Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue

Article abstract of DOI:10.1021/acschembio.7b00403

Irreversible enzyme inhibitors and covalent chemical biology probes often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine residues are not always available for site-specific protein labeling, and therefore new approaches are needed to expand the toolkit of appropriate electrophiles ("warheads") that target alternative amino acids. We previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled the development of clickable probe technology to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-containing inhibitor (aryl fluorosulfate probe (FS-p1)) with excellent chemical and metabolic stability that reacts selectively with a noncatalytic serine residue in the same active site of DcpS as confirmed by peptide mapping experiments. Our results suggest that noncatalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chemical probes.

Full text of DOI:10.1021/acschembio.7b00403

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Letter
Covalent Enzyme Inhibition through Fluorosulfate
Modification of a Non-Catalytic Serine Residue
Olugbeminiyi O Fadeyi, Lise R. Hoth, Chulho Choi, Xidong Feng, Ariamala
Gopalsamy, Erik C. Hett, Robert E. Kyne, Jr., Ralph P. Robinson, and Lyn H. Jones
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00403 • Publication Date (Web): 18 Jul 2017
Downloaded from http://pubs.acs.org on July 20, 2017
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Tyr143
Ser272
•
•
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Metabolically stable
Water stable
Selective labelling
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Covalent Enzyme Inhibition through Fluorosulfate
Modification of a NonꢀCatalytic Serine Residue
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§
∫
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‡
Olugbeminiyi O. Fadeyi, * Lise R. Hoth, Chulho Choi, Xidong Feng, Ariamala Gopalsamy,
‡
,∫
§,†
§
‡#
Erik C. Hett Robert E. Kyne, Jr., Ralph P. Robinson and Lyn H. Jones *
‡
Medicine Design, Pfizer Inc, 610 Main Street, Cambridge, Massachusetts, 02139, USA
§
Medicine Design, Pfizer Inc, Eastern Point Road, Groton, Connecticut, 06340, USA
Abstract
Irreversible enzyme inhibitors and covalent chemical biology probes often utilize the reaction of a protein cysteine
residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine
residues are not always available for siteꢀspecific protein labeling and therefore new approaches are needed to
expand the toolkit of appropriate electrophiles (“warheads”) that target alternative amino acids. We previously
described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger
enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled
the development of clickable probe technology to measure drugꢀtarget occupancy in live cells. Here we describe a
fluorosulfateꢀcontaining inhibitor (aryl fluorosulfate probe (FSꢀp1)) with excellent chemical and metabolic stability
that reacts selectively with a nonꢀcatalytic serine residue in the same active site of DcpS as confirmed by peptide
mapping experiments. Our results suggest that nonꢀcatalytic serine targeting using fluorosulfate electrophilic
warheads could be a suitable strategy for the development of covalent inhibitor drugs and chemical probes.
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Cysteineꢀtargeted irreversible enzyme inhibitors have found considerable utility in drug
1
discovery, particularly in the development of kinase inhibitor drugs and chemical probes.
Covalent enzyme inhibitors possess the potential advantages of high potency and enhanced
pharmacodynamic duration. However, in cancer there are several examples of cysteineꢀtoꢀserine
mutations causing reductions in efficacy of cysteineꢀreactive drugs and drug candidates. Also,
many enzymes do not possess a targetable cysteine residue within the active site. Drugs that
specifically target other amino acid residues could expand the synthetic biology toolbox and
significantly enhance the opportunities for covalent inhibitor development.
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Our group was the first to rationally design irreversible inhibitors that target tyrosine residues
in the active site of an enzyme. Sulfonyl fluoride probes (e.g. aryl sulfonyl fluoride probe (SFꢀ
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,
p1), Figure 1) were designed to react with Tyr113 and Tyr143 in the binding site of the mRNA
decapping scavenger enzyme DcpS, a pyrophosphatase responsible for the hydrolysis of the
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m GTP cap from mRNA fragments, and involved in mRNA and microRNA metabolism.
Recently, inhibitors of DcpS have been under investigation for the treatment of spinal muscular
5ꢀ7
atrophy (SMA), all based on a 2,4ꢀdiaminoquinazoline (DAQ) template that we used as the
basis for probe design, including a clickable SF covalent probe, SFꢀp1ꢀyne (Figure 1) to report
2
, 8
on drugꢀtarget engagement in live cells for the first time.
Figure 1. Structures of diaminoquinazoline (DAQ) inhibitors of DcpS.
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Sulfonyl fluorides are somewhat privileged electrophiles for use in chemical biology studies as
they possess contextꢀdependent protein reactivity balanced with adequate water stability for most
applications. Sulfonyl fluorides have been shown to react with a number of amino acid residues
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(Tyr, Lys, Ser, Thr, Cys, His) in protein binding sites and probes containing this electrophile
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have found considerable utility in chemical biology research.
The application of a fluorosulfate covalent electrophile (or “warhead”), however, has been
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reported to a much lesser extent. Recent work by the Kelly and Sharpless groups has shown
that fluorosulfates are significantly less reactive in a whole cell proteome than the better known
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sulfonyl fluorides. They showed that an aryl fluorosulfate chemical probe selectively modified
a conserved tyrosine residue in the binding site of intracellular lipid binding proteins (iLBPs). A
selective covalent inhibitor bearing a biphenyl core structure labeled Tyr134 in retinoic acid
binding protein 2 (CRABP2) in cells and inhibited RARαꢀmediated retinoic acid signaling in a
breast cancer cell line.
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The presence of two sulfonyl fluorideꢀreactive tyrosine residues in the DcpS binding site
suggested that a fluorosulfateꢀcontaining diaminoquinazoline (DAQ) may covalently modify and
inhibit enzymatic function. Below we describe the preparation of the fluorosulfate congener (aryl
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fluorosulfate probe (FSꢀp1)) of the aryl sulfonyl fluoride probe SFꢀp1 and, surprisingly, it was
found to selectively label a nonꢀcatalytic serine residue in the mRNA capꢀbinding site of DcpS,
as shown by peptide mapping experiments (Figure S3). Further profiling of FSꢀp1 suggests that
fluorosulfate targeting of serine residues in protein binding sites could be a viable strategy for
covalent inhibitor and chemical biology probe design.
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Although sulfonyl fluorides such as SFꢀp1 and the clickable probe SFꢀp1ꢀyne are useful
chemical biology probes, the relatively high reactivity of the sulfonyl fluoride warhead presents a
challenge for the development of selective and targeted covalent inhibitor drug candidates. For
instance, the instability of SFꢀp1 prevents the measurement of metabolic turnover rate in human
liver microsomes (HLM) and shakeꢀflask LogD using wellꢀestablished assays developed at
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Pfizer (Supporting Information, Figure S1).
We decided to explore an alternative
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electrophile, aryl fluorosulfate, which would be predicted to be considerably more stable, and
thus potentially more suitable as a drugꢀcompatible moiety for targeted covalent inhibitor
discovery.
To further assess and compare the intrinsic potential for ‘offꢀtarget’ protein reactivity of aryl
sulfonyl fluoride and fluorosulfate electrophiles, the fragments benzene sulfonyl fluoride (BSF)
and benzene fluorosulfate (BFS) were exposed to human serum albumin (HSA) and the
propensity for adduct formation was measured using LCꢀMS of intact protein. Neither fragment
reacted with HSA even when incubated with the protein for 24 h at 23°C, in the absence of light
(1 ꢀM HSA, 10 ꢀM compound), reflecting the lack of broad, nonꢀspecific protein reactivity of
these electrophiles. However, multiple adducts with BSF were observed when the concentration
was increased (1 ꢀM HSA, 300 ꢀM compound, Figure 2, panel a) while BFS showed minimal
adduct formation at these concentrations. A similar trend occurred when fluorosulfate probe FSꢀ
p1 and sulfonyl fluoride probe SFꢀp1 were exposed to HSA. Under these conditions, FSꢀp1 does
not label HSA, but some labeling occurred with the SFꢀp1 probe (Figure 2, panel b). These
results suggest that an aryl fluorosulfateꢀcontaining ligand may be more discerning in its protein
reactivity relative to an aryl sulfonyl fluoride.
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Figure 2. a) Intact protein MS of the reaction of benzene sulfonyl fluoride (BSF, 300 ꢀM) or
benzene fluorosulfate (BFS, 300 ꢀM) with human serum albumin (HSA, 1 ꢀM) after 24 hours.
b) Intact protein MS of the reaction of FSꢀp1 (5 ꢀM) or SFꢀp1 (5 ꢀM) with human serum
albumin (HSA, 0.5 ꢀM) after 24 hours.
We therefore decided to explore the potential reactivity of a DAQ inhibitor of the DcpS
enzyme bearing the fluorosulfate warhead, in order to compare this with the SFꢀp1 derivative.
The aryl fluorosulfate FSꢀp1 was prepared using the chemistry outlined in Scheme 1 (details
provided in the Supporting Information). The route relies on the recently developed reaction of
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phenols with sulfuryl fluoride gas to form fluorosulfates. pꢀCresol (1) was converted to aryl
fluorosulfate 2, followed by benzylic bromination to form 3. Subsequent alkylation of known
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phenol 4 yielded 5 and Bocꢀdeprotection furnished fluorosulfate probe FS-p1.
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Scheme 1. Preparation of DcpS Covalent Inhibitor FS-p1.
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The superior hydrolytic stability of FSꢀp1 relative to SFꢀp1 was confirmed in aqueous buffer
solutions. Incubation of SFꢀp1 in phosphate buffered saline (pH 7.4) for just 2 hours resulted in
considerable hydrolysis (measured by HPLC) while FSꢀp1 was completely intact after 24 hours
(Figure 3).
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Hydrolysis of FS-p1 and SF-p1
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Figure 3. HPLC traces showing the hydrolytic stability of FSꢀp1 and SFꢀp1. Compounds were
dissolved in 100 ꢀM PBS (pH 7.4): 5 ꢀL of 30 mM DMSO stock solution was added to 895 ꢀL
of PBS solution. HPLC: 12.5 minute run; 5 ꢀL aliquot; MeCN/H O with 0.1% TFA; traces at
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254 nm.
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FSꢀp1 was screened in a DcpS ELISA assay using a biotinylated cap substrate (m GpppA),
and was found to inhibit enzymatic function with an IC = 3.2 nM (Table 1). This value is
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comparable to the unsubstituted derivative 5ꢀbenzyloxyꢀ2,4ꢀdiamino quinazoline (compound
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DAQ1, Table 1),
although FSꢀp1 was significantly less potent than the more reactive covalent
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inhibitor SFꢀp1 in this assay (IC < 0.02 nM). Fluorosulfate FSꢀp1 was incubated with DcpS
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for 24 hours and intact mass LCꢀMS analysis confirmed the presence of a small moleculeꢀprotein
adduct (with a concomitant loss of fluoride, Figure 4b and Supporting Information). However,
the main species detected by MS had lost 18 mass units, corresponding to a protein dehydration
event (Figure 4d). The mass of another minor product agreed with a combination of the potential
dehydration as well as adduct formation with FSꢀp1 (and loss of fluoride), that is DhaꢀDcpS
undergoing an additional reaction with FSꢀp1.
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Figure 4. a) LCꢀMS of DcpS (MS adduct is acetonitrile clusters of DcpS (adduct mass =
41.02655) seen in the LCT Premier mass spectrometer) and b) DcpS treated with FSꢀp1 (0.5 ꢀM
protein/5 ꢀM probe) for 24 h at 23°C, in the absence of light. c) Crystal structure of SFꢀp1 (blue)
and DcpS (PDB 4QDV) showing reaction with Tyr143 (pink). FSꢀp1 (gold) docked into the
DcpS binding site (based on 4QDV structure) where the diaminoquinazoline motif was
constrained to the SFꢀp1 binding conformation and the remainder of the ligand was energy
minimized in the pocket. The proximity of the fluorosulfate electrophile to Ser272 (green) is
apparent. Key basic residues proximal to Ser272 are also highlighted: histidine triad (magenta);
Arg294 (cyan); Lys142 (red). d) DcpSꢀDha formation via βꢀelimination of DcpSꢀFSꢀp1 adduct.
Interested by these observations, we decided to further elucidate the site of protein
modification using peptide mapping experiments. The DcpS/FSꢀp1 adduct was treated with
trypsin and the digested peptides were analyzed by LCꢀMS (Supporting Information, Figure S2).
This work confirmed that the major species following reaction with FSꢀp1 and MS analysis
corresponded to dehydration of the Ser272 residue, resulting in the formation of dehydroalanine
at this position. These results are reminiscent of the chemical mutagenesis conversion of serine
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to cysteine hydrolases using sulfonyl fluorides. This reaction creates a sulfonate ester that at
higher pH eliminates to form an intermediate dehydroalanine species (Figure 4d).
It is possible that the presence of the Dha272 is an artifact of the analytical technique, and we
therefore further investigated the potential covalent modification of DcpS using native
electrospray ionization (ESI) MS. This milder technique showed that DcpS exists as a dimer (as
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revealed previously) and the major species following reaction with FSꢀp1 corresponded to an
adduct with 2:2 FSꢀp1/DcpS stoichiometry (minus 2 fluorides, Figure S3). A minor species was
detected that corresponded to dimer with one intact FSꢀp1 adduct and one dehydrated DcpS
species that increased over time (Figure S3). Although more studies are required to understand
these observations, it is possible that the acidic nature of the LCꢀMS mobile phase accelerates
dehydroalanine formation, and that elimination can also occur slowly in solution at pH 7.4.
The labeling of Ser272 is an important finding as this residue is known to contact the βꢀ
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phosphate of the m GTP cap but is not involved in catalytic activity since S272A retains the
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same activity as wildꢀtype enzyme. Ligandꢀmediated covalent modification of a nonꢀcatalytic
serine residue in a protein binding site is extremely rare, whilst labeling of the activated catalytic
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serine in serine hydrolases is wellꢀknown. To our knowledge, the only other confirmed
example is that of irreversible inhibition of cyclooxygenase enzymes COXꢀ1 and COXꢀ2 by
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aspirin through acetylation of Ser530 near the active site. In this example, there is a charge
interaction between the aspirin carboxylate and Arg120, and hydrogen bonding between Tyr385
and the acetyl group that facilitate the transacylation reaction (the carboxylate also acting as a
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general base to abstract the proton from the Ser530 hydroxyl substituent).
Similarly, a closer inspection of DcpS crystal structures with DAQ ligands provides some
insight into our results. The very low intrinsic reactivity of aryl fluorosulfates puts a strong
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emphasis on the need for an appropriate orientation of the ligand to facilitate labeling with a
suitably reactive amino acid in the binding site. In our previous work, the rational design of
sulfonyl fluorideꢀcontaining DAQs optimally placed the electrophile proximal to the nucleophilic
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Tyr113 and Tyr143 residues. The addition of an oxygen atom tether from the aromatic ring to
the electrophilic sulfur atom of the fluorosulfate hinders a conformation that could provide a
similar trajectory for reaction with the same tyrosine residues. Interestingly, Ser272 contacts a
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DAQ reversible inhibitor, D156844 (PDB 3BL7, Figure S4), and work from our group has
confirmed that this region of the binding site can accommodate a wide variety of alternative
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ligand substituents. Docking of FSꢀp1 into the DcpS binding site suggests that a desirable
conformation of the electrophilic aryl fluorosulfate and nucleophilic serine residue is attainable
(Figure 4c). Ser272 is also in proximity to basic amino acid residues (Arg294, Lys142) and is
located in the vicinity of the histidine triad motif (His268, His277, His279 – Figure 4c and S6).
As a result, this region of the binding pocket not only accommodates small molecule ligands, but
may also perturb the pK of the serine hydroxyl and/or enhance the leaving group ability of the
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fluoride ion from FSꢀp1 through hydrogenꢀbonding interactions – further work is warranted to
explore the possible mechanisms at play. In order to obtain information about the extent and rate
of reaction between DcpS and FSꢀp1, time course experiments were conducted using an MS
readout. The reaction mixture of 0.5 ꢀM DcpS and 5 ꢀM FSꢀp was stored at roomꢀtemperature
and continuously analyzed after defined time intervals. Figure S5 shows the rate of reaction of
2
DcpS with FSꢀp1 (Figure S5) is slower than with SFꢀp1 (which is complete within 1 hour), as
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one might expect based on the higher intrinsic reactivity of sulfonyl fluorides. These results
may also explain why SFꢀp1 is considerably more potent than FSꢀp1 in the DcpS ELISA assay,
where the compounds were incubated with the enzyme for 45 minutes (see SI). Since FSꢀp1 was
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not specifically designed to react with Ser272, further optimization of the equilibrium binding
component of aryl fluorosulfate DcpS inhibitors could potentially enhance the templated
conjugation rate with Ser272. Consistent with nonꢀcovalent interactions catalyzing the
conjugation, we found that fragments BSF and BFS, which would not be expected to possess
significant binding affinity for DcpS, did not react with the protein under these conditions (0.5
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ꢀM DcpS, 10 ꢀM probe, 18 hours incubation).
Table 1. Potency, metabolic stability and cell permeability of diaminoquinazoline DcpS
inhibitors
Compound
DAQ1 FS-p1
DcpS IC (nM)
1.0
266
2.0
20
3.2
364
2.7
5
0
MW
SFLogD
HLM (ꢀL/min/mg)
10.6
2.0
ꢀ6
MDCK/MDR1 AB (10 cm/s) 7.9
ꢀ6
MDCK/MDR1 BA (10 cm/s) 12
6.0
Finally, we wanted to ascertain the suitability of the fluorosulfate electrophile as a motif that
may be used in the design of small molecule drugs, beyond its utility in chemical biology probes.
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Metabolic stability was assessed in human liver microsomes (HLM) and the rate of turnover of
FSꢀp1 was lower than the unsubstituted derivative DAQ1, even though the lipophilicity of FSꢀp1
is higher (ꢁLogD 0.7 units, Table 1). FSꢀp1 also possessed adequate membrane permeability, as
measured in the Madin Darby Canine Kidney (MDCK) epithelial cell assay (transfected with the
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Pꢀglycoprotein efflux protein MDR1), although flux was found to be lower than DAQ1, and
there appeared to be evidence of efflux (Table 1). The high hydrogen bond donor capacity of the
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DAQ head group, combined with the additional Hꢀbonding groups present in FSꢀp1 are the likely
23
reasons for these observations.
In conclusion, the selective labeling of a nonꢀcatalytic serine residue in the DcpS binding site
by FSꢀp1 combined with its excellent metabolic and chemical stability, illustrate that the
fluorosulfate motif is suitable for incorporation into covalent drug design strategies. It will be
interesting to assess the in vivo pharmacodynamic and pharmacokinetic profiles of such
compounds in the future. The electropositive nature of the microenvironment of the reactive
Ser272 in DcpS and the proximal nature of basic amino acid residues may provide insight into
the propensity of other nonꢀcatalytic serine residues in proteins to be targeted with fluorosulfate
electrophiles.
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The identification of a dehydrated (dehydroalanine) species is intriguing as it suggests that
elimination of the intermediate sulfate adduct could be optimized, potentially through further
design of the ligand. From the perspective of synthetic biology, the ability to siteꢀspecifically
convert a nonꢀcatalytic serine residue to a dehydroalanine amino acid using fluorosulfate
chemistry is tantalizing as it could provide a route to incorporate a general chemical mutagenesis
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handle in proteins. We have explored selective fluorosulfate and sulfonyl fluoride labeling of a
number of proteins and the presence of a dehydroalanine species in the LCꢀMS is diagnostic of
adduct formation with serine or threonine residues. A future direction for this work could
therefore be the siteꢀspecific formation of dehydroalanine derivatized proteins in live cells that
may provide useful avenues for biological engineering and chemical biology research.
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The results presented here, and recent advances by other groups,
suggest that a vast
unchartered opportunity space lies ahead for the development of nextꢀgeneration covalent
modulators that target natural protein residues beyond cysteine.
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AUTHOR INFORMATION
Corresponding Authors
*Email: lynhowardjones@gmail.com; ljones@jnanatx.com, olugbeminiyi.fadeyi@merck.com
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Present Addresses
#
Jnana Therapeutics, 50 Northern Avenue, Boston, MA, 02210, USA
∫
Merck – Exploratory Sciences Center, Cambridge, Massachusetts, 02141, USA
†
Celgene Corporation, 200 Cambridge Park Drive, Cambridge, Massachusetts, 02140, USA
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
All authors were employees and shareholders of Pfizer when the research described in this
manuscript was performed. Research was conducted in accordance with all acceptable Pfizer
policies including IRB/IEC approval.
ACKNOWLEDGMENT
We thank C. Menard, A. Narayanan and L. Roberts for helpful discussions.
ASSOCIATED CONTENT
Supporting Information. Metabolic stability of FSꢀp1 in human liver microsomes; human DcpS
biochemical assay; DcpS intact LCMS, peptide mapping and native ESI MS experiments;
analysis of DcpS Ser272 microenvironment; time course of the reaction of FSꢀp1 with DcpS;
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synthetic methods and preparation of FSꢀp1. This material is available free of charge via the
Internet at http://pubs.acs.org.
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