Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72

Article abstract of DOI:10.1021/acs.jmedchem.9b01709

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cy

Full text of DOI:10.1021/acs.jmedchem.9b01709

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Article
Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72
Jonathan Pettinger, Michael Carter, Keith Jones, and Matthew D. Cheeseman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01709 • Publication Date (Web): 14 Nov 2019
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Kinetic Optimization of Lysine-Targeting Covalent
Inhibitors of HSP72
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Jonathan Pettinger, Michael Carter, Keith Jones, and Matthew D. Cheeseman*
Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research,
London SW7 3RP, United Kingdom.
KEYWORDS kinetic, covalent, irreversible, lysine, HSP72.
Abstract
The covalent inhibition mechanism of action, which overcomes competition with high
affinity, high abundance substrates of challenging protein targets, can deliver effective
chemical probes and drugs. The success of this strategy has centered on exposed
cysteine residues as nucleophiles but the low abundance of cysteine in the proteome
has limited its application. We recently reported our discovery that lysine-56 in the
difficult to drug target, HSP72, could form a covalent bond with a small molecule
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inhibitor. We now disclose the optimization of these targeted covalent inhibitors using
rational design. Essential to our optimization was the development of a new covalent
fluorescence polarization assay, which allows for the direct measurement of the key
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kinetic parameter in covalent inhibitor design, kinact/K , extrapolation of the underlying
I
parameters, kinact and K, and direct comparison to reversible analogues. Using our
i
approach, we demonstrate a >100-fold enhancement in covalent efficiency and key
learnings in lysine selective electrophile optimization.
Introduction
Despite many of our most important drugs utilizing irreversible covalent inhibition of
1
an enzyme, concerns relating to idiosyncratic toxicity led to the near exclusion of this
mechanism of action (MOA) from drug discovery programmes.2 The recent
renaissance in covalent inhibitors is in large part due to their inherent advantage over
reversible counterparts for antagonising proteins that have high-affinity, high-
3
abundance natural substrates. Whilst the previous generation of irreversible inhibitor
drugs were discovered by serendipity or were natural products, the rational design
strategy for modern targeted covalent inhibitors (TCIs) focuses on exploiting high
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resolution small-molecule/protein X-ray crystal structures of high affinity reversible
ligands to target active site, solvent-exposed cysteine residues with sparingly reactive
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electrophiles. Unfortunately, the rarity of cysteine in the proteome has limited its
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application, leading to an increased interest in targeting other potentially nucleophilic
residues.6-8
Depending on the length of exposure and the concentration, TCIs utilize both
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reversible and irreversible occupancy of a protein (Figure 1B). Defining a TCI only via
an IC value can be limiting for rational design, as the inhibitor (I) will inevitably appear
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more potent with increasing pre-incubation time. TCIs typically react via a two-step
MOA (Figure 1A); initially binding to the protein (E) in a reversible manner to generate
a non-covalent complex (EI). The occupancy of the reversible complex is determined
by the free concentration of the TCI and the equilibrium constant K. The reversible
i
complex can then undergo covalent bond formation as determined by the first-order
rate constant kinact, to give the covalent complex (E-I). These fundamental parameters
describe the efficiency of the TCI but cannot be determined directly from assay data.
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Figure 1. Simulated data describing the MOA and kinetic parameters used to quantify
the activity of TCIs. (A) TCI two-step binding mechanism. (B) The two components of
TCI MOA, both reversible and covalent occupancy contribute to total target occupancy
at a given concentration and time. Left = covalent occupancy (%CO) and right total
-1
occupancy (%TO), both simulated using K = 1 µM and k
= 0.069 min . FO =
I
inact
fraction reversible occupancy (see supporting information for derivation). (C)
Determination of the essential second order rate constant kinact/K from the
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concentration dependent kobs pseudo-first-order rate constant. Left = example where
K < [I] so k
and K can be deconvoluted. Right = example where K >> [I] so the
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inact
I
I
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individual kinetic parameters cannot be distinguished. (D) Nucleoside-derived
reversible 1 and covalent inhibitor 2 of HSP72.
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TCI activity is described by an equation analogous to the Michaelis-Menten
equation. When normalized for protein concentration, the rate of covalent bond
formation can be quantified by the pseudo first order rate constant, kobs (Figure 1C).
Because of the reversible binding event, kobs is not a true constant, as its value is
dependent on the concentration of the TCI. At TCI concentrations approaching binding
site saturation, kobs tends to the constant, kinact, equivalent to the half-life of the reaction
at a theoretical infinite concentration (t1/2inf).10 [I] at kinact/2 determines the pseudo-
equilibrium constant K , equivalent to K when describing enzyme substrates. The true
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reversible equilibrium constant for the process, K, is often used interchangeably with
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K but this is only a fair assumption when kinact<<koff11 and this may not be true for tight-
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reversible binding TCIs. At concentrations much lower than K , the response of k to
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obs
changing TCI concentration becomes linear. The gradient of this relationship gives the
second order rate constant kinact/K , which is the key kinetic parameter that describes
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the efficiency of the reaction and its optimization is the primary goal of any TCI
discovery effort towards a chemical probe (Figure 1C).
Analysis of the kinact/K parameter clearly demonstrates that there are two strategies
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for TCI optimization: firstly, through the reduction of K by increasing the reversible
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affinity of the ligand for the target protein, and secondly by increasing kinact. The
optimization of kinact differs from simply increasing the intrinsic reactivity of the
electrophile, as this would likely lead to greater off-target activity; instead, kinact
optimization focuses on the particular environment within the protein binding site
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compared to bulk aqueous solvent. The effect of solvent dielectric constant, proximal
residues, perturbed pK and the tightly controlled bond angles and distances resulting
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from the reversible binding of the ligand, can lead to a dramatically enhanced rate of
covalent bond formation and high selectivity. This effect of binding site rate
enhancement has led to effective, highly selective and successful TCI design of KRAS
G12C inhibitors, which display very weak reversible affinity but exploit extremely high
kinact values with sparingly low intrinsic reactivity electrophiles, resulting in kinact/KI
values suitable for in vivo efficacy.13, 14
Heat shock 70 kDa protein 1 (HSP72) is a member of the HSP70 family of molecular
chaperones. It is an ATPase that binds misfolded proteins, stabilizing the cellular
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environment and allowing the cell to return to homeostasis. HSP72 is induced in an
HSF1 dependent manner when the cell is undergoing stress and is overexpressed in
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several cancer cell types.16 This overexpression is correlated with metastasis, poor
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prognosis and resistance to chemotherapy in patients. Because of the clear role of
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HSP72 in cancer, it has become an important target in drug discovery but despite
considerable research effort, there is currently no potent, selective, cellularly active
chemical probe to study the function of HSP72 in cancer cells.
The nucleotide-binding domain (NBD) of HSP72 (HSP72-NBD) can be reversibly
targeted with a series of bis-aryl nucleoside-derived inhibitors (Figure 1D), which
display very high apparent affinity in biochemical assays but suffer a steep drop off in
activity in cancer cells.18 We hypothesized that the poor cellular activity of this
chemotype was due to competition with the high affinity (K for ATP = ~1 µM) high
M
19
abundance (~5 mM) substrate of HSP72, ATP. We recently reported the discovery
that lysine-56 can be selectivity targeted with an acrylate-derived TCI 2 (Figure 1D).20
Targeting lysine residues with TCIs presents a number of unique challenges and is
21
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still in its infancy but the greater prevalence of lysine in the proteome could result
in more wide ranging applications of the irreversible inhibitor paradigm than has so far
been possible through the rational targeting of cysteine.
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As part of our continuing effort to develop strategies and techniques to discover
lysine-targeting covalent ligands, we now report the disclosure of a HSP72 TCI, where
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our approach led to a 108-fold improvement in kinact/K . Critical to the optimization was
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the development of a covalent fluorescence polarization (covalent FP) assay that can
distinguish between the reversible and covalent components of target occupancy,
allowing for direct comparison between covalent and reversible analogues, which is
essential for correct structure activity relationship (SAR) interpretation. The covalent
FP-assay will be widely applicable to the optimization of TCIs of other non-enzymatic
or poorly catalytic proteins.
Results and Discussion
Analysis of the first-generation lysine-56 TCI binding mode and optimization of Ki.
Exploiting extensive mass spectrometry studies, single-point mutant proteins and
through the design of several key control compounds, we had previously
demonstrated that our first-generation acrylate TCI 2 (Figure 1D) was both highly
selective for lysine-56 and that an initial reversible binding step was critical to its MOA.
However, the rate of reaction of 2 was very slow, requiring >24 h exposure, even at
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high concentrations, before the intact protein MS indicated complete formation of the
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covalent complex. Therefore, our aim was to develop a strategy to optimize lysine-
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56 targeting TCIs of HSP72 towards a potentially cell active chemical probe.
To redesign the second-generation TCI with increased potency, we analyzed our
two previously reported co-crystal structures of HSP72-NBD with acrylate TCI 2
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reversibly bound. These structures revealed two distinct binding modes and protein-
residue conformations, which we hypothesized were both essential for the efficiency
of the covalent inhibition MOA (Figure 2A/B).
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Figure 2. Analysis of Lys56-targeting TCI binding modes. The tertiary conformation of
the protein is maintained in the previously described semi-open form in all structures
and modeling. The polar interactions of the adenine-type base and ribose are
maintained in each binding mode. All diagrams were adapted from analysis using
MOE (2014.09) and PyMOL Molecular Graphics System, Version 2.0 Schrödinger,
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LLC. 2.2.3 A: Tyr15 up-conformation. The acrylate electrophile of 2 forms a -stack
interaction with Tyr15 and a hydrogen bond with Thr-37. In this reversible complex,
the electrophile is too far from Lys56 to form a covalent bond (PDB: 5MKR). B: Tyr15
down-conformation. Following rotation of Tyr15, rotation around the linker of 2 now
positions the acrylate electrophile in close proximity to Lys56, suitable for covalent
bond formation in this pre-covalent complex. The acrylate moiety was not observed in
the electron density due to flexibility so was modelled to estimate distances (PDB:
5MKS). C: Binding mode of the high affinity reversible HSP72 inhibitor 1 in the Tyr15
up-conformation (PDB: 4IO8). D: Model of a putative binding mode for a high affinity
Lys56-targeting TCI. With Tyr15 in a down-conformation, rotation of the benzylic ether
positions a potential para-electrophile at an appropriate distance from Lys56 in a pre-
covalent complex. E: Three-step MOA, TCI binds HSP72 to form the reversible
complex (EI) before transition to the pre-covalent complex (EI*) that can result in
formation of the covalent bond and the irreversible complex (E-I).
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In the putative reversible complex of acrylate TCI 2 (Figure 2A, PDB: 5MKR), Tyr15
was in an up-conformation, blocking any direct vector from the ligand to the
nucleophilic Lys56. The electrophilic acrylate moiety formed an eclipsed conformation,
resulting in a -stack interaction with Tyr15 and a hydrogen bond to Thr37. In the
putative pre-covalent complex (Figure 2B, PDB: 5MKS)20 the electrophile was
observed in an elongated conformation and with Tyr15 in a down-conformation.23, 24
The conformational flip of Tyr15 allowed the acrylate moiety to position proximally to
the Lys56 nucleophile, which is essential for covalent bond formation. The hydrogen
bonding array of the 8-aminoadenosine-motif and the position of the lipophilic p-
chlorophenyl substituent were maintained in both binding modes. From this analysis,
we proposed that the MOA of a HSP72 TCI proceeds via a three-step mechanism
(Figure 2E). Firstly, the TCI binds HSP72 to give a reversible complex (EI) that would
contribute to the reversible occupancy but could not lead directly to covalent
occupancy. Transition of the reversible complex (EI) to the pre-covalent complex (EI*)
would be essential for the formation of the covalent bond and the resulting irreversible
complex (E-I). Optimization of the affinity for the reversible complex would still result
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in increased potency of the covalent bond formation, although K would now describe
i
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a pseudo-equilibrium constant as a combination of the microscopic rate constants for
the formation of the encounter complex and conformational switch, in a manner
consistent with an induced fit binding MOA.25 This would also assume that the
conformational flexibility of Tyr15 is sufficient to allow transition to the pre-covalent
complex and access the Lys56 nucleophile, so that covalent bond formation is rate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
determining in kinact
.
To optimize the reversible affinity of our TCI, we aimed to exploit the high affinity 5’-
O-benzyl class of HSP72 inhibitors (Figure 2C). Analysis of the reversible complex
26
27, 28
(
PDB: 4IO8) of nitrile 1 (FP-Assay pK>6.70, K=<0.20 µM, N=3)
revealed that
i
i
this ligand forms similar polar and lipophilic interactions in the 8-aminoadenosine- and
p-chlorophenyl-regions of HSP72-NBD as our acrylate TCI 2. Importantly, the 5’-O-
benzyl substituent occupies the same pocket as the acrylate moiety, so we
hypothesized that a benzyl group with an appropriately positioned electrophile could
exploit both the high affinity reversible complex and could transition to the pre-covalent
complex (Figure 2D) in a similar putative three-step mechanism as our acrylate TCI 2.
To assess whether the proposed transition of the reversible to the pre-covalent
complex was viable (Figure 3B), we carried out a rapid overlay of chemical structures
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(
ROCS) ligand based analysis on the favorability of the linker torsional angles in this
conformationally restricted structure, as this would be critical if we were to incorporate
a 5’-benzylic substituent in our TCI design. Through alignment of the 5’-O-benzyl motif
of 1 to the pre-covalent complex acrylate conformation of 2 and by comparison with
known conformations of this chemotype in the Cambridge Structural Database
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
CSD)30 (Figure 3C), it was demonstrated that a second generation inhibitor could
adopt an acceptable benzylic torsional angle, resulting in a viable conformation for
covalent bond formation. Finally, in this conformation the para-position gave the
shortest distance to Lys56 (Figure 3D), so an electrophile at this position was
incorporated to complete our rational design hypothesis for the second generation
HSP72 TCI.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Conformation analysis of the proposed second generation HSP72 TCI. A:
Overlay of the acrylate TCI 2 (green) and the high affinity reversible inhibitor 1 (cyan).
B: Overlay of the acrylate TCI 2 (green) and the proposed conformation of the second-
generation TCI (cyan) in the pre-covalent complex. The generic electrophile is
represented as a purple sphere. C: Frequency distribution of torsional angles for 5’-O-
adenosine derivatives observed in the CSD, the green line represents the desired
torsional angle. D: At the desired torsional angle, the modelled distances to Lys56
from different ring positions.
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Selection and synthesis of a lysine-targeting warhead and optimization of kinact. Our
first-generation TCI 2 utilized an acrylate warhead to form the covalent bond, which
we hypothesized was sub-optimal for targeting lysine residues in proteins. Lysine is a
hard nucleophile so should display an enhanced rate of reaction with hard
electrophiles but few lysine-selective electrophiles have so far been described in the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
literature. A recent study by Campos et al. successfully exploited activated phenolic
32
esters as hard electrophiles to target the catalytic lysine of PI3K. To incorporate this
concept into the design of our second generation TCI, we developed a synthetic
strategy that added an activated ester with a p-fluorophenol leaving group into the 5’-
para-benzylic vector we had identified from our TCI MOA analysis.
The synthesis of the second-generation TCI began with benzylation of the 5’-
hydroxyl of 2’,3’-acetonide protected 6-chlororiboside 3 (Scheme 1). The order of
addition is essential in this transformation to avoid oligomerization at the 6-chloro
position; 3 was first treated with 4-(bromomethyl) benzonitrile, followed by exposure
o
18
to NaH at 0 C, which gave the 5’-ether 4 in 56% yield. S Ar displacement with
N
ammonia at the 6-position before selective oxidation with bromine at the 8-position,
gave 5 in 59% yield over two steps as single regioisomer. A second S Ar displacement
N
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with 4-chlorobenzylamine gave the key covalent precursor 6 in 66% yield. To
synthesize the lysine-targeting warhead, the nitrile moiety of 6 was hydrolyzed under
basic conditions to give benzoic acid 7, which then underwent coupling with 4-
fluorophenol using standard HATU conditions, and following acetonide deprotection,
gave the second-generation TCI 8 in 7 steps and 5% overall yield. Deprotection of the
intermediate 6 gave the reversible molecular matched pair (MMP) 9 in 47% yield.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Scheme 1. Synthesis of the second-generation lysine-56 targeting TCI.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and conditions: (i) 4-(bromomethyl) benzonitrile, NaH (60% in mineral oil),
o
DMF, RT, 1h, 56%; (ii) 7M NH /MeOH, 110 C, MW, 2 h, 82%; (iii) Br , K HPO .3H O,
3
2
2
4
2
o
H O, 1,4-dioxane, RT, 1 h, 72%; (iv) 4-chlorobenzylamine, EtOH, 160 C, MW, 1 h,
2
o
66%; (v) 2M NaOH, EtOH, 100 C 3 h, 58%; (vi) 4-fluorophenol, HATU, DIPEA, DMF,
RT, 18 h, 47%; (vii) TFA:H O (5:2), RT, 0.5 h, 85%; (viii) 5:2 TFA/H O, RT, 0.5 h, 47%.
2
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Characterization of the second-generation TCI 8. To investigate the reversible
affinity and the potential to form a covalent bond with Lys56 in HSP72-NBD with our
second-generation TCI 8 (Table 1, Entry 3 and Table 2, Entry 2) we repeated our
previously described analysis using the nucleotide-derived FP-assay, comparing the
data to the first-generation TCI 2 (Table 1, Entry 1 and Table 2, Entry 1). Briefly,
displacement of the nucleotide derived FP-probe by the ligand was used to determine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27
an apparent (App.) K. Because the bound fraction of the probe is dependent on the
i
effective concentration of the protein, a time-dependent decrease following covalent
bond formation should result in a shift in the binding curve for the TCI. The time-
dependent FP-assay data revealed that the initial reversible binding affinity of
activated ester 8 displayed a 7-fold improvement over our first-generation TCI 2 but
was >13-fold weaker than the tight binding reversible nitrile MMP 9 (Table 1, Entry 2;
pK>6.70, K=<0.20 µM, N=3). Disappointingly, there was no clear time-dependence in
i
i
the App. K
i
and consistent with this result, analysis of the intact protein MS data for 8
also revealed no evidence of specific and selective covalent bond formation.
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Table 1. Kinetic and Affinity Analysis of Covalent and Non-Covalent Inhibitors of
HSP72
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
N
N
R1
aApp
ckinact
(x10-4 s-1
dInitial
N
bkinact/KI
dt1/2inf
(min)
N
Time
Entry
Cd
)
. K
i
O
(M-1s-1
(h)
)
i
K (M)
O
R3
(M)
R2
O
R2
0
.08
17
O
3
0.86±0.2
2
R3=
1
2
3
4
5
2
9
0.16
720
18
O
19
7.2
3.7
46
CN
<
0.2
0
NA
O
R3=
2
R =H
0.08
F
2.5
O
3
8
O
O
2.0
2.0
1.4
R3=
24
Cl
R1=
CN
NA
NH
15
13
R3=
O
NA
3.5
O
0.08
3
>
200
R2=
1
.0
.0
>200
>200
2
0
.08
6.9
3
6
14
35±1.7
3.6
32
10
1
.0
0.52
0.25
O
2.0
ND
F
S
O
7
8
16
17
R1=H
ND
0.5±0.02
3.3±0.03
ND
3.7
ND
31
>600
110
O
O
ND
ND
12
2
R1= NH
R =H
0.33
1.0
8.0
0
.08
3.3
3
9
18
N
0.33
1.0
0.22
93±2.9
4.4
26
4.7
NH
<
0.2
0
All data was processed and analyzed using GraphPad Prism 7.04. All values are
a
quoted to 2 significant figures. NA=Not Applicable, ND=Not Determined. App. K=
i
Apparent K. Each concentration represent an n=3 statistical repeats, arithmetic
i
mean±SEM. Each time course was generated from continuous measurements of each
assay and assumes no significant TCI depletion. App. K values were calculated from
i
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the fitted IC curve using non-linear regression. (4 parameters) using the method in
50
b
reference 27, see supporting information. Calculated using the method described in
Figure 4. Each value represents the arithmetic mean±SEM of n=3 biological repeats.
cCalculated from the respective kinact/K and initial K values using the method
I
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
described in Figure 4. dt1/2inf=ln2/kinact. Calculated from the IC curves using non-
d
50
linear regression (4 parameters) using the method described in Figure 4.
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Table 2. HSP72 TCI Analysis
Entr Comp
aApp. Ki
bIntact Protein MS
ckinact/KI
y
d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
140
120
100
80
0
.015
0.010
0.005
0.000
t = 5 min
t = 19 h
t = 46 h
1
2
6
0
0
4
0
5000
10000 15000 20000
Cd] / nM
3
4
5
[
log[Cd (nM)]
140
120
100
80
t = 5 min
t = 2 h
2
3
4
8
t = 24 h
NA
NA
6
0
0
4
3
4
5
log[Cd (nM)]
1
40
20
00
80
1
1
t = 5 min
t = 1 h
t = 2 h
13
14
6
0
0
4
3
4
5
log[Cd (nM)]
140
0
.015
0.010
0.005
0.000
120
00
1
t = 5 min
t = 1 h
8
0
60
t = 2 h
4
0
0
2
0
2000
4000
6000
3
4
5
[
Cd] / nM
log[Cd (nM)]
200
160
120
0.015
0.010
0.005
0.000
t = 5 min
t = 20 min
t = 1 h
5
6
16
17
ND
8
0
0
4
0
50000
100000
150000
3
4
5
[Cd] / nM
log[Cd (nM)]
200
160
120
0.015
0.010
0.005
0.000
t = 5 min
t = 20 min
t = 1 h
8
0
0
4
0
20000
40000
60000
3
4
5
[Cd] / nM
log[Cd (nM)]
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
00
0
.015
.010
160
120
80
0
t = 5 min
t = 20 min
t = 1 h
7
18
0.005
0.000
4
0
0
1000
2000
3000
3
4
5
[
Cd] / nM
log[Cd (nM)]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
All data was processed and analyzed using GraphPad Prism 7.04. App. K= Apparent
i
K. Each concentration represents n=3 statistical repeats, arithmetic mean±SEM. Each
i
time course was generated from continuous measurements of each assay and
assumes no significant TCI depletion. App. K values were calculated from the fitted
i
IC curve using non-linear regression. (4 parameters) using the method in reference
5
0
b
2
7, see supporting information. Intact protein mass spectrometry. Entry 1: HSP72-
NBD [2.3 µM] and TCI [200 µM] incubated for the time indicated. Entries 2-4: HSP72-
NBD [2.0 µM] and TCI [20 µM] incubated for the time indicated. The MS of the resulting
protein/TCI adducts were analyzed using Agilent MassHunter Qualitative B.06.
ckinact/K values calculated from the covalent FP assay. The gradient of each slope was
I
calculated from the linear regression, representative example of N=3 independent
biological repeats, see supporting information for details.
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From these data, we concluded that although our TCI design was successful in
predicting that the binding site could accommodate the activated ester and maintain
reversible affinity, we had failed to account for the stereoelectronic requirements of the
electrophile. Efficient nucleophilic addition to the carbonyl must satisfy the correct
Bürgi-Dunitz33 and Flippin-Lodge angles34 at appropriate reaction distances. This
could not be achieved with p-fluorophenolate leaving group adopting the necessary
vector-to-solvent in the conformationally restrictive TCI reversible binding mode, thus
blocking covalent bond formation and the E-I complex.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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Design and synthesis of the third generation HSP72 TCI. The aryl sulfonyl fluoride
electrophile has recently become popular in both synthetic chemistry and chemical
biology.35, 36 Sulfonyl fluorides are stable in water under physiologically relevant
conditions and have previously been shown to react readily with lysine residues in
proteins.35, 36 A recent study by Grimster et al. demonstrated that the electrophilicity of
the moiety displays a strong dependence on the electronics of the attached aromatic
ring and can be modulated to give an intrinsic reactivity against glutathione,
comparable to chemical probe-relevant N-arylacrylamide electrophiles.37 The
solvation-dependent fluoride leaving group is less likely to form a steric clash and the
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proposed S N associative mechanism
should allow for a less restrictive
A
stereoelectronic requirement for the reaction in the conformationally rigid protein
binding site. We therefore hypothesized that incorporation of an aryl sulfonyl fluoride
electrophile would be effective in our Lys56-targeting HSP72 third generation TCI.
Unfortunately, our current methodology for the synthesis of 5’-benzylriboside ethers
proved incompatible with the incorporation of the sulfonyl fluoride electrophile, so we
adapted our TCI design to include an ester linker.
0
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2’,3’-acetonide protected adenosine 10 was selectively oxidized with bromine to give
1
1 in moderate yield. S Ar displacement with 4-chlorobenzylamine gave 12, which
N
then underwent selective esterification with 4-(fluorosulfonyl)benzoyl chloride to give
1
3 in 56% yield. The sulfonyl fluoride electrophile proved stable to the acetonide-
deprotection conditions and gave the third generation TCI 14, following treatment with
TFA/H O, in 4 steps and 13% overall yield. To determine the effect of the 5’-ester
2
linker on the reversible affinity, we synthesized the non-covalent ester MMP 15 of ether
9
in two steps from the primary alcohol intermediate 12 in 16% yield using 4-
cyanobenzoyl chloride (Scheme 2).
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Scheme 2. Synthesis of third generation aryl sulfonyl fluoride HSP72 TCIs
NH2
NH2
N
N
N
N
N
N
N
NH
NH
N
(ix), (x) (xii), (xiii)
N
O
S
O
S
F
F
O
(xi)
(xiv)
O
HO
HO
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
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0
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0
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0
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0
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2
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8
9
0
O
O
O
O
HO
HO
O
O
17
18
NH2
NH2
NH2
NH2
Cl
N
N
Cl
N
N
N
N
N
N
N
N
N
N
Br
NH
NH
(i)
(ii)
(v)
N
N
N
O
N
O
N
O
O
O
(vi)
O
HO
O
OH
OH
OH
O
O
O
O
HO
O
10
11
12
15
(vii) (viii)
(iii)
NH2
NH2
NH2
N
N
N
Cl
N
N
Cl
N
N
N
NH
NH
N
(iv)
N
N
N
O
O
S
O
S
O
F
F
F
O
O
O
S
HO
HO
O
O
O
O
O
O
O
HO
HO
O
O
O
16
14
13
Reagents and conditions: (i) Br , K HPO .3H O, 1,4-dioxane, H O, RT, 0.5 h, 74%; (ii)
2
2
4
2
2
4-chlorobenzylamine, EtOH, 160 ºC MW, 1h, 97%; (iii) 4-(fluorosulfonyl)benzoyl
o
chloride, Et N, DMF, 0 C, 4 h; 56% (iv) 5:2 TFA/H O, RT, 0.5 h, 32%; (v) 4-
3
2
o
cyanobenzoyl chloride, Et N, DMF, 0 C, 3 h; (vi) 5:2 TFA/H O, RT, 0.5 h, 16% over
3
2
two steps; (vii) 4-(fluorosulfonyl)benzoic acid, HBTU, Et N, DMF, RT, 2 h; 66%; (viii)
3
5:2 TFA/H O, RT, 0.5 h, 74%; (ix) 33% MeNH in EtOH, 160 ºC, MW, 1 h, 77%; (x) 4-
2
2
(
fluorosulfonyl)benzoic acid, HBTU, Et N, DMF, RT, 2 h; 35%; (xi) 5:2 TFA/H O, RT,
3
2
0.5 h, 40%; (xii) quinoline-6-yl methanamine, EtOH, 160 ºC, MW, 2 h, 8%; (xiii) 4-
(
fluorosulfonyl)benzoic acid, HBTU, Et N, DMF, RT, 2 h; 40%; (xiv) 5:2 TFA/H O, RT,
3
2
0.5 h, 11%.
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6
Characterization of the third generation TCI. The reversible 5’-ester analogue 15
displayed a binding affinity of K=3.5 μM (pK=5.45±0.01, N=3), >18-fold less potent
i
i
0
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than the tight-binding ether MMP 9 but sufficiently potent to investigate the role of the
electrophile in HSP72 TCI design. Therefore, the third-generation ester sulfonyl
fluoride TCI 14 was analyzed in the HSP72-NBD FP-assay. Pleasingly, 14 displayed
a clear time-dependent shift in the probe displacement curve, consistent with covalent
bond formation. The App. K=17 µM observed after 5 min exposure of TCI 14 was
i
comparable to the reversible ester analogue 15. The App. K appeared to increase in
i
activity 24-fold over 2 h. The MMP irreversible control 13 showed no reversible binding
affinity and no time-dependent displacement of the FP-probe. The analysis was
20
repeated using the HSP72-NBD K56A mutant (see supporting information): no time-
dependent shift in the probe displacement curve was observed suggesting no
significant formation of the covalent adduct with TCI 14 under the same conditions as
the WT-HSP72-NBD, confirming the reaction specificity and requirement for an initial
reversible binding event.
To confirm these results were due to covalent bond formation we then analyzed the
reaction by intact-protein MS. A solution of HSP72-NBD and p-sulfonyl fluoride (SF)
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TCI 14 (20 μM 14 and 2.0 μM HSP72-NBD) were incubated at 21 ºC (room
temperature) for 2 h. The experiment was repeated with irreversible control 13 under
the same conditions. These data revealed that SF TCI 14 formed a covalent bond with
HSP72-NBD, with the reaction going apparently to completion within three hours of
exposure using this semi-quantitative assessment. The irreversible control MMP 13
gave no reaction under these conditions.
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The covalent FP-assay. The timeframe of the App. K shift and intact protein MS with
i
the third generation SF TCI 14 strongly indicated that it was far more efficient than the
first-generation acrylate TCI 2 (Table 2, Entry 1 vs Table 2, Entry 4), as the formation
of the covalent adduct was reduced from days to hours. However, using these data
alone, it was not possible to quantify this optimization or to determine whether the
increased activity was due to an increase in the reversible binding affinity K, an
i
increase in efficiency of the covalent reaction kinact or a mixture of the two, although
analysis of the early time point App. K values did suggest the two analogues might
i
possess comparable reversible affinity. To deconvolute the TCI optimization, it would
be necessary to develop a new method to determine the kinetic parameters involved
in the irreversible inhibition of HSP72-NBD.
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