Inhibition of Macrophage Migration Inhibitory Factor by a Chimera of Two Allosteric Binders

Article abstract of DOI:10.1021/acsmedchemlett.9b00351

Human Macrophage Migration Inhibitory Factor (MIF) is a trimeric cytokine implicated in a number of inflammatory and autoimmune diseases and cancer. We previously reported that the dye p425 (Chicago Sky Blue), which bound MIF at the interface of two MIF t

Full text of DOI:10.1021/acsmedchemlett.9b00351

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Letter
Inhibition of Macrophage Migration Inhibitory
Factor by a Chimera of Two Allosteric Binders
Pier F. Cirillo, Oluwatoyin A Asojo, Uday Khire, Yashang Lee, Sara Mootien, Peter Hegan, Alan
G Sutherland, Elizabeth Peterson-Roth, Michel Ledizet, Raymond A Koski, and Karen G Anthony
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00351 • Publication Date (Web): 19 Nov 2019
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Inhibition of Macrophage Migration Inhibitory Factor by a Chimera
of Two Allosteric Binders
Pier F. Cirillo,*a,b Oluwatoyin A. Asojo,*c Uday Khire,d Yashang Lee,a± Sara Mootien,a^ Peter Hegan,a†
Alan G. Sutherland,a Elizabeth Peterson-Roth,a Michel Ledizet,a Raymond A. Koski,a and Karen G. An-
thonya
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a) L2 Diagnostics, LLC, 300 George Street, New Haven, CT 06511, U.S.A.
b) Department of Chemistry and Chemical Engineering, University of New Haven, 300 Boston Post Road, West Haven, CT
06516, U.S.A.
c) Department of Chemistry and Biochemistry, Hampton University, 200 William R. Harvey Way, Hampton, VA 23668,
U.S.A.
d) CheminPharma, LLC, 4 Research Drive, Woodbridge, CT 06525, U.S.A.
KEYWORDS: MIF, CD74, inhibitor, allosteric, structure-based drug design, fragment-based drug design, X-ray.
ABSTRACT: Human Macrophage Migration Inhibitory Factor (MIF) is a trimeric cytokine implicated in a number of inflammatory
and autoimmune diseases and cancer. We previously reported that the dye p425 (Chicago Sky Blue), which bound MIF at the interface
of two MIF trimers covering the tautomerase and allosteric pockets, revealed a
unique strategy to block MIF’s pro-inflammatory activities. Structural liabilities,
including the large size, precluded p425 as a medicinal chemistry lead for drug
development. We report here a rational design strategy linking only the fragment
of p425 that binds over the tautomerase pocket to the core of ibudilast, a known
MIF allosteric site-specific inhibitor. The chimeric compound, termed L2-4048,
was shown by X-ray crystallography to bind at the allosteric and tautomerase
sites as anticipated. L2-4048 retained target binding and blocked MIF’s tautomer-
ase CD74 receptor binding, and pro-inflammatory activities. Our studies lay the
foundation for the design and synthesis of smaller and more drug-like compounds
that retain the MIF inhibitory properties of this chimera.
MeO
Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic
protein involved in the regulation of immune1,2 and inflammatory
processes,3 as well as in the induction of apoptosis.4-6 MIF is prom-
inently expressed by macrophages, T cells, and platelets, and hence
found in tissues throughout the body.7 MIF has been shown to have
cytokine-like activities,8,9 as well as to act as a chemokine,10 a hor-
mone,11 and a chaperone.12 Recombinant MIF exists mostly as a
trimer in solution and is likely recognized in this form by the CD74
receptor,13,14 though the oligomeric species interacting with
CXCR4, CXCR2, and CXCR7 receptors are not yet fully estab-
lished.15,16 MIF also possesses what is considered, in humans, to be
vestigial enzymatic activity as a tautomerase, since no physiologic
substrate has been identified to date.17 The tautomerase active site,
including an N-terminal proline,18 is a cylindrical pocket located at
the interface of monomer subunits, just beneath the CD74 binding
region.
H2N
HO3S
SO3H
HO
N
N
N
R
N
OH
HO3S
SO3H
NH2
OMe
p425
O
ibudilast (AV411): R = Me
AV1013: R = NH2
N
N
SO3H
H2N
O
HO
N
Because of its central and many-faceted roles, MIF has attracted
considerable interest by the drug discovery community.7 The ma-
jority of MIF inhibitors reported to date act by binding at the tau-
tomerase site.19-23 We have previously reported that the dye p425
(also known as Chicago sky blue 6B or Pontamine sky blue) (Fig-
ure 1), discovered through a high-throughput screening effort, is a
non-covalent, allosteric inhibitor of MIF.24
N
SO3H
N
N
1 (L2-4048)
Figure 1. Chemical structures of allosteric MIF inhibitors p425,
ibudilast, and new chimeric compound L2-4048 (1).
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NH2
NO2
NHBoc
NHBoc
NHBoc
1
2
3
4
5
6
7
8
Boc anhydride
1) nBuLi,
THF, -78 o
C
TBAF.3H2O
THF, -20 o
(59%)
K2CO3
SnCl2
O
MeOH/DCM
(97%)
C
THF/H2O
(85%)
2)
H
3
4
5
2
6
SiMe3
SiMe3
SiMe3
HO
-78 oC -rt
(70%)
NHBoc
NHBoc
TEMPO
KBr
9
1) TFA/ CH2Cl2
OH NH2
N
I
NH2
NaHCO3
NaOCl
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2) NaNO2, HCl
SO3H
N
N
-
DCM / H2O
C
3)
SO3
DBU, MeCN
0 oC - rt
N
N
O
O
0 o
SO3H
(28% 3 steps)
N
N
(41% 2 steps)
O
8
1
7
-
SO3
OH NH2
Scheme 1. Synthesis of chimeric compound L2-4048 (1).
Co-crystal structures of MIF-p425 complexes revealed that the
inhibitor lies on the surface at the interface of two MIF trimers,
covering the entrance to the tautomerase pocket, and engaging in
hydrogen bonds with Lys32, Asn109, and Asn110.24 P425 inhibits
not only the tautomerase activity of recombinant human MIF
(rhMIF), but also its binding to CD74 receptor (IC50 0.81 µM), as
well as its bioactivity, including dose-dependent induction of pro-
inflammatory mediators such as IL-6, IL-8, and MMP3 in fore-
skin fibroblasts, and p53-dependent apoptosis in HeLa cells.24
Despite impressive MIF inhibitory activities, the large size
(948.8 MW), highly charged nature brought about by four sul-
fonates, and the presence of two diazo linkages, rendered p425
non-viable as a starting point for medicinal chemistry efforts.
Therefore, we explored the possibility of creating a chimeric mol-
ecule with more drug-like characteristics that would retain MIF
binding features of p425. To this end, we devised a unique strategy
to link the MIF-binding features of p425 with that of ibudilast, a
clinically marketed phosphodiesterase inhibitor and a well-charac-
terized allosteric inhibitor of MIF.25 Ibudilast (AV411) and its
closely-related analog AV1013, bind MIF at an allosteric site adja-
cent to the tautomerase pocket in a region surrounded by aromatic
residues such that the inhibitor binding causes a conformational
change in Tyr36.25 This allosteric site has independently been iden-
tified by McLean et al. through a fragment screening of inhibitors
of MIF tautomerase26 (for other allosteric inhibitors, see ref 7 and
27). Ibudilast inhibits MIF’s tautomerase activity (ki 30.9 µM) in a
non-competitive manner and decreases MIF-mediated chemo-
taxis.25 Herein we report the design, synthesis, structural elucida-
tion, and MIF inhibitory activities of L2-4048, a unique chimera
of p425 and ibudilast allosteric MIF inhibitors.
according to the procedure reported by Ye et al.28 Following a
standard reduction, protection, and deprotection sequence, terminal
alkyne 5 was achieved in moderate yield. Deprotonation to form
the acetylide anion and reaction with isobutyraldehyde afforded
secondary propargylic alcohol 6, which could be oxidized by
bleach and TEMPO catalysis to alkynyl ketone 7. Compound 7
could be converted to the pyrazolo[1,5-a]pyridine system 8 by a
standard procedure.25 Deprotection of 8 to reveal the aniline, dia-
zotization, and direct reaction with 4-amino-5-hydroxynaphtha-
lene-1,3-disulfonic acid afforded L2-4048 in 9 steps from 2 and an
overall yield of 3.9%.
The binding of L2-4048 to MIF was investigated by X-ray crys-
tallography. We determined a high-resolution structure and have
deposited the coordinates and structure factors in the Protein Data
Bank under accession code 6PEG (Supporting Information). As we
observed for p425, there is only one molecule of inhibitor bound
per trimer of MIF (Figure 2A). A comparison of the three co-crystal
structures of MIF with allosteric inhibitors reveals that the allo-
steric binding site of L2-4048 is consistently at the interface of two
of the crystallographic trimers (i.e. or tightest packing trimers). In-
terestingly, the allosteric site involves three monomers that form an
alternate trimer (Figure 2B). The network of ligand interactions
includes residues from three MIF monomers, two in one trimer and
one in the second trimer, as was the case with the structure of the
complex with p425 (Figure 2C). L2-4048 interacts with residues in
the allosteric site, forming a π-stacking interaction with Trp108,
and causing the expected rotation of Tyr36 as was observed for
AV1013 (Figure 2C). Additionally, there is an edge-to-π interac-
tion between the benzylic aromatic ring and Phe113. The ketone
carbonyl is exposed to solvent.
Comparison of the co-crystal structures of MIF with p425 and
ibudilast showed proximity, but no overlap between the two inhib-
itors, which suggested that a chimeric, structure-based approach
utilizing binding features of both molecules would be effective. We
envisioned linking only the half of p425 which occludes the tau-
tomerase pocket with a portion of ibudilast, leading to structure 1
(L2-4048) (Figure 1). While still too large (MW 623.7 Da) and pos-
sessing two sulfonates and one diazo linkage, L2-4048 represents a
step towards the discovery of a potential medicinal chemistry lead.
Access to the tautomerase pocket is blocked by L2-4048 in the
same fashion as with p425. The sulfonate ion ortho- to the amine
is able to interact electrostatically with the charged ammonium side
chain of Lys32 (2.7 Å distance, Figure 3). Probably because of a
high desolvation penalty, this extra charged interaction does not in
any way improve binding over ibudilast alone. A similar finding
involving the engagement of this same Lys32 was reported by
Jorgensen et al.29 The other sulfonate ion is surrounded by a shell
of water molecules, just as in the p425 structure. The portion of the
naphthalene bearing the diazo and hydroxyl groups is involved in
hydrophobic interactions with Ile64 and Ala103 (Figure 3).
L2-4048 was synthesized according to Scheme 1. The meta-sub-
stituted propynyl nitrobenzene 2 could be synthesized in good yield
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Figure 2. MIF-L2-4048 co-crystallization results: (A) L2-4048 (gray sticks) binds at the interface of two of the crystallographic MIF trimers
(shown in ribbon). (B) Superposition of the rhMIF-4048 complex with both the p425-rhMIF complex (PDB 3U18) as well as the ternary
complex of 4-hydroxyphenylpyruvate (ENO) with the allosteric analogue of ibudilast (AV1013) (PDB 3IJJ) reveals a shared allosteric site
that lies in proximity to the active site where 4-hydroxyphenylpyruvate binds. (C) Stereo-representation of the network of amino acids
interacting with L2-4048.
similar extent as p425 as revealed by decreased IL-8 secretion in
MIF-stimulated foreskin fibroblasts. Thus, while L2-4048 main-
Key MIF residues that control activation of CD74, identified by
tains a fairly consistent level of activity across the various assays,
functional analyses of alanine mutants, include Tyr36, Lys66,
Asn109, Ile64, and Trp108.23 Figure 4 shows a comparison of our
the outlier in Table 1 is the tautomerase IC50 for p425. The reasons
for this are unclear at present, though it has been suggested that
binding mode in relation to the binding of AV1013 in the allosteric
p425 binds potently only as an aggregate.32 An evaluation of the
propensity for L2-4048 to form aggregates in solution is therefore
on-going.
site only, and the binding of the McLean et al quinolone,26 one of
the few MIF inhibitors that occupies both the allosteric site and the
tautomerase pocket. By not only occupying the allosteric pocket
resulting in the conformational change of Tyr36, but also extending
over the surface of MIF, our inhibitor may provide advantages over
the other inhibitors whose binding sometimes results in agonistic
rather than inhibitory activity with respect to CD74 or other pro-
teins.30
MIF binding efficiency and select activity data of L2-4048 com-
pared to p425 are summarized in Table 1. Surface Plasmon Reso-
nance revealed a Kd of binding very similar to that of p425, and
approximately 6.4- to 10-fold lower affinity than ibudilast, which
was 1.4 micromolar in this assay. The Binding Efficiency Index
(BEI = pkd/MW)31 for L2-4048 is approximately 25% higher than
for p425.
When viewing L2-4048 as an ibudilast analog, the addition of
the naphthyl-diazo-phenyl groups has not improved binding affin-
ity. Furthermore, compared with p425, L2-4048 displays a 20-fold
lower potency in the tautomerase assay and two fold less in inhib-
iting recombinant MIF-CD74 interaction. The interference with the
MIF-CD74 interaction, rather than the blocking of the vestigial en-
zymatic activity, should have the highest biological relevance and
should therefore be most predictive for results in cellular assays.
Indeed, L2-4048 attenuates MIF’s pro-inflammatory activity to a
Figure 3. View of the tautomerase active site of the complex of
MIF with L2-4048 (orange). Key amino acid side chains that are in
close proximity with the inhibitor are highlighted: Lys 32 (green),
Ile64 (blue) and Ala103 (aqua). Pro1 from the active site is evident
in the background between Ile64 and Lys32
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Present Address
Details from the MIF-L2-4048 co-crystal structure will be used
in future efforts to find suitable replacements of the diazo and sul-
fonate moieties. Furthermore, our analysis provides a new starting
point for the potential discovery of chimeric molecules of ibudilast
with tautomerase-site inhibitors, as in the McLean et al. quinolone,
if a way to bypass the Phe113 side chain is found. These efforts will
be reported in due course.
1
2
3
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^ Azitra Inc. 400 Farmington Ave. Farmington, CT 06032, U.S.A.
† Arvinas, 395 Winchester Ave, New Haven, CT 06511, U.S.A.
± Boehringer Ingelheim Corp., Old Ridgebury Rd., Ridgefield,
CT 06877, U.S.A.
5
6
7
8
9
Notes
The authors are submitting a patent application on compound L2-
4048 and analogs derived from it. The authors claim no other con-
flicts of interest.
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ACKNOWLEDGMENTS
The authors wish to thank Dr. Sukyeong Lee at the Baylor College
of Medicine for access to the X-ray facility. This work was sup-
ported by NIH grant R44 AR067569 from the National Institute of
Arthritis and Muscoskeletal and Skin Diseases.
ABBREVIATIONS
BEI, Binding Efficiency Index; Boc, tert-butoxycarbonyl; CD74,
Cluster of Differentiation 74; CXCR2, Chemokine Receptor 2;
CXCR4, Chemokine Receptor 4; CXCR7, Chemokine Receptor 7;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloro-
methane; ENO, 4-hydroxyphenylpyruvate enol form; HPP, 4-hy-
droxyphenylpyruvate; IL-8, Interleukin-8; MIF, Macrophage Mi-
gration Inhibitory Factor; SPR, Surface Plasmon Resonance;
TBAF, tetrabutylammonium fluoride; TEMPO, 2,2,6,6-tetra-
methylpiperidin-1-yloxy; TFA, trifluoroacetic acid; THF, tetrahy-
drofuran.
Figure 4. Comparison of MIF allosteric pocket ligands from super-
imposing of co-crystal structures of MIF complexed with AV1013
(Blue, PDB 3IJG), the McLean et al quinolone (ref. 26) (Green,
PDB 3L5T), which extends into the tautomerase active site, and
L2-4048 (Orange, PDB 6PEG).
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Table 1: Binding affinity and inhibition of MIF-mediated activities
by p425 and L2-4048 (see Supporting Information).
Compound
p425
L2-4048
(2) Calandra, T.; Roger, T. Macrophage Migration Inhibitory Factor: a
Regulator of Innate Immunity. Nature Reviews 2003, 3, 791-800.
MW (Da)
904.9
623.7
SPR Biacore Kd (µM)
Tautomerase IC50 (µM)
CD74 binding IC50
9.04 ± 0.83
0.37 ± 0.32
0.40 ± 0.04
6.86 ± 0.77
7.59 ± 0.21
0.93 ± 0.71
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(µM)
IL-8 induction IC50 (µM)
3.90 ± 0.22
4.42 ± 0.85
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ASSOCIATED CONTENT
Supporting Information
Supporting Information is available free of charge on the ACS Pub-
lications website at DOI:
(5) Guo, Y.; Hou, J.; Luo, Y.; Wang, D. Functional Disruption of Mac-
rophage Migration Inhibitory Factor Suppresses Proliferation of Hu-
man H460 Lung Cancer Cells by Caspase-Dependent Apoptosis. Can-
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Synthetic procedures; analytical data for intermediates 3-8 and fi-
nal L2-4048 (1); biophysical assays; in vitro cellular activity stud-
ies; crystallization; and structure determination data.
(6) Kim, J.Y.; Kwok, S.-K.; Hur, K.-H.; Kim, H.-J.; Kim, N.-S.; Yoo,
S.-A.; Kim, W.-U.; Cho, C.-S. Up-Regulated Macrophage Migration
Inhibitory Factor Protects Apoptosis of Dermal Fibroblasts in Patients
with Systemic Sclerosis Clin. Exp. Immunol. 2008, 152(2), 328-335.
Accession Codes
The crystal structure for the complex of L2-4048 with MIF was
deposited in the RSCB Protein Data Bank with PDB ID 6PEG.
(7) Trivedi-Parmar, V.; Jorgensen, W.L. Advances and Insights for
Small Molecule Inhibition of Macrophage Migration Inhibitory Factor.
J. Med. Chem. 2018, 61, 8104-8119.
AUTHOR INFORMATION
Corresponding Authors
* Email: pcirillo@newhaven.edu and oluwatoyin.asojo@hamp-
tonu.edu and asojo@bcm.edu, previously at National School of
Tropical Medicine, Baylor College of Medicine, 1102 Bates Ave-
nue, Houston Texas 77030
(8) Bloom, J.; Sun, S.; Al-Abed, Y. MIF, a Controversial Cytokine: a
Review of Structural Features, Challenges and Opportunities for Drug
Development. Exp. Op. Ther. Targ. 2016, 20(12), 1463-1475.
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ACS Medicinal Chemistry Letters
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Inhibition of Macrophage Migration Inhibitory Factor by a Chimera of Two Allo-
steric Binders
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Pier F. Cirillo,* Oluwatoyin A. Asojo,* Uday Khire, Yashang
Lee, Sara Mootien, Peter Hegan, Alan G. Sutherland, Elizabeth
Peterson-Roth, Michel Ledizet, Raymond A. Koski, and Karen
G. Anthony
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ACS Medicinal Chemistry Letters 2019, 10, x, xxx-xxx (Letter)
Publication Date (Web): , 2019
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