Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70

Article abstract of DOI:10.1021/acs.jmedchem.5b02001

HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.

Full text of DOI:10.1021/acs.jmedchem.5b02001

Article
pubs.acs.org/jmc
Exploiting Protein Conformational Change to Optimize Adenosine-
Derived Inhibitors of HSP70
Matthew D. Cheeseman,† Isaac M. Westwood,†,‡ Olivier Barbeau,† Martin Rowlands,† Sarah Dobson,†,‡
Alan M. Jones,† Fiona Jeganathan,† Rosemary Burke,† Nadia Kadi,† Paul Workman,† Ian Collins,†
Rob L. M. van Montfort,†,‡ and Keith Jones*,†
†Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SW7 3RP, U.K.
‡Division of Structural Biology, The Institute of Cancer Research, London SW7 3RP, U.K.
S
* Supporting Information
ABSTRACT: HSP70 is a molecular chaperone and a key
component of the heat-shock response. Because of its proposed
importance in oncology, this protein has become a popular target
for drug discovery, efforts which have as yet brought little success.
This study demonstrates that adenosine-derived HSP70 inhibitors
potentially bind to the protein with a novel mechanism of action,
the stabilization by desolvation of an intramolecular salt-bridge
which induces a conformational change in the protein, leading to
high affinity ligands. We also demonstrate that through the
application of this mechanism, adenosine-derived HSP70 inhib-
itors can be optimized in a rational manner.
proteins and the nucleotide exchange factor BAG family
molecular chaperone regulator 1 (BAG1) protein.6 While this
complexity presents numerous opportunities to antagonize the
refolding activity of HSP70, the clearest strategy remains ATP-
competitive binding of inhibitors to the conserved nucleotide-
binding domain of the protein. Unfortunately, this approach
has proven particularly challenging. There remains only one
published chemotype which displays ATP-competitive sub-
micromolar inhibition of HSP70 and has been shown to be
effective in cellular assays, a chemotype derived from adenosine
(Figure 1).7−10
INTRODUCTION
■
Heat-shock proteins are a highly conserved family of molecular
chaperones that facilitate the folding, stability, and cellular
localization of their substrate proteins.1 Up-regulation of the
pathways associated with the heat-shock response has been
implicated in a number of disease areas, including cancer.2
Recent focus has been on the inhibition of the molecular
chaperone heat-shock protein 90 (HSP90) using adenosine
triphosphate (ATP) competitive inhibitors, an approach that
has resulted in considerable success as several compounds have
now entered clinical trials.3 The heat-shock protein 70
(HSP70) family of molecular chaperones represents another
potential target for small-molecule mediated antagonism of the
heat-shock response pathway. The HSP70 isoform, heat-shock
cognate 70 (HSC70), is ubiquitously expressed in tissues, while
the inducible isoform, heat-shock protein 72 (HSP72), is
largely expressed in response to stress, including treatment with
HSP90 inhibitors, and aids cell survival through inhibition of
several apoptotic pathways.4 We have previously shown that
dual knockdown of these two HSP70 isoforms in human colon
and ovarian tumor cell lines results in apoptosis, which was in
contrast with nontumorigenic cell lines where apoptosis was
not observed, indicating a potential therapeutic window for
HSP70 inhibitors.5
The ATPase domain of HSP70 is a member of the actin
ATPase family of proteins, a target class which has delivered
very little success in the discovery of high affinity ligands.11
A
recent study12 to assess the potential of the HSP70-ATP
binding site for antagonism with small molecules using
SiteMap13 described the target as “difficult”,14 while a separate
analysis using a fragment-based screening approach returned a
very low hit rate (0.4%),12 a result generally associated with low
ligandability.15 Several studies into the biochemical mechanism
of HSP70 refolding activity and ATP hydrolysis have
demonstrated that the ATP binding site of HSP70 in solution
is highly flexible in nature, undergoing numerous conforma-
tional changes.16
With the challenge of finding ATP-competitive hit matter
against HSP70 hindering the potential development of
inhibitors for this important target, we sought to investigate
To execute their refolding activity, the HSP70 proteins utilize
the hydrolysis of ATP to adenosine diphosphate (ADP) and
inorganic phosphate (ADP/Pi) in a complex catalytic cycle
involving a number of protein conformational changes and
through a process which is tightly regulated by various
cochaperones such as the heat-shock protein 40 (HSP40)
Received: December 24, 2015
© XXXX American Chemical Society
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Figure 1. Adenosine-derived ATP-competitive inhibitors of HSP70. The affinity of three known HSP70 inhibitors derived from adenosine and
measured by SPR, see ref 7 for details.
the binding mechanism of adenosine-derived ligands to the
ATP site of HSP70. The aim was to improve our understanding
of how high affinity ligands bind to this region of the protein so
that this knowledge could be applied to future inhibitor design.
to adenosine 1. We decided to investigate this substituent in
order to better understand its role in the improved affinity of
these compounds (Table 1).
Table 1. 8- and 5′-Substituted Adenosine Based HSP70
RESULTS AND DISCUSSION
Ligands
■
Development of Toyocamycin Derived Ligands. The
slow turnover of ATP by HSP70, and the potent product
inhibition by ADP/Pi,17 means that using functional assays is a
challenge for the characterization of HSP70 ligand binding.
Therefore, we focused on surface plasmon resonance (SPR) as
a biophysical method to assess the affinity of ligands.
Unfortunately, full-length human HSP72 gave poor SPR data
in our hands, displaying erratic and difficult to interpret
sensorgrams. Therefore, the nucleotide-binding domain (NBD)
of human HSC70 (HSC70-NBD residues 1−381)18 was used
in all SPR experiments. Adenosine 1 is a relatively weak ligand
for HSC70-NBD, displaying a pKD= 3.95 0.01 (KD = 110
μM, n = 3),19 when measured by SPR, but we decided to use
this compound as a starting point for our investigations into the
binding mechanisms of this chemotype to the HSP70 proteins.
We began by analyzing the importance of the ribose motif to
the binding affinity of adenosine 1. Removing either the 2′- or
3′-hydroxyl groups20 from the sugar motif or changing their
relative and absolute stereochemistry resulted in no measurable
binding being observed with concentrations up to 1 mM (see
Supporting Information). Removal of either the 6-amino group
or the 3-nitrogen of the adenine ring also resulted in the loss of
all measurable affinity. These results demonstrate the
importance of the ribose motif and the adenine amino-
pyrimidine motif to binding of adenosine-derived ligands to the
hydrophilic region of the protein. In contrast, removal of the 5′-
hydroxyl was well tolerated, as compound 2 retained its affinity
in the SPR assay with a pKD = 3.88 0.02 (KD = 130 μM, n =
3) (Figure 2 and Supporting Information Table 1).21
a
All results are quoted as the geometric mean standard error of the
mean (SEM) of three independent experiments unless otherwise
b
stated, pKD = −log10(KD(μM) × 10−6). All values are quoted to 2
significant figures.
Methyl substitution on the 8-amino group (entry 2) gave
ligand 4 and resulted in a 2.5-fold drop in affinity,22 while
dimethyl analogue 5 (entry 3) displayed a KD greater than 1
mM. Replacement of the 8-amino substituent with a methoxy
group to give 6 (entry 4) also resulted in a complete loss of
activity. This dramatic effect on binding suggested that one of
the hydrogens of the 8-amino group was involved in hydrogen
bonding. To assess whether an intramolecular hydrogen bond
to the 5′-hydroxyl was important for affinity, we removed this
group to give 7 (entry 5). However, only a 1.6-fold drop in
affinity was observed with analogue 7 when compared with 8-
N-methylaminoadenosine 4 (entry 2). These results suggest
that an intramolecular hydrogen bond between the 8-amino
substituent and the 5′-hydroxyl group cannot rationalize the
increased affinity observed with the 8-aminoadenosine series.23
We speculated that the role of the 8-amino substituent as a
hydrogen bond donor is through interactions with the cyclic
ribose oxygen to stabilize a gauche conformation of adenosine,
which facilitates binding by reducing the energy barrier to the
initial binding event.24
It has previously been shown by Massey et al. that addition of
a primary or secondary amine to the 8-position of adenosine
resulted in a significant increase in affinity for these ligands.7 In
our hands, 8-aminoadenosine 3 gave a pKD = 5.16 0.01 (KD
= 7.0 μM, n = 3), a 16-fold increase in affinity when compared
Figure 2. 5′-Hydroxyl has little effect on affinity. The heteroatoms and
stereochemistry highlighted in red are important for affinity to HSP70
and could not be replaced by hydrogen or CH or the stereochemistry
changed.
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Finally, we sought to investigate the role of the imidazole
ring of adenosine to the binding of these ligands to HSP70
(Table 2).
Scheme 1. Natural Product Derived Ligands of HSP70
Table 2. Natural Product Nucleoside Derived HSP70
Ligands
methylsangivamycin 13 gave a pKD = 4.52 (KD = 30 μM, n =
1),29 which is a 7-fold drop in affinity compared to
sangivamycin 10 (Table 2, entry 3). We rationalized this
structure−activity relationship (SAR) through the potential
effect of the 7-substituent on the hydrogen bonding ability of
the key 8-N-methylamino group. The primary amide
substituent of 13 can form an intramolecular hydrogen bond
with the 8-N-methylamino group to give a resonance stabilized
six-membered intramolecular hydrogen bond,30 which would
mask the hydrogen bond donor effect of the 8-N-methylamino
substituent and block the binding of this ligand.
a
All compounds were purchased from the relevant commercial
b
suppliers and used without further purification. All results are quoted
as the geometric mean
SEM of three independent experiments
c
unless otherwise stated, pKD = −log10(KD(μM) × 10−6). All values
are quoted to 2 significant figures.
We screened three commercially available bacterial natural
products, all based on the replacement of the purine of
adenosine with a pyrrolopyrimidine scaffold, exchanging the
nitrogen at the 7-position25 with carbon.26 Comparing this
change in scaffold to adenosine 1 (entry 1): the more lipophilic
derivative tubercidin 8 (entry 2) displayed a 4-fold improve-
ment in affinity, substitution at the 7-postion was well tolerated,
with the nitrile derivative toyocamycin 9 (entry 3) displaying
comparable affinity to adenosine 1, and the primary amide
derivative sangivamycin 10 (entry 4) gave a 35-fold increase in
affinity at 3.3 μM.
With knowledge of the increased affinity observed with the
pyrrolopyrimidine scaffold in hand, we planned to combine the
scaffold hop with the improved affinity previously described for
8-amino substitution of the adenosine scaffold. 8-Amino
substitution of tubercidin proved synthetically intractable due
to the absence of an electron-withdrawing group at the 7-
position, making aromatic substitution at the 8-position
challenging. Therefore, we focused our efforts on the synthesis
of 8-aminotoyocamycin 12 and 8-aminosangivamycin 13
(Scheme 1).
Tribenzoyl intermediate 11 was prepared in four steps and
20% yield using a previously described procedure.27 Despite
repeated attempts, we were unable to introduce an ammonia
equivalent to the 8-position. However, we were successful using
methylamine as a nucleophile to give 8-N-methylaminotoyoca-
mycin 12, which underwent in situ deprotection of the benzoyl
groups under the reaction conditions. Treatment of inter-
mediate 11 with basic hydrogen peroxide resulted in hydrolysis
of the nitrile group, and subsequent addition of methylamine
gave 8-N-methylaminosangivamycin 13 in low yield and
moderate purity.28 8-N-Methylaminotoyocamycin 12 gave a
pKD = 5.47 0.02 (KD = 3.3 μM, n = 3) against HSC70-NBD.
This result represented a 27-fold improvement in activity
compared to toyocamycin 9 (Table 2, entry 1) and a 5-fold
increase in affinity when compared to the corresponding 8-N-
methyladenosine 12 (Table 1, entry 2). In contrast, 8-N-
To develop the toyocamycin scaffold further, we sought to
introduce a benzylic substituent to the 8-position amine.
Previously, Massey et al. have shown that N-benzyl substitution
at the 8-amino position could improve the affinity of adenosine
derived inhibitors of HSP70 (Figure 1).7 Because our
toyocamycin derived scaffold 12 had displayed a 5-fold
improvement in affinity compared to the corresponding
adenosine scaffold 4, we hypothesized that addition of an 8-
N-benzyl substituent to toyocamycin would result in an
improved chemotype for HSP70 inhibition. 8-N-Benzyltoyoca-
mycin 14 was prepared via a similar method to 12 using
benzylamine as a nucleophile. For comparison, 8-N-Benzyla-
denosine 15 was prepared in one step via a literature procedure
from commercially available 8-bromoadenosine (Scheme 2).31
8-N-Benzyladenosine derivative 15 gave a pKD = 5.84 0.02
(KD = 1.5 μM, n = 3), representing a 12-fold improvement in
binding affinity compared to the 8-N-methyladenosine
analogue 4 (Table 1, entry 2); however, the 8-N-benzyltoyo-
camycin analogue 14 gave a pKD = 5.56 0.02 (KD = 2.8 μM, n
= 3), representing little change in affinity compared to 8-N-
methyltoyocamycin derivative 12 despite the increase in
molecular weight and lipophilicity. Even though toyocamycin
9 was apparently an improved scaffold, when compared to
adenosine, for the inhibition of HSP70, we had been unable to
develop the compounds beyond a micromolar affinity ligand.
We believed that improving our understanding of the complex
SAR surrounding the nucleoside core and lipophilic 8-N-benzyl
substituent through analysis of ligand/HSP70 co-crystal
structures would be crucial to the discovery and development
of small-molecule inhibitors of HSP70.
HSP70 Conformations. In 1995, kinetic studies by McKay
and Ha using changes in tryptophan fluorescence indicated that
the HSP70 isoform, HSC70, undergoes a number of conforma-
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Scheme 2. 8-N-Benzyl Derivatives of Adenosine Analogues
Figure 3. Crystal Structure of ADP/Pi bound to HSP72. PDB 3ATU,
important hydrogen bonding interactions, with their distances in Å,
and residues are indicated. The key salt-bridge interaction between
lysine-56 and glutamic acid-268 was measured at 2.8 Å (2 SF). Orange
and turquoise spheres represent sodium and magnesium ions,
respectively. Only selected residues are shown and solvent has been
omitted for clarity.
tional changes during its catalytic cycle.32 The energy released
from ATP hydrolysis drives subsequent conformational changes
in the substrate binding domain, allowing HSP70 to carry out
its refolding function on client proteins. However, McKay and
Ha discovered that ATP and ADP have two distinct binding
mechanisms. While ADP binds and then dissociates in an
apparent single-step mechanism with slow-off kinetics, the
binding of ATP is a two-step process, requiring an additional
conformational change of the nucleotide-binding domain prior
to ATP hydrolysis. Because it had been demonstrated that the
two nucleotide substrates of HSP70 can jump between binding
mechanisms, we hypothesized that the complexity surrounding
the apparent SAR of the nucleoside derived HSP70 inhibitors
was due to a change in mechanism between a one-step and an
induced conformational change two-step mechanism, even if
kinetically this would be difficult to observe.33
To investigate whether a two-step binding model is
consistent with the observed nucleoside SAR, we decided to
probe HSP70 ligand binding by X-ray crystallography. There is
currently no crystal structure of full-length human HSP70;
therefore, we focused our crystallography efforts on the NBD of
the human HSP70 isoform HSP72 (HSP72-NBD).
In our hands, ADP 16 gave an affinity of pKD = 6.49 (KD =
0.32 μM, n = 1) when measured by SPR against the HSC70-
NBD.34 The co-crystal structure of ADP/Pi bound to the
HSP72-NBD has previously been solved16a and analysis of this
structure clearly revealed a closed conformation of the protein.
The two α-helices flanking the sides of the ATP binding site,
closed around the adenosine motif of ADP, forming multiple
hydrogen bonds with the ribose and adenine moieties of ADP
(Figure 3).
From analysis of this structure, the two phosphate groups of
ADP form multiple hydrogen bonds within the phosphate
binding region of the HSP72-NBD. It is also clear that the
HSP72-NBD would need to undergo a conformational change
in order for ADP/Pi to dissociate, resulting in the previously
described slow-off kinetics.32 It has been suggested previously
that the closed conformation is apparently stabilized by the
formation of a solvent exposed salt-bridge between glutamic
acid-268 and lysine-56.35 Intrigued by the formation of this
intramolecular interaction, we then sought to generate a co-
crystal structure with the bacterial natural product sangivamycin
10 (KD = 3.3 μM) (Figure 4).
Figure 4. Co-crystal structure of sangivamycin 10 bound to HSP72.
PDB 5AQZ, key hydrogen bonding interactions and their distances in
Å are indicated. The key salt-bridge interaction between lysine-56 and
glutamic acid-268 is absent. Only selected residues are shown and
solvent has been omitted for clarity.
The crystal structure of sangivamycin 10 revealed a similar
hydrogen bonding framework to the adenosine motif of the
ADP/Pi structure (Figure 3). The pyrrolopyrimidine ring
interacts with serine-275 and the 2′- and 3′-hydroxyls of the
ribose interact with lysine-271. However, in contrast to the
ADP/Pi HSP72-NBD structure, sangivamycin 10 co-crystal-
lized in a more open conformation. The two α-helices of the
nucleotide binding domain are no longer in close proximity,
and the solvent exposed salt-bridge between glutamic acid-268
and lysine-56 is absent. It is this difference in the HSP70-NBD
conformation which we hypothesize is an important factor in
the complexity of SAR observed for nucleoside-derived
inhibitors. The binding of both ADP/Pi and sangivamycin 10
are dominated by the formation of multiple hydrogen bonds to
key residues in the nucleoside binding cleft but the crucial
difference between the two ligands is their ability to induce and
stabilize the closed conformation of the HSP72-NBD. It is this
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ligand-driven induced conformational change that leads to high
affinity for HSP70. The HSP72-NBD closed conformation
observed in the ADP/Pi bound structure is presumably brought
about by the interactions of the β-phosphate group with the
two glycine-rich loops of the phosphate binding region. We
speculated that this conformational change could force water
molecules from the nucleotide binding domain cleft,
strengthening the many hydrogen bond contacts surrounding
the adenosine due to the more hydrophobic environment. The
absence of the phosphate groups, and in particular the β-
phosphate group, in sangivamycin 10 means there is no
induced conformational change so the affinity is only
dependent on the multiple hydrogen bonds (Figure 5).
Scheme 3. Synthesis of Quinoline Adenosine Derivative
Although several 8-N-substituted HSP70 inhibitors have
been described in the literature,7 the 8-N-quinoline adenosine
derived ligand 17 was chosen because it was reported to be the
highest affinity ligand which was only substituted at the 8-
position. In our hands, 17 gave a pKD = 6.14 0.01 (KD = 0.72
μM, n = 3) against HSC70-NBD when measured by SPR,
which was consistent with the data reported in the literature.37
8-N-Quinoline aminoadenosine derived ligand 17 was then
submitted to our co-crystallization protocol with HSP72-NBD
(Figure 6).
Figure 5. Induced open and induced closed conformations of HSP72.
PDB 3ATU and 5AQZ, the copper-colored structure represents the
co-crystal structure of ADP/Pi bound to the HSP72-NBD in the
induced closed conformation due to interactions of the phosphate
groups through hydrogen bonds with the glycine rich loops and
stabilized by the salt-bridge. The gray-colored structure represents the
co-crystal structure of sangivamycin 10 bound to the HSP72-NBD.
The overlay shows sangivamycin 10 bound to a more open NBD
conformation, in which the formation of the Glu-Lys salt-bridge is not
possible. Only selected residues are shown and solvent has been
omitted for clarity.
Figure 6. Co-crystallization of 8-N-quinoline aminoadenosine derived
ligand 17 bound to HSP72. PDB 5AR0, the HSP72 structure clearly
demonstrates the formation the key salt-bridge when co-crystallized
with 8-N-quinolineadenosine 17. Important hydrogen bonding
interactions are indicated with their distances in Å. Only selected
residues are shown and solvent has been omitted for clarity.
An Alternative Approach to Inducing the Closed
Conformation of HSP70. Mimicking phosphate groups with
druglike ligands represents a significant challenge in medicinal
chemistry.36 However, if it were possible to induce the closed
conformation of the HSP70 nucleotide binding domain via an
alternative mechanism, then mimicking the β-phosphate would
be unnecessary. Because the solvent exposed salt-bridge
between glutamic acid-268 and lysine-56 was the only
additional enthalpic intradomain interaction that we observed
in the HSP70-NBD closed conformation structure, when
compared with the more open sangivamycin 10 structure, we
hypothesized that a ligand stabilizing this interaction would
stabilize the closed conformation of HSP70, leading to
increased affinity. We therefore proposed that the previously
described potent 8-N-benzyladenosine derived ligands were
able induce a conformational change in HSP70 but via a
lipophilic mechanism, which would be more amenable to drug
discovery. To investigate this hypothesis, we synthesized the
known quinoline derived HSP70 inhibitor 17 in one step and
68% yield using our previously described method (Scheme 3).7
Co-crystallization of 17 with the HSP72-NBD showed the
NBD in a closed conformation (Figure 5), nearly identical to
the conformation of the HSP72-NBD in the ADP/Pi 16-bound
structures (Figure 6). To our knowledge, this is the first
example of a co-crystal structure demonstrating a non-
nucleotide ligand binding to the closed conformation of
HSP70. The similarity of the respective HSP72 conformations
is consistent with a potential role of the induced conformational
change in enhancing the affinity of the 8-N-benzyl nucleoside
derived HSP70 ligands. However, the mechanism by which the
inhibitor induced conformational change occurs must be
distinctly different from the mechanism of NBD closure upon
nucleotide binding because the quinoline moiety of 17 forms
no interactions within the phosphate binding region.
To rationalize the induced conformational change observed
with 17, we further analyzed the closed structure of HSP72-
NBD to identify the key binding interactions. 8-N-Quinoline
adenosine 17 displays a similar hydrogen bonding network with
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the same key residues observed in both ADP/Pi and
sangivamycin 10, with an additional solvent exposed water
molecule bound to the quinoline nitrogen. However, in
contrast with the more open conformation observed in the
HSP72-NBD sangivamycin 10 complex, the key salt-bridge
between glutamic acid-268 and lysine-56, which is present in
the nucleotide-bound HSP70-NBD structures, is clearly formed
in the 17-bound HSP72-NBD structure (Figure 5). In the
ADP/Pi/ HSC70-NBD co-crystal structure, the salt-bridge is
solvent exposed, weakening its effect (Figure 7).38 By contrast,
Table 3. 8-Position Optimization of Adenosine Derived
HSP70 Ligands
a
All results are quoted as the geometric mean
SEM of three
independent experiments unless otherwise stated, pKD
=
b
−log10(KD(μM) × 10−6). All values are quoted to 2 significant
c
figures. The geometric mean of n = 9 experiments.
SPR against HSC70-NBD. The weaker activity observed with
the benzyl group 15 compared to quinoline 17 we rationalized
was due to its less efficient desolvation of the glutamic acid/
lysine salt bridge by the smaller lipophilic group. Therefore, we
decided to add a number of lipophilic substituents to assess
whether we could improve the desolvation effect, promote the
induced closed conformation, and improve the affinity of the
ligands. para-Chloro-substitution gave adenosine derivative 18
(entry 3) with a pKD = 6.55 0.01 (KD = 0.28 μM, n = 9), a 5-
fold improvement in affinity compared to compound 15 and a
60-fold improvement compared to 8-N-methylaminoadenosine
4 (Table 1, entry 2). Although the kinetics of the these ligands
binding to HSP70 were at the limit of what could be accurately
measured by SPR, analysis of the off-rate did reveal that the
quinoline ligand 17 and the para-chlorobenzyl derivative 18
possessed measurably slow off-rates compared to sangivamycin
10, whose half-life was too short to be observed with this
technique (see Supporting Information for details).41 Similar
improvements were also observed for the para-fluoro derivative
19 (entry 4, pKD = 6.34 0.01, KD = 0.46 μM, n = 3) and para-
methyl derivative 20 (entry 5, pKD = 6.53 0.01, KD = 0.30
μM, n = 3). Because the crystal structure of this ligand class
shows that the benzylic moiety resides in the cleft formed by
the two α-helices in the nucleotide binding domain of HSP70,
it is unlikely that the para-lipophilic substituents interact with
HSP70 via a lipophilic pocket, as the group is essentially solvent
exposed. This effect could not be explained by an increase in
the overall lipophilicity of the ligand to exploit the nonspecific
hydrophobic effect because dichlorobenzyl derivative 21 (entry
Figure 7. Overlay of ADP/Pi and 8-N-quinoline adenosine 17 co-
crystal structures with HSP72. PDB 3ATU and 5AR0, overlay of the
ADP/Pi-HSP72 co-crystal structure (copper) and the 8-N-quinoline
adenosine 17-HSP72 co-crystal structure (gray). Only selected
residues are shown and solvent has been omitted for clarity.
in the 17-bound HSP72-NBD co-crystal structure, the
quinoline moiety is able to form a π-stack with arginine-272,
although this interaction is solvent exposed so is only likely to
be weak;39 the result is to place the quinoline group directly in
front of the salt-bridge. Because the quinoline is highly
lipophilic, this creates a more lipophilic environment
surrounding the salt-bridge, protecting it from water and
strengthening the interaction, which leads to increased affinity
for 8-N-benzyl aminonucleoside derived ligands.40 We propose
the binding mechanism of these ligands to HSP70 is analogous
to a “door and latch”. The initial binding event is similar for all
nucleoside derived ligands of HSP70 and is dominated by
hydrogen bonds to serine-275 and lysine-271. The nucleotide
binding domain is then able to close around the ligand but for
this process to be favorable, when balanced by the entropy cost
of restricting the conformational freedom of the protein, it must
be stabilized by the ligand. ADP can achieve this through
interactions with the phosphate binding region, while quinoline
ligand 17 stabilizes the key salt-bridge through hydrophobic
desolvation. Sangivamycin 10 has neither of these substituents
so predominately binds to the open conformation.
6, pKD = 5.45
0.01, KD = 3.5 μM, n = 3) displayed a
significant drop in affinity.
CONCLUSIONS
■
Optimizing the 8-Position. To test this hypothesis, and to
generate more active ligands of HSP70 for further develop-
ment, we synthesized a series of 8-N-benzylaminoadenosine
analogues using our previously described method (Table 3).
The 8-N-benzyl derivative 15, as shown previously, gave a
pKD = 5.84 0.02 (KD = 1.4 μM, n = 3) when measured by
The ATP binding site of HSP70 is a challenging region of the
protein to target with small molecules due to its hydrophilic
nature and high flexibility. To target the nucleotide binding
domain it is important to understand the conformational
changes that this region of the protein undergoes. Using
protein/ligand X-ray crystallography, we have demonstrated
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3.03 (s, 6H); δC (126 MHz, MeOD) 157.19, 153.92, 149.92, 149.22,
116.86, 89.06, 86.97, 71.96, 71.79, 62.86, 41.89. HRMS (ESI)
C12H19N6O4 (M + H+) requires 311.1462, found 311.1464.
that non-nucleotide ligands of HSP70 can induce conforma-
tional changes in the protein and that these changes can play an
important role in the binding of HSP70 inhibitors. In solution,
kinetic studies suggest that this protein undergoes a number of
conformational changes of not just the nucleotide binding
domain but also the substrate-binding domain.32 Also,
interactions between these two domains and the role of
cochaperones in these conformational changes have yet to be
addressed. Better understanding of the flexibility of HSP70 and
its effect on the affinity of ligands will contribute to better assay
design and more efficient inhibitor optimization.
(2R,3R,4S,5R)-2-(6-Amino-8-methoxy-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol 6. To a solution of 8-
bromoadenosine (0.11 g, 0.33 mmol) in MeOH (4 mL) was added
NaOMe (0.18 g, 3.3 mmol), and the mixture was stirred at room
temperature for ∼12 h. After this time, the solvent was removed under
reduced pressure and the resulting residue was purified by silica gel
chromatography eluting with 2 M MeOH/NH3:EtOAc (9:1) to give
the desired product as a yellow oil (0.025 g, 26%). δH (500 MHz,
MeOD), 8.06 (s, 1H), 5.91 (d, J = 7.1 Hz, 1H), 4.93 (dd, J = 7.0, 5.2
Hz, 1H), 4.33 (dd, J = 5.2, 2.0 Hz, 1H), 4.21 (s, 3H), 4.16−4.09 (m,
1H), 3.86 (dd, J = 12.6, 2.5 Hz, 1H), 3.71 (dd, J = 12.6, 2.9 Hz, 1H);
δC (126 MHz, MeOD) 155.19, 154.01, 150.10, 148.34, 115.35, 87.65,
86.82, 72.43, 71.55, 62.62, 56.69. HRMS (ESI) C11H16N5O5 (M + H+)
requires 298.1146, found 298.1152.
(2R,3R,4S,5R)-2-(6-Amino-8-(methylamino)-9H-purin-9-yl)-5-
methyltetrahydrofuran-3,4-diol 7. To a solution of 5′-deoxyadeno-
sine (0.024 g, 0.096 mmol) in dioxane (0.48 mL) and water (0.48 mL)
was added KHPO4 (0.065 g, 0.29 mmol) and bromine (0.023 g, 0.14
mmol) as a solution in water (0.50 mL), and the mixture was stirred
for 15 min. The mixture was then quenched satd sodium thiosulfate
solution (5.0 mL), extracted with EtOAc, dried (MgSO4), and the
solvent removed under reduced pressure. The resulting residue was
used in the next step without further purification.
The product from the previous step (0.032 g, 0.097 mmol) was
dissolved in 2 M MeNH2 in EtOH (1.9 mL), and the mixture was
heated to 70 °C for ∼12 h. After this time, the mixture was cooled to
room temperature and the solvent removed under reduced pressure.
The resulting residue was purified by silica gel chromatography eluting
with 2 M MeOH/NH3:EtOAc (1:9) to give the desired product as a
white solid (0.009 g, 33% over two steps); δH (500 MHz, DMSO-d6)
7.90 (s, 1H), 6.75 (q, J = 4.5 Hz, 1H), 6.45 (s, 2H), 5.58 (d, J = 4.2
Hz, 1H), 5.23 (d, J = 5.0 Hz, 1H), 5.09 (q, J = 5.0 Hz, 1H), 4.99 (d, J
= 5.7 Hz, 1H), 4.14−4.04 (m, 1H), 3.83 (app p, J = 6.2 Hz, 1H), 2.88
(d, J = 4.5 Hz, 3H), 1.24 (d, J = 6.3 Hz, 3H); δC (126 MHz, DMSO-
d6) 152.97, 152.91, 150.19, 149.12, 117.95, 88.07, 79.10, 74.87, 71.05,
29.73, 18.96. HRMS (ESI) C11H17N6O3 (M + H+) requires 281.1357,
found 281.1354.
EXPERIMENTAL SECTION
■
Experimental Procedures (Chemistry). Unless otherwise stated,
reactions were conducted in oven-dried glassware under an
atmosphere of nitrogen using anhydrous solvents. All commercially
obtained reagents and solvents were used as received. Thin layer
chromatography (TLC) was performed on precoated aluminum sheets
of silica (60 F254 nm, Merck) and visualized using short-wave UV
light. Flash column chromatography was carried out on Merck silica
1
gel 60 (partial size 40−65 μm). H NMR spectra were recorded on
Bruker AMX500 (500 MHz) spectrometers using an internal
deuterium lock. Chemical shifts are quoted in parts per million
(ppm) using the following internal references: CDCl3 (δH 7.26),
MeOD (δH 3.31), and DMSO-d6 (δH 2.50). Signal multiplicities are
recorded as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), doublet of doublets (dd), doublet of doublet of doublets (ddd),
apparent triplet (app t) broad (br), or obscured multiplet (obs m).
Coupling constants, J, are measured to the nearest 0.1 Hz. 13C NMR
spectra was recorded on Bruker AMX500 spectrometers at 126 MHz
using an internal deuterium lock. Chemical shifts are quoted to 0.01
ppm, unless greater accuracy was required, using the following internal
references: CDCl3 (δC 77.0), MeOD (δC 49.0), and DMSO-d6 (δC
39.5). High resolution mass spectra were recorded an Agilent 1200
series HPLC and diode array detector coupled to a 6210 time-of-flight
mass spectrometer with dual multimode APCI/ESI source. Analytical
separation was carried out on a Merck Purospher STAR RP-18, 30
mm × 4 mm column using a flow rate of 1.5 mL/min in a 4 min
gradient elution, UV detection was at 254 nm. All compounds were
>95% purity by HPLC analysis unless otherwise stated.
(2R,3R,4S,5R)-2-(6-Amino-8-(methylamino)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol 4. To a solution of 8-
bromoadenosine (0.051 g, 0.15 mmol) in EtOH (1.5 mL) was added
methylamine solution (33% solution in EtOH, 0.25 mL, 3.0 mmol),
and the mixture was heated to 80 °C for ∼12 h. After this time, the
mixture was cooled to room temperature and the solvent removed
under reduced pressure. The resulting residue was purified by silica gel
chromatography eluting with 2 M MeOH/NH3:EtOAc (8:2) to give
the desired compound as a white solid (0.031 g 71%); δH (500 MHz,
DMSO-d6) 7.90 (s, 1H), 6.95 (d, J = 4.8 Hz, 1H), 6.57 (s, 2H), 5.92
(dd, J = 6.0, 4.0 Hz, 1H), 5.86 (d, J = 7.3 Hz, 1H), 5.26 (d, J = 6.7 Hz,
1H), 5.16 (d, J = 4.0 Hz, 1H), 4.68 (td, J = 7.0, 5.3 Hz, 1H), 4.12 (ddd,
J = 5.6, 4.1, 2.1 Hz, 1H), 3.97 (d, J = 2.3 Hz, 1H), 3.70−3.58 (m, 2H),
2.89 (d, J = 4.5 Hz, 3H); δC (126 MHz, DMSO-d6) 152.86, 152.52,
150.28, 148.89, 117.64, 86.99, 86.13, 71.42, 71.20, 62.15, 29.59. HRMS
(ESI) C11H17N6O4 (M + H+) requires 297.1306, found 297.1299.
(2R,3R,4S,5R)-2-(6-Amino-8-(dimethylamino)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol 5. To a solution of 8-
bromoadenosine (0.064 g, 0.19 mmol) in EtOH (1.9 mL) was added
dimethylamine (40% solution in water, 0.42 mL, 3.70 mmol), and the
mixture was heated to 80 °C for ∼12 h. After this time, the mixture
was cooled to room temperature and the solvent removed under
reduced pressure. The resulting residue was purified by silica gel
chromatography eluting with 2 M MeOH/NH3:EtOAc (9:1) to give
the desired product as a white solid (0.027 g, 47%); δH (500 MHz,
MeOH) 8.05 (d, J = 1.1 Hz, 1H), 5.91 (d, J = 7.5 Hz, 1H), 5.16 (dd, J
= 7.5, 5.3 Hz, 1H), 4.36 (dd, J = 5.2, 1.4 Hz, 1H), 4.14 (d, J = 1.9 Hz,
1H), 3.86 (dd, J = 12.6, 2.3 Hz, 1H), 3.73 (dd, J = 12.6, 2.7 Hz, 1H),
(2R,3R,4R,5R)-2-(4-Amino-6-bromo-5-cyano-7H-pyrrolo[2,3-d]-
pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl Di-
benzoate 11. Prepared via a previously described procedure in four
steps and 20% overall yield. The spectroscopic data matched that
previously described.42
4-Amino-7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-6-(methylamino)-7H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile 12. (2R,3R,4R,5R)-2-(4-Amino-6-bromo-5-
cyano-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)-
tetrahydrofuran-3,4-diyl dibenzoate 11 (120 mg, 0.17 mmol) was
dissolved in 2 M methylamine in ethanol (5.0 mL), and the reaction
was heated in a microwave reactor at 140 °C for 1 h. After this time,
the mixture was cooled to room temperature and the solvent removed
under reduced pressure. The resulting residue was purified by silica gel
chromatography with the Biotage SP1 purification system (column,
10+S; flow rate, 15 mL/min; gradient starting with 100% DCM from 0
to 1 CV then from 100% DCM to 2% NH4OH/18% MeOH/80%
DCM from 1 CV to 21 CV) to give the desired compound as a white
solid (0.045 g, 80% yield); δH (500 MHz, MeOD) 3.22 (s, 3H), 3.83−
3.84 (m, 2H), 4.14 (q, J = 1.9 Hz, 1H), 4.27 (dd, J = 5.6, 2.0 Hz, 1H),
4.65 (dd, J = 7.6, 5.6 Hz, 1H), 6.29 (d, J = 7.9 Hz, 1H), 8.01 (s, 1H);
δC (126 MHz, MeOD) 29.55, 56.92, 61.31, 70.91, 71.19, 86.12, 87.35,
101.19, 118.75, 148.27, 149.87, 151.26, 154.16. HRMS (ESI)
C13H17N6O4 (M + H+) requires 321.1306, found 321.1303.
4-Amino-7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-6-(methylamino)-7H-pyrrolo[2,3-d]-
pyrimidine-5-carboxamide 13. To a solution of (2R,3R,4R,5R)-2-(4-
amino-6-bromo-5-cyano-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-
((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl dibenzoate 11 (87 mg,
0.13 mmol) in THF (1.0 mL) was added hydrogen peroxide (33%, 1.0
mL) and 1 M NaOH (1.0 mL). The reaction was stirred at room
G
DOI: 10.1021/acs.jmedchem.5b02001
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
temperature for ∼18 h before 1 M HCl was added until the mixture
reached pH 6, when the solvent was removed under reduced pressure.
The resulting residue was dissolved in 2 M methylamine in ethanol
(5.0 mL), and the reaction was heated to 120 °C in a microwave
reactor for 50 min. After this time, the mixture was cooled to room
temperature and the solvent removed under reduced pressure. The
resulting residue was purified by silica gel chromatography using the
Biotage SP1 purification system (column, 10+S; flow rate, 15 mL/min;
gradient starting with 100% DCM from 0 to 1 CV then from 100%
DCM to 2% NH4OH/18% MeOH/80% DCM from 1 CV to 21 CV)
to give the desired product as a yellow solid (0.009 g, 10% yield); δH
(500 MHz, MeOD) 2.89 (s, 3H), 3.78 (dd, J = 12.2, 1.9 Hz, 1H), 3.86
(dd, J = 12.2, 2.2 Hz, 1H), 4.18 (q, J = 1.7 Hz, 1H), 4.33 (dd, J = 5.5,
1.3 Hz, 1H), 4.85 (m, 1H), 6.12 (d, J = 7.9 Hz, 1H), 8.02 (s, 1H); δC
(126 MHz, MeOD) 35.35, 62.23, 71.62, 72.28, 86.70, 87.49, 98.51,
101.02, 146.72, 147.31, 150.61, 157.30, 167.61. HRMS (ESI)
C13H19N6O5 (M + H+) requires 339.1411, found 339.1408.
4-Amino-6-(benzylamino)-7-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile 14. (2R,3R,4R,5R)-2-(4-Amino-6-bromo-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)-
tetrahydrofuran-3,4-diyl dibenzoate 11 (31 mg, 0.045 mmol) was
dissolved in EtOH (2.0 mL), and benzylamine (29 mg, 0.27 mmol)
was added to the solution. The reaction was heated in a microwave
reactor at 160 °C for 1 h. After this time, the mixture was cooled to
room temperature and the solvent removed under reduced pressure.
The resulting residue was dissolved in methanol (0.5 mL), and 0.5 M
sodium methoxide (1.0 mL) was added. The reaction was stirred at
room temperature for 1 h, then the solvent was removed under
reduced pressure. The resulting residue was purified by silica gel
chromatography with the Biotage SP1 purification system (column,
10+S; flow rate, 15 mL/min; gradient starting with 100% DCM from 0
to 1 CV then from 100% DCM to 2% NH4OH/18% MeOH/80%
DCM from 1 CV to 21 CV) to give the desired compound as a white
solid (0.007 g, 39% yield); δH (500 MHz, MeOD) 3.80−3.86 (m,
2H), 4.18 (q, J = 1.8 Hz, 1H), 4.28 (dd, J = 5.6, 1.7 Hz, 1H), 4.73 (dd,
J = 7.8, 5.7 Hz, 1H), 4.78 (d, J = 10.3 Hz, 2H), 6.37 (d, J = 7.9 Hz,
1H), 7.25−7.27 (m, 1H), 7.33−7.36 (m, 2H), 7.41−7.43 (m, 2H),
8.01 (s, 1H); δC (126 MHz, MeOD) 46.28, 61.40, 71.14, 71.29, 86.27,
86.81, 87.38, 101.06, 118.30, 126.72, 126.98, 128.25, 138.41, 148.25,
149.94, 150.10, 154.22. HRMS (ESI) C19H21N6O4 (M + H+) requires
397.1619, found 397.1614.
8.02 (s, 1H), 7.95 (d, J = 1.8 Hz, 1H), 7.86 (dd, J = 8.8, 2.0 Hz, 1H),
7.54 (dd, J = 8.3, 4.3 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.92−4.83
(obs m, 3H), 4.32 (dd, J = 5.4, 1.7 Hz, 1H), 4.21 (q, J = 1.9 Hz, 1H),
3.87 (dd, J = 12.0, 2.3 Hz, 1H), 3.80 (dd, J = 11.9, 1.7 Hz, 1H); δC
(126 MHz, MeOD) 151.98, 151.94, 149.72, 149.52, 148.44, 146.69,
137.89, 136.94, 129.30, 128.46, 127.90, 125.07, 121.35, 116.59, 87.38,
86.47, 71.70, 71.53, 61.75, 45.29. HRMS (ESI) C20H22N7O4 (M + H+)
requires 424.1728, found 424.1725.
(2R,3R,4S,5R)-2-(6-Amino-8-((4-chlorobenzyl)amino)-9H-purin-9-
yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 18. To a solution of
8-bromoadenosine (0.078 g, 0.23 mmol) in n-butanol (2.3 mL) was
added iPr2NEt (0.12 mL, 0.68 mmol) and (4-chlorophenyl)-
methanamine (0.055 mL, 0.45 mmol), and the mixture was heated
to 120 °C for ∼12 h. After this time, the mixture was cooled to room
temperature and the resulting residue was purified by silica gel
chromatography eluting with EtOAc:MeOH (95:5) to give the desired
product as a white solid (0.033 g, 36%); δH (500 MHz, MeOD) 8.00
(s, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 6.10 (d, J =
7.5 Hz, 1H), 4.79 (dd, J = 7.6, 5.4 Hz, 1H), 4.66 (d, J = 15.9 Hz, 1H),
4.62 (d, J = 16.0 Hz, 1H), 4.30 (dd, J = 5.4, 1.7 Hz, 1H), 4.18 (q, J =
1.9 Hz, 1H), 3.85 (dd, J = 11.9, 2.3 Hz, 1H), 3.79 (dd, J = 12.0, 1.7 Hz,
1H); δC (126 MHz, MeOD) 152.19, 151.83, 149.68, 148.72, 137.67,
132.42, 128.22, 128.16, 116.54, 87.30, 86.40, 71.65, 71.50, 61.73,
44.80. HRMS (ESI) C17H20ClN6O4 (M+H+) requires 407.1229, found
407.1237.
(2R,3R,4S,5R)-2-(6-Amino-8-((4-fluorobenzyl)amino)-9H-purin-9-
yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 19. To a solution of
8-bromoadenosine (0.048 g, 0.14 mmol) in n-butanol (1.4 mL) was
added (4-fluorophenyl)methanamine (0.032 mL, 0.28 mmol) and
iPr2NEt (0.073 mL, 0.42 mmol), and the mixture was heated to 120
°C for ∼12 h. After this time, the mixture was cooled to room
temperature and the solvent removed under reduced pressure. The
resulting residue was purified by silica gel chromatography eluting with
EtOAc:MeOH (9:1) to give the desired product as a white solid
(0.043 g, 79%); δH (500 MHz, MeOD) 7.99 (s, 1H), 7.50 (dd, J = 8.3,
5.3 Hz, 1H), 7.44−7.38 (m, 2H), 7.23−7.16 (m, 1H), 7.09−7.04 (m,
2H), 6.09 (d, J = 7.6 Hz, 1H), 4.78 (dd, J = 7.6, 5.4 Hz, 1H), 4.65 (d, J
= 15.7 Hz, 1H), 4.60 (d, J = 15.7 Hz, 1H), 4.29 (dd, J = 5.4, 1.6 Hz,
1H), 4.17 (q, J = 1.9 Hz, 1H), 3.83 (dd, J = 12.0, 2.2 Hz, 1H), 3.78
(dd, J = 11.9, 1.7 Hz, 1H); δC (126 MHz, MeOD) 164.14, 163.00,
161.06, 152.14, 151.85, 149.66, 148.65, 134.75, 134.72, 130.99, 130.92,
128.53, 128.46, 116.55, 115.67, 115.50, 114.79, 114.62, 87.26, 86.41,
71.60, 71.50, 61.72, 44.79.43 HRMS (ESI) C17H20FN6O4 (M + H+)
requires 391.1525, found 391.1517.
(2R,3R,4S,5R)-2-(6-Amino-8-(benzylamino)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol 15. To a solution of 8-
bromoadenosine (0.061 g, 0.18 mmol) in n-butanol (1.76 mL) was
(2R,3R,4S,5R)-2-(6-Amino-8-((4-methylbenzyl)amino)-9H-purin-
9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 20. To a solution
of 8-bromoadenosine (0.062 g, 0.18 mmol) in n-butanol (1.79 mL)
i
added Pr2NEt (0.092 mL, 0.53 mmol) and benzylamine (0.038 mL,
0.35 mmol), and the mixture was heated to 120 °C for ∼16 h. After
this time, the mixture was cooled to room temperature and the
resulting residue was purified by silica gel chromatography eluting with
EtOAc:MeOH (95:5) to give the desired product as a white solid
(0.017 g, 26%); δH (500 MHz, MeOD) 8.00 (s, 1H), 7.45−7.38 (m,
2H), 7.37−7.32 (m, 2H), 7.28−7.23 (m, 1H), 6.11 (d, J = 7.7 Hz,
1H), 4.81 (dd, J = 7.6, 5.4 Hz, 1H), 4.69 (d, J = 15.8 Hz, 1H), 4.64 (d,
J = 15.8 Hz, 1H), 4.30 (dd, J = 5.6, 1.6 Hz, 1H), 4.18 (q, J = 1.9 Hz,
1H), 3.85 (dd, J = 12.0, 2.3 Hz, 1H), 3.79 (dd, J = 11.9, 1.7 Hz, 1H);
δC (126 MHz, MeOD) 152.12, 151.99, 149.65, 148.63, 138.75,
128.11, 126.68, 126.52, 116.56, 87.29, 86.41, 71.64, 71.52, 61.75,
45.42. HRMS (ESI) C17H21N6O4 (M + H+) requires 373.1619, found
373.1623.
(2R,3R,4S,5R)-2-(6-Amino-8-((quinolin-6-ylmethyl)amino)-9H-
purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 17. To a
solution of 8-bromoadenosine (0.099 g, 0.29 mmol) in n-butanol
(2.9 mL) was added quinolin-6-ylmethanamine (0.045 g, 0.29 mmol)
and iPr2NEt (0.15 mL, 0.85 mmol), and the mixture was heated to 120
°C under N2 for 24 h. After this time, the mixture was cooled to room
temperature and the solvent removed under reduced pressure. The
resulting residue was purified by chromatography eluting with
EtOAc:2 M MeOH/NH3 (80:20) to give the desired product as a
white solid (0.008 g, 7%); δH (500 MHz, MeOD) 8.83 (dd, J = 4.4,
1.7 Hz, 1H), 8.37 (dt, J = 8.4, 1.2 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H),
i
was added p-tolylmethanamine (0.045 mL, 0.36 mmol) and Pr2NEt
(0.093 mL, 0.54 mmol), and the mixture was heated to 120 °C for
∼48 h. After this time, the mixture was cooled to room temperature
and the solvent removed under reduced pressure. The resulting
residue was purified by silica gel chromatography eluting with
EtOAc:MeOH (9:1) to give the desired product as a white solid
(0.042 g, 61%); δH (500 MHz, MeOD) 7.99 (s, 1H), 7.28 (d, J = 7.8
Hz, 2H), 7.16 (d, J = 7.7 Hz, 2H), 6.09 (d, J = 7.6 Hz, 1H), 4.80 (dd, J
= 7.6, 5.4 Hz, 1H), 4.64 (d, J = 15.6 Hz, 1H), 4.58 (d, J = 15.6 Hz,
1H), 4.29 (dd, J = 5.4, 1.7 Hz, 1H), 4.18 (d, J = 1.9 Hz, 1H), 3.84 (dd,
J = 12.0, 2.3 Hz, 1H), 3.78 (dd, J = 11.9, 1.7 Hz, 1H), 2.32 (s, 3H); δC
(126 MHz, MeOD) 152.08, 152.01, 149.64, 148.59, 136.39, 135.64,
128.70, 126.57, 116.57, 87.27, 86.40, 71.61, 71.50, 61.74, 45.26, 19.70.
HRMS (ESI) C18H23N6O4 (M + H+) requires 387.1775, found
387.1779.
(2R,3R,4S,5R)-2-(6-Amino-8-((3,4-dichlorobenzyl)amino)-9H-
purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 21. To a
microwave vial was added 8-bromoadenosine (0.30 g, 0.87 mmol)
and 3,4-dichlorobenzylamine (0.29 mL, 2.2 mmol) in ethanol (1.7
mL). The reaction mixture was irradiated at 140 °C for 90 min. The
reaction mixture was concentrated, and the crude residue was purified
by silica gel column chromatography eluting with EtOH:DCM (3:7)
to give the desired product as a white solid (0.28 g, 73%); δH (500
H
DOI: 10.1021/acs.jmedchem.5b02001
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Journal of Medicinal Chemistry
Article
MHz, DMSO-d6) 7.90 (s, 1H), 7.70−7.53 (m, 3H), 7.37 (dd, J = 8.3,
2.0 Hz, 1H), 6.55 (s, 2H), 5.99−5.85 (m, 2H), 5.32 (d, J = 6.6 Hz,
1H), 5.18 (d, J = 4.0 Hz, 1H), 4.72 (td, J = 7.0, 5.3 Hz, 1H), 4.56 (d, J
= 6.0 Hz, 2H), 4.12 (ddd, J = 5.5, 3.9, 1.9 Hz, 1H), 3.99 (q, J = 2.3 Hz,
1H), 3.62 (dt, J = 7.0, 3.2 Hz, 2H) 3.45−3.35 (obs. m, 1H); δC (126
MHz, DMSO-d6) 153.01, 151.48, 150.25, 149.17, 141.70, 131.27,
130.86, 129.66, 129.58, 128.05, 117.38, 87.00, 86.30, 71.53, 71.38,
62.23, 44.76. HRMS (ESI) C17H19Cl2N6O4 (M + H+) requires
442.0867, found 442.0859.
(2R,3R,4S,5R)-2-(6-Amino-8-((4-methoxybenzyl)amino)-9H-
purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 22. To a
solution of 8-bromoadenosine (0.073 g, 0.21 mmol) in n-butanol
(4.2 mL) was added (4-methoxyphenyl)methanamine (0.083 mL, 0.63
mmol) and iPr2NEt (0.22 mL, 1.3 mmol), and the mixture was heated
to 120 °C for ∼12 h. After this time, the mixture was cooled to room
temperature and the solvent removed under reduced pressure. The
resulting residue was purified by silica gel chromatography eluting with
EtOAc:MeOH (95:5) to give the desired product as a white solid
(0.049 g, 58%); δH (500 MHz, MeOD) 7.98 (s, 1H), 7.32 (d, J = 8.8
Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.07 (d, J = 7.6 Hz, 1H), 4.78 (dd, J
= 7.6, 5.4 Hz, 1H), 4.60 (d, J = 15.3 Hz, 2H), 4.55 (d, J = 15.3 Hz,
1H), 4.28 (dd, J = 5.6, 1.7 Hz, 1H), 4.16 (q, J = 1.9 Hz, 1H), 3.83 (dd,
J = 12.0, 2.3 Hz, 1H), 3.78 (s, 3H), 3.77 (obs. dd, J = 11.9, 1.7 Hz,
1H); δC (126 MHz, MeOD) 158.96, 152.09, 151.98, 149.63, 148.59,
130.63, 127.94, 116.59, 113.49, 87.27, 86.38, 71.61, 71.48, 61.73,
54.26, 45.05. HRMS (ESI) C18H22N6NaO5 (M + Na+) requires
425.1544, found 425.1540.
Experimental Procedures (Surface Plasmon Resonance
Spectroscopy). All surface plasmon resonance (SPR) experiments
were carried out on a Biacore T100 enhanced to T200 sensitivity (GE
Life Sciences, Amersham Place, UK). Amine coupling chemistry was
used to immobilize the truncated HSC70-NBD domain (residues 1−
381) on a research grade CM5 sensor chip. The running buffer used in
the immobilization step consisted of 1× phosphate buffered saline (10
mM NaHPO4/NaH2PO4 pH 7.4, 2.7 mM KCl, 137 mM NaCl), and
the chip surface was activated for 10 min using a 1:1 mixture of 100
mM N-hydroxysuccinimide and 400 mM 1-ethyl-3-(3-
(dimethylamino)propyl)-carbodiimide. HSC70-NBD was injected at
a concentration of 70 μg/mL in a 10 mM acetate buffer pH 5.0 with
750 μM ADP as protection for the active site lysines. The reaction was
monitored in real time and stopped when a target immobilization level
of ∼5000RU was obtained. Finally, the surface was blocked via an
injection of 1 M ethanolamine at pH 8.5 for 7 min. The flow rate was
maintained at 10 μL/min for all the above procedures. Flow cell one
was left unmodified and used as the reference surface.
Experimental Procedure (HSP72-NBD Protein Preparation).
The human HSPA1A (HSP72) gene was amplified from the IMAGE
clone 3345864 (accession no. BC002453) by PCR using the respective
forward and reverse primers 5′-GATCGACCATATGGCCAA-
AGCCGCGGCGA-3′ and 5′-ACAGAATTCCTAATCTAC-
CTCCTCAATGG-3′. The HSPA1A gene was cloned into a
pTWOE vector, which is a modified version of pET-17b (Merck
Chemicals Ltd., Nottingham, UK) encoding a N-terminal 6xHis-tag
followed by a human rhinovirus 3C protease cleavage site. BL21-AI
cells (Invitrogen, Paisley, UK) transformed with the vector containing
the HSPA1A gene were grown in Luria−Bertani medium to an optical
density at 600 nm of 0.6 and induced with 0.5 mM isopropyl-β-D-1-
thiogalactopyranoside and 0.2% (w/v) arabinose at for 16 h at 20 °C.
Cells were harvested by centrifugation at 6000 rpm for 40 min at 4 °C
using an Avanti centrifuge J-26XP (Beckman Coulter, High Wycombe,
UK) with a JLA 8.100 rotor. Cell pellets were resuspended in 3
volumes of lysis buffer consisting of 25 mM Tris, 50 mM NaCl, 5% (v/
v) glycerol, 1× cOmplete EDTA-free protease inhibitors (Roche,
Basel, Switzerland), 25 U/mL benzonase nuclease (Merck Chemicals
Ltd.) at pH 7.5. Cell lysis was performed by sonication using a Vibra-
Cell VCX500 (Sonics & Materials Inc., Newtown, CT, USA) with a 13
mm solid probe for 24 cycles of 5 s on, 55 s off with amplitude set at
50%. The lysate was clarified by centrifugation at 20000 rpm for 30
min at 4 °C using an Avanti centrifuge J-26XP (Beckman Coulter)
with a JA 25.50 rotor.
The supernatant was passed through a 1.2 μm syringe filter
(Sartorius Stedim, Germany) and loaded onto a 5 mL Histrap FF
column (GE Healthcare, Chalfront St. Giles, UK) equilibrated in
buffer A, comprising 25 mM Tris, 50 mM NaCl, pH 7.5, and eluted
with a gradient of 0−100% buffer B (buffer A + 250 mM imidazole)
over 10 column volumes (CV). Fractions containing HSP72 were
pooled, concentrated, and loaded onto a Superdex 200 (16/60) size
exclusion column (GE Healthcare) equilibrated in 25 mM Tris, 400
mM NaCl, 2 mM EDTA 5% (v/v) glycerol, pH 7.5, for further
purification. Fractions containing HSP72 were pooled and further
purified to remove contaminating nucleotides using a 6 mL Resource
Q column (GE Healthcare) equilibrated in 20 mM Tris, 2 mM EDTA,
5% (v/v) glycerol, pH 7.5. Following a 10 CV wash with the same
buffer, HSP72 was eluted using a gradient from 0 to 500 mM NaCl
over 6 CV and loaded onto a Superdex 200 16/60 column (GE
Healthcare) equilibrated in a buffer containing 25 mM Tris, 400 mM
NaCl, 15 mM EDTA, 5% (v/v) glycerol, pH 7.5. The removal of
contaminating nucleotides was followed by measuring the ratio of
absorbance at 260 and 280 nm (A260/A280) using a NanoDrop ND-
1000 UV spectrophotometer (Thermofisher, Wilmington, DE, USA).
Samples with A260/A280 below 0.6 were regarded as nucleotide free.
Experimental Procedure (X-ray Crystallography). Purified
HSP72-NBD protein was thawed; buffer exchanged into fresh 100
mM HEPES pH 7.5 and incubated with 5 mM of the inhibitor for 30
min on ice prior to crystallization. HSP72-NBD/inhibitor co-crystals
were grown at 18 °C in sitting drops by mixing equal volumes of
protein solution (5−12 mg/mL) and precipitant solution containing
17−28% (v/v) PEG3350, 0.1 M HEPES pH 7.5, 2 mM MgCl2, and 2
mM NaH2PO4. Co-crystals of approximate dimensions 100 × 100 ×
300 μm3 typically formed overnight. Crystals were in liquid nitrogen,
using 22.5% (v/v) ethylene glycol for the co-crystals with
sangivamycin 10 and 25% (v/v) glycerol for compound 17.
Following protein immobilization, the running buffer was changed
to 1× phosphate buffered saline containing 0.05% (v/v) Tween20 and
5% (v/v) DMSO in order to reduce the nonspecific binding and
increase solubility of the compounds.
All liquid handling was carried out using an ECHO 550 acoustic
liquid dispenser (Labcyte, Dublin, Ireland), and compounds were
added to 384-well polypropylene V-bottomed plates (Greiner,
Stonehouse, UK), which were used as sample plates for the SPR
experiments. For each compound, an eight-point dose response
experiment was carried out using a concentration range of 50−2000
μM, 5−200 μM or 0.0625−2.5 μM, depending on the potency of the
compound. The buffer mix was made compatible with the Biacore
running buffer. The experiments were performed at a flow rate of 30
μL/min, a sample injection time of 60 s, and a dissociation time of 120
s. The CM5 surface was not regenerated between sample injections.
KD values were calculated from the background normalized binding
curve generated from the sensorgrams under equilibrium conditions,
using the 1:1 binding model in the Biacore software version 2 (GE Life
Sciences, Amersham Place, UK). All results are reported as the
geometric mean standard error of the mean (SEM) of at least three
independent measurements unless otherwise stated. The ratio of
experimental to theoretical RUmax varied from 0.84 to 1.40. Examples
of sensorgrams and binding curves can be found in the Supporting
Information.
X-ray diffraction data were collected at 100 K at Diamond Light
Source (Oxfordshire, UK; beamlines I04−1 and I24). Data were
integrated with XDS. All data were imported to MTZ format with
POINTLESS, then scaled and merged with AIMLESS and the CCP4
suite. The structures were solved by molecular replacement with
PHASER, with the public domain HSP72-NBD structure 1S3X as the
search model after removal of all nonprotein atoms. Structures were
refined in iterative cycles of model building with COOT and
refinement with BUSTER. TLS groups were selected with PHENIX
phenix.find_tls_groups. Ligand restraints were generated with GRADE
and MOGUL. The final structure quality was checked with
MOLPROBITY. The data collection and refinement statistics are
presented in Supporting Information Table S15, and for Fo − Fc
I
DOI: 10.1021/acs.jmedchem.5b02001
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
electron density figures for all structural data, see Supporting
Information Figure S14. Fo − Fc electron density figures were
generated with CCP4MG.
(3) (a) Garcia-Carbonero, R.; Carnero, A.; Paz-Ares, L. Inhibition of
HSP90 molecular chaperones: moving into the clinic. Lancet Oncol.
2013, 14, e358−e369. (b) Butler, L. M.; Ferraldeschi, R.; Armstrong,
H. K.; Centenera, M. M.; Workman, P. Maximizing the Therapeutic
Potential of HSP90 Inhibitors. Mol. Cancer Res. 2015, 13, 1445−1451.
(4) Powers, M. V.; Jones, K.; Barillari, C.; Westwood, I.; van
Montfort, R. L. M.; Workman, P. Targeting HSP70: the second
potentially druggable heat shock protein and molecular chaperone?
Cell Cycle 2010, 9, 1542−1550.
(5) Powers, M. V.; Clarke, P. A.; Workman, P. Dual targeting of
HSC70 and HSP72 inhibits HSP90 function and induces tumor-
specific apoptosis. Cancer Cell 2008, 14, 250−262.
(6) (a) Mayer, M. P.; Bukau, B. HSP70 Chaperones: cellular
functions and molecular mechanism. Cell. Mol. Life Sci. 2005, 62, 670−
684. (b) Laufen, T.; Mayer, M. P.; Beisel, C.; Klostermeier, D.; Mogk,
A.; Reinstein, J.; Bukau, B. Mechanism of regulation of HSP70
chaperones by DNAJ cochaperones. Proc. Natl. Acad. Sci. U. S. A. 1999,
96, 5452−5457.
(7) (a) Williamson, D. S.; Borgognoni, J.; Clay, A.; Daniels, Z.;
Dokurno, P.; Drysdale, M. J.; Foloppe, N.; Francis, G. L.; Graham, C.
J.; Howes, R.; Macias, A. T.; Murray, J. B.; Parsons, R.; Shaw, T.;
Surgenor, A. E.; Terry, L.; Wang, Y.; Wood, M.; Massey, A. J. Novel
adenosine-derived inhibitors of 70 KDa heat shock protein, discovered
through structure-based design. J. Med. Chem. 2009, 52, 1510−1513.
(b) Macias, A. T.; Williamson, D. S.; Allen, N.; Borgognoni, J.; Clay,
A.; Daniels, Z.; Dokurno, P.; Drysdale, M. J.; Francis, G. L.; Graham,
C. J.; Howes, R.; Matassova, N.; Murray, J. B.; Parsons, R.; Shaw, T.;
Surgenor, A. E.; Terry, L.; Wang, Y.; Wood, M.; Massey, A. J.
Adenosine-derived inhibitors of 78 KDa glucose regulated protein
(GRP78) ATPase: insights into isoform selectivity. J. Med. Chem.
2011, 54, 4034−4041.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b02001.
Details of commercially available adenosine analogues,
table of HSP70 affinities, NMR spectra of final
compounds, representative SPR sensorgrams and bind-
ing curves, Fo −Fc electron density for ligand-bound
HSP72 crystal structures, data collection and refinement
statistics for HSP72 co-crystal structures with ligands,
overlays of the co-crystal structures highlighting key
residues (PDF)
Molecular formula strings (CSV)
Accession Codes
Atomic coordinates and structure factors for the crystal
structures of HSP72-NBD with compounds can be accessed
using PDB codes: 10 5AQZ, 17 5AR0. Authors will release the
atomic coordinates and experimental data upon article
publication.
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0) 20 8722 4334. E-mail: keith.jones@icr.ac.uk.
■
(8) Several non-ATP competitive inhibitors have been described in
the literature see: (a) Fewell, S. W.; Day, B. W.; Brodsky, J. L.
Identification of an inhibitor of HSC70-mediated protein translocation
and ATP hydrolysis. J. Biol. Chem. 2001, 276, 910−914. (b) Fewell, S.
W.; Smith, C. M.; Lyon, M. A.; Dumitrescu, T. P.; Wipf, P.; Day, W.
W.; Brodsky, J. L. Small molecule modulators of endogenous and co-
chaperone stimulated HSP70 ATPase activity. J. Biol. Chem. 2004, 279,
51131−51140. (c) Rousaki, A.; Miyata, Y.; Jinwal, U. K.; Dickey, C. A.;
Gestwicki, J. E.; Zuiderweg, E. R. Allosteric drugs: the interaction of
antitumor compound MKT-077 with human HSP70 chaperones. J.
Mol. Biol. 2011, 411, 614−632. (d) Williams, D. R.; Ko, S. K.; Park, S.;
Lee, M. R.; Shin, I. An apoptosis-inducing small molecule that binds to
heat shock protein 70. Angew. Chem., Int. Ed. 2008, 47, 7466−7469.
(e) Jinwal, U. K.; Miyata, Y.; Koren, J.; Jones, J. R.; Trotter, J. H.;
Chang, L.; O’Leary, J.; Morgan, D.; Lee, D. C.; Shults, C. L.; Rousaki,
A.; Weeber, E. J.; Zuiderweg, E. R. P.; Gestwicki, J. E.; Dickey, C. A.
Chemical manipulation of HSP70 ATPase activity regulates tau
stability. J. Neurosci. 2009, 29, 12079−12088. (f) Miyata, Y.; Rauch, J.
N.; Jinwal, U. K.; Thompson, A. D.; Srinivasan, S.; Dickey, C. A.;
Gestwicki, J. E. Cysteine reactivity distinguishes redox sensing by the
heat inducible and constitutive forms of heat shock protein 70
(HSP70). Chem. Biol. 2012, 19, 1391−1399. (g) Howe, M. K.;
Bodoor, K.; Carlson, D. A.; Hughes, P. F.; Alwarawrah, Y.; Loiselle, D.
R.; Jaeger, A. M.; Darr, D. B.; Jordan, J. L.; Hunter, L. M.; Molzberger,
E. T.; Gobillot, T. A.; Thiele, D. J.; Brodsky, J. L.; Spector, N. L.;
Haystead, T. A. J. Identification of an allosteric small-molecule
inhibitor selective for the inducible form of heat shock protein 70.
Chem. Biol. 2014, 21, 1648−1659. (h) Dal Piaz, F.; Cotugno, R.;
Lepore, L.; Vassallo, A.; Malafronte, N.; Lauro, G.; Bifulco, G.;
Belisario, M. A.; De Tommasi, N. Chemical proteomics reveals HSP70
1A as a target for the anticancer diterpene oridonin in jurkat cells. J.
Proteomics 2013, 82, 14−26. (i) Rodina, A.; Patel, P. D.; Kang, Y.;
Patel, Y.; Baaklini, I.; Wong, M. J.; Taldone, T.; Yan, P.; Yang, C.;
Maharaj, R.; Gozman, A.; Patel, M. R.; Patel, H. J.; Chirico, W.;
Erdjument-Bromage, H.; Talele, T. T.; Young, J. C.; Chiosis, G.
Identification of an allosteric pocket on human HSP70 reveals a mode
of inhibition of this therapeutically important protein. Chem. Biol.
2013, 20, 1469−1480. (j) Hassan, A. Q.; Kirby, C. A.; Zhou, W.;
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Cancer Research UK grant nos.
C309/A8274 and C309/A11566. We acknowledge NHS
funding to the NIHR Biomedical Research Centre at The
Institute of Cancer Research and the Royal Marsden Hospital.
We thank Dr. Nora Cronin and the staff of DIAMOND Light
Source for their support during data collection.
ABBREVIATIONS USED
■
HSP90, heat-shock protein 90; HSP70, heat-shock protein 70;
HSP72, heat-shock protein 72; HSC70, heat-shock cognate 70;
HSP40, heat-shock protein 40, chaperone DnaJ; BAG1, BAG
family molecular chaperone regulator 1; ATP, adenosine
triphosphate; ADP, adenosine diphosphate; Pi, inorganic
phosphate; KD, equilibrium dissociation constant; pKD, negative
Log10 of the equilibrium dissociation constant; SEM, standard
error of the mean; SAR, structure−activity relationship; μM,
micromolar; n, number of statistical repeats; NBD, nucleotide-
binding domain; SPR, surface plasmon resonance
REFERENCES
■
(1) Hartl, F. U.; Bracher, A.; Hayer-Hartl, M. Molecular chaperones
in protein folding and proteostasis. Nature 2011, 475, 324−332.
(2) Powers, M. V.; Workman, P. Inhibitors of the heat-shock
response: biology and pharmacology. FEBS Lett. 2007, 581, 3758−
3769.
J
DOI: 10.1021/acs.jmedchem.5b02001
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Schuhmann, T.; Kityk, R.; Kipp, D. R.; Baird, J.; Chen, J.; Chen, Y.;
Chung, F.; Hoepfner, D.; Movva, N. R.; Pagliarini, R.; Petersen, F.;
Quinn, C.; Quinn, D.; Riedl, R.; Schmitt, E. K.; Schitter, A.; Stams, T.;
Studer, C.; Fortin, P. D.; Mayer, M. P.; Sadlish, H. The novolactone
natural product disrupts the allosteric regulation of HSP70. Chem. Biol.
2015, 22, 87−97.
(9) Recently, a set of novel piperidine derivatives as inhibitors of
HSP70 but is unclear whether the cellular effects can be attributed
HSP70 inhibition. Zeng, Y.; Cao, R.; Zhang, T.; Li, S.; Zhong, W.
Design and synthesis of piperidine derivatives as novel human heat
shock protein 70 inhibitors for the treatment of drug-resistant tumors.
Eur. J. Med. Chem. 2015, 97, 19−31.
(10) The validity of two HSP70 inhibitors have recently been
investigated: (a) Schlecht, R.; Scholz, S. R.; Dahmen, H.; Wegener, A.;
Sirrenberg, C.; Musil, D.; Bomke, J.; Eggenweiler, H. M.; Mayer, M. P.;
Bukau, B. Functional analysis of HSP70 inhibitors. PLoS One 2013, 8,
e78443. (b) Evans, L. E.; Cheeseman, M. D.; Yahya, N.; Jones, K.
Investigating apoptozole as a chemical probe for HSP70 inhibition.
PLoS One 2015, 10, e0140006.
(11) (a) Bork, P.; Sander, C.; Valencia, A. An ATPase domain
common to prokaryotic cell cycle proteins, sugar kinases, actin, and
HSP70 heat shock proteins. Proc. Natl. Acad. Sci. U. S. A. 1992, 89,
7290−7294. (b) Flaherty, K. M.; McKay, D. B.; Kabsch, W.; Holmes,
K. Similarity of the three-dimensional structures of actin and the
ATPase fragment of a 70-KDa heat shock cognate protein. Proc. Natl.
Acad. Sci. U. S. A. 1991, 88, 5041−5045. (c) Chene, P. ATPases as
drug targets: learning from their structure. Nat. Rev. Drug Discovery
2002, 1, 665−673.
(12) Massey, A. J. ATPases as drug targets: insights from heat shock
proteins 70 and 90. J. Med. Chem. 2010, 53, 7280−7286.
(13) SiteMap, version 2.2; Schrodinger LLC: 120 West 45th Street,
New York, NY 10036, 2008.
theoretical RUmax was approximately 1. Representative examples of
sensorgrams and binding curves can be found in the Supporting
Information.
(20) Standard adenosine and adenine numbering system is used
throughout this publication.
(21) This data can be observed in the Supporting Information.
(22) All fold differences that are quoted are statistically significant at
the 95% level using Student’s t-test, see: Cumming, G.; Fidler, F.;
Vaux, D. L. Error bars in experimental biology. J. Cell Biol. 2007, 177,
7−11.
(23) Stumpfe, D.; Hu, Y.; Dimova, D.; Bajorath, J. Recent progress in
understanding activity cliffs and their utility in medicinal chemistry. J.
Med. Chem. 2014, 57, 18−28.
(24) Jiang, L.; Mao, X.-A. Conformations of adenosine-5′-
triphosphate in the presence of Mg2+ at different pH. Polyhedron
2002, 21, 435−438.
(25) Adenosine numbering system used throughout for comparison.
(26) See Supporting Information for purchasing details of
commercially available natural products used in this study.
(27) Gunic, E.; Girardet, J.; Pietrzkowski, Z.; Esler, C.; Wang, G.
Synthesis and cytotoxicity of 4′-C- and 5′-C-substituted toyocamycins.
Bioorg. Med. Chem. 2001, 9, 163−170.
(28) 8-N-Methylaminosangivamycin 13 could only be isolated in
80% purity and was tested at this level of purity in subsequent assays.
(29) Because of the low yield and purity in the synthesis of 13, there
was only sufficient material to do a single SPR analysis to measure the
compound’s affinity to HSP70.
(30) Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding
in medicinal chemistry. J. Med. Chem. 2010, 53, 2601−2611.
(31) Minamoto, K.; Fujiki, Y.; Shiomi, N.; Uda, Y.; Sasaki, T.
Systematic general synthesis of purine 8,5′-imino and substituted
imino cyclonucleosides. J. Chem. Soc., Perkin Trans. 1 1985, 11, 2337−
2346.
(32) Ha, J.; McKay, D. B. Kinetics of nucleotide-induced changes in
the tryptophan fluorescence of the molecular chaperone HSC70 and
its subfragments suggest the ATP-induced conformational change
follows initial ATP binding. Biochemistry 1995, 34, 11635−11644.
(33) For a discussion on the effect of binding mechanism and
conformational change in drug discovery, see: Holdgate, G. A.; Gill, A.
L. Kinetic efficiency: the missing metric for enhancing compound
quality? Drug Discovery Today 2011, 16, 910−913.
(34) This result is comparable to the previously described affinity of
ADP for HSP70 (KD = 0.5 μM. See ref 7. The affinity of ADP for
HSP70 is known to be highly dependent on the assay conditions, see:
Arakawa, A.; Handa, N.; Shirouzu, M.; Yokoyama, S. Biochemical and
structural studies on the high affinity of HSP70 for ADP. Protein Sci.
2011, 20, 1367−1379.
(14) HSP70 is quoted as returning a Dscore = 0.95, classifying the
target as “difficult”, see reference 12 and Halgren, T. A. Identifying and
characterizing binding sites and assessing drugability. J. Chem. Inf.
Model. 2009, 49, 377−389.
(15) This analysis was carried out by Vernalis using their fragment
screening deck; see reference 12 and Hajduk, P. J.; Huth, J. R.; Fesik,
S. W. Drugability indices for protein targets derived from NMR-based
screening data. J. Med. Chem. 2005, 48, 2518−2525.
(16) (a) Arakawa, A.; Handa, N.; Ohsawa, N.; Shida, M.; Kigawa, T.;
Hayashi, F.; Shirouzu, M.; Yokoyama, S. The C-terminal BAG domain
of BAG5 induces conformational changes of the HSP70 nucleotide-
binding domain for ADP-ATP exchange. Structure 2010, 18, 309−319.
(b) Vogel, M.; Bukau, B.; Mayer, M. P. Allosteric regulation of HSP70
chaperones by a proline switch. Mol. Cell 2006, 21, 359−367. (c) Rist,
W.; Graf, C.; Bukau, B.; Mayer, M. P. Amide hydrogen exchange
reveals conformational changes in HSP70 chaperones important for
allosteric regulation. J. Biol. Chem. 2006, 281, 16493−16501.
(d) Mayer, M. P.; Bukau, B. HSP70 chaperones: cellular functions
and molecular mechanism. Cell. Mol. Life Sci. 2005, 62, 670−684.
(17) The off-rate of ADP/Pi from HSP72 has been calculated using
MABA-ADP and is ∼0.05 s−1, the steady state ATPase rate in the
absence of co-chaperones has been calculated as ∼4.5 × 10−4 s−1, see:
Gassler, C. S.; Wiederkehr, T.; Brehmer, D.; Bukau, B.; Mayer, M. P.
BAG-1M accelerates nucleotide release for human HSC70 and HSP70
and can act concentration-dependent as positive and negative cofactor.
J. Biol. Chem. 2001, 276 (35), 32538−32544.
(35) The salt-bridge between glutamic acid-268 and lysine-56 can be
observed in several other published HSP70/ADP structures, see: (a)
PDB 3ATU reference 34;. (b) PDB 3JXU: Wisniewska, M.; Karlberg,
T.; Lehtio, L.; Johansson, I.; Kotenyova, T.; Moche, M.; Schuler, H.
Crystal structures of the ATPase domains of four human HSP70
isoforms: HSPA1L/HSP70-hom, HSPA2/HSP70−2, HSPA6/
HSP70B′, and HSPA5/BiP/GRP78. PLoS One 2010, 5, e8625. (c)
PDB 1S3X: Sriram, M.; Osipiuk, J.; Freeman, B.; Morimoto, R.;
Joachimiak, A. Human HSP70 molecular chaperone binds two calcium
ions within the ATPase domain. Structure 1997, 5, 403−414. (d) PDB
2E8A: Shida, M.; Arakawa, A.; Ishii, R.; Kishishita, S.; Takagi, T.;
Kukimoto-Niino, M.; Sugano, S.; Tanaka, A.; Shirouzu, M.; Yokoyama,
S. Direct inter-subdomain interactions switch between the closed and
open forms of the HSP70 nucleotide binding domain in the
nucleotide-free state. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 223−232.
(18) For more details see the Supporting Information.
(19) All SPR data was measured with the Biacore T200 instrument
using the analysis software (version 2) provided. HSC70-NBD was
loaded on to the series S CM5 sensor chip as per the manufacturer’s
instructions and in the presence of 750 μM ADP to protect active site
lysines. pKD = −log10[KD(μM) × 10−6]. All results are the geometric
mean SEM of three independent experiments measured at similar
protein binding levels (∼5000 RU). The KD values were determined
from analysis of the background normalized binding curve generated
from the sensorgrams under equilibrium conditions using a 1:1
binding model. For all experiments, the ratio of experimental RUmax to
(36) Rye, C. S.; Baell, J. B. Phosphate isosteres in medicinal
chemistry. Curr. Med. Chem. 2005, 12, 3127−3141.
(37) 8-N-Quinolineaminoadenosine derived ligand 17 was previously
reported to have a KD = 0.23 μM as measured by SPR against the
HSP70 isoform GRP78, see ref 7.
K
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Journal of Medicinal Chemistry
Article
(38) Luo, R.; David, L.; Hung, H.; Devaney, J.; Gilson, M. K.
Strength of solvent-exposed salt-bridges. J. Phys. Chem. B 1999, 103,
727−736. (b) Dong, F.; Zhou, H. Electrostatic contributions to T4
lysozyme stability: solvent-exposed charges versus semi-buried salt
bridges. Biophys. J. 2002, 83, 1341−1347.
(39) Berry, B. W.; Elvekrog, M. M.; Tommos, C. Environmental
modulation of protein cation-π interactions. J. Am. Chem. Soc. 2007,
129 (17), 5308−5309.
(40) For a discussion on the effects of “burying” solvent exposed
intramolecular hydrogen bonds and salt-bridges of proteins, see:
(a) Hendsch, Z. S.; Tidor, B. Do salt bridges stabilize proteins? a
continuum electrostatic analysis. Protein Sci. 1994, 3, 211−226.
(b) Efimov, A.; Brazhnikov, E. V. Relationship between intramolecular
hydrogen bonding and solvent accessibility of side-chain donors and
acceptors in proteins. FEBS Lett. 2003, 554, 389−393.
(41) Murray, J. B.; Roughley, S. D.; Matassova, N.; Brough, P. A. Off-
rate screening (ORS) by surface plasmon resonance. An efficient
method to kinetically sample hit to lead chemical space from
unpurified reaction products. J. Med. Chem. 2014, 57, 2845−2850.
(42) (a) Porcari, A. R.; Townsend, L. B. An improved synthesis of
the versatile heterocycle, 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]-
pyrimidine. Synth. Commun. 1998, 28, 3835−3843. (b) Dang, Q.;
Gomez-Galeno, J. E. An efficient synthesis of pyrrolo[2,3-d]-
pyrimidines via inverse electron demand Diels-Alder reactions of 2-
amino-4-cyanopyrroles with 1,3,5-triazines. J. Org. Chem. 2002, 67,
8703−8705. (c) Taylor, E. C.; Hendess, R. W. Synthesis of
pyrrolo[2,3-d]pyrimidines. the aglycone of toyocamycin. J. Am.
Chem. Soc. 1965, 87, 1995−2003. (d) Sharma, M.; Bloch, A.; Bobek,
M. A practical synthesis of the antibiotic toyocamycin. Nucleosides
Nucleotides 1993, 12, 643−8.
(43) Complex due to F19-coupling.
L
DOI: 10.1021/acs.jmedchem.5b02001
J. Med. Chem. XXXX, XXX, XXX−XXX