A Phosphoramidate Strategy Enables Membrane Permeability of a Non-nucleotide Inhibitor of the Prolyl Isomerase Pin1

Article abstract of DOI:10.1021/acsmedchemlett.0c00170

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.

Full text of DOI:10.1021/acsmedchemlett.0c00170

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Letter
A Phosphoramidate Strategy Enables Membrane Permeability of a
Non-nucleotide Inhibitor of the Prolyl Isomerase Pin1
Daniel M. C. Schwarz, Sarah K. Williams, Maxwell Dillenburg, Carston R. Wagner,
and Jason E. Gestwicki*
Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00170
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ABSTRACT: The membrane permeability of nucleotide-based drugs, such as
sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy,
termed “ProTide”, masks the anionic phosphate through an N-linked amino ester and an
O-linked aromatic phospho-ester, such that release of the active drug requires
consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as
Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the
ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that
caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its
permeability. Moreover, this compound was processed by both esterase and
phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in
cells. Thus, Hint1 appears to recognize a broader set of substrates than previously
appreciated. It seems possible that other potent, but impermeable, phosphate-containing
inhibitors might likewise benefit from this approach.
KEYWORDS: ProTide, Hint1, pro-drug, phosphate-containing, PPIase, oncology
restrictive substrate envelope that is dominated by polar
hosphate groups are important in molecular recognition
Pthroughout biology. However, inhibitors that incorporate interactions around the phosphate (Supporting Information
Figure 1).8 However, we noted that an adjacent, hydrophobic
phosphates or phospho-mimetics are often too anionic to be
passively permeable to biological membranes.1−3 Accordingly,
pocket, which is normally involved in accommodating the
nucleobase, was potentially more amenable to alternative
substrates (Supporting Information Figure 1). A search of
appropriate, phosphate-containing inhibitors in the literature
turned our attention to inhibitors of the peptidyl-prolyl
isomerase, Pin1.
Pin1 is considered an attractive cancer target, owing to its high
expression in breast and prostate tumors and the strong
antitumor effects of Pin1 knockdown.17 Pin1 is a two-domain
protein composed of a catalytic peptidyl-prolyl isomerase
domain and noncatalytic WW domain. Both of these domains
bind selectively to prolines that are adjacent to phosphorylated
Ser/Thr (e.g. the pS/T-Pro motif). Only the catalytic domain,
however, can isomerize this bond, facilitating interconversion
between cis and trans peptidyl−prolyl bonds.18 Potent inhibitors
of Pin1’s catalytic activity, such as 1-(R)-phosphate, were
described by Pfizer and the phosphate was found to impart
significant affinity, but these molecules were too polar to be
many prodrug strategies have been developed to mask
phosphates,4−6 allowing better balance between potency,
selectivity, and permeability. One of the most successful of
these prodrug approaches is the phosphoramidate-based
“ProTide” technology (Figure 1).7 This caging group relies on
the consecutive action of esterase activity on the O-carboxy
ester, followed by liberation of the phospho-ester by intra-
molecular nucleophilic attack and then hydrolysis of the N-
linked amino ester by a intracellular phosphoramidase, often the
histidine triad nucleotide binding protein 1 (Hint1).8 Together,
these activities deliver the active, phosphate-bearing molecule to
the cytosol. ProTide approaches have proven especially
successful in enhancing the cellular delivery of nucleotide-
based antivirals, including blockbuster drugs (i.e., sofosbu-
vir9,10), clinical candidates (i.e., NUC-103111), and tool
molecules (i.e., 4Ei-1012). However, the applicability of ProTide
technology to non-nucleotides is nascent; while phosphorami-
dates have been shown to improve the plasma lifetime and clogP
values for a handful of non-nucleotides,13−16 it is not yet clear
whether they can be enzymatically liberated in cells. Indeed,
Hint1 is a selective, metabolic enzyme, which might not be
considered likely to accept non-nucleotides that are structurally
distinct from its natural substrates. Crystal structures of
substrate-bound Hint1 have supported this idea, revealing a
Received: April 5, 2020
Accepted: July 30, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00170
© XXXX American Chemical Society
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A

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Figure 1. Schematic of phosphoramidate liberation in the cytosol. Phosphoramidates, such as sofosbuvir, are enzymatically liberated by a
carboxylesterase (i.e., CES1) and subsequent phosphoramidase activity (i.e., Hint1) to reveal the free phosphate form. This mechanism has been
exploited in multiple nucleotide-based drugs but has not yet been demonstrated to work for non-nucleotides.
a
Scheme 1. Synthesis of the Pro-drug 1-(R)-Phosphoramidate and Its Free Phosphate Form 1-(R)-Phosphate
a
Reaction conditions: (i) DIPEA, DCM, −78 °C to rt, 2 h; (ii) DIPEA, DCM, −78 °C to rt, 2 h;29 (iii) DIPEA, THF, rt, 18 h; (iv) (1) t-BuMgCl,
THF, rt, 1 h; (2) b, 18 h; (v) NBuHSO4, DMF, 80°C, 6 h.28.
membrane permeable.19 Attempts to improve these compounds
focused on replacing the phosphate and optimizing nonpolar
contacts.19−23 Alternatively, previous work has overcome the
poor permeability of phosphate-bearing Pin1 inhibitors using
bis-POM masking groups.24 Likewise, cyclic peptides25 and
covalent inhibitors lacking the phosphate have been ex-
plored.26,27 While these efforts yielded important insights into
the potential of Pin1 as a drug target, we envisioned a
complementary approach, in which ProTide technology might
B
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Figure 2. 1-(R)-Phosphate, but not the control, binds to Pin1’s catalytic domain in vitro. (A) Schematic of the constructs used in this study: a truncated
Pin1 lacking the WW domain (Pin1-Cat) and full length Pin1 (Pin1-FL). (B) 1-(R)-Phosphate, but not 1-(S)-phosphate, competed with a labeled
tracer (FITC-WFYpSPFLE; Pintide) for binding to Pin1-Cat, as measured by FP. Importantly, neither 1-(R) or 1-(S)-phosphoramidates bind Pin1.
Results are the average of triplicates and the error bars represent SD. (C) ITC experiment confirming that 1-(R)-phosphate binds Pin1-FL, and with a
stoichiometry ∼1, consistent with preferential binding to the catalytic domain. Importantly, 1-(S)-phosphate did not have detectable binding.
be used to increase the permeability of Pfizer’s molecule, 1-(R)-
phosphate (Figure 1).
human Pin1 (Pin1-Cat; residues 45−163) by fluorescence
polarization (FP). In these experiments, we estimated inhibition
constant (IC50) values, based on competition with a fluorescent
peptide FITC-WFYpSPFLE (PinTide) that is known to bind
and inhibit the Pin1 catalytic site. As expected, we found that the
1-(R)-phosphate (IC50 < 300 nM), but not 1-(S)-phosphate
(IC50 > 10,000 nM), bound to Pin1-Cat (Figure 2B). Also, we
confirmed that neither of the pro-drugs, 1-(R)-phosphoramidate
RESULTS AND DISCUSSION
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To probe this possibility, we first synthesized 1-(R)-phosphor-
amidate and its enantiomer, 1-(S)-phosphoramidate, as well as
the free phosphates: 1-(R)-phosphate and 1-(S)-phosphate
(Scheme 1), guided by reported routes.19,28,29 It is important to
note that the phosphoramidates contain an additional stereo-
center about the phosphorus atom and, in these proof-of-
concept studies, these compounds were used as a ∼1:1 mixture
of diastereomers (Supporting Information Figure 5). Based on
cocrystal structures, we anticipated that 1-(R)-phosphate would
bind Pin1, while 1-(S)-phosphate would be an important,
inactive control (Supporting Information Figure 2). To test this
idea, we measured binding to the purified catalytic domain of
or 1-(S)-phosphoramidate, were able to bind Pin1-Cat (IC50
>
10,000 nM). Having confirmed that 1-(R)-phosphate binds the
catalytic site, we turned to studying the full-length protein (Pin1-
FL). As mentioned above, Pin1-FL also contains a noncatalytic
WW domain, which has been shown to bind phosphorylated
peptides containing a trans-proline.30 We expected that 1-(R)-
phosphate might be selective for the catalytic site over the WW
domain, because it mimics the twisted-amide transition state
that is only preferred by that site.19,31 Indeed, using isothermal
C
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titration calorimetry (ITC), we found that 1-(R)-phosphate
bound Pin1-FL with a stoichiometry ∼1 (N = 0.94 0.03),
suggesting that it primarily interacts with the catalytic site
(Figure 2C). We also noticed that a dissociation constant (Kd)
of 1-(R)-phosphate for Pin1-FL (72 37 nM) was enhanced
over the value measured for binding the truncated Pin1-Cat (see
above); an improvement that was expected because similar
effects have been previously observed for Pin1 substrates.32
Together, these binding studies showed that 1-(R)-phosphate
binds Pin1 with the expected affinity and domain preference in
vitro.
Next, we measured the relative hydrophobicity and
permeability of 1-(R)-phosphoramidate and 1-(R)-phosphate.
Using octanol−water partitioning, the phosphoramidate was
calculated to be significantly more hydrophobic than the
phosphate (Table 1). Consistent with this difference, the
that 1-(R)-phosphate was indeed being processed by intra-
cellular enzymes, we repeated the extractions in media lacking
cells. In these controls, neither the intermediate nor 1-(R)-
phosphate were identified, confirming that enzymatic activity
was required. Together, these results suggest that 1-(R)-
phosphoramidate is cell-permeable and that it is converted to
its active form in cells.
The next question is whether the liberated 1-(R)-phosphate
might engage Pin1. Due to the low permeability of 1-(R)-
phosphate itself (see Table 1), this question could not previously
be addressed. To test it, we performed a cellular thermal shift
assay (CETSA). Specifically, K562 cells were treated with 1-(R)-
phosphoramidate (25 μM) or solvent alone (0.25% DMSO) for
5 h to allow for liberation of the active molecule. Then, cells were
heated on a temperature gradient, lysed, and the soluble fraction
assayed for Pin1 abundance by Western blot. We found that
Pin1 was partially protected by the compound treatment (Figure
4A), consistent with binding of 1-(R)-phosphate to Pin1. This
result was also repeated in MDA-MB-231 cells, a model of
metastatic breast cancer. This experiment was important
because Pin1 has been specifically implicated in both prostate
and breast cancers.17 In these experiments, we leveraged the
findings from the K562 studies and performed the CETSA near
the most sensitive, half-maximal temperature (48 °C). As in the
K562 cells, Pin1 was stabilized (Figure 4B). Together, these
results suggest that 1-(R)-phosphate is released from 1-(R)-
phosphoramidate and that it binds Pin1 in two cancer cell types.
To explore whether 1-(R)-phosphoramidate was converted
by the known, enzyme-based mechanism in the cytosol, we
employed an inhibitor of Hint1, TrpGc.33−35 In early experi-
ments, we had noticed that, at time points longer than 24 h,
treatment of MDA-MB-231 cells with 1-(R)-phosphoramidate
led to a dose-responsive increase in Pin1 levels, even at normal
temperatures (Figure 5A). Using this biomarker, we found that
cotreatment with TrpGc (100 μM) blocked the cellular activity
of 1-(R)-phosphoramidate (Figure 5B), supporting an essential
role for Hint1.
Table 1. Installation of a Phosphoramidate Dramatically
a
Improves Hydrophobicity and Permeability
Compound
logP
logPe (cm/s)
1-(R)-phosphoramidate
1-(R)-phosphate
0.27
<−4.6
−4.6
<−6.3
a
Partitioning coefficient (logP) values were determined by equili-
brium octanol water partitioning and permeability constant (logPe)
by PAMPA. The levels of 1-(R)-phosphate were near limit of
detection (LOD).
phosphoramidate was also more permeable (Table 1).
Encouraged by this result, we then explored whether 1-(R)-
phosphoramidate might be enzymatically liberated to 1-(R)-
phosphate in cells. To ask this question, K562 cells were treated
for 5 h under serum-free conditions, followed by extensive
washing, centrifugation, ethyl acetate extraction, and measure-
ment of the reaction products by ultrahigh-performance liquid
chromatography mass spectrometry (UPLC-MS). Satisfyingly,
both the intermediate product of esterase activity and the 1-(R)-
phosphate product were detected in the treated K562 cell lysate
(Figure 3A). In addition, a small amount of the dephosphory-
lated metabolite was also present and its identity confirmed with
an authentic standard. The free phosphate peak increased with
time, consistent with enzymatic turnover (Figure 3B). To ensure
Finally, we wanted to ask whether pharmacological inhibition
of Pin1 would replicate the effects seen in knockdown studies.
Because Pin1 has both catalytic and WW domains that bind to
pS/T-Pro motifs, it is not clear from the knockdown
experiments which subset of its cellular roles might be mediated
Figure 3. Cellular liberation of 1-(R)-phosphate by cytoplasmic enzymes. K562 cells treated with 1-(R)-phosphoramidate were washed, pelleted, and
extracted with EtOAc. (A) The extract was analyzed by UPLC-MS to yield the base peak chromatogram (black). Then, the peaks corresponding to the
mass of the phosphoramidate (purple), the phosphate (green), and the dephosphorylated metabolite (gray) were identified in the treated sample and
compared to the approximate elution window of the authentic standards (bottom). (B) To understand release of the phosphate product over time, a
time course experiment was conducted and the peak area quantified. A solvent control (DMSO) was used to subtract the background. The averages of
duplicate experiments are shown, with the full range.
D
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Figure 4. Treatment with 1-(R)-phosphoramidate leads to Pin1 target engagement, by CETSA. (A) K562 cells were treated with 1-(R)-
phosphoramidate, heated at temperatures between 38 to 60 °C, and the soluble fraction assayed for Pin1 abundance by Western blot. Treatment with
1-(R)-phosphoramidate led to stabilization of Pin1, compared to the mock treated (quantified below). Results are the average of three independent
experiments, and error bars represent SEM. (B) Similar results were observed in MDA-MB-231 cells, treated at a fixed temperature (48 °C) in
biological quadruplicates (quantified below). **p value <0.01.
Figure 5. 1-(R)-Phosphoramidate is released in cells, leading to Pin1 binding and inhibition. (A) Treatment of MDA-MB-231 cells with 1-(R)-
phosphoramidate for 72 h results in a dose-responsive increase in Pin1 abundance. (B) Treatment with a Hint1 inhibitor, TrpGc, suppresses this 1-
(R)-phosphoramidate activity. (C) 1-(R)-phosphoramidate, but not the control, inhibited colonogenic activity in treated PC3 cells. Results are the
average of experiments performed in triplicate, and the error bars represent SD. A representative dose series is shown below. **p value <0.01.
by catalytic peptidyl−prolyl isomerization. One of the best
characterized biological roles for Pin1 is in tumor colony
formation; as Pin1 knockdown is reported to inhibit
colonogenic potential.36 Using PC3 neuroendocrine prostate
cancer cells, we tested whether treatment with 1-(R)- or 1-(S)-
phosphoramidate might reduce colony formation. We found
that 1-(R)-phosphoramidate, but not 1-(S)-phosphoramidate,
inhibited colony formation (Figure 5C), suggesting that
isomerase activity is indeed important for this role. Importantly,
treatment with 1-(R)-phosphoramidate did not significantly
reduce proliferation of more confluent PC3 cells after 72 h,
based on either ATP-Glo or MTT assays (Supporting
Information Figure 4). Thus, Pin1 plays a role in colony
formation at low density, but it does not seem to have a major
function in survival signaling at high cell density. Indeed,
knockdown of Pin1 or inhibition by other approaches shows a
similar result.27,36,37
Together, these results suggest that 1-(R)-phosphoramidate is
liberated by Hint1 in cells, releasing 1-(R)-phosphate, which
then binds Pin1’s catalytic site and inhibits some of its functions.
Thus, we propose that 1-(R)-phosphoramidate will be a useful
chemical probe, enabling future studies into Pin1’s enigmatic
roles in cancer. Such future studies would also benefit from
understanding which phosphoramidate diastereomer is a better
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substrate for Hint1, as well as quantitative measurements of its
cytosolic conversion kinetics.
chromatography; MS, mass spectrometry; CETSA, cellular
thermal shift assay; PAMPA, parallel artificial membrane
permeability assay
More generally, this work provides initial evidence that the
“ProTide” approach is more broadly applicable than previously
appreciated. We speculate that additional evaluation of Hint1’s
substrate envelope might open this approach to additional
scaffolds. For example, molecular recognition in kinase/
phosphatase signaling, 14−3−3 scaffolding and nucleoside
metabolism involves selective binding of phosphates.38 As
envisioned, this pro-drug approach rebalances the interplay
between permeability and potency, allowing chemical probes to
include the natural phosphate that is so important in molecular
recognition.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00170.
■
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Experimental details, supporting figures (S1−S5), and
analytical characterization. (PDF)
AUTHOR INFORMATION
Corresponding Author
Jason E. Gestwicki − Department of Pharmaceutical Chemistry,
University of California San Francisco, San Francisco, California
94158, United States; orcid.org/0000-0002-6125-3154;
Email: Jason.gestwicki@ucsf.edu
■
Authors
Daniel M. C. Schwarz − Department of Pharmaceutical
Chemistry, University of California San Francisco, San Francisco,
California 94158, United States
Sarah K. Williams − Department of Pharmaceutical Chemistry,
University of California San Francisco, San Francisco, California
94158, United States
Maxwell Dillenburg − Department of Medicinal Chemistry,
University of Minnesota, Minneapolis, Minnesota 55455, United
States
Carston R. Wagner − Department of Medicinal Chemistry,
University of Minnesota, Minneapolis, Minnesota 55455, United
States; orcid.org/0000-0001-7927-719X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00170
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a predoctoral fellowship (1 F31
CA232325-01A1 to D.M.C.S.) and training program Cancer
Cell Map Initiative (U54CA209891 to D.M.C.S.) and grants
from the University of California Drug Discovery Consortium
(UCDDC; to J.E.G.), the UCSF InVent Fund (to J.E.G.), and
the University of Minnesota Foundation (to C.R.W.). The
authors thank Bryan Dunyak for early contributions to the
project.
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ABBREVIATIONS
■
Hint1, histidine triad nucleotide binding protein 1; Pin1,
petidiyl−prolyl isomerase NIMA interacting 1; CES1, carbox-
ylesterase 1; FP, fluorescence polarization; ITC, isothermal
titration calorimetry; UPLC, ultrahigh performance liquid
F
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