An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56

Article abstract of DOI:10.1002/anie.201611907

The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.

Full text of DOI:10.1002/anie.201611907

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International Edition: DOI: 10.1002/anie.201611907
German Edition: DOI: 10.1002/ange.201611907
Medicinal Chemistry
An Irreversible Inhibitor of HSP72 that Unexpectedly Targets
Lysine-56
Jonathan Pettinger, Yann-Vaꢀ Le Bihan, Marcella Widya, Rob L. M. van Montfort, Keith Jones,
and Matthew D. Cheeseman*
Abstract: The stress-inducible molecular chaperone, HSP72, is
an important therapeutic target in oncology, but inhibiting this
protein with small molecules has proven particularly challeng-
ing. Validating HSP72 inhibitors in cells is difficult owing to
competition with the high affinity and abundance of its
endogenous nucleotide substrates. We hypothesized this could
be overcome using a cysteine-targeted irreversible inhibitor.
Using rational design, we adapted a validated 8-N-benzylade-
nosine ligand for covalent bond formation and confirmed
targeted irreversible inhibition. However, no cysteine in the
protein was modified; instead, we demonstrate that lysine-56 is
Scheme 1. Synthesis of the HSP72-NBD nucleotide-competitive tar-
geted covalent inhibitor 8.
the key nucleophilic residue. Targeting this lysine could lead to
a new design paradigm for HSP72 chemical probes and drugs.
H
eat shock 70 kDa protein 1 (HSP72) is a stress-inducible
binds reversibly, forming a non-covalent complex, before
covalent bond formation to give the irreversible complex
[Equation (1)]. This process means the reactivity of the
electrophilic warhead can be reduced, so the reaction is only
fast once the complex has formed.[8] We hypothesized that
the validated nucleotide-competitive 8-N-benzyladenosine
1 (Scheme 1),[9] which is a potent targeted reversible inhibitor
[Equation (2)], fulfills these criteria and could be modified for
targeted covalent inhibitor design.
ATPase molecular chaperone, which stabilizes and refolds
substrate proteins to maintain cellular homeostasis.[1] HSP72
is a well-established target in oncology, as upregulation is
associated with poor clinical outcomes[2] and drug resist-
ance.[3]
A significant hurdle to cellular activity for nucleotide-
competitive inhibitors of HSP72 is the high affinity for its
endogenous nucleotide substrates (ADP, KD ꢀ 110 nm).[4,5]
Irreversible inhibition is an important strategy for proteins
with high-affinity substrates,[6] with the recent renaissance led
by drugs targeting the tyrosine kinase EGFR, which circum-
vent the increased ATP affinity resulting from the T790M
resistance mutation.[7] Owing to the clear clinical potential
that a HSP72 inhibitor could offer and with few cell-active
chemical probes to study the role of HSP72 in cancer, we
proposed that a nucleotide-competitive targeted covalent
inhibitor could overcome many of these challenges.
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Proteins typically react through solvent-exposed nucleo-
philic cysteine residues.[8] Focusing only on the nucleotide-
binding domain (NBD), analysis of the HSP72 co-crystal
structure, bound with the validated nucleotide-competitive
inhibitor Ver-155008, (Figure 1, PDB: 4IO8) revealed three
residues: Cys17, Cys267 and Cys306 (see the Supporting
Information).[4] Three irreversible inhibitors of HSP70 have
been reported; YK5 2[10] and oridonin 3[11] are proposed to
target Cys267 of the NBD, while the natural product
novolactone[12] targets Glu444 in the substrate-binding
domain. Cys267 is distal from the validated targeted rever-
sible inhibitor 1 binding site and is buried deeply in a hydro-
phobic region, requiring significant protein conformational
change to become solvent-exposed, so is incompatible with
rational targeted covalent inhibitor design.[13] Of the remain-
ing reactive cysteine residues, Cys306 is also positioned too
far from the binding site. However, Cys17 is at the bottom of
the binding cleft with an unhindered vector pointing directly
towards the 5’-position of the reversibly-bound ligand. We
Crucial to the success of targeted covalent inhibitors is
their two-step process for inhibition.[8] The inhibitor first
[*] J. Pettinger, Dr. Y.-V. Le Bihan, Dr. R. L. M. van Montfort,
Prof. K. Jones, Dr. M. D. Cheeseman
Cancer Research UK Cancer Therapeutics Unit, The Institute of
Cancer Research
London, SW7 3RP (UK)
E-mail: matthew.cheeseman@icr.ac.uk
Dr. Y.-V. Le Bihan, Dr. R. L. M. van Montfort
Division of Structural Biology, The Institute of Cancer Research
London, SW7 3RP (UK)
M. Widya
Proteomics Core Facility, The Institute of Cancer Research
London, SW7 3RP (UK)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201611907.
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89% yield over two steps. Acylation of the primary alcohol
with acryloyl chloride and finally deprotection of the aceto-
nide gave our targeted covalent inhibitor 8 in seven steps and
6% overall yield. As controls, targeted reversible inhibitor
matched-pair 9, without the electrophilic warhead, and
acetonide-protected 10, which was predicted to be a non-
binding but still electrophilic analogue, were synthesized in
a similar manner (Figure 3 and the Supporting Information).
Figure 1. Targeting Cys17 at the base of the targeted reversible
inhibitor binding site of HSP72-NBD (PDB: 4IO8, residues 3–379) only
key residues are shown, solvent and hydrogens omitted for clarity,
carbon=grey, oxygen=red, nitrogen=blue, chlorine=green, sulfur=
yellow.
believed Cys17 could potentially be targeted as the key
nucleophilic protein residue and that the linker and electro-
phile could be developed by rational design (Figure 1).
HSP72 is a highly flexible protein, which complicates
inhibitor design.[9] The distance between Cys17 and the 5’-
position of the nucleoside analogues depends on the protein
conformation, ranging from 9.2–10.7 ꢀ (see Supporting
Information). Our design strategy required a versatile syn-
thesis of 5’-adenosine derivatives, so that the linker could span
the flexible gap to the nucleophilic residue, while retaining
the 8-N-benzyl moiety to maintain reversible affinity
(Figure 2). From analysis of our model, we predicted that
a 3-carbon flexible linker at the 5’-position would span the
distance in the open conformation of the HSP72-NBD.
Typically, the electrophile in a targeted covalent inhibitor is
an N-arylacrylamide[14] but our irreversible inhibitor would
require an aliphatic electrophile, so we incorporated an
acrylate group to maintain reactivity (Scheme 1).
Figure 3. HSP72-NBD tool compounds and irreversible inhibitor assay
controls.
To establish whether our rationally designed targeted
covalent inhibitor 8 could form a reversible complex, its
affinity was assessed using a fluorescence polarization (FP)
assay to measure the inhibition of binding of a fluorescent
nucleotide-probe (Figure 4 and the Supporting Informa-
tion).[15] The initial Ki values for 8-N-benzyladenosine 1 and
the reversible matched pair 9 were 1.9 mm and 42 mm,
respectively.[16] The acetonide-protected irreversible 10 dis-
played no affinity, while 8 gave an apparent initial affinity of
17 mm. The time-dependence of the Ki value was then assessed
for each ligand, as irreversible inhibitors should show
apparent increasing affinity over time.[8] As expected, 8-N-
benzyladenosine 1 and the reversible matched pair 9 dis-
played no time-dependence but 8 displayed an increase in its
affinity over 46 h, consistent with covalent bond formation
(Figure 4). The putative HSP70 irreversible inhibitors, YK5 2
and oridonin 3, displayed no initial activity in this assay
(200 mm), reflecting their low potency as nucleotide-compet-
Two-carbon homologation of 3’,4’-acetonide adenosine 4
gave primary alcohol 5 in 51% yield over three steps.
Selective C8-bromination then gave the key intermediate 6,
which was treated with para-chlorobenzylamine to give 7 in
Figure 4. a) Targeted reversible inhibitor 9 shows no time-dependent
displacement of the FP-probe. b) Time-dependent inhibition of the
HSP72-NBD with targeted covalent inhibitor 8 assessed using dis-
placement of a nucleotide-derived FP-probe; each point is carried out
in triplicate, error bars show the arithmetic meanꢂSEM.
Figure 2. HSP72-NBD Cys17 irreversible inhibitor model generated
using MOE 2013.0801, only key residues are shown, solvent and
hydrogens omitted for clarity, carbon=grey, oxygen=red, nitro-
gen=blue, chlorine=green, sulfur-=yellow.
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itive targeted reversible inhibitors. YK5 2 also displayed no
time-dependence in the FP-assay (22 h), in contrast to
oridonin 3, which despite no initial affinity did show displace-
ment of the probe over time (16 h), consistent with activity as
a non-specific affinity label [Equation (3), see the Supporting
Information for details].
To ensure that the irreversible inhibition observed with 8
was not due to the compound acting as a non-specific affinity
label, the non-binding irreversible matched pair 10 and the
electrophile, O-methyl acrylate, were assayed over the same
extended time period but continued to display no activity.
However, even though the potent electrophile iodoacetamide
displayed no apparent reversible binding, it did displace the
FP-probe over time, presumably due to its greater reactivity
compared to O-methyl acrylate (see the Supporting Informa-
tion).[13]
Figure 6. a) Trypsin-digest MS/MS indicating modification of HSP72-
NBD with 8 at residue Cys267 (precursor ion 356.47563+, error
ꢁ0.84 ppm) b) Proposed schematic of fragmentation of 8 upon MS/
MS.
was observed and the MS2 spectrum confirmed the modifi-
cation present on buried Cys267, the residue proposed to be
the target of YK5 2 and oridonin 3.
To confirm the formation of a covalent bond, a solution of
8 (200 mm, ꢀ 12 ꢁ initial Ki) and HSP72-NBD (2.3 mm) in tris-
buffer were incubated together and the samples analyzed by
intact-protein mass-spectrometry (MS, Figure 5). The MS
The trypsin-digest MS/MS data appeared to contradict
our conclusion that the targeted covalent inhibitor 8 was
actually acting in a specific manner, as it was unclear how the
compound could react with Cys267 without a significant
conformational change that should greatly disrupt the small-
molecule binding site. Therefore, we generated a cysteine-to-
alanine mutant (C267A) of HSP72-NBD, which bound 8 with
a similar initial affinity to the wild-type (FP-assay, Ki = 16 mm,
see the Supporting Information). However, when this mixture
was analyzed by intact-protein MS, 8 still formed a covalent
adduct, eliminating Cys267 as the key nucleophilic residue.
The C306A and C17A HSP72-NBD mutants displayed
similar results (see the Supporting Information).
We speculated that because the trypsin-digest MS/MS
data was not quantified, our original analysis had identified
a minor adduct and that the modification of Cys267 only
occurs readily once the protein denatures.[13] As it was not
possible to analyze all the nucleophilic residues with trypsin-
digest MS/MS, we used X-ray crystallography in an attempt to
predict which nucleophilic residues were sufficiently proximal
to react with the electrophile, while conserving the 8-N-
benzyladenosine binding mode (Figure 7).[17]
Figure 5. Intact-protein MS trace for the time-dependent modification
of HSP72-NBD with 8, incubation was performed at 218C.
data revealed a time-dependent increase in a peak at
43630 Da (corresponding to HSP72-NBD + 490 Da), con-
sistent with covalent bond formation with 8, and a minor bis-
adduct of HSP72-NBD + 980 Da. Control compounds 9 and
10 displayed no significant modification over the same period.
YK5 2 displayed no significant modification in this assay and
oridonin 3 appeared to react with HSP72-NBD multiple times
(see the Supporting Information).
Despite significant efforts, no structure could be solved
that describes the covalent complex. However, two co-crystal
Although it was clear that 8 acts as a targeted covalent
inhibitor of HSP72, we had yet to confirm that Cys17 was the
nucleophilic residue responsible, as predicted (Figure 2). To
determine which reactive residue was forming the adduct, the
pre-incubated HSP72-NBD/8 mixture was subjected to tryp-
sin-digest MS/MS. Owing to the limitations of the MS
analysis, it was necessary to focus the second MS fragmenta-
tion only on the three cysteine residues (Figure 6 and the
Supporting Information). The MS1 and MS2 data unexpect-
edly revealed no evidence of Cys17 modification. However,
a mass consistent with modification of the peptide TAC267ER
Figure 7. Targeted covalent inhibitor 8 reversibly bound to HSP72-NBD
at pH 8.8 (PDB: 5MKS, 2.0 ꢀ), only key residues are shown, solvent
and hydrogens omitted for clarity, carbon=grey, oxygen=red, nitro-
gen=blue, chlorine=green.
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Figure 8. a) Extracted ion chromatogram of the MS1 from the trypsin digest of K56A HSP72-NBD with 8. Mass consistent with 8-modified 22mer,
Leu50–Lys71, z=charge-state. b) Intact-protein MS trace of K56A HSP72-NBD with 8 showing no significant time-dependent modification. c) FP
assay of K56A HSP72-NBD with 8 showing no time-dependent displacement of the fluorescent nucleotide probe.
structures were solved, which represent two different rever-
sible binding modes of the ligand in the open conformation of
HSP72-NBD.[9] In both modes, 8 demonstrated the expected
hydrogen-bonding array of the adenine and ribose moieties,
with the lipophilic para-chlorobenzylamine moiety parallel
with the two a-helices of the binding cleft.[4] Interestingly, the
acrylate electrophile was observed in two conformations,
dependent on the crystallization conditions. At pH 4.1, the
moiety points towards the front of the pocket, with the
flexible Tyr15 in an up-conformation (PDB: 5MKR, see the
Supporting Information). At pH 8.8, Tyr15 is in a down-
conformation with the acrylate moiety now visible on a vector
parallel with the base of the binding cleft (PDB: 5MKS,
Figure 7 and the Supporting Information). The terminal
portion of the acrylate itself could not be detected in the
electron density, reflecting a high mobility. The closest
potentially nucleophilic residue to the electrophilic warhead
described in this structure is Lys56 (3.8 ꢀ). To determine if
Lys56 was the nucleophilic residue responsible for the
irreversible inhibition,[18] we repeated the trypsin-digest MS/
MS assay and found an MS1 mass consistent with modifica-
tion of 22mer peptide Leu50–Lys71 (Figure 8). Unfortunately,
no reliable MS2 spectrum could be acquired to confirm the
site of modification within this peptide. Therefore, a K56A
HSP72-NBD mutant was used to confirm Lys56 as the key
nucleophilic residue. This mutant displayed comparable
apparent initial affinity for 8 (Ki = 11 mm)[18] but in contrast
to wild-type HSP72-NBD and the cysteine mutants, time-
dependence (24 h) was no longer observed in the FP-assay
and no significant modification was observed in the intact-
protein MS (Figure 8).
This study began as the rational design of an irreversible
inhibitor of HSP72 that would target Cys17 but ended with
the identification of Lys56 as the key reacting nucleophilic
residue. The involvement of non-catalytic lysine residues as
nucleophiles in covalent bond formation with targeted
covalent inhibitors is rare.[19] The discovery that Lys56,
which is involved in a crucial salt-bridge in HSP72,[9] can
undergo specific covalent bond formation with a validated
inhibitor, opens up a new approach for antagonizing this
challenging but important protein. We are currently exploring
the potential of this new strategy through the design and
synthesis of inhibitors that possess improved reversible
affinity for HSP72 and electrophiles that are better matched
to the lysine nucleophile.[19] Once we have inhibitors with an
acceptable profile, they will be tested in cellular assays to
increase our understanding of the role of HSP72 in oncology.
Acknowledgements
This work was funded by an ICR Chairmanꢂs Studentship
Award (JP) and Cancer Research UK grants (C309/A8274,
C309/A11566). We thank Dr. Nora Cronin for support during
crystallographic data collection, Meirion Richards for help
with intact-protein mass spectrometry, Dr. Thomas Matthews
for the synthesis of YK5 2, Dr. Fiona Jeganathan for help with
the FP assay, and Dr. Norhakim Yahya and Dr. Craig
McAndrew for help with protein preparation.
Conflict of interest
The authors declare no conflict of interest.
Keywords: fluorescence polarization · irreversible inhibitors ·
mass spectrometry · medicinal chemistry · structural biology
[1] F. U. Hartl, A. Bracher, M. Hayer-Hartl, Nature 2011, 475, 324 –
332.
[2] M. V. Powers, P. Workman, FEBS Lett. 2007, 581, 3758 – 3769.
[3] G. Jego, A. Hazoume, R. Seigneuric, C. Garrido, Cancer Lett.
2013, 332, 275 – 285.
[4] a) D. S. Williamson, et al., J. Med. Chem. 2009, 52, 1510 – 1513;
b) R. Schlecht, et al., PLoS One 2013, 8, e78443.
[5] A. J. Massey, J. Med. Chem. 2010, 53, 7280 – 7286.
[6] C. H. Yun, K. E. Mengwasser, A. V. Toms, M. S. Woo, H.
Greulich, K. K. Wong, M. Meyerson, M. J. Eck, Proc. Natl.
Acad. Sci. USA 2008, 105, 2070 – 2075.
[7] S. Wang, S. Cang, D. Liu, J. Hematol. Oncol. 2016, 9, 34.
[8] J. Singh, R. C. Petter, T. A. Baillie, A. Whitty, Nat. Rev. Drug
Discovery 2011, 10, 307 – 317.
[9] M. D. Cheeseman, et al., J. Med. Chem. 2016, 59, 4625 – 4636.
[10] A. Rodina, et al., Chem. Biol. 2013, 20, 1469 – 1480.
[11] F. Dal Piaz, R. Cotugno, L. Lepore, A. Vassallo, N. Malafronte,
G. Lauro, G. Bifulco, M. A. Belisario, N. De Tommasi, J.
Proteomics 2013, 82, 14 – 26.
[12] A. Q. Hassan, et al., Chem. Biol. 2015, 22, 87 – 97.
4
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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[13] Y. Miyata, J. N. Rauch, U. K. Jinwal, A. D. Thompson, S.
significant (t-test, p < 0.05). See the Supporting Information for
Srinivasan, C. A. Dickey, J. E. Gestwicki, Chem. Biol. 2012, 19,
1391 – 1399.
[14] M. E. Flanagan, et al., J. Med. Chem. 2014, 57, 10072 – 10079.
[15] L. E. Evans, M. D. Cheeseman, N. Yahya, K. Jones, PLoS One
2015, 10, e0140006.
[16] X. Y. Huang, J. Biomol. Screening 2003, 8, 34 – 38.
[17] Owing to fragmentation of targeted covalent inhibitor 8 follow-
ing formation of the covalent adduct the standard automated
analysis cannot identify this modification.
full analysis.
[19] a) E. Anscombe, et al., Chem. Biol. 2015, 22, 1159 – 1164; b) G.
AkÅay, et al., Nat. Chem. Biol. 2016, 12, 931 – 936; c) H. P.
Morgan, et al., Biochem. J. 2012, 448, 67 – 72; d) U. P. Dahal,
et al., MedChemComm 2016, 7, 864 – 872; e) A. Narayanan,
L. H. Jones, Chem. Sci. 2015, 6, 2650 – 2659.
[18] All Ki values quoted herein are the geometric mean of at least
three independent experiments. Comparisons are only inter-
preted when the differences in their respective pKi values are
Manuscript received: December 7, 2016
Revised: January 4, 2015
Final Article published: && &&, &&&&
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Communications
Medicinal Chemistry
Simply irreversible: The stress-inducible
molecular chaperone, HSP72, is an
important target in oncology but few
validated inhibitors have emerged. To
overcome the high affinity and abun-
dance of its endogenous substrates, an
irreversible nucleotide-competitive inhib-
itor, which unexpectedly targeted lysine-
56, was discovered by rational design.
J. Pettinger, Y.-V. Le Bihan, M. Widya,
R. L. M. van Montfort, K. Jones,
M. D. Cheeseman*
&&&& — &&&&
An Irreversible Inhibitor of HSP72 that
Unexpectedly Targets Lysine-56
6
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!