Small Molecule NF-κB Inhibitors as Immune Potentiators for Enhancement of Vaccine Adjuvants

Article abstract of DOI:10.3389/fimmu.2020.511513

Adjuvants are added to vaccines to enhance the immune response and provide increased protection against disease. In the last decade, hundreds of synthetic immune adjuvants have been created, but many induce undesirable levels of proinflammatory cytokines including TNF-α and IL-6. Here we present small molecule NF-κB inhibitors that can be used in combination with an immune adjuvant to both decrease markers associated with poor tolerability and improve the protective response of vaccination. Additionally, we synthesize a library of honokiol derivatives identifying several promising candidates for use in vaccine formulations.

Full text of DOI:10.3389/fimmu.2020.511513

Small molecule NF-kB inhibitors as immune
potentiators for enhancement of vaccine adjuvants
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Aaron P. Esser-Kahn , Brittany A. Moser , Yoseline Escalante-Buendia , Rachel C.
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Steinhardt , Matthew G. Rosenberger , Britteny J. Cassaidy , Nihesh Naorem , Alfred C.
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Chon , Minh Nguyen , Ngoctran Tran
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University of Chicago, United States, University of California, Irvine, United States
Submitted to Journal:
Frontiers in Immunology
Specialty Section:
Molecular Innate Immunity
ISSN:
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664-3224
Article type:
Original Research Article
Received on:
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1 Nov 2019
Accepted on:
5 Aug 2020
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Provisional PDF published on:
5 Aug 2020
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Frontiers website link:
www.frontiersin.org
Citation:
Esser-kahn AP, Moser BA, Escalante-buendia Y, Steinhardt RC, Rosenberger MG, Cassaidy BJ, Naorem
N, Chon AC, Nguyen M and Tran N(2019) Small molecule NF-kB inhibitors as immune potentiators for
enhancement of vaccine adjuvants. Front. Immunol. 11:2333. doi:10.3389/fimmu.2020.511513
Copyright statement:
©
2020 Esser-kahn, Moser, Escalante-buendia, Steinhardt, Rosenberger, Cassaidy, Naorem, Chon,
Nguyen and Tran. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is
permitted, provided the original author(s) or licensor are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms.
This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF
and full text (HTML) versions will be made available soon.

Frontiers in Immunology | www.frontiersin.org

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Small molecule NF-kB inhibitors as immune potentiators for
enhancement of vaccine adjuvants
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Brittany A. Moser , Yoseline Escalante-Buendia , Rachel C. Steinhardt , Matthew G.
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Rosenberger , Britteny J. Cassaidy , Nihesh Naorem , Alfred C. Chon , Minh H. Nguyen ,
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Ngoctran T. Tran , Aaron P. Esser-Kahn
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1Pritzker School for Molecular Engineering, University of Chicago, 5640 South Ellis Avenue,
Chicago, Illinois 60637, USA.
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2Department of Chemistry, Chemical Engineering & Materials Science, Biomedical Engineering,
University of California, Irvine, California 92697, USA*
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Correspondence:
Aaron Esser-Kahn
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aesserkahn@uchicago.edu
Keywords: Adjuvants, vaccines, NF-kB, honokiol, capsaicin. (Min.5-Max. 8)
Abstract
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Adjuvants are added to vaccines to enhance the immune response and provide increased
protection against disease. In the last decade, hundreds of synthetic immune adjuvants have
been created, but many induce undesirable levels of proinflammatory cytokines including TNF-
 and IL-6. Here we present small molecule NF-B inhibitors that can be used in combination
with an immune adjuvant to both decrease markers associated with poor tolerability and
improve the protective response of vaccination. Additionally, we synthesize a library of
honokiol derivatives identifying several promising candidates for use in vaccine formulations.
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Introduction
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Vaccines remain one of the most effective ways of preventing disease. Despite their immense
success in preventing diseases such as polio, tetanus, and small pox, diseases such as HIV and dengue
present challenges that current clinical vaccine technologies cannot provide. To solve this problem,
one strategy that has been explored is to include adjuvants, molecules that enhance the immune
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response. Although novel adjuvants generate higher quality immune responses that cannot be achieved
with current approved adjuvants, to date, very few have been approved for use in human vaccines. This
disconnect is due, in part, to the challenge of balancing the proinflammatory immune response with
the protective, adaptive immune response.2 We recently reported that vaccines could be improved
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through the use of a peptide NF-B inhibitor, SN50 in combination with an immune adjuvant. The
addition of SN50 to adjuvanted vaccines led to increased safety of the adjuvant while enhancing
protection against disease. Although this method proved both general across a wide range of adjuvants
and effective against antigens of a variety of diseases, it still required a large amount of the peptide to
enable optimal safety and protection. Scale-up of peptides present synthetic challenges and can result
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in expensive production costs, limiting their potential in a clinical setting. Peptides might also induce
an immune response against themselves leading to a potential for decreased enhancement in subsequent

NF-kB inhibitors as immune potentiators
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vaccinations. We chose to explore other small molecule NF-B inhibitors as immune potentiators to
overcome these challenges.
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Here we demonstrate that select small molecule NF-B inhibitors are effective at reducing
adjuvant-induced inflammation while also increasing the adaptive humoral immune response. At the
same time, we demonstrate that not all NF-B inhibitors are effective immune potentiators. Of the
molecules we tested, honokiol and capsaicin proved to be effective at both limiting inflammation and
potentiating the protective response. Through knockout studies, we demonstrate that the increase in
antigen specific antibodies is independent from the anti-inflammatory activity, which is congruent with
our previous studies . To determine if these small molecules could be improved by chemical synthesis,
we explored derivatives of honokiol and found several promising candidates for potential use in
vaccines.
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Results and discussion
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Exploration of small molecule NF-B inhibitors in vitro
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To begin exploring alternative NF-B inhibitors, we examined the literature for promising
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candidates. Due to the strong correlation between NF-B activation and sepsis , cancer
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autoimmune disorders , a large library of NF-B inhibitors have been identified. Small molecule
NF-kB inhibitors often do not act on the NF-kB subunits themselves, but rather inhibit particular
proteins in the NF-kB pathway, upstream of NF-kB translocation to the nucleus (see supplementary
table 1). We first wanted to analyze the potential of a variety small molecule NF-B inhibitors to
inhibit inflammation in vitro in combination with lipopolysaccharide (LPS), a TLR4 agonist. We chose
several common commercially available NF-B inhibitors and tested them in RAW macrophages. We
chose to examine: Cardamonin (40 µM), Caffeic acid phenethyl ester (CAPE) (100 µM), Withaferin
A (WA) (400 nM), Resveratrol (10 µM), Salicin (100 nM), 5Z-7-Oxozeaenol (5-z-O) (5 µM),
Parthenolide (20 µM), Honokiol (20 µM), Capsaicin (200 µM), PDK1/Akt/Flt dual pathway inhibitor
(PDK1) (1 µM), and GYY 4137 (GYY) (200 µM). To determine if immune potentiation was specific
to NF-B or general to all anti-inflammatory molecules, we included the most common FDA approved
anti-inflammatory drugs, acetaminophen (10 mM) and ibuprofen (800 µM).13,14 We treated RAW
macrophages with inhibitors and LPS and assayed the supernatant for IL-6 secretion (Fig. 1a). CAPE,
WA, 5-z-O, honokiol and capsaicin demonstrated the greatest reduction in IL-6 levels.
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Exploration of small molecule NF-B inhibitors in vivo
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We next wanted to examine how these inhibitors would alter safety and protection in vivo. To
test this in vivo, we chose three of the small molecule inhibitors that were the most effective at
inhibiting IL-6 expression in vitro, capsaicin, honokiol and withaferin A (WA) and ran them alongside
acetaminophen and ibuprofen. We chose to vaccinate mice using CpG, a TLR9 agonist. For our in vivo
vaccination, we used ovalbumin (OVA) as a model antigen to examine the changes in humoral
response. We vaccinated mice with 100 µg OVA, 50 µg CpG, and inhibitor (800 µg ibuprofen, 2 mg
acetaminophen, 400 µg honokiol, 20 µg capsaicin or 600 µg WA). Due to the difficulty in solubility
of the inhibitors, all vaccines were formulated in Addavax, a squalene-based oil-in-water nano-
emulsion, to enable effective vaccine suspensions. To enable comparison between groups PBS and
CpG controls were also formulated in Addavax. We chose to analyze systemic levels of TNF- and
IL-6 because high levels of these cytokines are pyrogenic and have been correlated with undesirable
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vaccine-related side effects.
We previously determined that CpG-induced TNF- and IL-6 peak at
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NF-kB inhibitors as immune potentiators
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1 h post-vaccination. Mice vaccinated with CpG demonstrated high levels of TNF- (1067 pg/mL)
(Fig. 1b). Addition of an NF-B inhibitor decreased the level of TNF-. Ibuprofen decreased to the
mean level of TNF- to 738 pg/mL (1.4 fold), acetaminophen 584 pg/mL (1.8 fold), honokiol 464
pg/mL (2.3 fold), capsaicin 38 pg/mL (28 fold, equivalent to background levels), and WA 580 pg/mL
(1.8 fold). The systemic levels of IL-6 were also high with CpG vaccination (941 pg/mL). The groups
that included an NF-B inhibitor did not always decrease the level of IL-6 (Fig. 1c). Ibuprofen,
acetaminophen and WA did not alter the cytokine profile statistically significantly compared to CpG
alone. Ibuprofen (1001 pg/mL) increased the level of IL-6 by 1.06 fold. Acetaminophen (866 pg/mL)
decreased the level by 1.08 fold. WA increased the level of IL-6 to 967 pg/mL (1.02 fold increase).
However, honokiol and capsaicin dramatically reduced the systemic levels of IL-6 to 206 pg/mL (3.5
fold) and 47 pg/mL (20 fold), respectively.
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To broadly establish how the addition of these inhibitors impacts the antibody levels, we chose
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to analyze the total Ig(G+A+M) produced after 21 days. On day 21, we analyzed the anti-OVA
antibody levels (Fig. 1d). CpG was 1.6 fold (2312 U/mL) higher than PBS (1365 U/mL). Ibuprofen
(708 U/mL) and acetaminophen (955 U/mL) were 3.2 and 2.4 fold lower that CpG alone. CpG +
honokiol (12286 U/mL) was 5.3 fold more than CpG alone. CpG + capsaicin (8413 U/mL) was 3.6
fold higher than CpG alone. CpG + WA (3459 U/mL) was 1.5 fold higher than CpG alone.
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These results demonstrate that honokiol and capsaicin are capable of both mitigating the
systemic proinflammatory cytokines, TNF- and IL-6, while also increasing the adaptive humoral
response. WA demonstrated a decrease of systemic TNF- while maintaining a similar antibody level
as CpG alone. We were unable to formulate vaccines using CAPE and 5-z-O due to solubility issues;
however, we believe they are worth exploring in future studies using alternative formulations.
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Dose-dependence of capsaicin and honokiol
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Of the candidates, Capsaicin and honokiol demonstrated exceptional promise in these studies
so we examined them further. To better understand how these molecules are altering the immune
response over time, we vaccinated mice as described above and analyzed a larger variety of cytokines
at various timepoints. We analyzed 13 cytokines: IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23,
IL-27, MCP-1, IFN-β, IFN-γ, TNF-α, and GM-CSF. Of these, only 6 cytokines demonstrated
detectable levels in our assay: TNF-α, IL-6, IL-10, IL-1α, MCP-1 and IFN-γ (Fig. 2a-f). Consistent
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with our previous findings , CpG induced TNF- and IL-6 expression peaked at 1 h. Interestingly,
CpG combined with either capsaicin or honokiol had increased IFN-γ levels at 24 h compared to CpG
alone (8 fold and 9 fold, respectively) and slightly elevated MCP-1 levels (2.5 and 2 fold, respectively),
demonstrating that both capsaicin and honokiol are acting to potentiate the immune response and are
not simply suppressing immune activation. We next wanted to understand how changing the dose
would alter innate and adaptive humoral immune responses. For honokiol, we tested a concentration
2-fold higher (800 µg) and 2- fold lower (200 µg) than the original dose (400 µg). Mice vaccinated
with our original dose of capsaicin (20 µg) appeared lethargic for 30 min post-injection, therefore we
wanted to examine if we could lower the dose, but maintain adequate anti-inflammatory activity and
antibody-boosting potential. We chose to test a dose 4-fold lower (5 µg) and 20-fold lower (1 µg) than
the original dose (20 µg). Unfortunately, mice vaccinated with all doses of capsaicin appeared lethargic
post-injection, however mice vaccinated with the lowest dose of capsaicin (1 µg) only appeared
lethargic for ten minutes. All doses of honokiol demonstrated a significant decrease in TNF-
expression compared to CpG alone, however there was no significant difference between the different
doses (Fig. 2g). Capsaicin decreased TNF- levels significantly across all doses compared to CpG
alone. Capsaicin doses of 5 µg and 20 µg decreased levels of TNF- significantly more than 1 µg (Fig.
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NF-kB inhibitors as immune potentiators
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2g). The level of IL-6 was only decreased with 400 µg and 800 µg honokiol and 20 µg capsaicin (Fig.
2h). Twenty-one days later, we analyzed differences in anti-OVA antibody level and found that all
doses of honokiol increased levels of anti-OVA antibodies compared to CpG alone and the highest
level was found with 400 µg honokiol (Fig. 2i). 1 µg and 5 µg of capsaicin did not change level of
anti-OVA antibodies in the serum compared to CpG alone, however 20 µg significantly increased
serum levels. These results indicate that honokiol can only limit TNF- and IL-6 to a certain extent at
which higher doses do not provide additional decreases in these systemic inflammatory cytokines.
Additionally, the highest dose of honokiol decreased the antibody level, indicating that there is an
optimal dose at which this inhibitor can function as an immune potentiator. Capsaicin demonstrated a
dose-dependent response, where higher doses of capsaicin led to lower systemic TNF- and IL-6 levels
and higher antibody levels. This would point to a very promising candidate as an immune potentiator,
however, these mice experienced other undesirable side effects (lethargy).
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Determining the TRPV1-mediated effects of capsaicin
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The primary in vivo target for capsaicin is the transient receptor potential cation channel
subfamily V member 1 (TRPV1). TRPV1 modulates the immune response in a variety of ways, and
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importantly, has been implicated in dampening systemic inflammation associated with sepsis.
However, it has never been explored in a vaccine setting. To understand how activation of TRPV1 may
be modulating the effects of the adjuvant, we compared the immediate inflammatory response of the
vaccination in wild type mice (WT) and TRPV1 knockout mice. We vaccinated WT and TRPV1 KO
mice with 100 µg OVA and: 50 µg CpG, 50 µg CpG + 20 µg capsaicin or PBS. We analyzed systemic
levels of TNF- and IL-6 1 h after vaccination. We found that CpG induced high levels of TNF- and
IL-6 in both WT and TRPV1 KO mice. Addition of capsaicin dramatically and significantly reduced
both TNF- levels and IL-6 levels in the WT mice (Fig. 3a, 3b, S1). Although the mean was slightly
lower for both TNF- and IL-6 in the TRPV1 KO mice, these differences were not statistically
significant. This demonstrated that TRPV1 activation is responsible for the capsaicin-induced decrease
in systemic cytokine levels. As expected, the TRPV1 KO mice did not experience the lethargy
experienced by the WT mice in response to capsaicin, indicating that activation of TRPV1 is
responsible for this response. To examine if the increased antibody level was due to TRPV1 activation
on day 21, we analyzed levels of anti-OVA antibodies in the serum (Fig. 3c, S1). Interestingly, we
found that anti-OVA antibody levels were increased in groups with Capsaicin + CpG in both WT and
KO mice. This implies that the antibody-boosting activity of capsaicin is separate from TRPV1-
dependent decrease in inflammatory cytokines. This result demonstrates both that the decrease in
inflammation is not responsible for the antibody-boosting activity of the NF-B inhibitor and also that
the enhancement of the adaptive humoral response is independent of TRPV1 activation. These results,
while not definitive, showed two separate, but correlated mechanisms for capsaicin that result in the
reduction in cytokines and increase antibody levels. As such, capsaicin did not warrant further
examination as a potential clinical immune potentiator. We will explore the mechanistic implications
of this for immune potentiators more broadly in future publications.
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Synthesis of honokiol derivative library
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With capsaicin possessing two parallel mechanisms and possessing well-established side
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effects , we wanted to explore honokiol for further development as a candidate. Oral intake of
honokiol has been well studied in humans and has been established as safe with little to no side
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effects. However, it is subject to glucuronidation, leading to fast clearance. Additionally, vaccines
for various diseases may require diverse potentiator activities. An important question for immune
potentiators and honokiol was if standard SAR methods would yield alteration in potentiation activity.
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We wanted to investigate if compound libraries of promising immune potentiator candidates could be
synthesized and provide alterations to activity. To further explore this idea, we synthesized a library
of honokiol derivatives. Honokiol derivative libraries have been synthesized previously and examined
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for their effects on neuroprotection , antimicrobial agents and anti-cancer among others.
However, to date no such study has examined the effects of honokiol analogs on vaccines or a
combination of anti-inflammatory activity and adaptive humoral immune response. Phenylphenols and
biphenols were prepared using Pd-catalyzed Suzuki coupling using corresponding iodophenols and
hydroxyphenylboronoic acids as starting materials. These compounds were O-allylated using allylBr.
Resulting compounds were subjected to Claisen rearrangement using diethyl aluminum chloride to
yield a variety of ring substitutions (Scheme 1, Fig. 4a).
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We analyzed how the honokiol derivatives altered IL-6 production in RAW macrophages. We
chose to analyze the hydroxybiphenyls and O-allylated derivatives in addition to the product from the
Claisen rearrangement to understand how these functional groups play a role in the anti-inflammatory
action or increase in adaptive immune response (Fig. 4b, S2, S3). We treated RAW macrophages with
honokiol derivatives and LPS and analyzed IL-6 expression. The addition of LPS alone without a
honokiol derivative gave high levels of IL-6 expression (6848 pg/mL). The addition of honokiol
decreased IL-6 levels to 260 pg/mL, a decrease of 26-fold. Several derivatives including compounds:
1, 2, 3, 4, 8, and 11 demonstrated similar reductions in IL-6 expression in vitro making them promising
candidates for in vivo analysis. As a small molecule, honokiol impacts multiple pathways aside from
NF-kB such as: STAT3, EGFR, mTOR and caspase-mediated common pathways. It is probable that
alterations to the structure will impact which pathways are modulated, altering the downstream
response. It is likely that different vaccine formulations will require potentiators with unique
modulatory characteristics as well as pharmacokinetic/pharmacodynamic properties. Compounds
lacking a free hydroxyl group (such as 11) are unable to form glucuronide conjugates, potentially
extending the half-life. Here we demonstrated that compound libraries can be synthesized and screened
in vitro for immune activity and that alterations to the structure change the downstream response. Such
libraries could be used for improving rational design of future immune potentiators through machine
learning. In future studies we will explore the effects of these inhibitors in vivo.
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Conclusion
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In summary, we present that select small molecule inhibitors of NF-B can decrease the
inflammatory effects of adjuvanted vaccination - potentially enabling safer vaccination while also
acting as immune potentiators and increasing the antibody level. We identified two such immune
potentiators, honokiol and capsaicin that effectively decrease inflammation while increasing the
adaptive humoral response. We additionally provide evidence that implies that the decrease in
inflammation is separate from the increase in antibody response, potentially enabling distinct tunability
of either response. This study also identifies that only select NF-B inhibitors can be used as immune
potentiators, this broadens the potential for further modulation of the immune response. We
additionally synthesized and examined a library of honokiol derivatives and found that several
honokiol derivatives are promising candidates for future testing in vivo. In the future, this information
can be used in modern screening methods involving machine learning to identify better immune
potentiators. The use of combining NF-kB inhibitors with vaccine adjuvants could find use in creating
next-generation prophylactic vaccines and immunotherapy applications. In conclusion, we have
demonstrated that using small molecule NF-B inhibitors in combination with common immune
adjuvants can decrease the production of pro-inflammatory cytokines TNF- and IL-6 while boosting
antibody levels.
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Materials and methods
In vitro assays
RAW macrophage cytokine analysis
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RAW 264.7 macrophages were passaged and plated in a cell culture treated 12- well plate at 0.5 x106
cells/ well in 1 mL DMEM containing 10% FBS. Cells were grown for 2 days. Media was exchanged
for 1 mL DMEM containing 10% HIFBS. Inhibitors were diluted in Addavax and then in PBS.
Inhibitors were added at indicated concentrations and incubated for 45 min. After 45 min, LPS was
added at 100 ng/mL and incubated at 37 °C and 5% CO for 24 h. Cell supernatant was removed and
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analyzed using BD Cytometric Bead Array Mouse Inflammation Kit.
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Cell viability assay
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RAW macrophages were plated at 100k cells/ well in 180 uL DMEM/10% HIFBS. Inhibitors were
diluted as described above and added at indicated concentrations and incubated for 45 min. After 45
min, LPS was added to a final concentration of 100 ng/mL and incubated at 37 °C and 5% CO for 24
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h. MTT reagent was made fresh at a concentration of 5 mg/mL in PBS and sterile filtered. 150 L cell
supernatant was removed and 150 L PBS was added. 10 L MTT reagent was added to each well and
incubated at 37 °C and 5% CO
with 150 L DMSO and incubated at 37 °C and 5% CO
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for 2 h. 150 L supernatant was removed from each well and replaced
for 1 h or until purple crystals dissolved. Plate
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was analyzed using Multiskan FC plate reader (Thermo Scientific) and absorbance was measured at
450 nm. Data was analyzed using Graphpad Prism.
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Flow cytometry
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RAW macrophages (2 x 10 ) were plated in a 12 well plate in DMEM/10% HIFBS. Inhibitors were
diluted as described above and added at indicated concentrations and incubated for 45 min. After 45
min, LPS was added to a final concentration of 100 ng/mL and incubated at 37 °C and 5% CO for 24
h. Cells were stained for CD86 using BD cytofix/cytoperm fixation /permeabilization solution kit
according to manufacter’s protocol. Cells were analyzed using NovoCyte flow cytometer (ACEA
Biosciences, Inc.).
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In vivo assays
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All animal procedures were performed under a protocol approved by the University of Chicago
Institutional Animal Care and Use Committee (IACUC). 6-8 week-old C57/B6 female mice were
purchased from Jackson Laboratory (JAX). 6-8 week-old C57/B6 female Trpv1tm1Ju mice were
purchased from JAX for TRPV1 KO experiment. All compounds were tested for endotoxin prior to
use. All vaccinations were administered intramuscularly in the hind leg. Blood was collected from
the submandibular vein at time points indicated.
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Antigens were purchased from Invitrogen (Vaccigrade Ovalbumin). Vaccigrade CpG ODN 1826 was
purchased from Adipogen. AddaVax™ was purchased from Invivogen.
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Vaccination
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Mice were lightly anesthetized with isoflurane and injected intramuscularly in the hind leg with 50 µL
containing ovalbumin (100 µg), adjuvant, inhibitor and PBS. Adjuvant doses: CpG, 50 µg. Inhibitor
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concentrations: Honokiol (400 µg), Capsaicin (20 µg), Withaferin A (600 µg), acetaminophen (2 mg),
ibuprofen (800 µg). All vaccines contained 25 µL AddaVax™ to enhance solubility.
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Plasma cytokine analysis
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Blood was collected from mice at time points indicated in 0.2 mL heparin coated collection tubes
(VWR Scientific). Serum was isolated via centrifugation 2000 x g for 5 min. Supernatant was collected
and stored at -80 °C until use. Serum was analyzed using BD Cytometric Bead Array Mouse
Inflammation cytokine kit or LEGENDplex™ Mouse Inflammation Panel (Biolegend) according to
manufacturer’s protocol.
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Antibody quantification
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Mice were vaccinated with indicated formulations. Blood was collected at time points indicated in 0.2
mL heparin coated collection tubes (VWR Scientific) for plasma or uncoated tubes for serum. Plasma
was isolated via centrifugation (2000 x g, 5 min). Serum was isolated by allowing blood to clot for 15-
30 min RT and centrifuging (2000 x g for 10 min) at 4 °C. Serum was analyzed using a quantitative
anti-ovalbumin total Ig’s ELISA kit (Alpha Diagnostic International) according to the specified
protocol. Data was analyzed using Graphpad Prism.
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Chemistry
Conditions for Suzuki Coupling
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Hydroxyphenol boronic acid (20 mmol) was dissolved in 100 mL water. Appropriate iodophenol (10
mmol) and K
2
CO (40 mmol) was added followed by Pd/C (2 mol %). Solution heated to 80 C for 3h.
3
Solution was acidified with 1M HCl and extracted with EtoAc and washed with brine. Solvent
evaporated in vacuo. Compound was purified by column chromatography.
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74
Conditions for O-allylations
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2
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Phenol (1 mmol) (Derivative 1-8) was dissolved in dry acetone (5 mL) and K
2
CO (2 mmol) added.
3
AllylBr was added dropwise and refluxed. Reaction was monitored by TLC until completion (5-12h).
Reaction mixture was cooled and volatiles were removed in vacuo. 10% NaOH was added to the
mixture and extraction was performed using ethyl acetate, washed with brine and organic layers dried
using MgSO . Solvent was removed in vacuo affording an oily material that was purified by column
4
chromatography to yield the O-allylated derivative.
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81
Conditions for Claisen rearrangement
2
2
2
2
2
82
83
84
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O-allylated derivatives (9-17) (1 mmol) were dissolved in dry hexane (10 mL). Et AlCl in dry hexane
2
(4 mL) was added dropwise under argon. Mixture was stirred at room temperature for 2h. The mixture
was cooled on an ice bath and quenched using 2M HCl (20 mL). Extraction was performed with EtOAc,
washed with brine and dried over MgSO4. Solvent was removed in vacuo affording an oily material
that was purified by column chromatography to yield the C-allyl derivative.
2
87
Statistics and replicates
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89
Data is plotted and reported in the text as the mean ± s.e.m. Sample size is as indicated in biological
replicates in all in vivo and in vitro experiments. The sample sizes were chosen based on preliminary
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NF-kB inhibitors as immune potentiators
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experiments or literature precedent indicating that the number would be sufficient to detect significant
differences in mean values should they exist. P values were calculated using a one-way ANOVA and
Tukey post hoc test. All experiments have been repeated (sometimes with minor variations due to
reagents and materials) and replication was successful.
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2
2
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Funding and acknowledgement: We would like to acknowledge support by the NIH
(1U01Al124286-01 and 1DP2Al112194-01, GM099594). Prof. Esser-Kahn thanks the Pew Scholars
Program and the Cottrell Scholars Program for generous support. B.A.M. thanks NSF-GRFP (DGE-
1321846). We would like to thank NSF instrumentation grant CHE-1048528. This work was
supported, in part, by a grant from the Alfred P. Sloan foundation.
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99
This manuscript has been released as a Pre-Print at ChemRxiv.29
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Author contributions: B.A.M. and A.E.K. conceived of and designed the project and experiments
and wrote the manuscript. B.A.M., Y.E.B, R.C.S., M.G.R., B.J.C., M.N., N.T. performed experiments.
B.A.M. synthesized materials. B.A.M., N.N., A.C.C. performed compound characterization.
Competing interests: The authors report no competing interests. Data and materials availability: All
data are available in the main text or the supplementary materials.
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NF-kB inhibitors as immune potentiators
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Figure 1. Small molecule inhibitor screen in vitro and in vivo. (A) IL-6 levels from RAW
macrophages 24 h post-stimulation with NF-B inhibitor and LPS. Significance is compared to LPS
alone. n = 3. (B) Systemic TNF-  expression 1h post-vaccination, n = 5(C) Systemic IL-6 expression
1
h post-vaccination, n = 5. (D) Anti-OVA antibody level 21 days post-vaccination, n = 5. Significance
is compared to CpG vaccination. * p < 0.05, **p < 0.01, ***p < 0.001.
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NF-kB inhibitors as immune potentiators
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Figure 2. Broader cytokine response and dose effects of honokiol and capsaicin.
(
(
A-F) Systemic cytokine levels at 1 h, 24 h and 48 h post-vaccination, n = 5. CpG
black line), CpG + Capsaicin (red line), CpG + Honokiol (blue line), PBS (purple
line) (G) Systemic TNF- levels 1h post-vaccination with varying doses of honokiol
and capsaicin, n = 5. (H) Systemic IL-6 levels 1h post-vaccination, n = 5. (C) Anti-
OVA antibody levels 21 days post-vaccination, n = 5. Significance is compared to
CpG alone. * p < 0.05, **p < 0.01, ***p < 0.001.
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This is a provisional file, not the final typeset article

NF-kB inhibitors as immune potentiators
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Figure 3. Role of TRPV1 of capsaicin induced anti-inflammatory and immune
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potentiation. (A) Systemic TNF- levels 1 h post vaccination in wild type (WT) mice and
TRPV1 KO (KO), n = 5. (B) Systemic IL-6 levels 1 h post-vaccination, n = 5. (C) Anti-
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OVA antibody level 21 days post-vaccination, n = 5. * p < 0.05, **p < 0.01, ***p < 0.001.
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NF-kB inhibitors as immune potentiators
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Scheme 1. Honokiol derivative synthesis.
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NF-kB inhibitors as immune potentiators
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Figure 4. Honokiol derivatives and their inhibitory activity on IL-6 expression. (A) Honokiol
derivative library (B) IL-6 expression of RAW macrophages treated with honokiol derivatives and
LPS, n = 3. Significance is compared to LPS alone. * p < 0.05, **p < 0.01, ***p < 0.001.
1
5

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