9H); Cꢀ
NMR (CDCl , 101 MHz) δ ppm: 171.6, 170.7, 170.6, 150.7, 138.4, 130.4, 129.7, 128.6,
28.4, 127.6, 71.2, 70.5, 70.3, 70.2, 67.2, 58.5, 57.3, 56.8, 50.7, 43.4, 35.8, 34.9, 26.5, 16.0.
3
1
+
MS calc. for C28
39 7 6
H N O S 601.3, found 602.3 [M+H ].
General procedure for synthesis of final PROTAC molecules. The azideꢀ(PEG)n
derivative of compound 1 (40
μmol) was dissolved in methanol (5 ml). Catalytic amount of
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Pd on charcoal (10 %, dry) was added and the reaction mixture stirred under an atmosphere
of hydrogen for 3 h at 25 °C. The reaction mixture was filtered through a plug of celite and
the resulting solution evaporated to dryness to obtain the desired amine. The resulting
amines (35
dissolved in DCM (2 ml). HATU (14.3 mg, 37.5
adjusted to >9 by adding DIPEA (17.5 l, 100
μ
mol, 1.4 eq.) and 4 or the carboxylic acid form of (+)ꢀ3 (25
mol, 1.5 eq.) was added and the pH
mol, 4 eq.). After stirring the reaction
μmol, 1 eq.) were
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μ
μ
μ
mixture at 25 °C for 18 h the solvent was removed in vacuum. The crude was purified by
preparative HPLC as described above.
(2S R)ꢀ1ꢀ((S)ꢀ2ꢀ(Tertꢀbutyl)ꢀ14ꢀ((S)ꢀ4ꢀ(4ꢀchlorophenyl)ꢀ2,3,9ꢀtrimethylꢀ6Hꢀ
,4
thieno[3,2ꢀf][1,2,4]triazolo[4,3ꢀa][1,4]diazepinꢀ6ꢀyl)ꢀ4,13ꢀdioxoꢀ6,9ꢀdioxaꢀ3,12ꢀ
diazatetradecanoyl)ꢀ4ꢀhydroxyꢀ
Nꢀ(4ꢀ(4ꢀmethylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀ
1
carboxamide (5). White amorphous powder. Yield: 22.2 mg (66 %); HꢀNMR (CDCl
3
, 400
MHz) δ ppm: 8.65 (s, 1H), 8.30–8.25 (m, 2H), 7.63 (d, J = 10.0 Hz, 1H), 7.36 (d, J = 8.7 Hz,
H), 7.24 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 4.94–4.86
(m, 2H), 4.63–4.57 (m, 2H), 4.21 (d, J = 5.8 Hz, 2H), 4.07–3.81 (m, 5H), 3.72–3.52 (m, 7H),
2
3
.44 (dd, J = 15.9 Hz, J = 3.0 Hz, 1H), 3.13–3.08 (m, 1H), 2.55 (s, 3H), 2.45 (s, 3H), 2.39–
13
2
.35 (m, 4H), 2.27ꢀ2.20 (m, 1H), 1.66 (s, 3H), 1.09 (s, 9 H); CꢀNMR (CDCl
3
, 101 MHz) δ
ppm: 172.1, 171.1, 170.6, 170.4, 163.3, 156.2, 150.2, 149.9, 148.4, 138.5, 136.9, 136.7,
131.9, 131.6, 131.4, 131.3, 131.2, 130.2, 130.1, 129, 128.8, 127.7, 71.5, 70.4, 70.3, 69.8,
59.3, 57.5, 56.5, 53.9, 42.7, 39.7, 38.3, 37.2, 36.3, 26.6, 16.2, 14.5, 13.3, 11.8. HRMS m/z
+
calc. for C H N ClO S expected 957.3505, found 958.3498 [M+H ].
46
57
9
7 2
S,4R)ꢀ1ꢀ((S)ꢀ1ꢀ(4ꢀ((2S,4R)ꢀ1ꢀAcetylꢀ4ꢀ((4ꢀchlorophenyl)amino)ꢀ2ꢀmethylꢀ1,2,3,4ꢀ
tetrahydroquinolinꢀ6ꢀyl)phenyl)ꢀ12ꢀ(tertꢀbutyl)ꢀ1,10ꢀdioxoꢀ5,8ꢀdioxaꢀ2,11ꢀ
diazatridecanꢀ13ꢀoyl)ꢀ4ꢀhydroxyꢀ
Nꢀ(4ꢀ(4ꢀmethylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀ
1
carboxamide (8). White amorphous powder. Yield: 14.5 mg (42 %). HꢀNMR (CDCl
3
, 400
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4
4
4
4
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5
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5
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6
MHz) δ ppm: 8.66 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.79–7.77 (m, 1H), 7.60–7.50 (m, 5H),
7
.32 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H), 6.63–6.57 (m,
3
H), 4.89 (br. s, 1H), 4.54–4.37 (m, 4H), 4.22–4.15 (m, 2H), 4.07–3.90 (m, 3H), 3.81–3.47
(m, 10H), 3.20 (br. s, 1H), 2.71–2.64 (m, 1H), 2.50 (s, 3H), 2.38–2.32 (m, 1H), 2.24 (s, 3H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
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8
9
0
13
1.99–1.93 (m, 1H), 1.36–1.25 (m, 1H), 1.18 (d, J = 6.4 Hz, 3H), 0.85 (s, 9H); CꢀNMR
CDCl , 101 MHz) δ ppm: 171.1, 170.7, 170.6, 169.4, 167.5, 150.3, 148.5, 145.8, 143.4,
37.8, 136.3, 133.4, 131.5, 131.0, 129.5, 129.4, 128.3, 128.1, 126.9, 126.6, 126.0, 122.9,
(
3
1
1
22.4, 114.5, 77.3, 77.0, 76.7, 71.7, 70.9, 70.7, 70.5, 70.0, 58.8, 57.0, 56.9, 50.5, 43.3, 41.1,
+
3
9.8, 36.2, 36.0, 26.4, 23.1, 21.3, 16.1; HRMS m/z calc. for C H ClN O S [M+H ]
53 63
7
8
992.4142, found 992.4091.
S,4R)ꢀ1ꢀ((S)ꢀ1ꢀ(4ꢀ((2S,4R)ꢀ1ꢀAcetylꢀ4ꢀ((4ꢀchlorophenyl)amino)ꢀ2ꢀmethylꢀ1,2,3,4ꢀ
tetrahydroquinolinꢀ6ꢀyl)phenyl)ꢀ15ꢀ(tertꢀbutyl)ꢀ1,13ꢀdioxoꢀ5,8,11ꢀtrioxaꢀ2,14ꢀ
diazahexadecanꢀ16ꢀoyl)ꢀ4ꢀhydroxyꢀ
N
ꢀ(4ꢀ(4ꢀmethylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀ
1
carboxamide (9). White amorphous powder. Yield: 17.1 mg (71 %). HꢀNMR (CDCl , 400
3
MHz) δ ppm: 8.66 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.53–7.48 (m, 4H), 7.36–7.30 (m, 6H),
7
.22–7.19 (m, 2H), 7.14 (d, J = 8.7 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 4.88 (br. s, 1H), 4.60
(t, J = 7.7 Hz, 1H), 4.52 (d, J = 19.7 Hz, 2H), 4.42 (br. s, 1H), 4.34–4.18 (m, 3H), 3.94–3.72
(m, 3H), 3.68–3.51 (m, 14H), 3.21 (br. s, 1H), 2.70–2.64 (m, 1H), 2.49 (s, 3H), 2.44–2.36
(m, 1H), 2.22 (s, 3H), 2.05–2.01 (m, 1H), 1.34–1.25 (m, 1H), 1.18 (d, J = 6.3 Hz, 3H), 0.94
13
(
s, 9H); CꢀNMR (CDCl , 101 MHz) δ ppm: 171.1, 170.9, 170.2, 169.5, 167.4, 150.3,
3
1
1
45.8, 143.2, 138.2, 137.7, 136.3, 133.4, 130.9, 129.5, 129.3, 128.1, 127.8, 126.9, 126.5,
25.8, 122.7, 122.6, 114.6, 77.4, 77.0, 76.7, 71.0, 70.4, 70.3, 70.1, 70.0, 69.6, 58.5, 56.9,
56.8, 50.2, 47.6, 43.2, 41.0, 40.0, 36.2, 35.4, 26.4, 23.1, 21.3, 16.1; HRMS m/z calc. for
+
C H ClN O S [M+H ] 1036.4404, found 1036.4356.
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(2
S,4R)ꢀ1ꢀ((S)ꢀ1ꢀ(4ꢀ((2S,4R)ꢀ1ꢀAcetylꢀ4ꢀ((4ꢀchlorophenyl)amino)ꢀ2ꢀmethylꢀ1,2,3,4ꢀ
tetrahydroquinolinꢀ6ꢀyl)phenyl)ꢀ18ꢀ(tertꢀbutyl)ꢀ1,16ꢀdioxoꢀ5,8,11,14ꢀtetraoxaꢀ2,17ꢀ
diazanonadecanꢀ19ꢀoyl)ꢀ4ꢀhydroxyꢀ
N
ꢀ(4ꢀ(4ꢀmethylthiazolꢀ5ꢀyl)benzyl)pyrrolidineꢀ2ꢀ
1
carboxamide (10). White amorphous powder. Yield: 14.6 mg (58 %). HꢀNMR (CDCl , 400
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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41
42
43
44
45
46
47
48
49
50
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53
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57
58
59
60
MHz) δ ppm: 8.66 (s, 1H), 7.84 (d, J = 8.1 Hz, 2H), 7.54–7.49 (m, 4H), 7.36–7.30 (m, 5H),
7.26–7.22 (m, 2H), 7.15–7.12 (m, 3H), 6.62 (d, J = 8.6 Hz, 2H), 4.89 (br. s, 1H), 4.69 (t, J =
7.9 Hz, 1H), 4.57–4.46 (m, 3H), 4.33 (dd, J = 5.6, 14.7 Hz, 1H), 4.24–4.15 (m, 2H), 4.00–
3.80 (m, 3H), 3.66–3.55 (m, 18H), 3.46 (br. s, 1H), 2.69–2.63 (m, 1H), 2.50–2.44 (m, 4H),
2.22 (s, 3H), 2.10–2.06 (m, 1H), 1.36–1.25 (m, 1H), 1.18 (d, J = 6.2 Hz, 3H), 0.93 (s, 9H);
13
CꢀNMR (CDCl , 101 MHz) δ ppm: 170.2, 169.5, 167.3, 150.3, 145.8, 143.2, 138.2, 137.8,
3
1
36.3, 133.4, 129.5, 129.3, 128.1, 127.8, 126.9, 126.5, 125.9, 122.6, 114.5, 71.0, 70.6, 70.2,
6
9.8, 58.6, 57.0, 56.8, 50.3, 47.5, 43.2, 41.1, 39.9, 36.1, 35.3, 26.4, 23.1, 21.3, 16.1; HRMS
+
m/z calc. for C57
H71ClN
7
O
10S [M+H ] 1080.4666, found 1080.4623.
B. BIOLOGY
HeLa cells were kept in DMEM medium (Gibco) supplemented with 10% fetal bovine
serum (FBS) (Gibco), Lꢀglutamine (Gibco), penicillin, streptomycin. MV4;11 and HL60
cells were kept in RPMI medium (Gibco) supplemented with 10% FBS, Lꢀglutamine,
penicillin and streptomycin. Cells were kept at 37 °C, 5% CO2.
5
Testing compounds in cells. HeLa cells were seeded at 3
×
10 per well on a standard 6ꢀ
well plate. After a day, cells were treated with compounds for the desired time. Cells were
washed with PBS twice and lysed with RIPA buffer (Sigma), supplemented with protease
2
inhibitor cocktail (Roche), Benzonase (Merck) and 0.5 mM MgCl . Lysate was briefly
sonicated and centrifuged at 20000
×
g for 10 min at 4 °C. Supernatant was collected and
7
protein concentration measured by BCA assay. For MV4;11 and HL60, 1.2
×
10 cells in 15
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mL medium were treated with compound for the desired time. Cells were washed with PBS
twice, and lysed with hypotonic buffer (10 mM HEPES, 10 mM KCl, protease inhibitor
cocktail, Benzonase and 0.5 mM MgCl
incubation period to disrupt cell outer membrane and release nuclei. Nuclei were pelleted by
centrifugation at 2000 g for 15 min. The pellet was resuspended in RIPA buffer,
supplemented with protease inhibitor cocktail, Benzonase and 0.5 mM MgCl . The
suspension was briefly sonicated and centrifuged at 20000 g for 10 min at 4 °C.
2
) for 30 min by vortexing twice in between the
0
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0
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0
×
2
×
Supernatant was collected and protein concentration measured by BCA assay.
Immunoblotting. Protein on gel was transferred to nitrocellulose membrane using
iBlot2 (Life technology) according to manufacturer guidelines. Blots were probed with antiꢀ
Brd4 (AbCam, ab128874), antiꢀBrd3 (AbCam, ab50818), antiꢀBrd2 (AbCam, ab139690),
antiꢀ
ab133741) antibodies. Blots were developed with secondary antiꢀMouse IgG (Licor, 926ꢀ
2210) or antiꢀRabbit IgG (Licor, 926ꢀ32213) antibodies from Licor and bands visualized
βꢀactin (Cell signaling, #4970), antiꢀcMyc (AbCam, ab32072), antiꢀlamin B1 (AbCam,
3
using Licor Odessey Sa imaging system.
Western blot quantification. Image processing and band intensity quantification were
performed using Licor Image Studio software v5.2.5. Reported band intensities are
normalized to loading control, i.e. βꢀactin for total lysates and lamin B1 for nuclear extracts.
DC50 values were determined by assuming a linear model between the two data points across
the 50% protein level mark. Dmax was determined as the highest protein depletion across the
concentrations tested.
Cell viability assay. MV4;11 or HL60 cells were incubated with compounds at the
desired concentration for 48 h on a clearꢀbottom 384ꢀwell plate. Cells were kept in RPMI
medium supplemented with 10% FBS, Lꢀglutamine, penicillin and streptomycin. Initial cell
5
density was 3
×
10 per mL. Cells were treated with various concentration of compound or
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0.05% DMSO. After treatment, cell viability was measured with Promega CellTiterꢀGlo®
Luminescent Cell Viability Assay kit according to the manufacturer instructions. Signal was
recorded on a BMG Labtech Pherastar luminescence plate reader with recommended
settings. Data was analyzed with Graphpad Prism software to obtain EC values of each test
50
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compound.
Protein expression and purification. Bromodomain and VCB complex constructs and
5
protein preparation were described in previous publication. Wildꢀtype version of human
proteins VHL (UniProt accession number: P40337) ElonginC (Q15369), ElonginB (Q15370),
Brd2 (P25440), Brd3 (Q15059) and Brd4 (O60885) were used for all protein expression. In
brief, the His ꢀtagged constructs were transformed into E. coli BL21(DE3) and induced with
6
IPTG to produce the desired proteins. E. coli cells were homogenized at 4 degree Celsius and
6
His ꢀtagged proteins were purified from the soluble lysate was by passing through a Ni
affinity column. After cleaving the Hisꢀtag by TEV protease, a second Ni affinity column
purification was performed to obtain tagꢀfree protein in the flowꢀthrough. VCB was then
additionally purified by anion exchange using MonoQ (GE Healthcare). For all proteins,
purity was further polished by gel filtration chromatography.
Isothermal titration calorimetry (ITC). Titrations were performed on an ITC200
5
microꢀcalorimeter (GE Healthcare) as previously reported. The titrations were in ITC buffer
20 mM Bis–Tris propane, 150 mM NaCl, 1 mM tris(2ꢀcarboxyethyl)phosphine (TCEP), pH
7
.4 supplemented with either 0.2% or 3% DMSO and consisted of 19 injections of 2 ꢁl
protein solution at a rate of 0.5 ꢁl/s at 120 s time intervals. An initial injection of protein (0.4
l) was made and discarded during data analysis. All experiments were performed at 25 °C,
whilst stirring at 600 r.p.m. PROTACs were diluted from a 10 mM DMSO stock solution to
0 ꢁM in ITC buffer with the final concentration of DMSO to be 0.2% for triazolodiazepineꢀ
based PROTAC or 3% for tetrahydroquinolineꢀbased PROTAC. Bromodomain protein in the
ꢁ
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same buffer was titrated into the PROTAC in the cell. At the end of the titration, the excess
of solution was removed from the cell, the syringe was washed and dried, VCB complex (168
ꢁM, in the same buffer) was loaded in the syringe and titrated into the complex of PROTAC–
bromodomain. The concentration of the complex in the cell (C) after the first titration (16.8
µM), was calculated as follows:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
where: C
0
is the initial concentration of the PROTAC in the cell (20 µM), Vcell is the
volume of the sample cell (200.12 µM) and Vinj is the volume of titrant injected during the
first titration (38.4 µM). Titrations for the binary complex PROTAC–VCB were performed in
the same manner with VCB titrated into 16.8 µM of PROTAC in the cell. For titration with 8
and 9, concentrations of PROTAC and proteins were halved due to compound solubility. The
data were fitted to a singleꢀbindingꢀsite model to obtain the stoichiometry n, the dissociation
constant Kd and the enthalpy of binding ΔH using the Microcal LLC ITC200 Origin
software provided by the manufacturer.
ASSOCIATED CONTENT
Supporting Information
Doseꢀdependent western blots and quantification of protein levels in HeLa cells; ITC titration
curves and results; antiꢀproliferative activities in HL60; Brd4 and cMycꢀsuppression in
MV4;11 and HL60; original blot images; NMR spectra of tetrahydroquinolineꢀbased
PROTACs.
Molecular Formula Strings (CSV)
This material is available free of charge on the ACS Publications website.
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AUTHOR INFORMATION
Corresponding Author
*
Phone: +44 (0)1382 386230. Eꢀmail: a.ciulli@dundee.ac.uk
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Author Contributions
All authors have given approval to final version of the manuscript.
Funding Sources
This work was supported by the European Research Council (ERCꢀ2012ꢀStGꢀ311460
DrugE3CRLs Starting Grant to A.C.); the UK Biotechnology and Biological Sciences
Research Council (BBSRC grant BB/J001201/2 to A.C.); the European Commission (H2020ꢀ
MSCAꢀIFꢀ2014ꢀ655516 Marie SkłodowskaꢀCurie Actions Individual Fellowship to KꢀH.C.);
and the Wellcome Trust (Strategic Awards 100476/Z/12/Z for biophysics and drug discovery
to the Division of Biological Chemistry and Drug Discovery).
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We are thankful to L. Finn for support with tissue culture (MRCꢀPPU), and the Ferguson lab
for access to LIꢀCOR equipment.
ABBREVIATIONS USED
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BET, bromodomain and extraꢀterminal; Brd2/3/4, bromodomainꢀcontaining protein 2/3/4;
CRBN,
cereblon;
DIPEA
N,Nꢀdiisopropylethylamine;
HATU
1ꢀ
[
bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium
3ꢀoxid
hexafluorophosphate; PROTAC, proteolysisꢀtargeting chimera; VHL, von HippelꢀLindau
0
1
2
3
4
5
6
7
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9
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2
3
4
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6
7
8
9
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2
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5
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9
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0
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Journal of Medicinal Chemistry
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