Identification of biologically active pyrimido[5, 4-b]indoles that prolong NF-κB activation without intrinsic activity

Article abstract of DOI:10.1021/acscombsci.7b00080

Most vaccine adjuvants directly stimulate and activate antigen presenting cells but do not sustain immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to identify compounds that would sustain NF-κB activation by a stimulus from the Tolllike receptor (TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-κB activation at 12 h. The dynamic range of the assay was confirmed in a pilot screen of 14 631 compounds and subsequently in a main extensive screen with 166 304 compounds. Hit compounds were identified using an enrichment strategy based on unsupervised chemoinformatic clustering, and also by a na?ve "Top X" approach. A total of 2011 compounds were then rescreened for levels of coactivation with LPS at 5 h and 12 h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed correlation of the kinetic profiles with the structural similarities led to identification of four chemotypes: pyrimido[5, 4-b]indoles, 4H-chromene-3-carbonitriles, benzo[d][1, 3]dioxol- 2-ylureas, and tetrahydrothieno[2, 3-c]pyridines, which were segregated by 5 h and 12 h kinetic characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce NF-κB but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial library was synthesized which led to identification of potent N3-alkyl substituted pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo when used as a coadjuvant. The novel HTS strategy led to identification of compounds that are intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby potentiate vaccine efficacy.

Full text of DOI:10.1021/acscombsci.7b00080

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Article
Identification of Biologically Active Pyrimido[5,4-b] indoles
that Prolong NF-#B Activation Without Intrinsic Activity
Michael Chan, Alast Ahmadi, Shiyin Yao, Fumi Sato-Kaneko, Karen Messer, Minya Pu, Brandon
Nguyen, Tomoko Hayashi, Maripat Corr, Dennis Carson, Howard B. Cottam, and Nikunj Shukla
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00080 • Publication Date (Web): 28 Jun 2017
Downloaded from http://pubs.acs.org on July 7, 2017
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Series 1
Series 2
Bio-activity
Toll-like Receptor4
Yes
No
Agonist
Intrinsic NF-κB
Activity
Yes
No
No
Sustain NF-κB
Activation by LPS
Yes
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Identification of Biologically Active Pyrimido[5,4-
b] indoles that Prolong NF-κB Activation Without
Intrinsic Activity
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Michael Chan,a Alast Ahmadi,a Shiyin Yao,a Fumi Sato-Kaneko,a Karen Messer,b Minya Pu,b
Brandon Nguyen,a Tomoko Hayashi,a Maripat Corr,c Dennis A. Carson,a Howard B. Cottam,a
Nikunj M. Shukla*,a
a. Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093, USA.
b. Division of Biostatistics, University of California San Diego, La Jolla, CA 92093, USA.
c. Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
KEYWORDS: adjuvant, pyrimidoindole, NF-κB, LPS, TLR4
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ABSTRACT
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Most vaccine adjuvants directly stimulate and activate antigen presenting cells, but do not sustain
immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to
identify compounds that would sustain NF-κB activation by a stimulus from toll-like receptor
(TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and
conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final
assay evaluated prolongation of LPS induced NF-κB activation at 12h (hours). The dynamic
range of the assay was confirmed in a pilot screen of 14,631 compounds, and subsequently in a
main extensive screen with 166,304 compounds. Hit compounds were identified using an
enrichment strategy based on unsupervised chemoinformatic clustering, and also by a naïve ‘Top
X’ approach. 2,011 compounds were then rescreened for levels of co-activation with LPS at 5h
and 12h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed
correlation of the kinetic profiles with the structural similarities led to identification of four
chemotypes: pyrimido[5,4-b]indoles; 4H-chromene-3-carbonitriles; benzo[d][1,3]dioxol-2-
ylureas; and tetrahydrothieno[2,3-c]pyridines, which were segregated by 5h and 12h kinetic
characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the
revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce
NF-κB, but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial
library was synthesized which led to identification of potent N3-alkyl substituted
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pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo
when used as a co-adjuvant. The novel HTS strategy led to identification of compounds that are
intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby
potentiate vaccine efficacy.
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Introduction: The dearth of adjuvants in human vaccines has motivated the search for effective
non-toxic immunomodulators that provide sustained immunoprotection particularly to high-risk
populations such as the elderly.1-5 The use of Toll-like receptor (TLR) ligands has gained interest
as sensors of innate immunity and stimulators of antigen-presenting cells (APCs) that prime
protective immune responses.6 The use of TLR ligands as adjuvants can provide a potent
activation stimulus to resident APCs, and promote further APC recruitment to the site of antigen
injection and subsequent delivery to the draining lymph nodes.2-3 In a prior high throughput
screen (HTS) using the Förster resonance energy transfer (FRET) based CellSensor® NF-κB-bla
reporter in the human monocytic leukemic cell line, THP-1, we identified pyrimido[5,4-b]indole
and 2-aminoquinazoline chemotypes that were characterized as TLR4/MD-2 ligands.7-8 These
compounds were directly immunostimulatory and provided the basis for developing a lead
compound shown to be an effective adjuvant in a murine model of influenza.9
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Other toll-like receptor ligands have also been developed as vaccine adjuvants as they are innate
immune stimulators and can directly activate APCs.2, 10-17 To initiate immune responses from
vaccines, antigen-loaded activated APCs travel from the site of injection to regional lymph nodes
and prime naïve T cells. During the time period that the APC travels from the vaccine injection
site to a regional lymph node, the activation status is down-regulated (Fig. 1A, red dashed line).
Activation of innate immune responses is regulated through multiple negative feedback loops.18-
20 Negative regulators of TLR signaling that have been reported to date include processing
pathways (ubiquitination21-23, degradation24, stabilization25), molecular competition20, promoter
regulation23, phosphatases26-27, and epigenetic regulators.28-32 For example, interleukin-1
receptor-associated kinase (IRAK)-M20, phosphatidylinositide (PI)3 kinase inhibitors33 and anti-
inflammatory cytokines (IL-1030 and transforming growth factor β34) have evolved to co-
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modulate innate immune responses and inflammation.35 These negative feedback systems may
adversely impact the ability of adjuvants to sustain APC activation, and to induce optimal T
lymphocyte memory responses.
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Most vaccine development approaches have been to directly activate and stimulate APCs.
However, prolonging the activation status of a cell by intercepting a negative regulatory pathway
would be as valuable an approach to allow the impetus of the primary stimulus to be sustained
during the transit time to the lymph node and to increase the number of T and B cell interactions
the activated cell may have in the lymphoid environment. A compound disrupting one or more of
the regulatory systems could sustain the activation status of a TLR stimulated APC. To date,
HTS campaigns related to NF-κB have focused on identifying pathway inhibitors36-41 or intrinsic
activators.7-8 However, no HTS campaign has focused on identifying compounds that sustain
initial NF-κB stimulation by a TLR agonist. We hypothesized that identification of such small
molecules could complement current TLR agonists as adjuvants and improve the efficacy of
vaccines that are locally administered.
Results and Discussion: Overall HTS strategy and design. Here, we aimed to identify
compounds that manipulate negative feedback systems to sustain innate immune activation
through a classic TLR4 ligand, lipopolysaccharide (LPS). We again used the CellSensor® NF-
κB-bla reporter in a human monocytic cell line, THP-1, as a model of functional monocyte-
derived APCs for HTS. This cell-based assay was chosen as many of the cellular functions of
APCs are retained and the dual fluorescence normalizes relative cell number and viability to NF-
κB activity in each well. Typically, LPS stimulates NF-κB activation within the APC and then an
increase in regulatory “deactivation” molecules (Fig. 1A, blue dashed line) results in a decline in
activation status. We sought to find small molecules that would prolong the LPS induced
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activation status by reducing or inhibiting these negative molecular regulators (Fig. 1A, red and
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blue lines, respectively).
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To identify compounds that prolonged the activation of APCs by LPS, we developed a tiered
HTS strategy (Fig. 1B). First, the conditions for the THP-1 CellSensor® NF-κB-bla assay were
optimized for a 12h stimulation assay with LPS as described below. An initial pilot screen was
performed with 14,631 representative compounds to evaluate the performance of the assay and
quality control. The main HTS was then performed with a collection of 166,304 compounds that
included the compounds used in the pilot screen to verify the reproducibility of the assay. We
utilized our previously reported structure-based cluster analysis to select 2,011 compounds for
confirmation.42 These compounds were tested in duplicate in a confirmation screen for prolonged
activation at 12h, but we also included an additional 5h screen to evaluate the kinetics of the
stimulatory response. Approximately 20% (407 compounds) were confirmed as hits and
substructure based clustering broadly classified some of these compounds into four chemotypes:
pyrimido[5,4-b]indoles; 4H-chromene-3-carbonitriles; benzo[d][1,3]dioxol-2-ylureas; and
tetrahydrothieno[2,3-c]pyridines. As we had prior success with developing a pyrimidoindole as a
lead compound, this class was selected for further structure-activity relationship (SAR) studies.
Assay optimization: For our LPS co-stimulation strategy we optimized the THP-1 CellSensor®
NF-κB-bla assay for variables including: 1) cell number; 2) preincubation time of thawed cells;
3) concentration of FBS; 4) incubation time; and 5) the dose and timing of a positive control
compound, wortmannin, a PI3 kinase inhibitor that has been reported to augment initial NF-κB
activity.43-45 In the first set of experiments, the tissue culture fetal bovine serum (FBS)
concentration was kept constant at 10% while cell numbers were varied between 10,000
cells/well and 20,000 cells/well, and the preincubation time of thawed cells was either -16h or 0h
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relative to test compound and LPS addition (Fig. 2A). In addition, FRET response ratios were
measured after 24h for 100ng/mL LPS co-incubated with graded concentrations of wortmannin
in the above-mentioned four different cell plating conditions. Higher FRET ratios were reliably
demonstrated with 20,000 cells/well and a 16h preincubation time of the thawed cells (Fig. 2A).
The potential for individual test compounds to bind to bovine albumin and other proteins in FBS
could lead to a false negative output, so we probed the effect of lowering the FBS concentration
(5%, 2% or 1%) on prolonged LPS stimulation in the presence of graded concentrations of
wortmannin (Fig. 2B). Response ratios decreased with reduced concentrations of FBS and also at
higher concentrations of wortmannin (Fig. 2B). Thus, 5% FBS concentration was determined to
be optimum for the assay.
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As the optimal incubation period of THP-1 Cellsensor® NF-κB recommended by the
manufacturer is 5h to 6h, a longer incubation time with THP-1 cells may lead to cellular stress
responses as well as degradation of compounds due to metabolism. We were therefore interested
to find the optimum incubation time to allow for natural decay of the LPS stimulation without
losing the ability to distinguish an increase in the FRET signal due to aberrations in cellular
metabolism or cell death. Using the previously optimized parameters (20,000 cells/well and 5%
FBS), response ratios were measured at 6, 12, 18 and 24h after stimulation with100ng/mL LPS
alone (Fig. 2C). In addition, LPS was added after 18h (LPS last) in untreated cells and the
response ratio was measured after 6h to verify retention of the FRET activity in the cells after a
long incubation time. LPS stimulation results in activation at 6h; however, the response ratio
shows a significant drop at 12h which reached a plateau at later time points. The response ratio
for LPS at 6h and for 'LPS last' are almost identical suggesting that THP-1 cells do not lose
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activity even after longer incubation time (Fig. 2C). As there was no difference at 12h and later
time points we chose 12h for the HTS.
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Next, we varied the dose and time of wortmannin (as a test compound) addition relative to LPS.
Wortmannin at graded doses was added either 1h before, or at 0, 2 and 4h after the addition of
LPS. The response ratios were higher when wortmannin was added before or with LPS, but the
signal decreased when wortmannin was added 2h or 4h after LPS (Fig. 2D). Thus, the addition
of 0.25µM wortmannin concurrent with 100ng/mL LPS was chosen as optimal for the assay. The
final optimized conditions for the HTS to identify compounds that prolong initial activation of
NF-κB by LPS are summarized in a schematic protocol (Fig. 2E).
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Pilot screen and HTS: Using the optimized assay conditions, a pilot screen was first performed
using 14,631 compounds selected to represent the diversity of the libraries in the chemical
collection at University of California San Francisco (UCSF) Small Molecule Discovery Center
(SMDC). The screen was performed using LPS alone (LPS 12h) and LPS + 0.25 µM
wortmannin as a positive control, 0.5% DMSO (no LPS) as a negative control and an additional
control included adding LPS at 5h before adding substrate (LPS 5h). The percent activation
calculated using LPS 5h as reference is shown in Fig. 3A. Statistical analysis showed good
quality for the different controls and we proceeded with the main screen, which included 166,304
test compounds on 527 plates. In addition, 27 plates containing 8,627 compounds from the pilot
screen were re-screened in triplicate within the main screen, in order to provide quality control
information and to estimate confirmation rates. The percent activation plot for the main screen
(Fig. 3B) showed inter-plate variability for the LPS control (LPS 12h). To mitigate for the
variability, we calculated values based on the LPS control within each plate. Intra-plate
calculations for 'distance to LPS' was calculated and used as the metric for compound selection.
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The main screen median Z' was 0.74 (0.43 to 0.90). As in prior work, we used cluster enrichment
methods including Murcko scaffold classes, Daylight functional clusters, and Top X approach, to
identify 2,011 compounds for the confirmation and kinetic profiling screens (Supporting
Information, Fig. S1).42
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Confirmation screen and kinetic profiling: The selected hits from the main screen were then
evaluated in duplicate in a confirmation screen at 12h and an additional screen at 5h for kinetic
profiling. Fig. 4A shows a scatter plot of the data (distance to LPS) for these compounds from
the 12h confirmation screen on the ordinate axis and from 5h kinetic profiling screen on the
abscissa axis. 407 compounds were identified as confirmed hits from the 12h screen using a data-
driven method that fits a mixture of two linear models utilizing both the primary and
confirmation screen data for these compounds. All of the confirmed hits enhanced NF-κB
induction activity of LPS at 12h and were broadly classified into four groups showing 4 general
kinetic patterns (Fig. 4B). Based on 5h screen, these compounds were either early inhibitors (5h
distance to LPS<0) or early enhancers (5h distance to LPS>0). Within the early inhibitors, the
compounds were grouped as either showing moderate increase or high increase in NF-κB
induction compared to LPS at 12h (green and orange symbols, respectively, Fig. 4A), where cut-
off used was mean+SD of the LPS 5h values from all the confirmation screen plates. Similarly,
within the enhancer groups, compounds showed either moderate or high increases in NF-κB
induction compared to LPS at 12h (red and blue symbols, respectively, Fig. 4A). Theoretical
activation profiles for these groups of compounds are shown in Fig. 4B with the same color
patterns. The structures of these compounds were then subjected to substructure based clustering
using the server based ChemMine tools (University of California, Irvine;
http://chemmine.ucr.edu/tools/launch_job/Clustering/) and binning clustering application with a
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similarity cutoff of 0.5. The application allowed us to group these compounds into several
chemotypes and correlation of kinetic profiles with structural similarities led us to identify four
major structural classes, namely: pyrimido[5,4-b]indoles; 4H-chromene-3-carbonitriles;
benzo[d][1,3]dioxol-2-ylureas; and tetrahydrothieno[2,3-c]pyridines (Fig. 4C). Most of the
compounds in pyrimido[5,4-b]indole and tetrahydrothieno[2,3-c]pyridine series were early
enhancers while the bulk of the compounds in the 4H-chromene-3-carbonitriles and
benzo[d][1,3]dioxol-2-ylureas series were early inhibitors, as described earlier. Fig. 4C shows
the distribution of the compounds in a chemotype connected to a calculated geometric centroid
from the coordinates of the individual compounds. The centroid for these four different
chemotypes clearly lies in the four different regions with distinct kinetic activation profiles as
shown in Fig. 4B.
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Structure-activity relationship studies on pyrimidoindoles: Pyrimidoindoles were identified
as TLR4/MD2 agonists in a previous HTS,7, 42 which was designed to identify compounds that
induced NF-κB activation. The identification of this chemotype here as prolonging NF-κB
induction warranted additional investigation into the structural differences to understand
functional characteristics. Overall, 536 pyrimidoindoles were evaluated in both the HTS
campaigns, the bulk of which consisted of 357 amide-linked compounds (66.6%) and a small
fraction of 27 ester-linked compounds (5%). Within the amide-linked compounds, there was a
relatively higher number of N3-aryl (297 compounds) versus N3-methyl (60 compounds)
substituted pyrimidoindoles pointing toward a bias for N3-aryl derivatives. A plot of potency
signatures for the current HTS (distance to LPS) on the y-axis against previous HTS42 (percent
activation) on the x-axis showed a clear demarcation for pyrimidoindoles designated as 'hits' in
the two different but related assays (Fig. 5). The amide-linked hits identified in the previous HTS
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showed high dominance for N3-aryl substitution (73%) nearly matching the ratio present in the
HTS library, however the amide-linked hits identified in the current HTS had a very high
dominance for N3-methyl substitution (62%), despite being low in number (Fig. 5). In addition,
the current HTS also identified two active ester-linked compounds. Here, it is important to
distinguish between the two HTS campaigns relative to the pyrimidoindoles chemotype. The first
campaign was designed to identify compounds that alone had intrinsic ability to activate NF-κB,
and as mentioned above, pyrimidoindoles bearing N3-aryl substitutions were found in this
category, whereas those compounds bearing the N3-methyl substituent were inactive in the prior
screen. In contrast, the present campaign was designed to identify compounds that could enhance
or prolong an already-present TLR4 innate immune stimulus (LPS), and in this category, several
pyrimidoindoles bearing N3-methyl substituent were found to be active. With this in mind, we
decided to pursue a combinatorial library of 42 compounds to explore a homologous series of
N3-alkyl substitutions including N3-methyl, -ethyl, -propyl, -butyl, -isopropyl, and N3-aryl
substitutions including N3-p-fluorophenyl and -p-methoxyphenyl. The other variation in the
combinatorial library consisted of three esters (methyl, ethyl and tert-butyl) and three amides
linked to 2-amino substituent of thiazole, benzothiazole and 6-methylbenzothiazole at the C2
position as these substituents were identified as most potent in both the main and the 12h
confirmation screens.
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The proposed compounds were synthesized as shown in Scheme 1. The common precursor ethyl
3-amino-1H-indole-2-carboxylate (1) was reacted with isothiocyanate chemset 2{1-7} to obtain
the intermediate thiourea chemset 3{1-7}, followed by intramolecular condensation to yield the
fused pyrimidine ring using acetyl chloride and ethanol to obtain chemset 4{1-7}. These
compounds were then reacted with three different 2-chloroacetates 5{1-3} and 2-
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chloroacetamides 5{4-6} to obtain the 42 compound combinatorial library of differently N3 and
C2 substituted pyrimidoindoles 6{1-7, 1-6}.
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These compounds were tested in the Cellsensor® THP-1 cells at 5µM concentration for
enhancement of NF-κB upregulation with LPS at 5h and 12h. The activity data was calculated as
percent activation (%act), which was measured as FRET response ratios relative to the LPS
alone control (Supporting Information, Table S1). None of the synthesized compounds
significantly enhanced NF-κB induction at 5h, while all the ester linked derivatives and thiazole
amide linked compounds 6{1-7, 1-4} showed negligible increases in NF-κB induction at 12h
compared to LPS alone. However, some of the amide-linked compounds with the 2-
aminobenzothiazole and 2-amino-6-methylbenzothiazole substituents (6{1-7, 5-6}) were very
active and showed a distinct relationship between the N3-alkyl chain length and the NF-κB
inducing activities in presence of LPS at 12h. In N3-alkyl substituted benzothiazole derivatives
6{3-7, 5} the percent activation value increases moderately for N3-methyl (6{4,5}, %act=100),
N3-ethyl (6{5,5}, %act=120), N3-propyl (6{6,5}, %act=119) derivatives; however, the N3-butyl
derivative (6{4,5}, %act=180) was found to be the most potent, and surprisingly the N3-
isopropyl derivative (6{3,5}, %act=75) showed NF-κB inhibitory effects with values lower than
that of LPS alone. A similar trend was observed with the N3-alkyl substituted
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methylbenzothiazole derivatives 6{3-7, 6} with both the N3-propyl and N3-butyl substituted
derivatives being very potent (6{6,6}, %act=190, 6{7,6}, %act=192 respectively). The N3-
isopropyl derivative (6{3,6}, %act=85) with LPS showed reduction in NF-κB activation
compared to the LPS alone suggesting that branched alkyl chain is not tolerated. In the N3-aryl
substituted derivatives, the 4-flouorphenyl substituted compounds (6{1,5-6}) exhibited better
potency than the corresponding 3-methoxyphenyl substituted analogs (6{2,5-6}). 
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Next, we assayed the toxicity of these compounds in THP-1 cells using an (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. Toxicity was measured as
percent viability relative to the vehicle, (0.5% DMSO). All of the weakly potent ester-linked
compounds were found to be non-toxic (%viability>90) at 5µM, while all of the thiazole
substituted amide-linked compounds were found to be relatively toxic suggesting an off-target
effect related to the presence of the thiazole group. Among the N3-alkyl substituted
benzothiazole or 6-methylbenzothiazole derivatives, all except the N3-propyl and -isopropyl
derivatives were relatively more toxic.
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In order to identify an optimum N3-alkyl chain length for maximal potency, we synthesized N3-
pentyl (9a and 10a) and hexyl (9b and 10b) derivatives for the benzothiazole and 6-
methylbenzothiazole substituted compounds as shown in Scheme 2. These higher chain analogs
showed declining potency with increasing chain length (9a; %act=154, 9b;%act=143, 10a;
%act=139, 10b; %act=142) and also were found to be relatively more toxic. We had earlier
reported that N5-methylation of pyrimidoindoles reduces their toxicity without abrogating the
activity at TLR4.7 Thus, we synthesized N5-methyl derivatives of the potent N3-butyl substituted
derivatives 6{7, 5-6}. These compounds, however, did not eliminate toxicity, but lost their
ability to sustain an LPS induced NF-κB response (11; %act=94, 12; %act=91).
Moreover, while these active N3-alkyl derivatives enhance the LPS induced NF-κB activation,
they appear to do so by a mechanism that is independent of TLR4 binding, unlike the derivatives
identified in the previous HTS. We evaluated the abilities these compounds have to activate the
NF-κB and TLR4 signaling pathways using THP-1 NF-κB Cellsensor® and human TLR4 HEK
reporter cell lines, respectively. We found that these compounds lack activity in either of these
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assays as compared to the potent pyrimidoindoles identified in the previous HTS (Supporting
Information, Fig. S2).
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For all the above reasons, we were interested to pursue vaccination studies in mice with these
novel compounds in the presence of a TLR4 stimulus to evaluate their potential co-adjuvant
effects. We chose MPLA (monophosphoryl lipid A), a known TLR4 agonist and Food and Drug
Administration (FDA) approved adjuvant, and ovalbumin (OVA) as antigen for the vaccination
studies. Examination of antigen-specific antibodies showed that co-immunization of MPLA with
compounds 6{7,5}, 6{6,6} or 6{7,6} induced statistically significant increases in antigen
specific IgG titers when compared to mice immunized with MPLA alone (p<0.05 compared to
MPLA plus antigen, Fig. 7).
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Conclusions: In conclusion, a novel HTS assay using THP-1 Cellsensor® reporter cells has been
standardized and used to identify compounds that prolong NF-κB activation when co-
administered with a TLR4 stimulus (LPS). Hit compounds were identified using an enrichment
strategy based on unsupervised chemoinformatic clustering of the compounds within the screen
library, and also by a naïve ‘Top X’ approach. Confirmation screening at 12h and kinetic profile
screening at 5h yielded 407 confirmed hits. The correlation of the kinetic profiles with the
structural similarities led to identification of four chemotypes including pyrimido[5,4-b]indoles.
A 42-member combinatorial library of pyrimidoindoles was synthesized that led to identification
of potent N3-alkyl substituted pyrimidoindole analogs. Although these potent compounds were
intrinsically inactive towards NF-κB induction or TLR4 activation, when co-adjuvanted with
MPLA, they augmented the level of antigen-specific IgG production in mice Many of the
negative regulatory pathways involve kinase; hence future studies to elucidate the mechanisms of
action of these derivatives will include kinase inhibitory assays among other pathways.
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Experimental Methods:
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Compound Library. A library of compounds was acquired from the UCSF Small Molecule
Discovery Center consisting of 166,304 chemical entities from eight suppliers (Supporting
Information, Fig. S3).
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High throughput screening and Statistical Analysis. HTS was performed by Life
Technologies Corporation at their commercial facility (Madison, WI) in 384 well plates using
Cellsensor® THP-1 cells. The screen was performed using LPS alone (LPS 12h) and LPS +
0.25µM wortmannin as positive controls, 0.5% DMSO as negative control and an additional
control included adding LPS at 5h before adding substrate (LPS 5h). There were 527 plates in
the main screen, and 18 plates each in the12h confirmation screen and in the 5h kinetic screen.
Emission ratios were computed as the ratio of fluorescence density values at two wavelengths,
fluorescein versus coumarin emission, after background subtraction using the mean of the cell-
free wells. Response ratios (RR) were computed as the emission ratio divided by the mean
emission ratio from no-lipid wells within the same plate. To optimize assay parameters, box plots
and bar graphs were used to describe data. For compound selection, percent activation by plate
was computed as the difference between RR from a particular well and the mean RR from no-
LPS (0.5% DMSO) wells, divided by the difference of the mean RRs between LPS 5h wells and
no LPS (0.5% DMSO) wells. Quality checks in the pilot screen included making dot plots of
percent activation values versus well number by well type, calculating Z' for each plate, making
scatter plots for duplicated compounds and outlier detection. In both the main screen and the
confirmation screen, to account for the variability from the LPS 12h wells (where LPS was
added at 0h), distance to LPS (standardized percent activation values against LPS 12h, calculated
as the difference between percent activation values for the test compound and mean of LPS 12h
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control wells divided by the standard deviation for the LPS 12h control wells within each plate)
was used as the metric for compound selection. In main screen, a cluster enrichment method42
was used to identify hits where Murcko scaffold classes, Daylight functional clusters, and Top X
approach (whether the distance to LPS first >3) were used together to select compounds to carry
forward. In the confirmation screen, compounds were identified as confirmed hits based on a
mixture of two linear models using data from both confirmation and primary screens.
R (www.r-project.org, version 3.3.1) and Prism 6 (GraphPad Software, San Diego, CA)
statistical software were used to carry out these analyses.46
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Chemistry. Reagents and solvents used were obtained as at least reagent grade from commercial
sources and used without further purification unless noted otherwise. Moisture or air-sensitive
reactions were conducted under argon atmosphere in oven-dried (120oC) glass apparatus. The
solvents were removed under reduced pressure using standard rotary evaporators. Flash
chromatography was carried out on a Biotage Isolera One (Charlotte, NC); while analytical thin-
layer chromatography (TLC) was performed using precoated TLC silica gel 60 F254 aluminum
sheets purchased from EMD (Gibbstown, NJ) and visualized using UV light. Reaction
monitoring, compound characterization, and purity analysis were performed using a 1260
Infinity/6420 Triple Quad (Agilent Technologies, Inc., Santa Clara, CA) with a Supelco
Discovery HS C18 column (Sigma-Aldrich). All the compounds were identified to be at least
98% pure by UV. Compounds of interest were analyzed by high resolution MS (HRMS) using an
Agilent 6230 ESI-TOFMS (Santa Clara, CA). 1H NMR spectra were obtained on a Varian
Mercury 400 (Varian, Inc., Palo Alto, CA). 13C HNMR spectra were obtained on a Varian VX
500 equipped with a Varian XSens 2 channel NMR Cold Probe (Varian, Inc., Palo Alto, CA).
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The chemical shifts are expressed in parts per million (ppm) using suitable deuterated NMR
solvents.
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General procedure for the synthesis of Compound 3{1-7}. To a solution of compound 1 (1
eq.) in warm ethanol was added the appropriate isothiocyanate 2{1-7} (1.1 eq.) dropwise with
stirring. The reaction mixture was refluxed for 6h and then allowed to cool at 4 oC overnight.
Precipitated solids were filtered, washed with ethanol and dried overnight under vacuum to yield
compounds 3{1-7} as white solids.
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General procedure for the synthesis of Compound 4{1-7}. A solution of sodium ethoxide (10
eq.) was prepared in anhydrous ethanol and stirred for 5 minutes. Separately, compound 3{1-7}
was dissolved in anhydrous ethanol and added to the sodium ethoxide solution. The reaction
mixture was further refluxed for 12h and then allowed to cool at 4 °C overnight. Precipitated
solids were filtered and washed with cold ethanol to obtain compound 4{1-7} as white solids.
General procedure for the synthesis of Compound 6{1-7,1-6}. To a solution of compound
4{1-7}(1 eq.) in anhydrous DMF were added, compound 5{1-6} (1.1 eq.) and triethylamine (2
eq.). The reaction mixture was heated to dissolve the components and then stirred at room
temperature for 10 minutes or until the reaction was complete. The solvent was then removed
under vacuum to obtain the residue which was suspended in methanol with sonication.
Precipitated solids were filtered and washed with excess methanol to give compound 6{1-7,1-6}
as white solids.
Syntheses of Compounds 9 and 10. These compounds were synthesized in a manner similar to
that of the combinatorial library using either pentyl isothiocyante (7a) for compound 8a or hexyl
isothiocyanate (7b) for compound 8b and then cyclized and alkylated as described for syntheses
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of compounds 6{1-6,1-7} to give pentyl derivatives 9a and 9b and hexyl derivatives 10a and
10b respectively.
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Syntheses of Compounds 11 and 12. To a solution of compound 6{7,5} for compound 11 or
compound 6{7,6} for compound 12 (1 eq.) in anhydrous DMF, was added sodium hydride (1.1
eq.) at room temperature. The reaction mixture was stirred for 5 min followed by the addition of
iodomethane (1.1 eq.) and stirred for additional 2h. The solvent was then removed under vacuum
and the residue was dissolved in ethylacetate, washed with water, dried over sodium sulfate and
concentrated to obtain the crude product which was purified using column chromatography to
obtain compound 11 or 12.
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Biology
Cell lines and reagents
The CellSensor® NFκB-bla human monocytic THP-1 cell line was purchased from Thermo
Fisher Scientific (Waltham, MA). This cell line contains a stably integrated beta-lactamase
reporter gene under the control of the Nuclear Factor kappa B (NF-κB) response element
(https://tools.thermofisher.com/content/sfs/manuals/CellSensor_NFkBbla_THP1_man.pdf). NF-
κB activation results in beta-lactamase production, which shifts the fluorescence emission of the
beta-lactamase substrate (LiveBLAzerTM-FRET B/G (CCF4-AM), Thermofisher) to favor
coumarin (460 nm emission) over fluorescein (530 nm emission). Murine or human TLR4 HEK
Blue™ cells were purchased from Invivogen. These cell lines were stably co-transfected with
human or mouse TLR4, MD-2, and CD14 co-receptor genes, and an inducible secreted
embryonic alkaline phosphatase (SEAP) reporter gene. QuantiBlue was purchased from
Invivogen, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyl tetrazolium bromide) was purchased
from Acros Organics, and ovalbumin was purchased from Thermo Fisher Scientific. For the HTS
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LPS E. coli 0111:B4 (Sigma Aldrich) was used and LPS-EB Ultrapure (cat# tlrl-3pelps,
Invivogen, San Diego CA) was used in the validation and subsequent SAR studies.
Wortmannin33, a non-specific, covalent inhibitor of PI3K, and MPLA was purchased from
Invivogen (San Diego, CA).
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Measurement of NF-κB activation using CellSensor® NF-κB-bla THP-1
CellSensor® NF-κB-bla THP-1 cells were plated in 96-well plates at 80 µl (5104 cells) per well
in RPMI supplemented with 10% dialyzed fetal bovine serum (FBS, Omega Scientific, Inc.,
Tarzana, CA), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin
and100 µg/ml streptomycin (additives from Thermo Fisher Scientific). 10 µl of 10x (50 µM) of
compound and 10 µL of 10x (100 ng/ml) LPS-EB in assay media were then added. Cells were
incubated for 5h and 20h in 5% CO2 at 37° C, after which 20 µL of 6xLiveBLAzerTM FRET B/G
Substrate (CCF4-AM) mixture (prepared according to the manufacturer's instructions) was added
to each well. Plates were incubated at room temperature in the dark for 2h. Fluorescence was
measured on a Tecan Infinite M200 plate reader (Männedorf, Switzerland) at an excitation
wavelength of 405 nm, and emission wavelengths of 465 nm and 535 nm. Background values
(cell free wells at the same fluorescence wavelength) were subtracted from the raw fluorescence
intensity values and the emission ratios were calculated as the ratio of background subtracted
fluorescence intensities at 465 nm to background subtracted fluorescence intensities at 535 nm.
The response ratio was then calculated using the formula: [(emission ratio of a test well)/average
emission ratio of wells with vehicle (0.5% DMSO)]. Compounds were ranked using "%act"
metrics, which was calculated as [response ratio of the compound/response ratio of LPS]. In each
assay, negative control wells were included that were cell-free and cells with vehicle (0.5%
DMSO).
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Cell viability assays
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THP-1 cells were dispensed in 96-well plates (105 cells/well) and treated with 5 µM of each
compound. After 18h of incubation, a solution of MTT in assay media (0.5 mg/ml) was added to
each well and further incubated for 4-6 hours, followed by addition of cell lysis buffer (15% w/v
SDS and 0.12% v/v 12N HCl aqueous solution), incubated overnight and then absorbance
measured at 570 nm using 650 nm as reference with a plate reader.
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In vitro assays using TLR4 reporter cell lines
Murine or human TLR4 HEK Blue™ cells (2.5  104 cells per well of a 96 well plate) were
incubated with 5 µM of each test compound. The culture supernatants were harvested after a 20-
24 h incubation period. SEAP activity in the supernatants was determined using QuantiBlue, and
absorbance read at 630 nm per the manufacturer’s instructions as previously reported.7
Animals
Seven to nine-week-old C57BL/6 (wild-type, WT) mice were purchased from The Jackson
Laboratories (Bar Harbor, MA). All animal experiments received prior approval from the UCSD
Institutional Animal Care and Use Committee.
In vivo adjuvant activity study
WT mice (n=5 per group) were immunized in the gastronemius muscle with ovalbumin (20
μg/animal) mixed with MPLA (10 μg/animal) and compound 6{7,5} or 6{6,6} or 6{7,6} (100
nmol/animal) on days 0 and 14. On day 21, immunized mice were bled and OVA-specific IgG
titers were measured by ELISA as previously described.47
Statistical analysis for in vitro and in vivo studies
Data for in vitro and in vivo studies are represented as mean ± standard error of the mean (SEM)
or mean ± standard deviation (SD). Prism 6 (GraphPad Software, San Diego, CA) statistical
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software was used to obtain p-values for comparison between groups (p<0.05 was considered
significant) for in vivo study. One-way ANOVA followed by Dunn’s post hoc test was used to
compare multiple groups.
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FIGURES
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(A)
(B)
12h
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Assay Optimization with
LPS alone
LPS + Compounds
De-activation Molecules
De-activation Molecules
+ Compounds
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THP-1 NF-B
Cellsensor® Cells
Pilot screening
(14,631)
Main screening
(166,304)
Time after stimulation
Confirmation screening
at 12h (2,011)
Additional screening
at 5h for kinetic
profiling (2,011)
Confirmed hits
(407)
Pyrimdo[5,4-
4H-chromene-3-
carbonitriles
(15)
b]indoles
(20)
SAR on
Pyrimidoindoles
Tetrahydrothieno
[2,3-c]pyridines
(15)
Benzo[d][1,3]dio
xol-2-ylureas
(15)
Figure 1. Theoretical kinetics of LPS activation and the HTS workflow strategy. (A) Theoretical
kinetic profiles showing activation by LPS (dashed red line) and subsequent deactivation at 12h
due to increase in deactivation molecules (dashed blue line). Addition of compounds that prolong
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the initial activation (solid red line) and also suppress the formation of deactivation molecules
(solid blue line) were identified through HTS. (B) The cell based assay using THP-1
CellSensor® NF-κB reporter cells was optimized for identification of compounds that prolonged
NF-κB activation when stimulated with LPS for 12h. The assay was tested in a pilot screen of
14,631 compounds to validate the approach. In the main library screen, 166,304 compounds
were screened in the presence of LPS for NF-κB activation at 12h. Cluster based statistical
analysis yielded 2,011 compounds, which were retested in a confirmation screen at 12h with LPS
in duplicate. An additional screen was performed on these 2,011 compounds for measuring NF-
κB activity at 5h to observe the activity kinetics. 407 compounds that had confirmed activity at
12h were structurally clustered using Tanimoto index at 0.5. Four large families of chemotypes
were identified as hits based on their kinetic profile of 5h and 12h that prolonged NF-κB
activation with LPS. The pyrimidoindole class of compounds was selected for further SAR
studies. The number in parentheses corresponds to number of compounds.
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(E)
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5%FBS
Cells preplating
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or test compound
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Add
100ng/mL
Add
Read
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12h
Time (hours)
Figure 2. Optimization of assay conditions in THP-1 CellSensor® assay. The assay conditions
were optimized for the following parameters: (A) Cell numbers/well and cells preplating time;
(B) FBS concentration; (C) incubation time; and (D) concentration of wortmannin and the time
of its addition relative to LPS. (E) The schematic shows the optimum assay parameters and the
assay protocol used for the HTS. Briefly, the cells were preplated at a density of 20,000
cells/well in 5% FBS 16h prior to addition of 100ng/mL LPS alone or LPS with 0.25µM
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wortmannin. An additional control (LPS 5h) was added 5h prior to the addition of FRET
substrate at 12h and the plates were read at 14h.
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LPS 12h +
Wortmannin
LPS
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No LPS
(0.5%DMSO)
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LPS 12h +
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LPS
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No LPS
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Well Number
Figure 3. Pilot and HTS main screen. Percent activation values for the controls and test
compounds from (A) the pilot screen and (B) the main screen. The histogram plot to the left for
each screen shows the intra-assay statistics on the percent activation values for No LPS (0.5%
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DMSO, negative control), LPS 12h (100ng/mL LPS control added at time 0h), LPS 12h +
wortmannin (positive control), and LPS 5h (100ng/mL LPS control added 5h before the assay
read out). The scatter plot to the right shows percent activation values for all the compounds and
controls used in each screen. Each black circle represents an individual test compound, while
controls are colored as in the histogram plot.
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(B)
(C)
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12h
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LPS
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4H-chromene-3-carbonitriles
Benzo[d][1,3]dioxol-2-ylureas
Tetrahydrothieno[2,3-c]pyridines
Pyrimdo[5,4-b]indoles
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Time after stimulation
5h (distance to LPS)
Figure 4. Confirmation and kinetic profile screens. (A) A scatter plot of distance to LPS values
for 2,011 compounds selected for confirmation and kinetic profile screens. The abscissa values
correspond to kinetic profile experiment while the ordinate values correspond to the confirmation
screen experiment. (B) The compounds were divided into four categories based on their
theoretical kinetic profiles for NF-κB activation with LPS. These compounds were either early
inhibitors (5h distance to LPS<0) or early enhancers (5h distance to LPS>0). Within the early
inhibitors, the compounds were grouped as either showing moderate increase or high increase in
NF-κB induction compared to LPS at 12h (green and orange symbols and lines, respectively).
Similarly, within the enhancer groups, compounds showed moderate or high increase in NF-κB
induction compared to LPS at 12h (red and blue symbols and lines, respectively). (C) Cluster
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binning of confirmed hits indicates segregation of chemotypes into distinct kinetic profiles.
Large clusters were colored based on chemotype, namely pyrimido[5,4-b]indoles (red circles),
tetrahydrothieno[2,3-c]pyridines(blue triangles), benzo[d][1,3]dioxol-2-ylureas (green triangles)
and 4H-chromene-3-carbonitriles (orange squares). The center of the chemotype cluster was
calculated as the geometric mean of the coordinates within a cluster. The colors are consistent
with the kinetic profiles shown in (B).
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