Rational design of peptide derivatives for inhibition of MyD88-mediated toll-like receptor signaling in human peripheral blood mononuclear cells and epithelial cells exposed to Francisella tularensis

Article abstract of DOI:10.1111/cbdd.13039

Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of MyD88-mediated toll-like receptor (TLR) signaling by Francisella tularensis. Fifty-three tripeptide derivatives were synthesized to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1β in human peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to LPS extracts from F. tularensis. From 53 compounds synthesized and tested, ten compounds were identified as effective inhibitors of F.?tularensisLPS-induced cytokines. Compound stability testing in the presence of human liver microsomes and human serum resulted in the identification of tripeptide derivative 7 that was a potent, stable, and drug-like small molecule. Target corroboration using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of 1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling represent a promising strategy toward host-directed adjunctive therapeutics for inflammation associated with biothreat agent-induced sepsis.

Full text of DOI:10.1111/cbdd.13039

DR. DANIEL ANGUS RYAN (Orcid ID : 0000-0003-3971-5399)
Article type : Research Article
Rational Design of Peptide Derivatives for Inhibition of
MyD88-Mediated TLR Signaling in Human PBMCs and Epithelial
Cells Exposed to F. tularensis
Ryan, D. A.;a ‡ Degardin, M.;a ‡ Alam, S.;b Kissner, T. L.;b Hale, M;b, Cameron, M. D.;c Rebek, M.;a
Ajami, D.;a Saikh, K. U.;b and Rebek, J. Jr.a*
‡ These authors contributed equally.
* Corresponding author
E-mail address: jrebek@scripps.edu (Julius Rebek, Jr.)
a Department of Chemistry,
The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Rd,
La Jolla, California, 92037, USA.
b Department of Immunology,
Army Medical Research Institute of Infectious Diseases,
1425 Porter Street,
Frederick, MD 21702, USA.
c Department of Molecular Therapeutics,
The Scripps Research Institute, 130 Scripps Way,
Jupiter, Florida, 33458, USA.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13039
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Keywords:
TIR domain inhibitors, protein-protein interactions, TLR signaling inhibitors, MyD88,
proinflammatory cytokines, Francisella tularensis, sepsis
ABSTRACT
Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of
MyD88-mediated TLR signaling by F. tularensis. Fifty-three tripeptide derivatives were synthesized
to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain
interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1β in human
peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to
LPS extracts from F. tularensis. From fifty-three compounds synthesized and tested, ten compounds
were identified as effective inhibitors of F. tularensis LPS-induced cytokines. Compound stability
testing in the presence of human liver microsomes and human serum resulted in the identification of
tripeptide derivative 7 that was a potent, stable and drug-like small molecule. Target corroboration
using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein
supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also
attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial
epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of
1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling
represent a promising strategy toward host-directed adjunctive therapeutics for inflammation
associated with biothreat agent-induced sepsis.
1. Introduction
Systemic Inflammatory Response Syndrome (SIRS) is classified by a set of clinical
manifestations of immunological disorder and inflammation that can be the result of trauma,
infectious or non-infectious diseases.[1, 2] When an infection is the cause of the inflammatory response,
the condition is normally termed sepsis.[3] In sepsis, an uncontrolled reaction of the innate immune
system and broader immunological dysfunction may progress to tissue damage, organ damage or
death.[4] Beyond primary infections, sepsis is a concern in many medical conditions when damaged or
dysfunctional tissues and organs become predisposed to infection, or through opportunistic infections
of immunocompromised hosts.[5, 6] Sepsis has been reported to affect more than 750,000 people per
year in the United States, a rising number,[7] and mortality rates have been reported in the range of 20-
50 %.[8, 9] In low-to-middle income countries the morbidity and mortality due to sepsis is considered
to be higher.[10] Despite advances in treatments that include antibiotics, intravenous fluids injection
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and/or vasopressors, dialysis, blood sugar level maintenance, respiratory support and surgery,[11, 12]
there is still no efficient therapy available.[13] A difficulty with treating sepsis is that a number of
inflammatory pathways become active simultaneously such as cytokine network and coagulation
system.[7] Under normal circumstances, bacterial infections are countered by host innate immunity,
which identifies pathogens and pathogen-associated molecular patterns (PAMPs) leading to the
release of proinflammatory cytokines that ultimately destroy the pathogens.[14] When there is
excessive production of cytokines and other inflammatory mediators including danger-associated
molecular patterns (DAMPs),[15] normal immune response is altered manifesting symptoms of sepsis
with potentially dire consequences to the host.
Bacterial infections are the most common cause of sepsis.[16] One such pathogen is
Francisella tularensis that is an endotoxin-positive, Gram-negative bacterium and the causative agent
of tularemia, also known as ‘rabbit fever’ or ‘tick fever’. Although the pathogenesis of F. tularensis is
complex, an initial induction period where the pathogen largely evades the immune system precedes a
‘cytokine storm’ associated with severe, systemic sepsis.[17-20] Respiratory infections of F. tularensis
leading to pneumonic tularemia and sepsis are considered among the most severe forms of infection
with the highest mortality rate.[21] According to the Centers for Disease Control and Prevention
(CDC), the incidence of tularemia in the United states has been in the range of 93-205 cases per year
from 2005 through 2014.[22] Despite the relatively low incidence of tularemia, F. tularensis has been
classified as one of six current Category A bioterrorism agents by the CDC due to its high virulence,
potential for aerosolized infection and history as an investigational warfare agent.[20] An infection of
only 10 bacteria have been reported as sufficient to cause tularemia in humans, although the minimal
threshold for infection also has been shown to depend on the route of infection.[23, 24] First-line
treatment of tularemia are antibiotics with streptomycin and gentamicin being the drugs of choice.[25]
However, there has been renewed interest in developing more effective therapies and strategies to
treat sepsis as a late-stage condition of tularemia.[26]
Toll-like receptors (TLRs) function as recognition elements of innate immune system and
mediate the proinflammatory effects of sepsis at a cellular level.[27, 28] The proinflammatory response
to F. tularensis in human cells is consistent with a TLR-mediated signaling mechanism, including
induction of NF-κb and increased TNF-α and IFN-γ as observed in peripheral blood mononuclear
cells (PBMCs) isolated from tularemia patients.[29] However, F. tularensis differs from other
endotoxins, such as E. coli, in that lipopolysaccharide (LPS) from F. tularensis is modified and
induces cytokines in both a TLR2- and TLR4-dependent manner, while being relatively more potent
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31]
for TLR2 that other endotoxins.[30,
Signal transduction from a ligand-activated TLR proceeds
through a cytosolic toll-like/interleukin-1 receptor recognition (TIR) domain that is common to TLR
and IL-1 receptors. Myeloid differentiation primary response protein 88 (MyD88) is involved in
intracellular signal transduction by binding of its TIR domain (i.e., a MyD88 TIR domain) to the
structurally analogous TIR domains of a TLR/IL-1 receptor or other adaptor proteins.[32, 33] MyD88-
mediated signaling has been ascribed to all TLR receptor subtypes, except TLR3 that is dependent on
a different adaptor protein (i.e., TIR domain-containing adaptor protein-inducing IFN-β, TRIF). A
conserved ‘BB-loop’ region has been characterized as a surface-exposed β-strand decisively involved
in TIR domain interactions and propagation of TLR/IL-1 receptor signaling with MyD88.[34-36] The
importance of the ‘BB-loop’ region in TIR domain interactions has been supported by structure-
function studies that exhibited dominant-negative effects on TLR receptor signaling. Point mutations
in ‘box 2’ motif of TIR domains have been shown to block TLR signaling and mice carrying point
mutations (e.g., P712H) in the TLR4 TIR domain were shown to be endotoxin resistant.[34, 37] The
downstream effects of TLR and IL-1R activation through MyD88 include activation of mitogen-
activated protein (MAP) kinases, activation of transcription factors NF-κb, AP-1 or IRF3 and
generation of proinflammatory cytokines.[38] Thus, TIR domain interactions represent a critical
junction for inflammatory signaling and pose a pharmacological target for attenuating late-stage
sepsis in tularemia by modulating the host inflammatory response.[39, 40]
The goal of this study was to develop small molecule inhibitors of proinflammatory cytokines
resulting from TLR signaling activation upon exposure to F. tularensis. Our group previously
reported compound AS1 (i.e., ‘hydrocinnamoyl-L-valinoyl-pyrrolidine’) that is a small molecule
mimetic of the Phe-Val/Ile/Leu-Pro conserved ‘box 2’ motif in the TIR domain ‘BB-Loop’ region
(Table 1). AS1 was effective at blocking proinflammatory cytokines in response to IL-1 receptor
activation.[39, 41] Dimeric analogs of AS1 were also developed that more potently disrupt protein-
protein interactions of MyD88.[42-44] AS1 attenuated in vitro IL-1β signaling in murine lymphocytes
and EL4 thymoma cells in a concentration range of 10-100 μM and reduced IL-1β-induced fever in
mice at 200 mg/kg (i.p.). The compound did not reduce LPS-mediated activation of TLR signaling up
to 100 μM, demonstrating relatively high specificity for IL-1 receptor signaling. Due to the high
sequence homology among the TIR domains of the TLR/IL-1 receptors, we hypothesized that small
molecule derivatives of ‘box 2’ motif peptides might be similarly prepared that attenuate
proinflammatory signaling through TLR signaling pathways. Here, we report the development of
tripeptide derivatives capable of decreasing proinflammatory cytokines in primary human cells
exposed to F. tularensis. These small molecules represent early-stage candidates for use as an
adjunctive therapy for TLR-mediated inflammation associated with sepsis.
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2.
Methods and Materials
2.1. Materials and Instrumentation
All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were used as
received without further purification unless noted below. Amino acids and peptide coupling agents
(i.e., EDC) were purchased from Oakwood Products Inc. (Estill, SC). Compounds 11, 14, 19 and 23
were purchased from Enamine Ltd (Monmouth Jct., NJ). Solvents were purchased from Fisher
Scientific (Waltham, MA) or J.T. Baker (Avantor Performance Materials Inc., Center Valley, PA).
Solvents dichloromethane, tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and triethylamine
were purified by passage through two columns packed with activated alumina under an argon
atmosphere encased in a glass contour systems from SG Water, USA. Deuterated solvents were
purchased from Cambridge Isotope Laboratories, Inc. For HPLC, water was obtained from a Milli-Q
water delivery system (EMD Millipore), while CH3CN and TFA were purchased in HPLC quality.
Analytical thin-layer chromatography (TLC) was performed on Silicycle 60 F254 glass-backed plates.
Hepatic microsomes were purchased from BioreclamationIVT (Chestertown, MD). Human serum was
purchased from BioWorld (Dublin, OH) and stored frozen until use. The Meso Scale Discovery
(MSD) multi spot array ultra-sensitive cytokine assay kit was purchased from MSD (Gaithersburg,
MD). Ficoll-Hypaque was purchased from GE Healthcare Biosciences (Piscataway, NJ). HEK 293
(TLR4-MD2- NF-κB-SEAPorter™ transfected) stable cell line was purchased from Imgenex (San
Diego, CA). Additional information on materials, instrumentation and methods can be found in the
Supporting Information.
2.2. General Synthetic Procedures
2.2.1. ‘Procedure A’ for EDC-mediated amide bond formation:
To a solution of an N-Boc protected amino acid (1 equiv, 100 mM), 1-hydroxybenzotriazole hydrate
(2 equiv) and amine coupling partner (1.2 equiv.) in dichloromethane was added 1(-3-
dimethylamino)propyl-3-carbodiimide hydrochloride (EDC, 1.2 equiv) and triethylamine (1.2 equiv)
at 21 °C. The reaction was stirred for 12-16 hours. At this time, the reaction was diluted with
dichloromethane and washed with 0.5 N HCl(aq), a saturated solution of NaHCO3(aq) and water. The
organic layer was dried (magnesium sulfate), filtered, and concentrated by rotary evaporation. For
synthetic intermediates, the crude product thus obtained was of sufficient purity to advance to the next
reaction. For final products, the crude product was purified by preparative HPLC and lyophilized to
obtain compounds of suitable purity for biological screening.
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2.2.2. ‘Procedure B’ for removal of Boc group:
To a solution of an N-Boc protected amide (1 equiv, 142 mM) in dichloromethane was added
trifluoroacetic acid (8 equiv) at 0°C. The reaction was stirred at this temperature and monitored by
TLC for consumption of starting material (typically 5-8 hours). At this time, the reaction was
concentrated by rotary evaporation. The oily residue was diluted with diethyl ether and then extracted
with water (3x). The combined aqueous layers were neutralized to pH 8 by portionwise addition of
solid NaHCO3 and solid Na2CO3. The aqueous layer was re-extracted with dichloromethane and ethyl
acetate multiple times using TLC (ninhydrin staining) to monitor recovery of the amine from the
aqueous phase as necessary. The combined organic layers were dried (magnesium sulfate), filtered
and concentrated by rotary evaporation and vacuum. The crude products (e.g., compound 3) thus
obtained were of sufficient purity to advance to the next reaction.
2.2.3. ‘Procedure C’ for N-sulfonylation:
Benzenesulfonyl chloride or an aryl-substituted benzenesulfonyl chloride (1.2 equiv) was added to a
solution of amine (1 equiv, 100 mM) and triethylamine (1.2 equiv) in dichloromethane at 21 °C. The
reaction was stirred for 12-16 hours. At this time, the reaction was diluted with dichloromethane and
washed with 0.5 N HCl(aq), a saturated solution of NaHCO3(aq) and water. The organic layer was dried
(magnesium sulfate), filtered, and concentrated by rotary evaporation and vacuum. The crude product
thus obtained was purified by preparative HPLC and then lyophilized to obtain compounds suitable
for biological screening.
2.2.4. ‘Procedure D’ for N-acylation:
Benzoyl chloride or an aryl-substituted benzoyl chloride (1.2 equiv) was added to a solution of amine
(1 equiv, 140 mM) and triethylamine (1.2 equiv) in dichloromethane at 21 °C. The reaction was
stirred for 12-16 hours. At this time, the reaction was diluted with dichloromethane and washed with
0.5 N HCl(aq), a saturated solution of NaHCO3(aq) and water. The organic layer was dried (magnesium
sulfate), filtered and concentrated by rotary evaporation and vacuum. The crude product thus obtained
was purified by preparative HPLC and then lyophilized to obtain compounds suitable for biological
screening.
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2.3. Compounds Synthesis and Characterization
All new compounds and compounds not previously characterized by comparable techniques in the
literature were characterized. The synthesis and characterization of compound 7 and synthetic
intermediates was described below; all other compound synthesis procedures and characterization
data was reported in the Supporting Information. Compound 14 was reported previously.[45] Synthetic
intermediates 3, S1-S7 and S12 were previously described,[46] or have been indexed in the Chemical
Abstracts Service.
(S)-tert-Butyl (4-methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-yl)carbamate (Intermediate S1). Reaction
of Boc-L-Leu (2.13 g, 9.21 mmol) and pyrrolidine by Procedure A provided compound S1 (3.04 g) as
a yellow oil in approximate 85-90 % purity by 1H NMR. Compound characterization data (1H NMR,
13C NMR and mass spectrometry analysis) were consistent with the literature report.[47] Product S1
was used in subsequent steps without further purification.
(S)-2-Amino-4-methyl-1-(pyrrolidin-1-yl)pentan-1-one (Intermediate 3). Reaction of S1 (3.04 g, 10.7
mmol) by Procedure B provided compound 3 (1.24 g, 6.73 mmol) in 73 % yield over two steps from
1
Boc-L-Leu. Compound 3 was obtained as a yellow oil in approximate 95 % purity by H NMR.
Compound characterization data (1H NMR, 13C NMR and mass spectrometry analysis) were
consistent with the literature report.[47] Compound 3 was used in subsequent steps without further
purification.
(S)-4-Ethyl-N-(4-methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-yl)benzamide (7). Reaction of 3 (202 mg,
1.10 mmol) and 4-ethylbenzoic acid by Procedure A provided compound 7 (219 mg, 0.69 mmol) in
1
63 % yield after preparative HPLC purification. Compound 7 was obtained as a white powder. H
NMR (600 MHz, CDCl3) δ 7.73 (d, J = 8.1 Hz, 2H), 7.42 (s, 1H), 7.23 (d, J = 8.1 Hz, 2H), 5.04 (m,
1H), 3.91 (m, 1H), 3.53 (m, 2H), 3.44 (m, 1H), 2.67 (q, J = 7.6 Hz, 2H), 2.02 (m, 2H), 1.88 (m, 2H),
1.74 (m, 2H), 1.51 (m, 1H), 1.23 (t, J = 7.6 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H)
ppm. 13C NMR (75 MHz, CDCl3) δ 171.98, 167.81, 148.74, 130.77, 128.13, 127.43, 49.80, 46.99,
46.58, 41.55, 28.26, 26.02, 24.95, 24.21, 23.41, 21.96, 15.33 ppm. HRMS (ESI) m/z for C19H28N2O2
calculated [M+H]+: 317.2229, found: 317.2222 [M+H]+.
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2.4. Hepatic Microsomal Compound Stability Assay
Compound stability in the presence of 1 mg/mL hepatic microsomes was evaluated by
incubating 1 μM compound in 100 mM potassium phosphate at pH 7.4. The reaction was initiated by
addition of NADPH to give a 1 mM final concentration. Aliquots were removed at 0, 5, 10, 20, 40 and
60 minutes and added to acetonitrile (5x volume) to stop the reaction and precipitate proteins.
NADPH dependence of the reaction was evaluated with the addition of no-NADPH control samples.
At the end of the assay, the samples were centrifuged through a Millipore Multiscreen Solvinter 0.45
micron low binding PTFE hydrophilic filter plate and analyzed on a ABSciex 5500 LC-MS/MS. Data
was log transformed and represented as half-life calculated by using the 0 minute sample as 100 %
compound remaining. Carbamazapine was used as an analytical internal standard for the mass
spectrometer. Sunitinib was run with the compounds as a historical standard for inter-assay
reproducibility.
2.5. Serum Compound Stability Assay
Compound stability in the presence of human serum was evaluated by adaptation of a
reported procedure.[48] Test compound was incubated at 200 μM in human serum at 37 °C in a
sealable glass vial. Aliquots were removed at 0, 15, 30 min, 1, 2, 4, 8, 16, 32 and 64 h. Each aliquot
was extracted with dichloromethane (5 x 0.5 mL) and the combined organic extracts were
concentrated. A solution of 2 % DMSO in water/acetonitrile (1/1) containing 200 μM of 4-
hydroxybenzoic acid internal standard was added, the sample was vortexed and analyzed by analytical
HPLC. Quantification of peak areas normalized to the internal standard provided the concentration of
test compound at each time point. ortho-Nitrobenezene trifluoroacetamide that was previously shown
to be a substrate for human cholinesterases was analyzed to validate serum amidase activity in the
samples used.[49]
2.6. Cell Isolation, Culture Conditions and Compound Treatment
Peripheral blood mononuclear cells (PBMC), obtained from consenting healthy donors in
accordance with Institutional Review Board-approved research donor protocol, were isolated by
standard density gradient centrifugation with Ficoll-Hypaque (GE Healthcare Biosciences AB,
Uppsala, Sweden), harvested from the interface, washed, and suspended in RPMI 1640 medium.
Purified hPBMCs were cryopreserved and on the day of use the cells were thawed and cultured for 2
hours prior to treatment.
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For application of test compounds to cell culture, stock solutions of compound in dimethyl
sulfoxide (DMSO) were freshly prepared for experiments and administered to cell cultures such that
the final concentration of DMSO vehicle was 1 % (v/v) at all concentrations tested. Control groups
(i.e., in the presence or absence of LPS) received DMSO vehicle at the same final concentration. For
application of LPS extract to cells, the LPS extract was administered as a solution in water 30 min
after addition of test compound unless noted otherwise. Statistical comparisons among experimental
groups were made using a one-way ANOVA with Bonferroni post-hoc test (α = 0.05).
2.7. Isolation of LPS from F. tularensis
Bacteria were grown on modified Thayer-Martin agar plates prepared with ingredients: GC
medium base (Difco Labs, 36 g/L), hemoglobin (GIBCO Labs, 10 g/L) and Isovitalex (Becton
Dickinson, 10 mg/L). A frozen stock (NDBR 101) of F. tularensis was thawed and 0.2 mL was spread
evenly on a plate. The inoculated plate was incubated at 37 °C for 2 days in an incubator with 5 %
CO2 and 95 % humidity. Colonies were plated on 3 additional plates and incubated for 16 h. Plates
with confluent growth were plated on 30 additional plates and incubated for 3 days in culture
conditions described above. Bacteria were harvested by adding 15 mL of PBS (pH 7.4) to each agar
plate and gently scrapping the agar surface to dislodge the bacteria. The bacteria remaining on the
agar surface were harvested by serial passage of the first 15 mL suspension. Bacteria were collected
by centrifugation at 10,000 x g for 30 min at 4 °C, and washed 5 times with PBS. The suspension of
bacteria was frozen at -70 °C and inactivated by gamma irradiation with 2.1 megarads absorbed dose.
Inactivated bacteria were dialyzed against sterile deionized water using a 3500 MW restriction
membrane and then lyophilized. The LPS fraction was obtained by extraction of whole bacteria in a
mixture of sterile deionized water and phenol (Sigma Chemicals, molecular biology grade) in a
modification of the method of Westphal and Jann.[50] The heat killed bacteria were suspended in a
mixture of water/phenol (1.2/1.0 v/v) and placed at 68 °C for 15 min in screw capped Teflon®
centrifuge tubes. Phase separation was accomplished by centrifugation at 10,000 x g for 30 min at
room temperature. The aqueous phase was collected. An equivalent volume of sterile deionized water
was added to the remaining phenol phase and phenol/water interface, placed at 68 °C for 10 min, and
the second aqueous phase collected by centrifugation as described above. This procedure was
repeated once more for a total of three aqueous phases. The three aqueous phases were pooled and
dialyzed against sterile deionized water for 4 days at 4 °C with daily changes of dialysate. The
dialyzed phenol-water extract was freeze dried. The yield of the phenol-water extract was 15 % of the
whole cells calculated as dry weight. A 50 mg (dry weight) portion of the extract was treated at 37 °C
for 16 hours in a 10 mL solution of 50 mM Tris (pH 7.4, BioRad Labs, Richmond, CA), 10 mM
MgC12, 0.2 % sodium azide, 2 mg deoxyribonuclease and 3.5 mg ribonuclease (Sigma Chemicals).
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Proteins were degraded by the addition of 10 mg proteinase K (Sigma Chemicals) for 16 hours at 45
°C. LPS was extracted from the proteinase digest by phenol/water extraction using the same protocol
as described above. The yield of purified LPS was 51.4 % (dry weight) of the phenol-water extract
preparation. LPS sample purity was examined by Western blots with polyclonal antibody ABE 335
(Critical Reagents. Program, Frederick, MD). Each sample was estimated to be >99 % pure. LPS
quantitation was performed using the LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher
Scientific, Waltham, MA).
2.8. Cytokine Assay
Cell cultures in the presence or absence of test compound and LPS were incubated (37 °C, 5
% CO2) for 16 h. Cytokines in culture supernatants were measured using a Meso Scale Discovery
(MSD) multi spot array ultrasensitive cytokine kit for human TNF-α, IFN-γ, IL-6, and IL-1β per the
manufacturer’s protocol. Briefly, the 96-well cytokine assay plate was blocked with ‘diluent 2’ for 30
min at room temperature with constant shaking at 400 rpm. The calibrators were processed per
manufacturer’s protocol. Aliquots (25 μL) of both calibrator and sample solutions were separately
added to the plate in triplicate and incubated for 2 h at room temperature with constant shaking at 400
rpm. After 2 h, the plates were washed 3 times with 1× PBS + 0.05 % Tween-20, and then 25 μL of
the detection antibody solution was added to each well of the plate. The plate was incubated for 2 h at
room temperature with constant shaking at 400 rpm. After 2 h, the plates were washed 3 times with
1×PBS + 0.05 % Tween-20. Finally, 150 μL of 2× Read Buffer T was added to each well of the plate
and analyzed on the SECTOR Imager. The assay results were read using an MSD SECTOR Image
2400 incorporating a CCD. Sample cytokine concentrations were determined with Softmax Pro
Version 4.6 software following manufacturer’s specifications. Averaged cytokine levels for TNF-α,
IFN-γ, IL-6, and IL-1β were expressed as percent inhibition relative to cytokine levels in untreated,
LPS-induced cells (i.e., % Inhibition = 100 – {100* [cytokine concentration in compound-treated
LPS-induced cells/cytokine concentration in LPS-induced cells]}). In certain examples, apparent
activation of cytokines in the presence of a test compound and LPS was observed and this data was
reported as having no inhibitory effect. Results were based on cytokine levels that occurred within the
linear range of the LPS-induced cytokine response curve. Concentration (i.e., dose)-response curves
were obtained by plotting the data using Graphpad Prism (La Jolla, CA) on a semi-log plot and IC50
values obtained by non-linear curve fitting.
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2.9. NF-κB Transcriptional SEAP-Reporter Assay
Stably-expressing TLR4/MD-2/NF-κB/SEAPorter™ HEK 293 cells (5×105 cells/ 0.5ml/well)
were cultured in 24-well plates with varying concentrations of test compound, or test compound and
TIR domain protein for competitive binding experiments. After 30 min, F. tularensis LPS extract (10
μg/mL final concentration) was added and the cells incubated at 37 °C for 16 h. The culture
supernatant was collected and centrifuged to remove any cell debris. The Great EscAPe SEAP Assay
from Clonetech was used to determine the amount of alkaline phosphatase secreted into the
supernatant following the manufacturer’s specifications. A 1× dilution buffer was prepared and a 75
μL aliquot was mixed with 25 μL of the supernatant, then incubated for 30 min at 65 °C to inactivate
endogenous alkaline phosphatase. The samples were placed on ice for 3 min and then equilibrated at
room temperature. SEAP substrate solution (100 μL) was added to each sample and light intensity
was read at 10 minute intervals using a chemiluminescent plate reader. Averaged values were
expressed as percent inhibition relative to cytokine levels in untreated, LPS-induced cells.
Concentration (i.e., dose)-response curves were obtained by replotting the data using Graphpad Prism
(La Jolla, CA) on a semi-log plot and IC50 values obtained by non-linear curve fitting.
2.10. Preparation of MyD88 TIR Domain Protein
Preparation of MyD88 TIR domain protein was adapted from a published protocol.[43] The
open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified
from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and
inserted into pDONR201 (Invitrogen, Carlsbad, CA,). The gene was sequence verified and then
inserted into the destination vector pDEST-HisMBP by Gateway recombinatorial cloning to generate
expression vector pPS2218.[51]
The recombinant His-MBP-MyD88(157–296) fusion protein was expressed in E. coli
BL21(DE3) Codon Plus-RIL cells (Stratagene, La Jolla, CA), which were grown in Luria broth and
induced at mid-log phase with 1 mM IPTG for 4 h at 30 °C. The cells were harvested by
centrifugation at 4 °C and frozen at -80 °C until use. The cell pellet was resuspended in 50 mM
sodium phosphate pH 7.1, 150 mM NaCl, 5 % glycerol, 25 mM imidazole, and then the cells were
disrupted using an APV Model G1000 homogenizer (Invensys, Roholmsvej, Denmark). The lysate
was centrifuged at 15,000 rpm at 4 °C, filtered, and the fusion protein was purified by immobilized
metal affinity chromatography (IMAC) as described.[52] Fractions containing the fusion protein were
pooled, cleaved overnight with hexahistidine-tagged TEV protease, and then subjected to another
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round of IMAC as described.[52] The flow-through fractions were pooled, concentrated to 5 mL and
applied to a 320 mL XK26/60 Sephacryl S-100 gel filtration column (GE Healthcare, Piscataway, NJ)
equilibrated in 25 mM 2-(N-morpholino)ethanesulfonic acid pH 6.3, 150 mM NaCl, and 2 mM tris(2-
carboxyethyl)phosphine hydrochloride. The peak fractions corresponding to MyD88 (157–296) were
pooled and concentrated to approximately 1 mg/mL.
2.11. F. tularensis Live Vaccine Strain Infection of hPBMCs
For compound treatment pre-infection, test compound was added to hPBMCs and after a 30
min incubation the cells were infected with F. tularensis live vaccine strain (LVS) obtained from
USAMRIID. Frozen aliquots of F. tularensis LVS were thawed and grown in chocolate blood agar
plate for 48 hours at 37 °C. The bacterial colonies were then resuspended in 5-10 mL liquid medium
BGEM (Lonza, MD). Optical Density (OD) was taken at 600 nm to calculate the Colony Forming
Unit (CFU). Mononuclear cells at 1x106/mL RPMI 1640 in the presence or absence of 5 % AB human
serum per tube were prepared then treated with a concentration-range of test compound at 500 µM,
100 µM, 10 µM, 1 and 0 µM. After 30 min incubation at 37 °C, cells were infected with F. tularensis
LVS at a MOI of 1:5 for 2 hours at 37 °C. After 2 hours, cells were isolated by centrifugation at 1200
rpm and the supernatant was carefully discarded. Fresh medium (1 mL) containing gentamycin (10
µg/mL) and the same test compound at specified concentration were added to the cells. Supernatants
were collected at 48 hours and analyzed with the MSD cytokine assay as described above.
For post-infection experiments, cells were infected with aliquots of F. tularensis LVS as
described above. After a 30 min incubation, the infected cells were isolated by centrifugation and
resuspended in media as described above, then treated with a concentration-range of test compound.
Supernatants were collected at 48 hours and analyzed with the MSD cytokine assay as described
above.
2.12. F. tularensis Live Vaccine Strain Infection of Broncho Epithelial cells
Human bronchial epithelial cells (16HBE14o-) were obtained from Dr. D. C. Gruenert,
California Pacific Medical Center Research Institute, San Francisco, CA. 16HBE14o- cells were
grown to 80 % confluence in 24-well plates and sub-cultured in EMEM supplemented with 10 %
FBS. All experiments in this study were performed on 16HBE14o- cells between 4-15 passage levels.
For development of polarized 16HBE14o- cells, the cells were trypsinized with the Reagent Pack
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subculture system (Lonza Biological, Walkersville MD), then resuspended in bronchial epithelial
growth medium (BEGM) at 5 x 105 cells/mL. Aliquots (500 µL) of the resulting cell suspension were
added to collagen-coated inserts (1.0 mm, 24-well plate, BD Biosciences, Farming MA). Medium in
both the apical and basal chambers was changed every 48 h. Cells were cultured until polarized
monolayers developed (approximately 8 days in culture) and apical cell layers were used in these
experiments.
For compound treatment pre-infection, media was removed and replaced with 300 µL of
either BEGM or BEGM containing test compound (final concentration 100 µM, 10 µM, 1 and 0 µM).
After a 30 min incubation, 300 µL of BEGM containing F. tularensis LVS (1µg/mL) was added to
give a MOI of 1:5 and incubated for 2 hours at 37o C. Cells were isolated by centrifugation 1200 rpm
and the supernatant was discarded. Fresh medium (0.5 ml) containing gentamycin (10 µg/ml) and the
same test compound at specified concentration were added to the cells. Supernatants were collected
after 48 hours and analyzed by MSD cytokine assay as described above.
For post-infection experiments, human bronchial epithelial cells were infected with aliquots
of F. tularensis LVS at a MOI of 1:5 as described above. After a 2 h incubation, the infected cells
were isolated by centrifugation and resuspended in media as described above, then treated with a
concentration-range of test compound. Supernatants were collected after 48 hours and analyzed by
MSD cytokine assay as described above.
3. Results
3.1. Chemistry
Compounds were synthesized to mimic a key tripeptide sequence of the BB-Loop region of
the TIR domains of human MyD88 and TLRs (Table 1). Three regions of the molecule were targeted
for structural optimization: i) an N-terminal phenylalanine present in TLRs 1, 2 and 4, ii) a central
region for leucine/isoleucine/valine present in TLRs and MyD88 and iii) a C-terminal proline present
in TLRs 1, 2, 4 and MyD88. All analogs were purified to >98 % by preparative HPLC. Compounds
were tested iteratively to identify structural features that conferred compound potency for inhibition of
cytokines.
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Solution phase peptide coupling chemistry readily provided the target tripeptide derivatives
(Figure 1). EDC-mediated coupling of N-Boc-L-Leu-OH (2) with secondary amines (e.g., pyrrolidine)
provided a C-terminal amide proline surrogate without appreciable loss of stereochemistry. Following
removal of the N-terminal Boc group to give amine 3, diverse N-terminal substituents were
introduced by reaction of amine 3 with either sulfonyl chlorides or carboxylic acids in the presence of
EDC. A dihydrocinnamyl group present in AS1 was incorporated in an analogous leucine scaffold
(i.e., 4), as well as analogs 5 and 6 with hydroxylated dihydrocinnamyl groups. Alternatively, the
ethylene tether present in dihydrocinnamyl was truncated in the preparation of benzoyl amides with
alkyl (7), hydroxyl (8), or cyano (9, 10) substituents on the aryl ring. Sulfonamides were also explored
as alternatives to amide bonds in the N-terminal region. Benzenesulfonamide analogs were prepared
that were either unsubstituted (11) or substituted with alkyl (12, 13, 14, 15), chloro (16), cyano (17,
18), alkoxy (19, 20, 21), acetamido (22) and methanesulfonyl (23) functional groups on the aryl ring.
These compounds represented a range of N-termainl linkers, aryl substituents and hydrogen bonding
groups designed to identify substructures in the N-terminal aryl region associated with compound
potency for inhibition of LPS-induced cytokines.
In the central region of the scaffold, non-peptidic substructures were investigated as an
alternative to the valine/leucine/isoleucine present in TIR domains of TLRs and MyD88 (Figure 2A).
A series of piperidine 2-, 3-, and 4-carboxylic acids (i.e., scaffolds A1, A2, and A3) were prepared to
test how altering the position of the C-terminal pyrrolidine amide would affect compound potency. An
octahydroindole scaffold (i.e., scaffold B1) was prepared to provide a more hydrophobic side chain in
the central region of the molecule. Tripeptide derivatives based on scaffolds A1, A2, A3 and B1 were
synthesized using EDC-mediated peptide chemistry as described above using either N-Boc protected
piperidine carboxylic acids or N-Boc protected octahydroindole carboxylic acid. Following removal
of the N-terminal Boc group, N-terminal aromatic substituents were introduced in the last step to
provide compounds 24-39 that contained either the dihydrocinnamyl group of AS1 or diverse N-
terminal aryl substituents (i.e., ‘R’ groups in Figure 2). A second series of compounds (i.e., 40-56)
based on scaffolds A1, A2, A3 and B1 was prepared by substituting the C-terminal pyrrolidine amide
for a larger and more lipophilic N-methyl-N-cyclohexylamide (Figure 2B). Together, fifty-three
compounds were prepared that represented a range of substructures in the three regions of the AS1
scaffold. Compounds were prepared and tested iteratively for inhibition of TLR-MyD88 signaling as
described below.
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3.2. Biological Evaluation of Compounds
3.2.1. Effect of Compounds on Cytokines Levels in Human Peripheral Blood Mononuclear Cells
Compounds 4-56 were tested for inhibition of cytokines in primary human peripheral blood
mononuclear cells (hPBMCs) treated with F. tularensis LPS extracts (10 μg/mL) using an adaptation
44]
of protocols previously described for other pathogens.[43,
Cells were pre-incubated with test
compound for 30 minutes and then exposed to LPS extracts from F. tularensis (10 μg/mL) for 16
hours. Cytokine response was measured using a MSD human proinflammatory fourplex ultrasensitive
kit (TNF-α, IFN-γ, IL-6, IL-1β) with electrochemiluminescent output.[44] The calibrated response
curve demonstrated that all four cytokines gave a linear response in the range of 1 pg/mL to 10 μg/mL
of LPS extracts. hPBMCs that were not treated with LPS averaged 51 pg/mL of TNF-α, 54 pg/mL of
IFN-γ, 18 pg/mL of IL-1β and 44 pg/mL of IL-6 (i.e., baseline levels). hPBMCs treated with F.
tularensis LPS extracts (10 μg/mL) averaged 9400 pg/mL of TNF-α, 470 pg/mL of IFN-γ, 13000
pg/mL of IL-6 and 3600 pg/mL of IL-1β. For compound-treated cells, percent inhibition of TNF-α,
IFN-γ, IL-6 and IL-1β levels were expressed as percent inhibition of cytokine levels relative to LPS-
treated cells in the absence of test compound (Figure 3).
Nineteen compounds were identified that provided greater than 10 % average inhibition of
the four cytokines in F. tularensis LPS-induced hPBMCs. Among compounds with diverse N-
terminal aryl substructures (i.e., compounds from Figure 1), compound 7 with a 4-ethylbenzoyl amide
was one of the most potent analogs and inhibited 99 % of TNF-α, 96 % of IFN-γ, 99 % of IL-6 and 21
% of IL-1β at 100 μM. By contrast, analogs of compound 7 that incorporated either a 4-hydroxy-
substituent in 8 or cyano-substituents in 9 and 10 were less effective or inactive at this concentration.
Substituted benzenesulfonamides were found to be effective alternatives for benzenecarboxamide
substructures in the N-terminal region as observed for compounds 13, 15, 16, 17 and 20. Alkyl-
substituted benzenesulfonamides 13 and 15 gave an average of 16 % and 19 % inhibition of the four
cytokines in F. tularensis LPS-induced hPBMCs, respectively. Compound 16 with a 4-chloro aryl
substituent emerged as the most potent benzenesulfonamide in this structural series and inhibited 98
% of TNF-α, 84 % of IFN-γ, >99 % of IL-6 and 4 % of IL-1β. Unsubstituted benzenesulfonamide 11
was inactive at this concentration (i.e., 100 μM). Other substituted benzenesulfonamides were
identified that inhibited LPS-induced cytokines in hPBMCs, including the 3-
cyanobenzenesulfonamide 17 and 3,4-dimethoxybenezenesulfonamide 20. Compound 17 was
marginally more potent than 20 for average inhibition of cytokines, and 17 inhibited 55 % of TNF-α,
56 % of IFN-γ, 40 % of IL-6 and 47 % of IL-1β. From this compound series (i.e., Figure 1
compounds), compounds 7 and 16 provided the greatest attenuation of cytokine levels with an average
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79 % and 71 % inhibition of the four cytokines at 100 μM, respectively. Τhe results supported that
non-polar alkyl- and chloro-substituted aryl groups were a structural feature in the N-terminal region
associated with compound potency, although this effect showed some dependency on substituent ring
position. Neither AS1 nor its leucine analog 4 attenuated LPS-induced cytokines in hPBMCs at these
concentrations.
Non-natural scaffolds in the core region of the tripeptide (i.e., scaffolds A1, A2, A3 and B1
from Figure 2) were tested for inhibition of cytokines in LPS-challenged hPBMCs. In the first series
of compounds (i.e., Figure 2A compounds), the C-terminal region of these non-natural scaffolds was
maintained as a pyrrolidine amide to mimic a proline amide in this region. For the 2-
piperidinecarboxylic acid series (i.e., scaffold A1), compounds 24 and 28 were identified as inhibitors
of cytokines in F. tularensis LPS-challenged hPBMCs. Compound 28 with an N-terminal 4-
chlorobenzenesulfonamide substructure was more potent than 24 with an N-terminal dihydrocinnamyl
substructure, which was consistent with SAR trends observed in the N-terminal region. Compound 28
inhibited 66 % of TNF-α, 51 % of IFN-γ, 33 % of IL-6 and 47 % of IL-1β at 100 μM. For the 3-
piperidinecarboxylic acid series (i.e., scaffold A2), only compound 32 attenuated cytokines with a
21% average inhibition of the four cytokines. These results supported a positional dependency of the
pyrrolidine amide on compound potency, where compounds with a pyrrolidine amide in either the 2-
or 4-position were more potent than 3-substituted piperidine carboxamide analogs. In the 4-
piperidinecarboxylic acid (i.e., scaffold A3) series, both compound 30 that contained an N-terminal 4-
chlorobenzenesulfonamide and compound 37 that contained an N-terminal 4-(tert-
butyl)benzenesulfonamide inhibited cytokines. These results with 30 and 37 were consistent with
prior structure-activity trends observed in the N-terminal region. Compound 30 was the most potent
compound among the piperidine carboxylic acid analogs tested in this series and inhibited 99 % of
TNF-α, 90 % of IFN-γ, 86 % of IL-6 and 97 % of IL-1β at 100 μM.
Compounds with non-natural core scaffolds (i.e., scaffolds A1, A2, A3 and B1 from Figure 2)
wherein the C-terminal pyrrolidine amide was changed to a more bulky and lipophilic N-methyl-N-
cyclohexylamide were also tested (i.e., Figure 2B compounds). A trend emerged that compounds with
a C-terminal N-methyl-N-cyclohexylamide substructure were generally more potent than analogous
C-terminal pyrrolidine amides. For the 2-piperidinecarboxylic acid series compounds (i.e., scaffold
A1) incorporating a C-terminal N-methyl-N-cyclohexylamide, compound 40 with an N-terminal
dihydrocinnamyl
amide
and
compound
45
with
an
N-terminal
4-
(methanesulfonyl)benzenesulfonamide were both potent inhibitors of cytokines, although analogous
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compounds in the pyrrolidine amide series (i.e., 24 and 31) were considerably less potent or inactive
at the same concentration. Compound 40 inhibited >99 % of TNF-α, 93 % of IFN-γ, >99 % of IL-6
and 99 % of IL-1β levels. Compound 45 inhibited 79 % of TNF-α, 54 % of IFN-γ, 40 % of IL-6 and
51 % of IL-1β levels. Also in the 2-piperidinecarboxylic acid series, compound 53 with an N-terminal
4-cyanobenzoyl amide was identified as one of the most potent inhibitors of cytokines and inhibited
>99 % of TNF-α, IFN-γ, IL-6 and 99% of IL-1β at 100 μM. For the 3-piperidinecarboxylic acid series
compounds (i.e., scaffold A2) incorporating a C-terminal N-methyl-N-cyclohexylamide, compounds
41, 50 and 56 were identified as inhibitors, unlike their C-terminal pyrrolidine amide analogs.
However, these compounds were of modest potency and inhibited TNF-α, IFN-γ, IL-6 and IL-1β in
the range of 0-57 %. Again, the results supported that the 3-piperidinecarboxylic acid derivatives were
less effective than the 2-piperidinecarboxylic acid analogs. Among 4-piperidinecarboxylic acid
analogs, none of the compounds tested with a C-terminal N-methyl-N-cyclohexylamide provided
greater than 10 % average inhibition of cytokines that marked a departure from structure-activity
trends observed in the C-terminal pyrrolidine amide series. Compound 52 based on the
octahydroindole scaffold inhibited cytokines at 100 μM in contrast to the analogous C-terminal
pyrrolidine amide 39 that was inactive at this concentration. Compound 52 inhibited 40 % of TNF-α,
47 % of IFN-γ, 66 % of IL-6 and 13 % of IL-1β levels. Overall, tripeptide analogs with an N-methyl-
N-cyclohexylamide in the C-terminal region were found to be among the most potent inhibitors of
proinflammatory cytokines in hPBMCs challenged with F. tularensis LPS extracts. The 2-
piperidinecarboxylic acid derivatives 40 and 53 identified in this structural series were the most potent
compounds identified in this study.
From these studies, compounds 7, 16, 17, 20, 28, 30, 40, 45, 52 and 53 were identified as the
most effective inhibitors of cytokines in F. tularensis LPS-challenged hPBMCs in vitro. These
compounds showed the greatest average inhibition of the cytokines tested and were effective across
TNF-α, IFN-γ, IL-6 and IL-1β. As AS1 and analog 4 were ineffective at attenuating cytokines in
response to exposure to F. tularensis LPS, the new compounds represented a significant advance in
using small molecules to block MyD88-mediated TLR signaling by F. tularensis LPS.
3.2.2. Compound Stability in Human Microsomes and Serum
The stability of potent compounds identified above was tested via a time-course study in the
presence of human liver microsomes.[53] Compounds were incubated in microsomal protein
preparations and the change in compound concentration was determined at specified time points by
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mass spectrometry. The half-life of the compound in the presence of human microsomes was
determined using a pseudo first-order kinetic analysis based on an observed decrease in compound
peak area (Table 2).
Microsomal stability studies confirmed that AS1 exhibited fast loss in the presence of human
liver microsomes with a half-life of 9 minutes. Some of the new molecules resulted in a significant
increase in compound half-lives in the presence of hepatic microsomes. Compounds 7 and 8 derived
from substituted benzoic amides in the N-terminal region had half-lives of 57 min and 120 min,
respectively. Among N-terminal sulfonamides, compound 20 with an N-terminal 3,4-
dimethoxybenzenesulfonamide had a half-life of 50 minutes. Compound 17 with an N-terminal 3-
cyanobenzenesulfonamide had a half-life of 26 minutes. However, many of the other potent
compounds in this series, such as compounds 13, 15, and 16 derived from alkyl- or chloro-substituted
benzenesulfonamides, were relatively unstable (i.e., t1/2 <16 min). Compounds derived from
piperidinecarboxylic acid substructures (i.e., 28, 30, 40, 50, 52, 53 and 56) all exhibited rapid loss in
the presence of human liver microsomes, irrespective of the position of the C-terminal amide on the
piperidine ring. The octahydroindole-derived compound 45 with a C-terminal N-methyl-N-
cyclohexylamide group was also rapidly lost in the presence of human liver microsomes (i.e., half-life
< 3 min). Thus, compound 7 emerged as the lead candidate due to its potent inhibition of all four
cytokines (i.e., TNF-α, IFN-γ, IL-6 and IL-1β) in LPS challenged hPBMCs, while showing good
stability in the presence of human liver microsomes. Compounds 16, 17 and 20 also exhibited
improved stability in the presence of human liver microsomes, but these compounds were less potent
than compound 7 for in vitro attenuation of cytokines. Subsequently, chemical stability of compound
7 was tested in human serum at 37 °C by adaptation of an established HPLC method.[48] No loss of 7
was observed for up to 64 h post incubation that confirmed excellent chemical stability of the
compounds to serum degradation, which included positive controls for serum amidase activity.[49]
3.2.3. Validation of TLR-MyD88 Signaling Inhibition by Compound 7: F. tularensis LPS-Induced
SEAP Reporter Gene Expression Driven by the Transcriptional Activation of NF-κB.
To further characterize the effect of compound 7 on blocking MyD88-mediated TLR
signaling in response to LPS stimulation, a secreted alkaline phosphatase (SEAP) reporter gene under
the transcriptional control of NF-κB was examined using an established reporter gene construct
(TLR4/MD-2/NF-κB/SEAPorter™) in HEK-293 cells. SEAP activity was determined using a pro-
chemiluminescent substrate (Figure 4). Cells treated with LPS extract from F. tularensis showed a
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significant induction of Nf-κB driven SEAP reporter expression compared to untreated cells (i.e., 3.7-
fold; p < 0.001). In the presence of compound 7 (i.e., 0, 1, 10, 100 or 500 μM), a concentration-
dependent inhibition of SEAP expression was observed (Figure 4A). Compound 7 was effective at
concentrations as low as 1 μM that gave 7 % inhibition of SEAP expression (p < 0.01) and inhibited >
98 % of SEAP expression at 500 μM. Compound 7 had an IC50 of 27 μM for F. tularensis LPS-
induced SEAP expression. Since the SEAP reporter construct expresses full length TLR4 and F.
tularensis LPS was previously shown to agonize the TLR4 receptor,[31] the results suggest that
compound 7 specifically blocked TLR4-MyD88-dependent signaling in F. tularensis LPS-challenged
cells.
Specific inhibition of TIR domain interactions by compound 7 was tested in a competition
experiment using MyD88 TIR domain protein. HEK-293 cells containing the NF-κB driven SEAP
reporter gene construct (TLR4/MD-2/NF-κB/SEAPorter™) were incubated with F. tularensis LPS
(10 μg/mL) and compound 7 that lowered SEAP expression to 45 % of the untreated LPS-induced
cells (i.e., 55 % inhibition). TIR domain protein from MyD88 was then added in a concentration range
(0-100 μg/mL) and the resulting SEAP activity was measured (Figure 4B). TIR domain protein
concentration-dependently lessened the inhibitory effect of 7 on SEAP reporter expression induced by
F. tularensis LPS. TIR domain protein at 100 μg/mL resulted in a return to 81 % SEAP expression
(i.e., 21 % inhibition) that represented a 2.6-fold lessening in the inhibitory effect of 7 at this
concentration. For comparison, bovine serum albumin (BSA) protein added in the same concentration
range (0-100 μg/mL) showed no obvious concentration-dependent effects in the presence of
compound 7 with 52 % of SEAP expression remaining (i.e., 48 % inhibition) at 100 μg/mL of BSA.
Control experiments showed that TIR domain protein had no significant effect on SEAP expression
induced by F. tularensis LPS in the absence of 7. In addition, neither TIR domain protein nor
compound 7 significantly affected basal levels of SEAP reporter expression in cells not treated with
LPS. These results supported that compound 7 specifically interacts with TIR domain protein for
inhibition of LPS-induced TLR signaling.
3.2.4. Investigation of Compound 7 with a Live Vaccine Strain of F. tularensis: Pre- and Post-
Exposure Scenarios with hPBMCs and Human Bronchial Epithelial Cells
Compound 7 was tested in hPBMCs or human bronchial epithelial cells exposed to a F.
tularensis LVS at a multiplicity of infection (MOI) of up to 1:5 (human cells: F. tularensis). A
concentration range of compound 7 (i.e., 0, 1, 10, 100 or 500 μM) was administered to hPBMCs 30
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minutes prior to exposure to F. tularensis LVS and cytokine levels were measured 48 hours following
exposure (Table 3). hPBMCs challenged with F. tularensis (MOI of 1:5) in the absence of 7 gave a
large induction of TNF-α (250-fold), IFN-γ (130-fold), IL-6 by (240-fold) and IL-1β (330-fold). In
the presence of compound 7, a concentration-dependent inhibition of cytokines was observed with
IC50 values for TNF-α of 49 μM, IFN-γ of 12 μM, IL-6 of 65 μM and IL-1β of 54 μM. Compound 7
was also effective at reducing cytokine levels in primary human bronchial epithelial cells. Human
bronchial epithelial cells exposed to F. tularensis LVS (MOI of 1:5) gave a significant induction of
cytokines, but the fold-change induction in cytokines was lower compared to hPBMCs. Human
bronchial epithelial cells exposed to F. tularensis LVS (MOI of 1:5) increased TNF-α (4.5-fold), IFN-
γ (3.0-fold), IL-6 (4.3-fold) and IL-1β (2.6-fold). In the presence of compound 7, a concentration-
dependent inhibition of cytokines was observed with IC50 values for TNF-α of 50 μM, IFN-γ of 21
μM, IL-6 of 12 μM and IL-1β of 7 μM. Accordingly, compound 7 inhibited TNF-α, IFN-γ, IL-6 and
IL-1β levels in a narrow range for both cell types (i.e., IC50 values ranged from 12-65 μM for
hPBMCs and 7-50 μM for bronchial epithelial cells).
Compound 7 was also tested in a post-exposure scenario. Compound 7 was administered to
hPBMCs or human bronchial epithelial cells after the cells had been exposed to F. tularensis LVS
(MOI = 1:5) and cytokine levels were measured 48 hours following exposure (Table 4). For hPBMCs,
compound 7 gave a concentration-dependent inhibition of cytokines albeit with lower potency than
for a pre-treatment scenario. hPBMCs challenged with F. tularensis LVS (MOI of 1:5) and then
treated with a concentration-range of 7 after 30 minutes provided IC50 values for TNF-α of 67 μM,
IFN-γ of 210 μM, IL-6 of 99 μM and IL-1β of 170 μM. Primary human bronchial epithelial cells were
challenged with F. tularensis LVS (MOI of 1:5) for a longer duration (i.e., 2h) to increase the relative
induction of cytokine levels. Primary human bronchial epithelial cells challenged with F. tularensis
LVS (MOI of 1:5) and then treated with a concentration-range of 7 after 2 hours provided IC50 values
for TNF-α of 13 μM, IFN-γ of 11 μM, IL-6 of 12 μM and IL-1β of 5 μM. For bronchial epithelial
cells, compound 7 inhibited TNF-α, IFN-γ, IL-6 and IL-1β cytokine levels in a comparable range as
the pre-exposure scenario (i.e., IC50 range of 7-50 μM for pre-exposure and 5-13 μM for post-
exposure). The results may suggest some differences in the efficacy of compound 7 compared among
hPBMCs and bronchial epithelial cells, however the induction of cytokines in bronchial epithelial
cells was also generally lower than observed for hPBMCs. Collectively, these in vitro results support
that targeting TIR domain interactions of the TLR/IL receptor and MyD88 was an effective approach
to attenuating cytokines in either a pre- or post-exposure scenario to a live vaccine strain of F.
tularensis.
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4.
Discussion
Sepsis is a complex condition that poses challenges to therapeutic development in part because it
remains unknown if targeting a single signaling pathway can lead to an effective treatment for the
immunological effects.[54] Given the important role of TIR domain interactions in MyD88-dependent
TLR/IL receptor signaling across multiple receptor subtypes, small molecules were developed to
inhibit proinflammatory cytokines by targeting this signaling junction. This strategy targets a host’s
immune response to inflammatory pathogens rather than the pathogen itself and may have potential
applications as an adjunctive therapy for sepsis.[39, 40, 55] With hPBMCs used in this study, exposure to
F. tularensis induced a marked increase in NF-κb and TNF-α, IFN-γ, IL-1β and IL-6 cytokines that
was consistent with the established role for MyD88-dependent TLR/IL receptor signaling for
56, 57]
induction of proinflammatory cytokines.[29,
Small molecule development was guided by
mimicking TIR domain regions known to be important to TLR/IL receptor interactions with MyD88.
Compound 7 emerged from testing several candidate tripeptide mimetics and 7 potently decreased
proinflammatory cytokines in hPBMCs exposed to F. tularensis with comparable potency across the
cytokines tested. Compound 7 was also effective in human bronchial epithelial cells that relate to the
respiratory effects of sepsis that are considered a particularly severe form of tularemia.[21] While it
remains to be seen if such a strategy can be effective in vivo, targeting the host inflammatory
signaling pathways with small molecule 7 attenuated cytokines in a pre-exposure or post-exposure
scenario for isolated cells.
Structural trends were identified among those compounds that most potently inhibited cytokines
in hPBMCs exposed to F. tularensis. Substitution of the central valine residue in AS1 for a leucine
residue in compound 7 was a structural feature associated with biological potency. This structural
modification was consistent with the presence of a leucine in the target region of the TIR domain of
MyD88 or an isoleucine in the TIR domains of TLR2 and TLR4.[58, 59] Other substitutions for the
central valine of AS1 resulted in the identification of piperidinecarboxylate-derived compounds (e.g.,
30, 40, 50 or 53) that potently inhibited proinflammatory cytokine levels. Although this represented
an interesting structural development by changing the spatial relation of the C-terminal and N-
terminal groups, these piperidinecarboxylate derivatives were rapidly lost in the presence of human
liver microsomes and so were not further pursued. In the N-terminal region, alkyl- or chloro-
substituted benzoyl amides or benzenesulfonamides were identified as structural features associated
with a marked increase in potency. Lipophilic aryl substituents in this phenylalanine mimetic region
may improve binding, but the positional-dependency of these substituents on compound potency
suggested that the effects were not solely due to indiscriminate binding by lipophilic molecules. Based
on both the structural resemblance of compound 7 to the target tripeptide sequence and interactions of
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7 with the MyD88 TIR domain, compound 7 may indeed function as a peptidomimetic inhibitor of
TIR domain interactions. However, further resolution of the binding site of 7 is still needed. One
approach might be to conduct competitive binding experiments using radiolabeled compounds and
isolated TIR domain proteins, where MyD88 inhibitors that have established binding sites may be
used to test for site-specificity of the new compounds. However, alternative binding modes for
compound 7 cannot be excluded and detailed characterization of a TIR domain protein complex with
compound 7 may be necessary to more precisely attribute the molecular basis for increased potency.
Co-crystallization of TIR domains and compound 7, or solution phase NMR binding experiments are
two possibilities to obtaining this information. Compound 7 was a potent inhibitor of cytokines, but
also resulted in much greater stability of the compound in the presence of human liver microsomes
and so represented a significant advance in development a drug-like small molecule.
Compound 7 and most analogs prepared in this study have not been previously reported, however
other classes of small molecules and glycolipids have been developed as modulators of
proinflammatory signaling at the TLR level (i.e., upstream in the signaling pathway).[60] Among TIR
domain inhibitors, AS1 that targeted the box 2 of TIR domain for inhibition of IL-1 receptor signaling
represents a close structural comparison to compound 7 in that both compounds are small tripeptide
derivatives intended to mimic similar regions of TIR domains.[39] The inhibitory effect of compound 7
on TLR signaling represents a key distinction among these molecules. Other molecules targeting box
2 of TIR domains have been developed that typically employed larger molecules designed to mimic
larger regions of the TIR domain proteins, including some molecules potentially capable of
mimicking dimeric interactions. Synthetic cell-penetrating oligopeptides based on TIR domain
sequences were reported to inhibit TIR domain interactions for MyD88 homodimerization,[61] and
MyD88-TLR heterodimerization,[62] for attenuation of proinflammatory signaling. Based on some of
the peptide work, compound ST2825 was developed as a peptidomimetic inhibitor of the MyD88 TIR
domain ‘BB-Loop’ region that encompassed the box 2 domain targeted by AS1.[63] Compound
ST2825 was reportedly based on a heptapeptide sequence of this domain and blocked TIR domain
interactions induced by IL-1β treatment and reduced IL-6 levels in C57BL/6 mice. Additionally, we
have reported a dimerization strategy for AS1 that gave compounds (i.e., EM163 and 4210) that were
significantly more potent than AS1, and compound 4210 attenuated proinflammatory cytokines
induced by several biothreat agents and was efficacious in a lethal shock challenge in BALB/c
mice.[42, 44] More recently, ortho-vanillin and certain derivatives (e.g., compound C29) were identified
as inhibitors of the TIR domain interactions of TLR2 and inhibited TLR2-mediated inflammation in
mice.[64] Direct comparisons of potency among compound 7 and the above TIR domain inhibitors are
difficult due to the different experimental conditions and proinflammatory stimulants used, but
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compound 7 appears to have comparable potency range as most of the larger peptide derivatives and
ortho-vanillin (i.e., NF-κb reporter data indicated inhibition in a range of 1-200 μM for most
molecules where data is available). Dimeric AS1 analogs such as 4210 have been shown to be a
strategy for increasing the potency of these small tripeptide mimetics.[44] Thus, the potency of
compound 7 relative to size of the molecule represents a possibly advantageous property. More
generally, the current work further supports that compounds that inhibit TIR domain interactions
associated with specific TLR subtypes (e.g., TLR4) might be used as a scaffold for developing
inhibitors of the related TIR domains based on structural analogy among these domains. Collectively,
these studies show that disrupting protein-protein interactions associated with TIR domain
interactions may be a promising strategy for controlling host-mediated inflammatory processes
.
Conclusions
Small molecule inhibitors of MyD88-mediated TLR signaling were developed that target TIR
domain interactions. Compound 7 inhibited cytokines in both primary human PBMCs and bronchial
epithelial cells challenged with F. tularensis, including a live vaccine strain. Compound 7 was also
effective at lowering cytokine levels in cells post-exposure to F. tularensis. Small molecules, such as
7, that inhibit proinflammatory signaling at the TLR-MyD88 signaling junction may represent a
potential strategy for a host-directed, adjunctive treatment for severe inflammatory reactions of sepsis,
including those associated with biothreat agents.
Disclosures
Peripheral blood mononuclear cells used in this study were obtained from healthy donors with written
consents, in accordance with guidelines of the Human Use Committee (HUC) and an institutional
(USAMRIID) review board-approved research donor protocol. Human bronchial epithelial cells
(16HBE14o-) were obtained from Dr. D. C. Gruenert, California Pacific Medical Center Research
Institute (San Francisco, CA) following institutionally approved protocols and in accordance with the
HUC guidelines. The views expressed in this paper are those of the authors and do not purport to
reflect official policy of the USAMRIID.
This article is protected by copyright. All rights reserved.

Acknowledgments
We are grateful for support from the Defense Threat Reduction Agency-Joint Science and Technology
Office, Translational Medical S&T Division. This research was supported by DTRA award
HDTRA1-10-C-003 (Scripps Research Institute, La Jolla). We are also grateful to Dr. Dave Waag
(Bacteriology Division, USAMRIID) for providing LPS extracts from F. tularensis and heat
inactivated F. tularensis for this study.
Competing Financial Interests
The authors declare no competing financial interests.
Supporting Information
Supplementary data related to this article can be found online.
Abbreviations:
CDC, Centers for Disease Control and Prevention; DAMPs, damage-associated molecular patterns;
DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole; hPBMCs, human peripheral
blood mononuclear cells; IFN, interferon; IL, interleukin; IMAC, immobilized metal affinity
chromatography; IRF, interferon regulatory factor; LPS, lipopolysaccharides; LVS, live vaccine
strain; MAP, mitogen-activated protein; MOI, multiplicity of infection; MyD88, myeloid
differentiation primary response protein 88; NF-κB, nuclear factor κB; PAMPs, pathogen-associated
molecular patterns; PBMC, peripheral blood mononuclear cells; SIRS, systemic inflammatory
response syndrome; TIR, toll-like/interleukin-1 receptor recognition; THF, tetrahydrofuran; TLR, toll-
like receptor; TFA, trifluoroacetic acid; TNF, tumor necrosis factor.
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Figure Legends and Tables
Table 1. Sequence alignment of the target BB-Loop region of human TIR domains of MyD88, TLRs
and IL-1 receptor.
Protein
Accession Number
(UniProtKB)
Sequence*
MyD88
TLR1
Q99836
Q15399
O60603
O00206
P14778
196 R D V L P G T
671 R N F V P G K
677 R D F I P G K
710 R D F I P G V
428 R D D Y V G E
TLR2
TLR4
IL-1R1
* AS1-mimetic sequence in bold.
Figure 1. Synthesized tripeptide derivatives 4-23 with diverse N-terminal aryl substituents. i)
pyrrolidine (1 equiv.), EDC (1.2 equiv.), HOBt (2.0 equiv.), CH2Cl2, 21 °C, 12-16 h, ii) TFA (8
equiv.), CH2Cl2, 0 °C, 5-8 h; NaHCO3(s) iii) RCO2H (1.2 equiv.), EDC (1.2 equiv.), HOBt (2.0 equiv.),
CH2Cl2, 21 °C, 12-16 h, iv) RSO2Cl (1.2 equiv.), NEt3 (1.2 equiv.), CH2Cl2, 21 °C, 12-16 h.
Figure 2. Synthesized tripeptide derivatives 24-56 with 2-, 3- or 4-piperidinecarboxylic acid
substructures (i.e., scaffolds A1, A2 or A3) or a 2-substituted L-octahydroindolecarboxylic acid
substructure (i.e., scaffold B1). A) Compounds synthesized with a C-terminal pyrrolidine amide, B)
Compounds synthesized with a C-terminal N-methyl-N-cyclohexylamide (B).
Figure 3. Effects of compounds (100 μM) on cytokine levels in human peripheral blood mononuclear
cells exposed to LPS extracts (10 μg/mL) from F. tularensis. Compounds were administered to cells
30 min prior to addition of LPS extracts and the concentration of TNF-α, IFN-γ, IL-6, and IL-1β was
measured after 16 h. Data was expressed as percent inhibition of cytokine levels relative to LPS-
treated cells in the absence of test compound, showing results for compounds that had greater than 10
% average inhibition of TNF-α, IFN-γ, IL-6, and IL-1β.
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Table 2. Half-lives of compounds in the presence of human hepatic microsomes.
Compound
t1/2 (min)
Compound
t1/2 (min)
AS1
7
9
57
120
5
28
30
40
45
50
52
53
56
5
2
2
2
7
2
2
7
8
13
15
16
17
20
2
16
26
50
Figure 4. Effect of compound 7 on SEAP reporter expression in HEK 293 cells expressing the
TLR4/MD-2/NF-κB/SEAPorter™ construct induced by F. Tularensis LPS (10 μg/mL). A. Compound
7 concentration-range (0, 1, 10, 100 and 500 μM) was administered to cells for 30 min prior to
addition of F. tularensis LPS (10 μg/mL) and SEAP activity was measured by chemiluminescence
after 16 h. Data was expressed as a percentage of SEAP activity compared to LPS-treated cells in the
absence of test compound. Values are the average (n = 3) showing error bars for standard deviation.
Error bars were obscured by data symbols in several instances. B. Competitive binding experiment
using MyD88 TIR domain protein with compound 7. Human MyD88 TIR domain protein or bovine
serum albumin protein control were administered in a concentration range (i.e., 0, 1, 5, 25 and 100
μg/mL) to cells incubated in the presence of compound 7 (100 μM), and F. tularensis LPS (10
μg/mL) was added after 30 min. Separately, TIR domain protein (100 mg/mL) was administered to
cells in the absence of compound 7 and F. tularensis LPS (10 μg/mL) was added after 30 min. SEAP
activity was measured by chemiluminescence after 16 h. Data was expressed as a percentage of SEAP
activity compared to LPS-treated cells in the absence of test compound. Values are the average (n = 3)
showing error bars for standard deviation. Error bars were obscured by data symbols in several
instances.
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Table 3. Compound 7 IC50 values for cytokines in hPBMCs or human bronchial epithelial cells
exposed to F. tularensis live vaccine strain 30 minutes after compound treatment.
IC50 (μM)
Cytokine
hPBMCs
Epithelial Cells
(MOI = 1:5)
(MOI = 1:5)
49
12
65
54
50
21
12
7
TNF-α
IFN-γ,
IL-6
IL-1β
Table 4. Compound 7 IC50 values for cytokines in hPBMCs or human bronchial epithelial cells
exposed to F. tularensis live vaccine strain for 30 min (hPBMCs) or 2 h (epithelial cells) prior to
compound treatment.
IC50 (μM)
Cytokine
hPBMCs
Epithelial Cells
(MOI = 1:5)
(MOI = 1:5)
67
210
99
13
11
12
5
TNF-α
IFN-γ,
IL-6
170
IL-1β
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