One-step Heck Reaction Generates Nonimmunosuppressive FK506 Analogs for Pharmacological BMP Activation

Article abstract of DOI:10.1021/acsmedchemlett.9b00144

FKBP12 ligands such as FK506 have been shown to activate the BMP signaling pathway and facilitate tissue regeneration. However, the immunosuppressive activity of FK506 limits its clinical application. Using Heck reaction, we generated nonimmunosuppressive

Full text of DOI:10.1021/acsmedchemlett.9b00144

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
One-step Heck Reaction Generates Nonimmunosuppressive FK506
Analogs for Pharmacological BMP Activation
Yuefan Wang,†,‡ Brandon J. Peiffer,†,‡ Qi Su,§ and Jun O. Liu*,†
†Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine and the SJ Yan and HJ Mao Laboratory
of Chemical Biology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States
§Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: FKBP12 ligands such as FK506 have been
shown to activate the BMP signaling pathway and facilitate
tissue regeneration. However, the immunosuppressive activity
of FK506 limits its clinical application. Using Heck reaction,
we generated nonimmunosuppressive analogs of FK506 by
fusing heterocycles to the calcineurin (CN) binding domain of
FK506. Structure−activity relationships provided novel
mechanistic insights into the FK506−CN interaction that
can be exploited for rational design of future analogs.
KEYWORDS: FKBP ligands, regenerative medicine, nonimmunosuppressive, BMP signaling, SMAD1/5, FKVP, calcineurin
FK506 (tacrolimus), a 23-membered macrocyclic natural
product isolated from Streptomyces tsukubaensis, has been
widely used as an immunosuppressant for prevention of
allogeneic transplant rejection.1,2 The immunosuppressive
activity of FK506 results from its inhibition of T cell
receptor-mediated signal transduction. At a molecular level,
FK506 binds FK506-binding proteins (FKBPs) upon entering
cells.3 The resultant FKBP12−FK506 complex forms a ternary
complex with the protein phosphatase calcineurin, allosterically
blocking access to its active site,4−6 preventing dephosphor-
Figure 1. Structures of FK-506 and FKVP.
ylation of the nuclear factor of activated T cells (NFAT) by
calcineurin and its ensuing nuclear translocation and tran-
scriptional activation of IL-2 and some other cytokine genes.4
accelerate wound healing in diabetic rats in a BMP-dependent
manner.13
In addition to facilitating the interaction between FK506 and
In the past, we and others have relied on ruthenium-
calcineurin, FKBP12 has been shown to interact with several
catalyzed cross metathesis (CM) reaction to directly modify
other proteins including TGF-β/BMP type 1 receptors.7−10
the terminal alkene of FK506 (C40) to generate non-
FK506 has been shown to exhibit unique pharmacological
immunosuppressive FK506 analogs.14−17 Unfortunately,
activities unrelated to immunosuppression and has potential
FKVP and other nitrogen-containing analogs could only be
obtained in very low yields (<10%) under CM reaction
conditions. This is most likely due to the nitrogen lone
for treating diseases such as pulmonary hypertension11 and
bladder cancer.12
Recently, we synthesized a novel nonimmunosuppressive
FK506 analog, named FKVP (FK506-C40-py), that retained
FKBP binding and lacked calcineurin inhibition activity
(Figure 1). FKVP was found to activate Bone Morphogenic
Protein (BMP) signaling in lymphocytes and endothelial cells
through disruption of the FKBP12−BMPR1 interaction.
Moreover, the combination of FKVP and AMD3100, an
antagonist of the CXCR4 receptor that is capable of mobilizing
hematopoietic stem cells from the bone marrow, was found to
electron pair that competitively coordinates to the ruthenium
metal center.18 The use of soluble tosylated salts of amines
could improve yields,19 but it did not help in the synthesis of
FKVP. Another reported functionalization method toward
FK506 terminal olefin is thiol-ene “click” reaction,20 which
Received: March 31, 2019
Accepted: August 19, 2019
Published: August 19, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00144
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ACS Medicinal Chemistry Letters
Letter
a
requires more than one step to synthesize the nitrogen-
containing FK506 analogs.
Table 1. Heck Reaction of FK-506 with Aryl Halides
Aside from the ruthenium-catalyzed CM reaction, the
palladium-catalyzed Heck reaction also allows for modifica-
tions of terminal alkenes, making the Heck reaction a viable
alternative for synthesizing FKVP. Importantly, palladium
catalysts used in the Heck reaction are compatible with
nitrogen-containing heterocycles such as pyridine.21−24 More-
over, halogen-containing heterocycles are commercially avail-
able and inexpensive. After optimizing the reaction conditions
for the production of FKVP (1b),25 we found that reacting
FK506 with 4-iodopyridine (2.0 equiv) in the presence of
Pd(OAc)2 (10 mol %) and P(o-tol)3 (20 mol %) in DMF at
100 °C gave the best yield (66%, Table S1). These conditions
can be applied toward production of several new FKVP
analogs.
It was previously reported that when the terminal olefin
(C40) of FK506 is linked to small aromatic groups such as
phenyl and β-naphthyl group, the resulting FK506 analogs
retained most of the inhibitory activity against calcinerurin.15
In contrast, FKVP lost most of its activity against calcineurin
with negligible effect at concentrations up to 10 μM,13 even
though pyridine is smaller than naphthalene. To further
explore this apparent paradoxical observation, we conducted a
structure−activity relationship study to gain new insight into
the interactions between FK506 analogs and calcineurin. Using
FKVP as a starting point, we selected three pyridinyl-halides,
three quinolyl-halides, and four aniline-halides as substrates for
the Heck reaction. To our delight, all halide substrates were
successfully coupled to FK506 with moderate-to-good yields
(Table 1).
The halide substrates displayed distinct reactivity in the
Heck reaction. First, bromides and iodides gave similar yields.
Second, electron-withdrawing groups on the pyridines and
quinolines appeared to increase the yields (entry 4,6,7). Third,
unprotected anilines gave the lowest yields (entry 9−11).
Importantly, the unreacted FK506 starting material in the
reaction mixture was easily separated from the more polar
nitrogen-containing products with flash chromatography,
which posed a major problem for the purification of other
related FK506 analogs.15
With FK506 analogs in hand, we assessed their effects on
cell viability, BMP activation, and NFAT activation at two
concentrations, 1 μM and 10 μM (Figure 2). In a cell viability
assay using donor-pooled human umbilical vein endothelial
cells (HUVEC), we found that quinoline analogs (5b−7b)
inhibited cell proliferation at 10 μM (Figure 2a), while other
compounds were comparable to FK506 in their cytotoxicity.
We next used a BMP-response-element (BRE) pathway
reporter (luciferase under the control of the ID1 promoter) in
Jurkat T cells to determine whether the new analogs were
capable of activating the BMP signaling pathway.11 Initial
screening of the compounds showed that most analogs had
similar activity as FK506 or FKVP. This is somewhat expected,
as we have previously shown that FKBP12 binding is necessary
and sufficient for activation of BMP signaling (Figure 2b).
To determine the effects of the analogs on calcineurin, we
employed a PMA/ionomycin-activated Nuclear Factor of
Activated T-cells (NFAT) reporter in Jurkat T cells15
(luciferase under the control of the IL-2 promoter) (Figure
2c). Two analogs (2b, 3b) did not cause significant inhibition
of the NFAT-Luciferase reporter at concentrations up to 10
μM, similar to FKVP (1b). Surprisingly, most other analogs
a
Reaction condition: FK-506 (0.050 mmol), aryl halide (0.10 mmol),
Pd(OAc)2 (0.0050 mmol), P(o-tol)3 (0.010 mmol), and Et3N (0.10
b
c
mL) in DMF (1.0 mL) at 100 °C under Ar. Isolated yield. Yield in
parentheses is based on FK-506 recovery.
showed either partial or nearly complete inhibition of the
NFAT reporter at 1 μM (Figure 2c). It is noteworthy that
some of the immunosuppressive analogs, including 6b and 7b,
have bulkier substituents than 2b and 3b due to the presence
of a fused aromatic ring. How those bulkier groups are
accommodated at the binding site of calcineurin remains to be
determined.
We determined the EC50 values of the three non-
immunosuppressive analogs (1b−3b) in the BMP luciferase
assay. All three analogs were found to be slightly more potent
than FK506 (Figure 3a, Table S2), likely attributable to
increased solubility of the more polar pyridine substituents. In
addition, we observed dose-dependent induction of SMAD1/5
phosphorylation by compounds 2b and 3b in Jurkat T cells,
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Figure 3. (a) Dose−response curves for BMP reporter activation by
three nonimmunosuppressive analogs (1b, 2b, and 3b) and FK506.
(b) Activation of SMAD1/5 phosphorylation in Jurkat T cells by
selected analogs. Cells were treated with the compounds for 2 h.
HSP90 was used as a loading control.
Figure 2. FK506 analogs display variable immunosuppressive
qualities. (a) Cell viability after 72 h analog treatment in HUVEC
cells. (b) All analogs activate a BMP pathway reporter in Jurkat cells
with similar potency to FK506. (c) Derivatives show structure-
dependent effects in NFAT reporter inhibition in Jurkat cells. Error
bars represent standard deviation from the mean for all measurements
(n = 3), and absorbance/luminescence values were normalized to
DMSO-treated cells.
Figure 4. Binding detail toward calcineurin. (a) Close-up of FK-506
terminal olefin with calcineurin. (b) Steric effect of FKVP with
calcineurin.
consistent with the previously observed effect of both FK506
and FKVP (Figure 3b).
remains active against calcineurin cannot be explained by steric
clash alone.
The structure of the FKBP12−FK506−calcineurin complex
was previously determined by X-ray crystallography.5,6,15 In
this complex, the terminal alkene of FK506 fits into a binding
pocket in calcineurin formed by hydrophobic amino acids
(Figure 4a). When modeled in place of FK506, the pyridine
moiety in FKVP (1b) shows a steric clash with the side chain
of M118 of calcineurin (Figure 4b), which may explain the
elimination of calcineurin binding by analogs 1b−3b. It was
previously shown that both phenyl and β-naphthyl substitution
at C40 of FK506 allowed for significant retention of
calcineurin inhibition.15 How the β-naphthyl analog, which
contains bulkier substituents than FKVP at the same position,
As steric hindrance cannot explain the difference in
calcineurin inhibitory activity among the existing analogs, we
turned our attention to electrostatic status of the basic
nitrogen-containing analogs. We noticed that heterocycles
with higher pKa values (1b, 2b, 3b, and 5b) showed less
calcineurin inhibition. Conversely, those with a lower pKa
caused by electron-withdrawing groups were all immunosup-
pressive at 1 μM (4b, 6b, and 7b).26 These observations
suggest that the formation of positively charged pyridinium
and quinolinium appendage at the terminal alkene of FK506
play a more important role in disrupting the interaction
C
DOI: 10.1021/acsmedchemlett.9b00144
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Notes
between the terminal alkene of FK506 and the hydrophobic
pocket in calcineurin. These observations reveal an alternative
and complementary mechanism for the loss of calcineurin
inhibition in nonimmunosuppressive FK506 analogs, which in
the past has been rationalized by a large molecular “bump” to
sterically hinder calcineurin binding.15 It is likely that the same
binding pocket in calcineurin has significant conformational
flexibility to accommodate noncharged bulky aromatic rings
such as those present in 4b, 6b, and 7b.
It is important to note that the predominant electrostatic
effects of C40 substituents on inhibition of calcineurin only
holds true within the same family of heterocycles containing
the pyridine core. The pKa value of aniline (9b−11b) is
slightly higher than methylaniline (8b), but methylaniline with
the highest pKa (8b) showed the strongest calcineurin
inhibition at 1 μM. It is possible that the additional methyl
group may forge favorable interaction with the hydrophobic
pocket of calcineurin, overcoming the unfavorable electrostatic
effect.
In summary, we have developed a one-step synthesis of
FK506 analogs to couple FK506 with small nitrogen-
containing heterocycles using the Heck reaction. We have
identified a new class of nonimmunosuppressive analogs with
diverse structures and high potency in activating the BMP
signaling pathway. Moreover, these basic nitrogen-containing
FK506 analogs are easy to separate from FK506, allowing for
bulk synthesis of the analogs for future preclinical studies.
Elimination of calcineurin inhibition rendered those analogs
not only nonimmunosuppressive but also free of nephrotox-
icity and neurotoxicity, which are caused by inhibition of
calcineurin.27,28 In the SAR study, we found that electrostatic
effect can play a dominant role in disrupting FK506−
calcineurin interaction among the heterocycle series. Given
the role of BMP signaling in wound healing and tissue
regeneration,13 our newly established method for synthesizing
and identifying nonimmunosuppressive BMP agonists will
facilitate the development of nonimmunosuppressive analogs
of FK506 for regenerative medicine and other BMP-related
diseases.
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00144.
Experimental procedures and characterization of com-
pounds 1b−11b (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: joliu@jhu.edu.
■
ORCID
Brandon J. Peiffer: 0000-0002-6783-2923
Jun O. Liu: 0000-0003-3842-9841
Author Contributions
‡These authors contributed equally.
Funding
This work was supported by the Liu Lab Discretionary Fund,
FAMRI (to J.O.L.) and an NIH-funded Chemistry-Biology
Interface Program at Johns Hopkins University (T32
GM080189) (to B.P.)
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in the protein microenvironment. pKa calculated for substituted
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3-fluoro pyridine, 3.47; quinoline, 4.87; o-aniline, 4.04.
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