Targeted Covalent Inhibition of Plasmodium FK506 Binding Protein 35

Article abstract of DOI:10.1021/acsmedchemlett.0c00272

FK506-binding protein 35, FKBP35, has been implicated as an essential malarial enzyme. Rapamycin and FK506 exhibit antiplasmodium activity in cultured parasites. However, due to the highly conserved nature of the binding pockets of FKBPs and the immunosuppressive properties of these drugs, there is a need for compounds that selectively inhibit FKBP35 and lack the undesired side effects. In contrast to human FKBPs, FKBP35 contains a cysteine, C106, adjacent to the rapamycin binding pocket, providing an opportunity to develop targeted covalent inhibitors of Plasmodium FKBP35. Here, we synthesize inhibitors of FKBP35, show that they directly bind FKBP35 in a model cellular setting, selectively covalently modify C106, and exhibit antiplasmodium activity in blood-stage cultured parasites.

Full text of DOI:10.1021/acsmedchemlett.0c00272

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Targeted Covalent Inhibition of Plasmodium FK506 Binding Protein
35
Thomas C. Atack,* Donald D. Raymond, Christa A. Blomquist, Charisse Flerida Pasaje,
Patrick R. McCarren, Jamie Moroco, Henock B. Befekadu, Foxy P. Robinson, Debjani Pal,
Lisl Y. Esherick, Alessandra Ianari, Jacquin C. Niles, and William R. Sellers*
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ABSTRACT: FK506-binding protein 35, FKBP35, has been
implicated as an essential malarial enzyme. Rapamycin and
FK506 exhibit antiplasmodium activity in cultured parasites.
However, due to the highly conserved nature of the binding
pockets of FKBPs and the immunosuppressive properties of these
drugs, there is a need for compounds that selectively inhibit
FKBP35 and lack the undesired side effects. In contrast to human
FKBPs, FKBP35 contains a cysteine, C106, adjacent to the
rapamycin binding pocket, providing an opportunity to develop
targeted covalent inhibitors of Plasmodium FKBP35. Here, we
synthesize inhibitors of FKBP35, show that they directly bind
FKBP35 in a model cellular setting, selectively covalently modify
C106, and exhibit antiplasmodium activity in blood-stage cultured
parasites.
KEYWORDS: FK506-binding protein, FKBP35, plasmodium, antimalarial, targeted covalent inhibition
alaria is caused by parasites of the genus Plasmodium.
MWorldwide, there are over 200 million cases every year.
Despite progress in antimalarial therapies and in mosquito-
control, over 400,000 people die annually, the majority
resulting from Plasmodium falciparum (Pf).1 Additionally,
Plasmodium vivax (Pv) causes relapsing infection due to latent
liver-stage parasites which remains a challenge for malaria
eradication.1,2 Finally, resistance to the frontline antimalarials
is emerging, accelerating the need for new therapeutics with
novel mechanisms of action.3−5
FK506-binding proteins (FKBP) are conserved throughout
evolution and found across the Plasmodium genus.6−8
Immunosuppression mediated by inhibitors of calcineurin
and mTOR requires recruitment of FKBPs.9−11 Specifically,
FK506 and rapamycin (Figure 1) induce heterodimers
between human FKBP12 and calcineurin or mTOR,
respectively. As a result, FKBPs became targets in drug-
discovery.12,13
Figure 1. Structures of FKBP ligands with IC50s in blood-stage
antiplasmodium assays (SLF is untested against cultured parasites).
The pipecolate binding motif common to the natural product ligands
is in red.
In contrast to the 14 human FKPB isoforms, Plasmodium
species have a single FKBP, FKBP35.7,8 Interestingly, FK506
and rapamycin have antiplasmodium activity in blood-stage
assays6,14,15 and it has been hypothesized that inhibition of
FKBP35 alone might suffice for antiparasitic activity.6,14
Several groups have synthesized FKBP35-targeting compounds
(Figure 1). Wandless et al. developed chimeric molecules
consisting of the synthetic ligand for FKBP (SLF) conjugated
Received: May 21, 2020
Accepted: September 1, 2020
Published: September 1, 2020
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to methotrexate to deliver methotrexate to parasites through
FKBP12 binding but did not report the antiplasmodium
activity.16 Compounds SRA and D44 exhibited good
antiplasmodium and PPIase activity despite lacking the
pipecolate core common to many FKBP-binders (Figure 1,
in red).17,18 More recently, a library of novel [4.3.1]-aza-
bicyclic sulfonamides ([4.3.1]-ABS) exhibited broad anti-
microbial activity, including activity against Plasmodium.19
To date, all reported inhibitors show higher binding affinity
for human FKBP12. This lack of selectivity is amplified by the
cellular abundance of FKBP12 (4−5 μM in erythrocytes)
versus FKBP35 (50−100 nM in Plasmodium).16 A difficulty in
achieving selectivity among different FKBPs is the highly
conserved proline binding pocket where 13 out of 16 residues
within 5 Å of rapamycin are identical between FKBP12 and
Pf FKBP35. Interestingly, just outside of the binding pocket,
Pf FKBP35 has a cysteine (C106) whereas FKBP12 has a
histidine at this position. This cysteine is conserved across the
human-infecting Plasmodium species and thus provides the
opportunity to develop FKBP35 covalent inhibitors.20 Such
therapeutics might provide improved selectivity and prolonged
duration of action and could be interesting for targeting the
liver hypnozoite stage of Plasmodium vivax.
Figure 3. Docking studies of covalent SLF analogues. (A) Structures
of representative compounds from Series 1 and Series 2. (B) Model
structure of 1a (purple) covalently bound to C106 (indicated by
arrow) and docked in FBD35 with SLFb (gray) as a reference. (C)
Model structure of 2a (yellow) covalently bound to C106 (indicated
by arrow) and docked in FBD35 with SLFb (gray) as a reference.
Docking model based on alignment of SLFb-bound FKBP51 binding
domain (PDB 4DRK) and rapamycin-bound FKBP35 binding
domain (PDB 4QT2).
We began by modifying D44 (Figure 1) to generate three
analogues (Figure 2) and modeled binding of these analogues
Next, we investigated both series to probe the reactivity of the
cysteine and determine how the vector of the covalent bond
between the ligand and FKBP35 influences compound
binding.
For Series 1 (Scheme 1), a convergent synthesis was
employed between pipecolic acid 3 or 4 with chiral benzylic
alcohols 6a−d. Compounds 5a−d were synthesized via
Claisen−Schmidt condensation between methylvanillin and
2′-, 3′-, or 4′-nitroacetophenone followed by hydrogenation.
meta-Aniline 5b was Boc-protected to generate compound 6b.
Coupling of 3 and 6b followed by Boc-deprotection furnished
SLF, which was acylated with acryloyl chloride to produce 1a
or propionyl chloride to produce 1b. Compounds 1c and 1d
required an alternate synthetic pathway since the free anilines
resulted in rapid decomposition. In order to avoid this
decomposition pathway, intermediates 5a and 5c were acylated
with 3-chloropropionyl chloride and then coupled with 3.
Elimination of the β-chlorine with NEt3 generated compounds
1c and 1d. SLFb was synthesized by coupling benzylic alcohol
6d with 3 followed by deprotection of the acid. Compounds 1e
and 1f were synthesized similarly to 1a and 1c using 4 as the
coupling partner with 5b and 5a, respectively.
Figure 2. Structures of D44 derivatives from pilot study.
using the D44-FKBP35 structure (PDB 4J4N).18,21 D44a, in
which the terminal ethyl is substituted with a benzylic
acrylamide, was predicted to occupy the same position as
D44 and bind favorably to C106. D44b replaces the
acrylamide with a propionamide to serve as a noncovalent
control, and in D44c the acrylamide is placed in an unfavorable
position as a control for nonspecific cysteine-reactivity. By
differential scanning calorimetry (DSC), none of these
compounds showed a thermal shift against full-length
FKBP12 or the Pf FKBP35 binding domain (FBD35)
indicating absent binding compared to rapamycin. Addition-
ally, we observed only trace, nonspecific covalent binding to
C106 by mass spectrometry (Table S1). Together these data
suggest that D44 does not bind to FKBP35.
We investigated the binding of these compounds in two
orthogonal assays: DSC and fluorescence polarization (FP,
Table 1). All compounds were screened against recombinant
FBD35 and FKBP12 to investigate selectivity.
Next, we turned to SLF as a starting point.16 As there are no
structures of SLF-bound FKBP35 we used the structure of the
related compound SLFb bound to human FKBP51(PDB
4DRK)22 aligned with rapamycin-bound Pf FKBP35 (PDB
4QT2)23 as the basis for docking. We noted two groups
proximal to C106: the aryl ring of the benzylic ester (for Series
1) and the tert-pentyl group adjacent to the ketoamide (for
Series 2, Figure 3a), and compounds from both series were
modeled (Table S2). As a prototypical compound from Series
1, the model predicts covalently bound 1a has good overlap
with SLFb in the FKBP35 binding pocket (Figure 3b).
Interestingly, covalently bound compounds from Series 2 show
a greater degree of overlap with SLFb (Figure 3c) resulting in
better docking scores than Series 1 compounds (Table S2).
Previous studies have shown that SLF has >10-fold lower
affinity for FKBP35 than FKBP12;16 however, we observe a
less profound difference in affinities. The addition of the
covalent warhead to this compound (1a) does not drastically
alter the IC50 as measured by FP. However, we observe a large
thermal shift of +13.4 °C by DSC. Saturation of the acrylamide
to the propionamide (1b) improves the binding by FP slightly
but reduces the thermal shift to +2.9 °C, suggesting that the
ability to form a covalent bond makes for a more
thermodynamically stable complex. Moving the acrylamide
warhead to the ortho position (1c) showed >10-fold weaker
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a
Scheme 1. Synthesis of Series 1 Compounds
Table 1. Binding of Series 1 Ligands to FBD35 and FKBP12
a
Reagents and conditions: (a) Methyl oxalyl chloride, DIPEA, DCM,
22 °C, 6 h; (b) 1,1-dimethylpropylmagnesium chloride, THF, −78
°C, 2 h; (c) LiOH, MeOH, 0−25 °C, 12 h; (d) SeO2, pyridine, 110
°C, 3 h; (e) oxalyl chloride, DMF, DCM, 0 °C, 30 m then: pipecolic
acid methyl ester, NEt3, DCM, 0 °C, 2 h; (f) KOH, EtOH, 0 °C, 12 h;
(g) H2, Pd/C, EtOAc, 2−24 h; (h) 3-chloropropionoyl chloride OR
tert-butyl bromoacetate, K2CO3, acetone, 22 °C, 16 h; (i) (+)-DIP-
chloride, THF, −45 °C, 16 h then: diethanolamine, Et2O, 22 °C, 2 h;
(j) Boc2O, 1,4-dioxane, 150 °C, 4 h; (k) DCC, DMAP, DCM, 22 °C,
2 h; (l) TFA, DCM, 22 °C, 2 h; (m) acryloyl chloride OR propionyl
chloride, NEt3, DCM, 0 °C, 2 h; (n) NEt3, MeCN, 80 °C, 6−18 h.
a
Determined by fluorescence polarization and fitting the data to a
binding by FP but retained a large thermal shift of +9.6 °C. To
rule out the possibility of nonspecific covalent bond formation,
compound 1d was synthesized where the acrylamide is
predicted to angle away from C106. 1d maintains good
binding by FP and a thermal shift close to other noncovalent
controls, suggesting that the large thermal shifts we observe
result from covalent bond formation.
Concerned about the lipophilicity and solubility of these
compounds (1a cLogP = 5.0, PBS pH 7.4 solubility = 0.31 μM;
1c cLogP = 4.4, PBS pH 7.4 solubility = 3.5 μM), we
investigated swapping the tert-pentyl moiety for a 3,4,5-
trimethoxybenzyl as ligands with this modification retain good
affinity for human FKBPs.24 As predicted, this change resulted
in a decrease in the cLogP (3.6 for 1e and 3.0 for 1f) and a
concomitant increase in PBS solubility (3.1 μM and 23 μM,
respectively). However, no binding to FBD35 was detected by
FP, and only 1e displayed weak binding by DSC. Compound
1f resulted in a negative DSC shift indicating a destabilizing
interaction with FBD35. Both 1e and 1f were able to bind to
FKBP12 albeit with lower affinity when compared to 1a or 1c.
These data indicate that despite the high homology between
the binding pockets, FKBP35 cannot accommodate large aryl
rings.
b
competitive inhibition model (nM). Determined by differential
scanning calorimetry (°C). Incomplete displacement of fluorescent
probe.
*
Holt et al.,25 4,4-dimethyldihydrofuran-2,3-dione was heated
with 8a or 8b in the presence of DMAP to form intermediates
9a and 9b. Acylation of the resultant alcohol with acryloyl
chloride or propionyl chloride generated esters 2c, 2d, and 2h.
Inspired by this strategy, aryl acrylamides 2f and 2g were
synthesized by the reaction between 8a and a Boc-protected
isatin derivative followed by Boc-deprotection and acylation of
the resultant aniline. Attempts to convert the alcohol of 9a into
an amine in order to access 2a and 2b were unsuccessful. In
order to place an acrylamide in this position, 8a was coupled
with Boc-protected gamma-aminobutyric acid to synthesize 2e,
which lacks the ketone and geminal dimethyl groups that were
previously shown to enhance FKBP-binding affinity.26
Recognizing that the precursor to 2a and 2b was a beta-
amino carbonyl, we envisioned that a Mannich reaction could
provide access to these compounds. To test this, intermediate
11 was generated by tert-butyl protection of Cbz-protected
pipecolic acid, Cbz-deprotection, and subsequent coupling
with 3-methyl-2-oxobutanoic acid. 11 was treated with para-
methoxybenzylamine and aqueous formaldehyde to form the
Mannich adduct, which was acylated with either 3-
chloropropionoyl chloride or propionyl chloride to generate
12a and 12b. Oxidative deprotection of the benzylic amide
followed by TFA treatment gave intermediates 13a and 13b
For Series 2, we examined both alkyl (2a−2e, 2h) and aryl
(2f, 2g) acrylamides and acrylates. The synthesis of Series 2
required a change in strategy compared to Series 1 (Scheme
2). Compounds 2c−2g were synthesized from common
intermediate 8a, which was generated from the coupling of
6e with Boc-protected pipecolic acid. 2h was similarly
synthesized from intermediate 8b. Following the strategy of
C
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a
Scheme 2. Synthesis of Series 2 Compounds
Table 2. Binding of Series 2 Ligands to FBD35 and FKBP12
a
Reagents and conditions: (a) Pd(OAc)2, NEt3, DMF, 80 °C, 16 h;
(b) (+)-DIP-chloride, THF, −45 °C, 6 h then: diethanolamine, Et2O,
22 °C, 2 h; (c) DCC, Boc-Pip-OH, DMAP, DCM, 0 °C, 2 h; (d)
TFA, DCM, 22 °C, 2 h; (e) 4,4-dimethyldihydrofuran-2,3-dione,
DMAP, toluene, 80 °C, 16 h; (f) acryloyl chloride OR propionyl
chloride, NEt3, DCM, 0 °C, 2−18 h; (g) DCC, tBuOH, DMAP,
DCM, 0 °C, 2 h; (h) H2, Pd/C, EtOH, 1 h; (i) DCC, 3-methyl-2-
oxobutanoic acid, DMAP, DCM, 0 °C, 2 h; (j) formaldehyde, para-
methoxybenzylamine, MeOH, H2O, 50 °C, 44 h; (k) CAN, MeCN/
H2O, 16 h; (l) EDC, DMAP, DCM, 0 °C, 12 h; (m) Boc2O, DMAP,
THF, 6 h; (n) 8a, DMAP, toluene, 80 °C, 2 h.
a
Determined by fluorescence polarization and fitting the data to a
b
competitive inhibition model (nM). Determined by differential
scanning calorimetry (°C).
which were then coupled with 6e to complete the syntheses of
2a and 2b.
We predict that this large shift in Tm is a result of a covalent
bond between these ligands and C106. In support of this, all of
the covalent compounds that were able to bind to FBD35 by
FP also exhibited much larger thermal shifts (>9 °C vs 2−4 °C,
generally) relative to their noncovalent controls (1a vs 1b, 2a
vs 2b, 2c vs 2d, and 1c, 2e, and 2h). Notably, we observed two
distinct melting points in the DSC traces for putative covalent
compounds that were recorded after short incubations with
FBD35. To highlight this phenomenon, compound 1c was
measured by DSC after a short incubation time with FBD35
and FKBP12. When incubated with FBD35, we observe two
overlapping melting curves with distinct ΔTm at 1.6 and 9.6 °C
relative to the apoprotein (Figure 4a). Repeating the same
experiment with FKBP12 results in a single ΔTm of 3.2 °C
(Figure 4b). The first ΔTm is likely indicative of the overall
weaker affinity of 1c for FBD35, which is corroborated by our
FP results. This ΔTm is within the same range of all the
noncovalent controls, which we interpret as resulting from a
reversible ligand−protein interaction. With extended incuba-
tion times (>24 h), we observe only the second, larger thermal
shift with the covalent compounds whereas the noncovalent
controls remain unchanged (data not shown). All compounds
showed only a single thermal shift when tested against human
FKBP12, regardless of incubation time.
In our covalent docking model, 2a and 2c were predicted to
form more stable covalent complexes than 1a or 1c (Table S2).
While the IC50s overall did not change significantly between
the two series, we observed larger thermal shifts in the DSC. As
observed in Series 1, this large shift was dramatically reduced
upon removal of the Michael acceptor (2b and 2d) indicating
that Series 2 compounds can likely form a covalent bond with
FBD35. Removal of the ketone and geminal dimethyl from 2a
(2e) significantly lowered binding affinity but maintained the
large thermal shift. Despite the predictions of robust binding
with 2f or 2g in the docking model (Table 2), we saw no
binding in the FP or DSC assays for either protein providing
additional evidence that FKBP35 cannot accommodate aryl
rings within the binding pocket. Similar to the solubility issues
in Series 1, several of the Series 2 compounds have poor
solubility and high cLogP values. In particular, 2c has a cLogP
of 4.4 and a PBS solubility of <0.1 μM. By contrast, SLFb has a
similar cLogP of 4.1 but a PBS solubility of 59 μM. Adding this
free carboxylate to 2c to generate 2h reduced the cLogP to 3.5
while PBS solubility improved to 70 μM. Further, this
compound exhibited a very good IC50 and the largest thermal
shift recorded.
D
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bioluminescent resonant energy transfer (nanoBRET) assay to
measure target engagement.28 We generated HEK293T cells
stably expressing either nanoluciferase-tagged (nLuc) full
length Pf FKBP35 or nLuc-tagged FKBP12 (Figure S3).
Using a BODIPY-conjugated rapamycin tracer (Rap-Gly-
BDP), we were able to measure the interaction with both
FKBPs and measure its displacement in cells with unlabeled
compounds to infer direct target engagement (Figure S4).
As a positive control we measured displacement of the tracer
with unmodified rapamycin (Figure 5a,b black squares) and
Figure 5. Intracellular binding of compounds to FKBP35 and
FKBP12 in a NanoBRET cellular target engagement assay. (A)
Normalized BRET ratio of covalent and noncovalent matched pairs in
HEK-293T cells expressing nLuc-tagged FKBP35. (B) Normalized
BRET ratio of covalent and noncovalent matched pairs in HEK-293T
cells expressing nLuc-tagged FKBP12.
Figure 4. Evidence for covalent bond formation between 1c and
FBD35 observed in DSC and confirmed by LC-MS. (A) DSC trace of
1c measured on FBD showing two distinct melting curves at Δ = 1.6
°C and Δ = 9.6 °C. (B) DSC trace of 1c on FKBP12 showing a single
melting curve shift at Δ = 3.2 °C. (C) Extracted ion chromatogram of
iodoacetamide-treated, chymotrypsin-digested FBD35 found the
expected carbamidomethylated peptide (GYGDEGCGESIPGN, m/z
= 855.9, 2nd charge state) at RT = 3.5 min. (D) Mass spectrum of the
peak at 3.5 min in panel C. (E) Extracted ion chromatogram of 1c-
treated, chymotrypsin-digested FBD35 found the expected 1c-
modified peptide (GYGDEGC*GESIPGN, m/z = 1116.5, 2nd charge
state) at RT = 5.9 min. (F) Mass spectrum of the peak at 5.9 min in
panel E.
found robust tracer displacement with IC50s of 34 nM
(FKBP35) and 20 nM (FKBP12), respectively (Table S3).
As a negative control, we measured tracer displacement using
GPI-1046 (Figure S5), an early FKBP12 ligand29 of similar
structure to SLF whose ability to bind FKBPs was
subsequently called into question.22,30 We proceeded to
measure matched pairs 1a and 1b, 2a and 2b, and 2c and
2d in the assay. After a 2 h incubation all compounds displace
the fluorescent tracer, providing evidence that these com-
pounds not only engage with FKBPs but are also cell
permeable. Despite submicromolar IC50 values against both
FKBPs in the FP assay SLFb displayed markedly reduced
probe displacement likely due to reduced cell permeability as a
result of the free carboxylate. Similar to the FP assay, there
were no significant differences between covalent and non-
covalent compounds in this assay. This suggests the intrinsic
binding of the compounds remains a major driver of target
affinity at 2 h. Nonetheless, the NanoBRET assay confirms that
these compounds are not only cell-permeable but can also
directly engage the target FKBP in a cellular setting.
Given the success of our biochemical assays, we investigated
the activity of compounds 1a and 1b on live, cultured parasites
(Figure 6). Using a luciferase reporter NF54 P. falciparum
parasite line, both 1a (IC50 = 1.4 μM) and 1b (IC50 = 1.9 μM)
showed dose-dependent growth-inhibition (Figure 6a). Grat-
ifyingly, these compounds performed similarly to unmodified
rapamycin (IC50 = 1.1 μM). Despite displaying poorer binding
in the FP assay, 1c shows similar antiplasmodium activity as 1a
(IC50 = 1.9 μM), possibly linked to faster adduct formation
with FKBP35 (Figure S2c). As we observed in the NanoBRET
assay, SLFb (IC50 = 33.7 μM) performs poorly compared to 1a
and 1b (Figure 6b). Despite similar affinity and higher warhead
reactivity, 2c also performs poorly in this assay (IC50 = 13 μM,
Figure S6a). This discrepancy is likely due to differences in
plasma stability as 2c is rapidly degraded by plasma where 1a−
c are not (Figure S6b). Additionally, these compounds were
not cytotoxic in HEK293T cells across the relevant
To confirm the formation of a covalent adduct, we analyzed
FBD35 by LC-MS alone or after 24 h of incubation with 1c
(Figure S1). After extended incubation with 1c, we observed a
nearly 1 min shift in the retention time of the protein complex
vs the apo protein with an increase of mass to 14.5 kDa,
consistent with the expected mass of the apo protein (13.9
kDa) plus the ligand (579 Da). Additionally, we subjected both
the FBD35 apoprotein and the 1c-FBD35 adduct to enzymatic
digestion to generate the peptide containing C106 (GYG-
DEGCGESIPGN, Figure 4c−f, Figure S1). In the extracted ion
chromatogram of the apoprotein, the carbamidomethylated
peptide was found at 3.5 min (Figure 4c) with the correct mass
(m/z = 855.9, second charge state, Figure 4d). When
incubated with 1c, there was some residual unmodified peptide
found at 3.5 min (Figure S1c−d) as well as the predicted 1c-
inclusion peptide at RT = 5.9 (Figure 4e) with the correct
mass (m/z = 1116.5, second charge state, Figure 4f). This mass
was not found in the untreated protein (Figure S1e−f).
We next determined the relative rates of covalent adduct
formation and FKBP35 consumption (Figure S2a,b) for
compounds 1a−c, 2a, and 2c via LC-MS. Measuring the
reaction half-life reveals a rank order of 2c > 1c > 2a > 1a and
no binding by 1b (Figure S2c). Given their highly conserved
structures, the stark difference in reaction half-lives between 2c
(t1/2 = 0.78 h) and 2a (t1/2 = 11.7 h) is likely a result of the
difference in reactivities between acrylates and acrylamides,27
highlighting the importance of fine-tuning warhead electro-
philicity.
We next were interested in comparing the interaction of
compounds with FKBP35 and human FKBP12 in a consistent
cellular setting. To this end we employed a nanoluciferase
E
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Cancer Institute, Boston, Massachusetts 02215, United States;
Harvard Medical School, Boston, Massachusetts 02115, United
States; Email: wsellers@broadinstitute.org
Authors
Donald D. Raymond − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Christa A. Blomquist − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Charisse Flerida Pasaje − Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
Patrick R. McCarren − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Jamie Moroco − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; orcid.org/
0000-0001-8250-5923
Figure 6. P. falciparum strain NF54 growth-inhibition assay. (A)
NF54 parasites were incubated with 1a, 1b, SLFb, or rapamycin in a
dose-dependent manner. Proliferation was measured by luminescence
after a complete life-cycle and normalized to DMSO (100%) and
chloroquine (100 nM, 0%). (B) Structural differences between
synthetic ligands and compound IC50s (μM).
Henock B. Befekadu − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Foxy P. Robinson − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Debjani Pal − Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States
Lisl Y. Esherick − Department of Biological Engineering,
Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
Alessandra Ianari − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Jacquin C. Niles − Department of Biological Engineering,
Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States; orcid.org/0000-0002-
6250-8796
concentration range indicating that the Plasmodium assay data
are not a result of general cytotoxicity (Figure S7).
While the in vitro data confirm these compounds covalently
modify C106 and the NanoBRET assay demonstrates direct
target engagement of FKBP35 in a cellular context, additional
genetic studies will be necessary to fully establish whether
FKBP35 inhibition is responsible for their antiplasmodium
activity. Despite this limitation, our results suggest that
covalent inhibition of FKBP35 could be an avenue for the
development of antimalarial therapeutics. The presence of
C106 provides a functional handle for high-throughput
screening of covalent fragment libraries for the development
of novel chemical matter that could provide improved
selectivity between FKBP35 and the human FKBPs. While
we predominantly examined simple acrylamides, the slow rate
of covalent-bond formation indicates more electrophilic
warheads might prove beneficial for additional selectivity and
potency. In fact, switching to a more reactive acrylate
drastically accelerated the rate of covalent bond formation
but at the cost of plasma stability. Additional experiments
examining warhead reactivity should be a fruitful endeavor.
Alternatively, it is possible that the pipecolic acid core does not
provide an optimal foundation upon which to covalently attack
C106. Thus, screening efforts to uncover new high affinity
FKBP-binding chemotypes optimized for the covalent
modification strategy presented here have the potential to
create more potent and selective compounds for this highly
conserved enzyme. Such compounds will be of significant
interest for testing against liver-stage disease.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00272
Author Contributions
T.C.A., A.I., and W.R.S. generated the concept and experi-
ments. T.C.A. designed and synthesized all compounds,
analyzed data, and prepared the manuscript. D.D.R. prepared
and isolated recombinant proteins and ran DSC experiments
and mass spec assays. C.A.B. performed cell culture and
nanoBRET experiments. C.F.P. and L.Y.E. performed and
analyzed parasite viability experiments. P.R.M. performed
computational modeling. T.C.A. and H.B.B. performed FP
experiments. J.M. ran and with T.C.A. analyzed mass spec
experiments. F.P.R. and D.P. performed cell viability experi-
ments. A.I. and J.C.N. facilitated the collaboration. All authors
discussed the results and commented on the manuscript. All
authors have given approval to the final version of the
manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00272.
■
sı
Funding
Synthesis and characterization of all compounds and
biophysical, biochemical, and biological assay proce-
dures (PDF)
This research was supported by The Broad Institute (SPARC
grant (T.C.A., D.D.R., W.R.S.) and Broad Next10 (J.C.N))
and The Bill and Melinda Gates Foundation (OPP1158199
and OPP1162467 (C.F.A.P., L.Y.E., J.C.N.)).
AUTHOR INFORMATION
Corresponding Authors
■
Notes
The authors declare the following competing financial
interest(s): W.R.S. is a Board or SAB member and holds
equity in Peloton Therapeutics, Ideaya Biosciences, Civetta
Therapeutics, and Bluebird and has consulted for Array, Astex,
Dynamo Therapeutics, Ipsen, PearlRiver Therapeutics, Sanofi,
Thomas C. Atack − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; orcid.org/
0000-0001-5953-2261; Email: tatack@broadinstitute.org
William R. Sellers − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; Dana-Farber
F
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ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Featured Letter
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Isomerases (Immunophilins) and Their Roles in Parasite Biochem-
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and Servier and receives research funding from Pfizer
Pharmaceuticals and Deerfield Management.
Biographies
Thomas Atack received his Ph.D. degree from Indiana University
Bloomington where he developed novel iron- and manganese-
catalyzed cross-coupling reactions under Prof. Silas Cook. He then
transitioned to the Broad Institute of MIT and Harvard where he
joined the research group of Dr. William R. Sellers to study
therapeutic development for diseases with unmet medical need. His
research interests revolve around the design, synthesis, and character-
ization of novel chemical matter for therapeutic development.
William Sellers is a physician-scientist, Core Institute Member at the
Broad Institute and a Professor of Medicine at the Dana-Farber
Cancer Institute and Harvard Medical School. He directs a research
group focused on utilizing functional genomic technologies to
discover new targets and new therapeutics in cancer. He is a co-
founder of Civetta Therapeutics. Previously, Dr. Sellers directed
cancer drug discovery and early cancer clinical development at the
Novartis, led the Cancer Cell Line Encyclopedia project, co-
discovered EGFR mutations in lung cancer and served on the
National Cancer Advisory Board.
ACKNOWLEDGMENTS
■
The authors thank Eamon Comer for thoughtful chemistry
discussions; Dale Porter for helping secure internal funding
sources; and Christian Stephan Meyners and Wei Jiang for
assistance with the fluorescence polarization assay.
(16) Braun, P. D.; Barglow, K. T.; Lin, Y. M.; Akompong, T.;
Briesewitz, R.; Ray, G. T.; Haldar, K.; Wandless, T. J. A Bifunctional
Molecule That Displays Context-Dependent Cellular Activity. J. Am.
Chem. Soc. 2003, 125 (25), 7575−7580.
(17) Harikishore, A.; Leow, M. L.; Niang, M.; Rajan, S.; Pasunooti,
K. K.; Preiser, P. R.; Liu, X.; Yoon, H. S. Adamantyl Derivative as a
Potent Inhibitor of Plasmodium FK506 Binding Protein 35. ACS Med.
Chem. Lett. 2013, 4 (11), 1097−1101.
(18) Harikishore, A.; Niang, M.; Rajan, S.; Preiser, P. R.; Yoon, H. S.
Small Molecule Plasmodium FKBP35 Inhibitor as a Potential
Antimalaria Agent. Sci. Rep. 2013, 3, 2501.
ABBREVIATIONS
■
FKBP, FK506 binding protein; FBD, FKBP binding domain;
SLF, synthetic ligand for FKBP; PPIase, peptidyl prolyl
isomerase; DSC, differential scanning calorimetry; FP,
fluorescence polarization; nLuc, nanoluciferase; BRET, bio-
luminescent resonant energy transfer
̈
(19) Pomplun, S.; Sippel, C.; Hahle, A.; Tay, D.; Shima, K.; Klages,
A.; Unal, C. M.; Rieß, B.; Toh, H. T.; Hansen, G.; Yoon, H. S.;
̈
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