A novel inhibitor of inducible NOS dimerization protects against cytokine-induced rat beta cell dysfunction

Article abstract of DOI:10.1111/bph.14388

Background and Purpose: Beta cell apoptosis is a major feature of type 1 diabetes, and pro-inflammatory cytokines are key drivers of the deterioration of beta cell mass through induction of apoptosis. Mitochondrial stress plays a critical role in mediating apoptosis by releasing cytochrome C into the cytoplasm, directly activating caspase-9 and its downstream signalling cascade. We aimed to identify new compounds that protect beta cells from cytokine-induced activation of the intrinsic (mitochondrial) pathway of apoptosis. Experimental Approach: Diabetogenic media, composed of IL-1β, IFN-γ and high glucose, were used to induce mitochondrial stress in rat insulin-producing INS1E cells, and a high-content image-based screen of small molecule modulators of Casp9 pathway was performed. Key Results: A novel small molecule, ATV399, was identified from a high-content image-based screen for compounds that inhibit cleaved caspase-9 activation and subsequent beta cell apoptosis induced by a combination of IL-1β, IFN-γ and high glucose, which together mimic the pathogenic diabetic milieu. Through medicinal chemistry optimization, potency was markedly improved (6–30 fold), with reduced inhibitory effects on CYP3A4. Improved analogues, such as CAT639, improved beta cell viability and insulin secretion in cytokine-treated rat insulin-producing INS1E cells and primary dispersed islet cells. Mechanistically, CAT639 reduced the production of NO by allosterically inhibiting dimerization of inducible NOS (iNOS) without affecting its mRNA levels. Conclusion and Implications: Taken together, these studies demonstrate a successful phenotypic screening campaign resulting in identification of an inhibitor of iNOS dimerization that protects beta cell viability and function through modulation of mitochondrial stress induced by cytokines.

Full text of DOI:10.1111/bph.14388

A Novel Inhibitor of iNOS Dimerization Protects Against
Cytokine-Induced Rat -cell Dysfunction
Linlin Zhong1, 4, Tuan Tran1, 4, Tyler D. Baguley1, Sang Jun Lee1, Adam Henke1, Andrew
To1, Sijia Li1, Shan Yu1, Fabio A. Grieco2, Jason Roland1, Peter G. Schultz1,3, Decio L.
Eizirik2, Nikki Rogers1, Arnab K. Chartterjee1, Matthew S. Tremblay1 and Weijun Shen1
1California Institute for Biomedical Research (Calibr), 11119 North Torrey Pines Road, La
Jolla, California 92037, United States, 2ULB Center for Diabetes Research, Universite´ Libre
de Bruxelles (ULB), Route de Lennik 808, 1070 Brussels, Belgium, 3Department of Chemistry,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States.
4These authors contributed equally to this work.
Correspondence should be addressed to W.S. and M.S.T. (wshen@calibr.org;
mtremblay@calibr.org)
Running Title: An iNOS dimerization inhibitor rescues ER stress
Keywords: high throughput screening • intrinsic apoptosis • β-cells • iNOS dimerization
Abbreviations: NO, nitric oxide; iNOS, inducible nitric oxide synthase; HCA, high content
analysis; Apaf-1, apoptotic protease activating factor 1; GSIS, glucose stimulated insulin
secretion; LOPAC, Library of Pharmacologically Active Compounds; HTRF, Homogeneous
Time Resolved Fluorescence; CHOP, C/EBP-homologous protein; BiP, binding
immunoglobulin protein; PDI, protein disulfide isomerase; sXBP1, spliced X-box binding
protein 1; UPR, unfolded protein response; ER, endoplasmic reticulum; ATF, activating
transcription factor; RT-qPCR, real-time quantitative polymerase chain reaction; MMP, matrix
metalloproteinase; SAR, structure-activity relationship;
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/bph.14388
This article is protected by copyright. All rights reserved.

Abstract
Background and Purpose -cell apoptosis is a major feature of type 1 diabetes and pro-
inflammatory cytokines are key drivers of the deterioration of β-cell mass through induction of
apoptosis. Mitochondrial stress plays a critical role in mediating apoptosis by releasing
cytochrome C into the cytoplasm, directly activating caspase-9 and its downstream signaling
cascade. We aim to identify new compounds that protect β cells from cytokine-induced
activation of the intrinsic (mitochondrial) pathway of apoptosis.
Experimental Approach Diabetogenic media, comprised of IL-1β, IFN-γ and high glucose
were used to induce mitochondrial stress in rat insulin-producing INS1E cells and High content
image-based screen of small molecule modulators of Casp9 pathway was performed.
Key Results We report a novel small molecule, ATV399, identified from a high content image-
based screen for compounds that inhibit cleaved caspase-9 activation and subsequent β-cell
apoptosis induced by a combination of IL-1β, IFN-γ and high glucose, which together mimic
the pathogenic diabetic milieu. Through medicinal chemistry optimization, potency was
markedly improved (6-30 fold), with reduced activity on CYP3A4 inhibition. Improved
analogs, such as CAT639, exhibit improved β-cell viability and insulin secretion in the rat
insulin-producing INS1E cells and primary rat dispersed islet cells. Mechanistically, CAT639
reduced the production of nitric oxide (NO) by allosterically inhibiting dimerization of the
inducible nitric oxide synthase (iNOS) without affecting its mRNA levels.
Conclusion and Implications Taken together, these studies demonstrate a successful
phenotypic screening campaign resulting in identification of an inhibitor of iNOS dimerization
that protects β-cell viability and function through modulation of mitochondrial stress induced
by cytokines.
This article is protected by copyright. All rights reserved.

caspase-9 -
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1625
HDAC - http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=848
GSK3β -
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2030
IL-1 receptor antagonists -
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5878
inducible nitric oxide synthase (iNOS) -
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1250
Baricitinib -
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7792
Flumazenil -
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4192
IκB kinase -
http://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId=578
Janus kinase (JAK)-1/2 -
http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=581
GABA receptor -
http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=72
nuclear factor kappa B (NF-κB) -
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2039
sarcoendoplasmic reticulum pump Ca2+ ATPase (SERCA) -
http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=159#841
Thapsigargin (TG) -
http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5351
This article is protected by copyright. All rights reserved.

Introduction
Type 1 diabetes is ultimately caused by cellular stress, which activates apoptosis (programmed
cell death) of β-cells and results in progressive reduction of β-cell mass and insulin insufficiency.
Pro-inflammatory cytokines, such as interleukin (IL)-1β and interferon (IFN)-γ, induce
dysfunction of the mitochondrial membrane potential (Barbu et al., 2002; Papaccio et al., 2005),
leading to mitochondrial stress and β-cell death (Gurzov et al., 2011). Mitochondria play crucial
functions in aerobic eukaryotic cells, such as ATP production, cellular differentiation,
proliferation and apoptosis (Green et al., 1998). During apoptotic signaling, mitochondria release
the pro-apoptotic signal cytochrome C to the cytosol and form an apoptosome complex with
apoptotic protease activating factor 1 (Apaf-1) and pro-caspase-9 to induce production of cleaved,
activated caspase-9 (Li et al.; Pop et al.; Renatus et al., 2001), which then activates caspase-3/7
and triggers apoptosis (Gurzov et al., 2011).
Current diabetes therapies mostly focus on relieving symptoms, such as insulin replacement,
decreasing hepatic glucose production and increasing peripheral glucose uptake. Drugs
preventing β-cell apoptosis and preserving β-cell function represent a novel therapeutic
approach for type 1 diabetes. Recently, small molecules targeting Na+ or Ca2+ ion channels,
HDAC and GSK3β were shown to prevent β-cell apoptosis with variable effects and high risks
of side effects due to broad target inhibition (Yang et al., 2014)-(Lundh et al., 2013). IL-1
receptor antagonists increased insulin sensitivity in patient with type 1 diabetes in clinical trials
(Cavelti-Weder et al., 2012),(Sloan-Lancaster et al., 2013),(Larsen et al., 2007),(van
Asseldonk et al., 2015),(Rissanen et al., 2012). Herein, we describe a high content image-
based phenotypic screen and the discovery of a small molecule (ATV399) that inhibits caspase-
9 and caspase 3/7 activation induced by cytokines in cultured rat insulin-producing INS1E
cells. Medicinal chemistry optimization generated analogues CAT639 and CBD504 with
improved potency. Mechanistic studies showed that this series of compounds improve β-cell
viability and insulin secretion in rat insulin-producing INS1E cells and primary rat dispersed
islet cells by inhibiting dimerization of inducible nitric oxide synthase (iNOS) and reducing
the production of NO.
This article is protected by copyright. All rights reserved.

Materials and methods
Materials
Antibodies against CHOP, cleaved caspase-3, caspase-3, Phospho-stress-activated protein
kinase (SAPK)/JNK, SAPK/JNK, Phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK and -
actin were purchased from Cell Signaling (Boston, MA). Cell lysate buffer was obtained from
Cell signaling (Boston, MA). Antibody against iNOS was purchased from Abcam (Boston,
MA). Pierce LDS Sample Buffer, Non-reducing (4X) was purchased from Thermo Fisher
(Dallas, TX). Nitric oxide assay kit was obtained from Cayman Chemical (Chicago, IL).
High throughput screening protocol
Primary screen:
Rat insulin-producing cells that were maintained in growth medium (Roswell Park Memorial
Institute (RPMI) 1640 medium containing 1X antibiotics, 1X HEPES (Thermo Fisher, Dallas,
TX), 1 mM sodium pyruvate (Thermo Fisher, Dallas TX), 1X Glutamax, 1X MEM NEAA
(ThermoFisher, Dallas, TX), 1 mM β–mecaptoethanol (Thermo Fisher, Dallas, TX) and 10%
fetal bovine serum (FBS, ThermoFisher, Dallas TX) were detached using trypsin. After
removing excess trypsin by gentle centrifugation (1000 rpm for 5 min), the cells were
resuspended in growth medium at a density of 250 cells/µL. 40 µL of this solution was then
dispensed to each well of 384-well clear bottom plates (Corning Inc., Corning, NY), which
were pre-spotted with 20 nL of compound (5 µM final concentration of 50 µL total volume).
The plates were incubated for 24 h in an incubator at 37 °C with constant supply of 5% CO2,
and 95% humidity. After the incubation period, each well was supplemented with 10 µL of
growth medium containing cytokines and glucose (25 ng/mL INF-γ, 2.5 ng/mL IL-1β, and 33
mM glucose final concentration). Plates were put back into the incubator for additional 24 h.
Cells were then fixed and permeabilized using paraformaldehyde (PFA; 5% final
concentration; 10 min) and triton-X100 (1% final concentration; 10 min), respectively.
Medium containing PFA and Triton-X100 were replaced with blocking buffer that contains
2.5% goat serum (Sigma Aldrich, St. Louis, MO) and 2% BSA (Sigma Aldrich, St. Louis, MO)
in PBS. The plates were incubated at room temperature (RT) for 30 min and blocking buffer
was then replaced with 25 µL of primary antibody (220X diluted from the manufacturer stock
solution) solution that contains 2% BSA in PBS. With gentle shaking, primary antibody was
incubated with the cells for 1 h. The cells were washed 2X with 50 µL PBS solution that
contains 2% tween-20 (PBST). The cells were then incubated with 25 µL of secondary antibody
(1000X diluted from the manufacturer stock solution) solution that contains 1 µg/mL Hoechst
This article is protected by copyright. All rights reserved.

33342 and 2% BSA in PBS for 1 h. Finally, the cell was washed 2X with 50 µL PBST solution
and was stored at 4 °C in 50 µL/well PBS until imaging using Cell Insight CX5 high content
imager (Thermo Fisher, Dallas, TX).
Counter screen:
Like primary screen, compounds were pre-spotted in 20 nL of DMSO and rat insulin-producing
INS1E cells (10000 cells/40 µL/well of 384 well white solid bottom plate; Corning) were
incubated in an incubator at 37 °C with constant supply of 5% CO2, and 95% humidity for 24
h. After the incubation, plates were taken out and each well was supplemented with 10 µL of
growth medium containing Cytokines and glucose (25 ng/mL INF-γ, 2.5 ng/mL IL-1β, and 33
mM glucose final concentration). Plates were put back into the incubator for additional 24 h.
Quantification of cleaved caspase-3/7, caspase-9 and cell viability was done by addition of 5
µL/well of Caspase-3/7 Glo (Promega, Madison, WI), Caspase-9 Glo (Promega, Madison, WI),
and Cell Titer-Glo (Promega, Madison, WI), respectively. The evolved luminescence signal
was read using Envision (0.1 second/well, Perkin-Elmer).
Insulin secretion assay
Rat insulin producing INS1E cells maintained in growth medium were detached using trypsin.
After removing excess trypsin by gentle centrifugation (1000 rpm for 5 min), the cells were
resuspended in growth medium at a density of 250 cells/µL. 40 µL of this solution was then
dispensed to each well of 384-well plates (Corning) that were pre-spotted with 20 nL of various
concentrations compound. The plates were incubated for 24 h in the incubator at 37 °C with
constant supply of 5% CO2, and 95% humidity. After the incubation, plates were taken out and
each well was supplemented with 10 µL of growth medium or 10 µL of growth medium
containing cytokines. Plates were put back into the incubator for additional 24 h.
Quantification of secreted insulin in medium was done by using HTRF Insulin Assay Kit
(Cisbio Assay, Bedford, MA) on 2 µL of the medium. Briefly, 8 µL antibodies solution
containing two monoclonal antibodies that recognize the insulin was added for each 2 µL of
the medium. These antibodies are labeled with fluorophores that are Fluorescence Resonance
Energy Transfer (FRET) pair. FRET signal was measured using Envision plate reader with
excitation at 320 nm and emission at 665 nm and 615 nm.
This article is protected by copyright. All rights reserved.

Rat insulin-producing INS1E cells were plated at a density of 5000 cells/well in 384-well
plates. CAT639 pre-treated cells for 4 h and then cytokines were added for 24 h. Cell viability
was tested using CTG assay. Activity of caspase-9 and caspase-3/7 was measured with
caspase-9 Glo and caspase-3/7 Glo assay kit individually.
Mitochondrial membrane potential assay
Mitochondrial membrane potential was evaluated by using JC-1 dye (Thermo Fisher, Dallas
TX). The treatment for the cells was the same as previously mentioned. After treatment, cells
were suspended in culture medium at a density of 1x106 cells/mL. Cells were incubated in JC-
1 dye for 20 min. Cells were washed once and resuspended in 200 L PBS and analyzed by
flow cytometry. During apoptosis, JC-1 dye aggregates in the interior of the membrane; a
fluorescence emission wavelength will shift from green (~529 nm) to red (~590 nm). A ratio
(red/green) decrease indicates mitochondrial membrane depolarization. Data was analyzed by
FlowJo software.
Assessment of nitric oxide release
The final breakdown products of NO are nitrite and nitrate. NO production was detected by the
sum of nitrite and nitrate levels measured with nitrite/nitrate fluorometric assay kit (Cayman
Chemical, Chicago IL). The assay was conducted by following the manual from manufacture.
Rat insulin-producing INS1E cells were incubated in DMEM/F12 medium during treatment to
prevent nitrite present in RPMI from interfering with the assay procedure.
Islet isolation and cell culture
Sprague Dawley rats were sacrificed under anesthesia using carbon dioxide. Pancreases were
removed after perfusing 1 mg/mL collagenase P (Sigma-Aldrich, St. Louis, MO) in HBSS.
Pancreases were incubated in a 37 °C water bath for 10 min, vortexed for 5 sec and incubated
in water bath for another 1-2 min. The collagenase digestion was terminated by adding HBSS
with 10% FBS. Islets were pelleted by centrifuging at 200 g for 1 min and washed in HBSS
for 3 times. Histopaque-1077 was used as purification islets derived from acinar cells. Islets
were re-suspended in 10 mL histopaque-1077 and 10 mL HBSS was slowly layered on the top.
After centrifugation at 1350 rpm for 5 min without break, islets were collected from the
interface. Islets were cultured in RPMI1640 supplemented 10% FBS and 1%
antibiotic/antimycotic (Thermo-Fisher, Waltham, MA) and 5.5 mM glucose. Islets were
This article is protected by copyright. All rights reserved.

trypsinized with 0.05% trypsin for 5-7 min and strongly pipetted several times. After
neutralizing the trypsin by adding RPMI 1640 with 10% FBS and filtered through 70 µm cell
strainer, the dispersed islet cells were pelleted by centrifuging for 5 min at 1500 rpm. After
overnight recovery, 5000 cells/well were plated in 384-well plates for cell viability assay and
in 96 well v-bottom plates for insulin stimulation (GSIS) assay. For GSIS, cells were pre-
incubated in 2.8 mM glucose for 1 h; concentration was then changed to 2.8 or 20 mM glucose
for 1 h. Medium was collected for measuring insulin amount by using HTRF insulin assay.
Low-temperature SDS PAGE
Dimerization of iNOS was separated by using low-temperature SDS-PAGE. Rat insulin-
producing INS1E cells were seeded at a density of 3 x 106 cells/well in 6-well plates. After
pretreatment with different concentrations of CAT639 for 4 h, cells were treated with 10 ng/mL
IL-1β for 24 h. Then cells were lysed on ice with cell lysis buffer (Cell signaling, Danvers,
MA) and sonicated for 5 sec. The supernatant was cleared by centrifugation at 4 °C and 16000
g. Protein samples were prepared with LDS non-reducing sample buffer without heating. SDS-
PAGE gel was run on ice at constant 125 mA for 5 h with pre-cooled NuPAGE SDS running
buffer. Proteins were transferred by wet transfer at constant 70 mA for 2.5 h. The following
western blot steps were done as usual.
Protein and mRNA analyses
Rat insulin-producing INS1E cells were harvested at different time points after treatment and
lysed in RIPA buffer. All of antibodies were used at 1:1000 dilution. Primary antibodies were
incubated overnight, and secondary antibodies were incubated for 1 h. The image was scanned
by LI-COR Odyssey. RNA was extracted by Trizol and reverse transcribed into cDNA by using
a Super Script II Reverse Transcriptase Kit (Invitrogen Inc., Carlsbad, CA). Gene expression
was quantified using RT-PCR and normalized against GAPDH. Primer sequences are shown
in Supplemental Table 6.
Data and statistical analysis
Data are represented as Mean ± SD (Standard Deviation). Student's t-test and one way analysis
of variance (ANOVA) were used for statistical analysis and P < 0.05 is considered as
significant difference. The data and statistical analysis comply with the recommendations on
experimental design and analysis in pharmacology (Curtis et al., 2015).
This article is protected by copyright. All rights reserved.

Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the
2017/18 (Alexander et al., 2017a; Alexander et al., 2017b).
Results
High content image-based phenotypic screen identified ATV399 as a putative
mitochondrial stress inhibitor
Recent advances in automated image analysis provide robust tools that enable the integration
of high content analysis (HCA) of phenotypic endpoints toward high throughput screening
identification of drug candidates (Philip et al., 2008). HCA allows for multi-parametric data
collection such that two or more targets can be recorded simultaneously (DeBiasio et al., 1987;
Giuliano et al., 1997; Korn et al., 2007). To this end, we developed an HCA-based phenotypic
assay to identify small molecules that inhibit mitochondrial stress-induced apoptosis by
monitoring cell number and cleaved caspase-9 levels as indicators of viability and health in the
presence of cellular stress. Specifically, rat insulin-producing INS1E cells were treated with
DMSO or compounds for 24 h, followed by a combination of cytokines (2.5 ng/mL of IL-1β
and 25 ng/mL IFN-γ) and high glucose (33 mM) for 24 h. This diabetogenic cocktail induces
cleaved caspase-9 levels > 3-fold over neutral control (DMSO), with a robust Z factor (Z’ ~
0.71) (Fig. 1B and C). We initially screened ~88,000 compounds, including compounds with
known mechanism of action (such as the Library of Pharmacologically Active Compounds
(LOPAC) from Sigma Aldrich) and compounds with unknown mechanism of action from
commercial vendors. Compounds inhibiting cytokine-induced caspase-9 activation at least
50% were considered as hits (hit rate ~ 0.1%). Hit compounds were counter-screened using
luminescent Caspase 3/7 Glo, Caspase 9 Glo and Cell Titer Glo (CTG) assays to ensure the
authenticity of the hits and remove cytotoxic compounds.
Certain compounds with known mechanism of action, such as IKK-16, Baricitinib and
Flumazenil, were identified (Fig. 1D). For example, IKK-16 and Baricitinib are known
inhibitors of IκB kinase (IKK) and Janus kinase (JAK)-1/2, respectively, which block signal
transduction induced by cytokines, thereby reducing cleaved caspase-9 levels and improving
cell viability (Marrero et al., 2006; Waelchli et al., 2006; Reilly et al., 2013; Negi et al., 2015).
This article is protected by copyright. All rights reserved.

In addition to validating the screening assay, the observation that Flumazenil can attenuate
activation of caspase-9 is interesting and suggests this GABA receptor antagonist may have
alternative applications. Importantly, ATV399 emerged as one of the most potent hit
compounds from the Life Chemicals Library screen, representing a novel small molecule with
unknown mechanism of action that could be further investigated as a potential drug candidate.
ATV399 improved β-cell survival and function impaired by cytokine stress
In hit validation studies, ATV399 was shown to dose-dependently inhibit caspase-9 activation
(IC50 = 3.3 μM, Fig. 2A). Phenotypically, ATV399 also improved β-cell survival, as
determined by CTG assay that measures intracellular ATP content as a surrogate for cell
viability. Addition of a pro-inflammatory cytokine cocktail reduced cell viability
approximately 60% in rat insulin-producing INS1E cells, while pretreatment with ATV399 for
24 h fully rescued viability (Fig. 2A). To test whether ATV399 attenuates loss of β-cell function
in the presence of cytokines, a Homogeneous Time Resolved Fluorescence (HTRF) assay
(Cisbio; Bedford) that quantitatively measures the level of secreted insulin in culture medium
was employed. Although cytokines markedly inhibited insulin secretion (from 480 ng/mL to
320 ng/mL), pre-treatment with ATV399 at 5 µM fully recovered insulin levels (Fig. 2A),
indicating that ATV399 can rescue insulin secretion impaired by cytokines.
In addition to causing mitochondrial stress, cytokines are also known to induce apoptosis by
triggering the unfolded protein response (UPR) and subsequent endoplasmic reticulum (ER)
stress(Cardozo et al., 2005; Chan et al., 2011). The UPR is initially activated to restore ER
homeostasis, while sustained UPR activation and unresolved ER stress can trigger
apoptosis(Hotamisligil; Laybutt et al., 2007; Eizirik et al., 2008). We assessed the ability of
ATV399 to modulate ER stress by monitoring ER stress-related gene and protein expression
levels using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting,
respectively. Treatment with 5 µM ATV399 in the presence of cytokines significantly reduced
expression levels of activating transcription factor 4 (ATF4), ATF6, binding immunoglobulin
protein (BiP), C/EBP-homologous protein (CHOP), protein disulfide isomerase (PDI) and
spliced X-box binding protein 1 (sXBP1) (Fig. 2B). We observed enhanced expression of these
genes upon treatment with cytokines that is markedly reduced with ATV399 treatment. Anti-
apoptotic effects of ATV399 were confirmed at the protein level, with CHOP and cleaved
caspase-3, two major pro-apoptotic proteins, up-regulated upon treatment with cytokines after
8 h or 16 h and dramatically decreased by ATV399 treatment (Fig. 2C).
This article is protected by copyright. All rights reserved.

Medicinal chemistry optimization led to analogues with improved potency
The interesting β-cell protection phenotype induced by ATV399 prompted us to explore
structural analogs to establish a structure-activity relationship (SAR). The activity of initial
ATV399 analogs from inventory led us to the pyridinylpiperidine portion of the molecule for
a focused SAR effort. Of the six active compounds, all but one contained this
pyridinylpiperidine functionality, while most analogs lacking this moiety displayed modest or
no activity. Additionally, because many types of substitution were tolerated at the benzamide
portion of the molecule, we decided to focus on the pyridinylpiperidine to both identify what
was important for activity and try to increase potency. After determining that the sulfonamide
was needed for activity, we discovered that, as in our initial hit ATV399, the 3-pyridine moiety
was needed adjacent to the nitrogen of the pyrimidine (Table 1, Entries 1-6). Other heterocycles
were investigated but were not tolerated. When investigating substitution of the pyridine ring,
it was discovered that mono-substitution ortho to the nitrogen was preferred (Table 1, Entries
7-10;), leading to compound CAT639 (Entry 8), which robustly inhibited caspase-9 activity at
sub-micromolar levels (IC50 = 0.48 µM) (Table 1). From initial ADME studies, we also knew
that ATV399 exhibited potent inhibition of CYP3A4, which is a key liability for drug
development. This potent inhibition was partially alleviated with the introduction of the ortho-
methyl group of CAT639. The potency of analogs emerging from further SAR studies tracked
with the electronic properties of the substituents on the pyridine ring, with electron-
withdrawing substituents being preferred (Table 1, Entries 11-14). Substitution of the methyl
group for a trifluoromethyl group led to CBD504 (Entry 11) with further increased potency
(IC50 = 0.098 µM). The lead compound CBD504 also has improved CYP profile comparing to
the ATV399 and CAT639 (Supplemental Table 1). Next, we evaluated the potential in vivo
application of mCBD504 in mouse pharmacokinetics studies, oral dosing of 50 mg/Kg
mCBD504 (n =3), formulated as 10 mg/mL clear solution in 75%PEG300/25%D5W, showed
a reasonable half-life (T1/2 = 10 hrs), although with a relatively low exposure (Cmax = 141
ng/mL) (Supplemental Fig. S6), which is not sufficient to support in vivo efficacy studies.
Further medicinal chemistry optimization is required to improve its exposure and potency.
This article is protected by copyright. All rights reserved.

CAT639 protected primary rat islet survival and function impaired by cytokines
Consistent with ATV399, CAT639 dose-dependently recovered cell viability and insulin
secretion impaired by cytokines (Supplementary Fig. S1A, B). We next examined expression
levels of ER relevant genes by qRT-PCR. Again, ER stress sensor genes (ATF4, ATF3, ATF6,
BiP, CHOP, and sXBP1) were down-regulated by CAT639 in the presence of cytokines
(Supplemental Fig. 1C). Effects of CAT639 on phenotypic activity and gene expression
followed the same trend as ATV399, supporting the notion that CAT639 and ATV399 are
likely to work via the same molecular mechanism. Furthermore, we also applied CAT639 6
hours after cytokine treatment and found that post-treatment of CAT639 also partially rescued
β-cell viability and function impaired by cytokine-induced ER stress, suggesting that CAT639
may have therapeutic effect in addition to the prophylactic effect (Fig. 3A, B).
Given the improved potency of CAT639 in protecting rat insulin-producing INS1E cell
viability and function in the presence of stressors, we investigated the effect of CAT639 on
dispersed rat primary islet cells. A combination of cytokines (5 ng/mL IL-1β and 100 ng/mL
IFN-γ) induced cellular stress and apoptosis, reducing cell viability to 47% in dispersed
primary rat islet cells. Importantly, CAT639 treatment mitigated effects of cytokines, with cell
viability increased to 60% at 0.6 µM and 78% at 10 µM (Fig. 4A). In addition, we measured
the effect of CAT639 on glucose stimulated insulin secretion (GSIS) in dispersed primary rat
islets. Cytokines dramatically inhibited insulin secretion at both 2.8 mM (3-fold) and 20 mM
glucose, which was rescued by pre-treating with CAT639 in a dose-dependent manner (Fig.
4B).
Mechanism of action for ATV399 and its analogs
Since several kinase inhibitors are reported to inhibit mitochondrial stress, we profiled the hit
compound ATV399 in a panel of 50 representative serine/threonine and tyrosine kinases
(Supplemental Table. 2). ATV399 did not inhibit any kinase targets at 10 µM, suggesting that
kinase inhibition was unlikely to be the primary mechanism of action.
This article is protected by copyright. All rights reserved.

Interestingly, the matrix metalloproteinase (MMP) family of enzymes has recently been shown
to be present within the cell in the cytoplasmic compartments as well as in the mitochondria
(Klein et al., 2011). MMPs are typically involved in the degradation of proteins in the
extracellular matrix, but they were reported to induce apoptosis by impairing the mitochondrial
membrane potential upon activation within the cell (Kowluru et al., 2011; Mohammad et al.,
2011). For this reason, we assessed gene expression levels of several MMPs (MMP1, MMP2,
MMP9) and pro-apoptotic genes such as BH3-interacting domain death agonist (BID), death
protein 5 (dp5), myeloid cell leukemia 1 (Mcl-1), and NADPH Oxidase Activator (NOXA).
CAT639 markedly down-regulated these genes in the presence of cytokines (Fig. 5B/5C). This
suggests that CAT639 is involved in the mitochondrial signaling pathway.
To further elucidate the mechanism of action of CAT639, we treated rat insulin-producing
INS1E cells with individual cytokines. IL-1β alone could decrease rat insulin-producing INS1E
cell viability and increase caspase-9 activity (Fig. 5A), while IFN-γ could not (data not shown).
Interestingly, CAT639 fully rescued these effects of IL-1β on rat insulin-producing INS1E
cells. (Fig. 5A), suggesting the mechanism of action involves downstream effectors of IL-1β.
Next, we investigated the ability of CAT639 to protect the mitochondria using JC-1, a
commonly used dye that provides a readout of mitochondrial membrane potential (Δψm)(Liu
et al., 2007; De Proost et al., 2008). CAT639 potently reversed IL-1β-induced mitochondrial
membrane depolarization (Fig. 5D). Specifically, IL-1β increased apoptotic cells from 8.4%
to 27.6%. CAT639 markedly reduced the percentage of apoptotic cells to 10% at 5 μM.
Two major canonical pathways of IL-1β are nuclear factor kappa B (NF-κB) signaling (Heimberg
et al., 2001; Eldor et al., 2006) and the Jun NH2-terminal kinase (JNK) / p38 mitogen-activated
protein kinase (p38MAPK) pathways (Lin et al., 2005). To evaluate potential impact of CAT639
on these pathways, we used the NF-κB luciferase reporter gene-based assay to assess the NF-ƙB
signaling pathway. Results showed that CAT639 did not reduce NF-κB-Luc signal induced by
IL-1β (Supplemental Fig. 2A). In addition, CAT639 had no effect on the phosphorylation of JNK
and only modestly attenuated the phosphorylation of p38 MAPK induced by IL-1β (Supplemental
Fig. 2B). Cytokines induced ER stress in pancreatic β-cells by downregulation of the
sarcoendoplasmic reticulum pump Ca2+ ATPase (SERCA) and depleting Ca2+ from endoplasmic
reticulum (Oyadomari et al., 2001). Thapsigargin (TG), a potent inducer of ER stress, specifically
binds to SERCA and inhibits its function (Xu et al., 2004). Cat639 protected from cell death,
This article is protected by copyright. All rights reserved.

insulin secretion and the expression of ER stress genes from cytokines stressor but not TG
stressor, suggesting CAT639 may target the upstream of the SERCA (Supplemental Fig. S5).
In many pro-inflammatory contexts, activation of NF-κB (Xie et al., 1994) leads to the
production of nitric oxide (NO), which is generated from L-arginine by nitric oxide synthases
(NOS). In β-cells, iNOS is the predominant source of NO (Eizirik et al., 1992) and most
dynamic in terms of response to cellular stress and has been shown to be a factor in models of
type 1 diabetes and acute pancreatitis (Qader et al., 2003; Torres et al., 2004; Fujimoto et al.,
2005; Muhammed et al., 2012). Indeed, large amounts of NO generated by iNOS plays a major
role in IL-1β-mediated rat β-cell apoptosis (Shimabukuro et al.; Welsh et al., 1991). Thus, we
examined the iNOS mRNA expression levels using qRT-PCR assay. IL-1β induced a 36,000-
fold increase of iNOS at mRNA levels as compared to DMSO treatment. CAT639 only mildly
reduced the iNOS mRNA levels (Fig. 6A). However, CAT639 markedly blocked NO
production that was induced by either IL-1β alone or IL-1β and IFN-γ (Fig. 6B). To assess
whether the ability of CAT639 to protect rat insulin-producing INS1E cells from apoptosis was
dependent on NO, we used a nitric oxide donor, (±)-S-Nitroso-N-acetylpenicillamine (SNAP).
SNAP reduced INS1E cell viability, however, CAT639 treatment did not improve cell viability.
Similarly, CAT639 treatment did not inhibit caspase-9 activity induced by SNAP (Fig. 6C).
These results suggest that CAT639 may be directly inhibiting iNOS, downstream of iNOS
mRNA induction but upstream of chemical NO donor like SNAP.
NO production depends on iNOS expression and activity. We first examined the effect of
CAT639 on total iNOS protein. The western blot results showed that iNOS was barely detected
under normal physiological conditions and significantly increased after induction with
cytokines. However, CAT639 did not block induction of iNOS protein expression (Fig. 6D).
Interestingly, iNOS only generates NO when the enzyme forms a homodimer (Baek et al.,
1993; Li et al., 2005; Daff, 2010). Therefore, we studied the effect of CAT639 on iNOS
dimerization. To do this, we detected the dimer and monomer of iNOS by low temperature
SDS-PAGE immunoblots. Indeed, a clear band of dimeric iNOS was detected when rat insulin-
producing INS1E cells were treated with IL-1β, while a reduction in dimeric enzyme induction
was observed in cells co-treated with CAT639 (Fig. 6D). These data support the notion that
CAT639 may be downregulating NO production by inhibiting iNOS dimerization. Similar to
CAT639, other iNOS inhibitors like 1400W also protects β-cell viability and function induced
by cytokine stress (Supplemental Fig. S3).
This article is protected by copyright. All rights reserved.

Discussion
Mitochondrial stress and β-cell death, induced by pro-inflammatory cytokines, plays a key role
in type 1 = diabetes. Through a high content image-based screening campaign, we have
identified a novel small molecule that inhibits cleaved caspase-9 activation and subsequent β-
cell apoptosis induced by diabetogenic media (a combination of IL-1β, IFN-γ and high
glucose), potentially through inhibition of iNOS by disrupting of its dimerization. Inhibition of
iNOS by small molecules or in vitro knockdown of iNOS is known to improve β-cell viability
and glucose-stimulated insulin secretion (Bai-Feng et al.; Hynes et al., 2011; Muhammed et
al., 2012). Moreover, iNOS inhibitors have been shown to attenuate fasting hyperglycemia
and fasting hyperinsulinemia and significantly improve insulin sensitivity in diabetic mice
(Bai-Feng et al.; Sugita et al., 2005). There are three major types of NOS inhibitors described
to date: (1) inhibitors that mimic the substrate arginine (Víteček et al., 2012); (2) inhibitors
that mimic the cofactor 6R-5,6,7,8-tetrahydrobiopterin (BH4) and inhibit NOS enzymatic
activity by interfering with the electron transfer and preventing the formation and release of
NO at the catalytic center of NOS (Klatt et al., 1995; Wei et al., 2008; Daff, 2010); and (3)
inhibitors of enzyme activation by targeting the phosphorylation and dimerization of iNOS
(Pan et al., 1996; Sennequier et al., 1999; Symons et al., 2011).Our data suggests that the
compounds ATV399, CAT639 and CBD504 inhibit the activation of iNOS by preventing iNOS
dimerization and reducing NO production. Assessment of the expression levels of ER and
mitochondrial stress-related genes revealed that ATV399 and CAT639 could rescue
detrimental effects induced by cytokines. However, the mRNA expression levels of iNOS were
only moderately modulated by the compounds. In conjunction, the production of NO was
inhibited with 5 µM treatment in the presence of cytokines. It has been reported that allosteric
inhibitors of iNOS and small molecule inhibitors that bind to the iNOS-heme cofactor and form
an irreversible complex with the monomer iNOS, which prevent it from being converted to
dimeric iNOS (McMillan et al., 2000; Nagpal et al., 2013). Low temperature SDS-
polyacrylamide gel electrophoresis clearly indicated the inhibition of dimeric iNOS by
CAT639 and analogs, however, the exact mode of binding of CAT639 to iNOS monomer and
dimer require further detailed biochemical and structural investigations.
This article is protected by copyright. All rights reserved.

Altogether, our data demonstrates a successful high content image-based screen, which was
aimed at identifying inhibitors of cleaved caspase-9 activation, and using this screen, we
identified compounds that are capable of inhibiting dimerization of iNOS. Although the series
of compounds potently recover rat insulin-producing INS1E cells from cell death, insulin
secretion and primary rat dispersed islet cells, we were not able to see similar level of protection
in human primary islets or β-cells, which is consistent to the report that changes of NO
production are not required for suppressing human islet function induced by cytokines (Eizirik
et al., 1994). Unfortunately, this limits the translatability of this discovery into human type 1
diabetes as this dominant signaling pathway in rodent β-cells turned out to be not crucial for
human β-cells. Nevertheless, iNOS dimerization inhibitors have the potential to treat a number
of other diseases. For example, iNOS dimerization inhibitors such as BBS-4, KLYP961 and
PPA250 have been studied for treatment of diseases associated with inflammatory pain and
neuropathic pain (Ohtsuka et al., 2002; Davey et al., 2007; Symons et al., 2011). Furthermore,
there are three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS) and
endothelial NOS (eNOS) (Förstermann et al., 2012) and they all activate through
homodimerization. To test whether CAT639 has similar effect on eNOS and nNOS, we induced
nNOS dimerization in SH-SY5Y neuronal cell lines with LPS and cytokine and eNOS
dimerization with VEGF in pulmonary artery endothelial cells, and interestingly, CAT639 also
inhibited nNOS and eNOS dimerization from the western blot assay (Supplemental Figure S4).
Thus, further evaluation of the CAT639 and its analogs on their in vivo safety and their potential
application in rheumatoid arthritis, neuroinflammatory and neurodegenerative diseases are
warranted.
Author Contribution
Conceptualization, L.Z., T.T., M.T.T. and W.S.; Methodology, L.Z., T.T., S.J.L., T.D.B., and
A.H.; Investigation, L.Z., T.T., A.T., S.L., S.Y. and F.A.G.; Writing – Original Draft, L.Z.
and T.T.; Writing – Review & Editing, L.Z., J.R., N.R., M.T.T. and W.S; Funding
Acquisition, P.G.S., M.T.T. and W.S.; Supervision, D.L.E., A.K.C., P.G.S, M.T.T. and W.S.
This article is protected by copyright. All rights reserved.

Acknowledgement
We would like to thank Jeff Janes, Mitch Hull, Hung Nguyen, Megan Wogan and Alex
Kretowicz for technical assistance and helpful discussions.
Conflict of interest
The authors declare no conflicts of interest.
References
Alexander SP, Fabbro D, Kelly E, Marrion NV, Peters JA, Faccenda E, et al. (2017a). THE CONCISE GUIDE
TO PHARMACOLOGY 2017/18: Enzymes. British journal of pharmacology 174 Suppl 1: S272-S359.
Alexander SP, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding SD, et al. (2017b). THE CONCISE
GUIDE TO PHARMACOLOGY 2017/18: Overview. British journal of pharmacology 174 Suppl 1: S1-S16.
Baek KJ, Thiel BA, Lucas S, Stuehr DJ (1993). Macrophage nitric oxide synthase subunits. Purification,
characterization, and role of prosthetic groups and substrate in regulating their association into a
dimeric enzyme. Journal of Biological Chemistry 268: 21120-21129.
Bai-Feng L, Yong-Feng L, Ying C Silencing inducible nitric oxide synthase protects rat pancreatic islet.
Diabetes Research and Clinical Practice 89: 268-275.
Barbu A, Welsh N, Saldeen J (2002). Cytokine-induced apoptosis and necrosis are preceded by
disruption of the mitochondrial membrane potential (Δψm) in pancreatic RINm5F cells: prevention by
Bcl-2. Molecular and Cellular Endocrinology 190: 75-82.
Cardozo AK, Ortis F, Storling J, Feng Y-M, Rasschaert J, Tonnesen M, et al. (2005). Cytokines
Downregulate the Sarcoendoplasmic Reticulum Pump Ca2+ ATPase 2b and Deplete Endoplasmic
Reticulum Ca2+, Leading to Induction of Endoplasmic Reticulum Stress in Pancreatic β-Cells. Diabetes
54: 452-461.
Cavelti-Weder C, Babians-Brunner A, Keller C, Stahel MA, Kurz-Levin M, Zayed H, et al. (2012). Effects
of gevokizumab on glycemia and inflammatory markers in type 2 diabetes. Diabetes Care 35: 1654-
1662.
Chan JY, Cooney GJ, Biden TJ, Laybutt DR (2011). Differential regulation of adaptive and apoptotic
unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic
beta cells. Diabetologia 54: 1766-1776.
Daff S (2010). NO synthase: Structures and mechanisms. Nitric Oxide 23: 1-11.
This article is protected by copyright. All rights reserved.

Davey DD, Adler M, Arnaiz D, Eagen K, Erickson S, Guilford W, et al. (2007). Design, Synthesis, and
Activity of 2-Imidazol-1-ylpyrimidine Derived Inducible Nitric Oxide Synthase Dimerization Inhibitors.
Journal of Medicinal Chemistry 50: 1146-1157.
De Proost I, Pintelon I, Brouns I, Kroese ABA, Riccardi D, Kemp PJ, et al. (2008). Functional Live Cell
Imaging of the Pulmonary Neuroepithelial Body Microenvironment. American Journal of Respiratory
Cell and Molecular Biology 39: 180-189.
DeBiasio R, Bright GR, Ernst LA, Waggoner AS, Taylor DL (1987). Five-parameter fluorescence imaging:
wound healing of living Swiss 3T3 cells. The Journal of Cell Biology 105: 1613-1622.
Eizirik DL, Cardozo AK, Cnop M (2008). The Role for Endoplasmic Reticulum Stress in Diabetes Mellitus.
Endocrine Reviews 29: 42-61.
Eizirik DL, Cagliero E, Bjorklund A, Welsh N (1992). Interleukin-1 beta induces the expression of an
isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated
macrophages. FEBS Lett 308: 249-252.
Eizirik DL, Sandler S, Welsh N, Cetkovic-Cvrlje M, Nieman A, Geller DA, et al. (1994). Cytokines suppress
human islet function irrespective of their effects on nitric oxide generation. J Clin Invest 93: 1968-
1974.
Eldor R, Yeffet A, Baum K, Doviner V, Amar D, Ben-Neriah Y, et al. (2006). Conditional and specific NF-
kappaB blockade protects pancreatic beta cells from diabetogenic agents. Proc Natl Acad Sci U S A
103: 5072-5077.
Förstermann U, Sessa WC (2012). Nitric oxide synthases: regulation and function. European Heart
Journal 33: 829-837.
Fujimoto M, Shimizu N, Kunii K, Martyn JAJ, Ueki K, Kaneki M (2005). A Role for iNOS in Fasting
Hyperglycemia and Impaired Insulin Signaling in the Liver of Obese Diabetic Mice. Diabetes 54: 1340-
1348.
Giuliano KA, DeBiasio RL, Dunlay RT, Gough A, Volosky JM, Zock J, et al. (1997). High-Content
Screening: A New Approach to Easing Key Bottlenecks in the Drug Discovery Process. Journal of
Biomolecular Screening 2: 249-259.
Green DR, Reed JC (1998). Mitochondria and Apoptosis. Science 281: 1309-1312.
Gurzov EN, Eizirik DL (2011). Bcl-2 proteins in diabetes: mitochondrial pathways of beta-cell death and
dysfunction. Trends Cell Biol 21: 424-431.
This article is protected by copyright. All rights reserved.

Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S, et al. (2018). The IUPHAR/BPS
Guide to PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to
IMMUNOPHARMACOLOGY. Nucleic acids research 46: D1091-D1106.
Heimberg H, Heremans Y, Jobin C, Leemans R, Cardozo AK, Darville M, et al. (2001). Inhibition of
cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-
repressor prevents beta-cell apoptosis. Diabetes 50: 2219-2224.
Hotamisligil GS Endoplasmic Reticulum Stress and the Inflammatory Basis of Metabolic Disease. Cell
140: 900-917.
Hynes SO, McCabe C, O’Brien T (2011). β Cell Protection by Inhibition of iNOS Through Lentiviral
Vector-Based Strategies. In: McCarthy OH, Coulter AJ (eds). Nitric Oxide: Methods and Protocols, edn.
Totowa, NJ: Humana Press. p^pp 153-168.
Klatt P, Schmidt K, Lehner D, Glatter O, Bächinger HP, Mayer B (1995). Structural analysis of porcine
brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an
SDS-resistant dimer. The EMBO journal 14: 3687-3695.
Klein T, Bischoff R (2011). Physiology and pathophysiology of matrix metalloproteases. Amino acids
41: 271-290.
Korn K, Krausz E (2007). Cell-based high-content screening of small-molecule libraries. Current Opinion
in Chemical Biology 11: 503-510.
Kowluru RA, Mohammad G, dos Santos JM, Zhong Q (2011). Abrogation of MMP-9 gene protects
against the development of retinopathy in diabetic mice by preventing mitochondrial damage.
Diabetes 60: 3023-3033.
Larsen CM, Faulenbach M, Vaag A, Volund A, Ehses JA, Seifert B, et al. (2007). Interleukin-1-receptor
antagonist in type 2 diabetes mellitus. N Engl J Med 356: 1517-1526.
Laybutt DR, Preston AM, Åkerfeldt MC, Kench JG, Busch AK, Biankin AV, et al. (2007). Endoplasmic
reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia 50: 752-763.
Li H, Poulos TL (2005). Structure–function studies on nitric oxide synthases. Journal of Inorganic
Biochemistry 99: 293-305.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. Cytochrome c and dATP-
Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell 91:
479-489.
This article is protected by copyright. All rights reserved.

Lin F-S, Lin C-C, Chien C-S, Luo S-F, Yang C-M (2005). Involvement of p42/p44 MAPK, JNK, and NF-κB
in IL-1β-induced ICAM-1 expression in human pulmonary epithelial cells. Journal of Cellular Physiology
202: 464-473.
Liu T, Hannafon B, Gill L, Kelly W, Benbrook D (2007). Flex-Hets differentially induce apoptosis in cancer
over normal cells by directly targeting mitochondria. American Association for Cancer Research 6:
1814-1822.
Lundh M, Scully SS, Mandrup-Poulsen T, Wagner BK (2013). Small-molecule inhibition of inflammatory
beta-cell death. Diabetes Obes Metab 15 Suppl 3: 176-184.
Marrero MB, Banes-Berceli AK, Stern DM, Eaton DC (2006). Role of the JAK/STAT signaling pathway in
diabetic nephropathy. American Journal of Physiology - Renal Physiology 290: F762-F768.
McMillan K, Adler M, Auld DS, Baldwin JJ, Blasko E, Browne LJ, et al. (2000). Allosteric inhibitors of
inducible nitric oxide synthase dimerization discovered via combinatorial chemistry. Proceedings of
the National Academy of Sciences of the United States of America 97: 1506-1511.
Mohammad G, Kowluru RA (2011). Novel role of mitochondrial matrix metalloproteinase-2 in the
development of diabetic retinopathy. In: Investigative ophthalmology & visual science Vol. 52, pp
3832-3841.
Muhammed SJ, Lundquist I, Salehi A (2012). Pancreatic β-cell dysfunction, expression of iNOS and the
effect of phosphodiesterase inhibitors in human pancreatic islets of type 2 diabetes. Diabetes, Obesity
and Metabolism 14: 1010-1019.
Nagpal L, Haque MM, Saha A, Mukherjee N, Ghosh A, Ranu BC, et al. (2013). Mechanism of inducible
nitric-oxide synthase dimerization inhibition by novel pyrimidine imidazoles. The Journal of biological
chemistry 288: 19685-19697.
Negi G, Sharma SS (2015). Inhibition of IκB Kinase (IKK) Protects Against Peripheral Nerve Dysfunction
of Experimental Diabetes. Molecular Neurobiology 51: 591-598.
Ohtsuka M, Konno F, Honda H, Oikawa T, Ishikawa M, Iwase N, et al. (2002). PPA250 [3-(2,4-
Difluorophenyl)-6-{2-[4-(1H-imidazol-1-ylmethyl) Phenoxy]ethoxy}-2-phenylpyridine], a Novel Orally
Effective Inhibitor of the Dimerization of Inducible Nitric-Oxide Synthase, Exhibits an Anti-
Inflammatory Effect in Animal Models of Chronic Arthritis. Journal of Pharmacology and Experimental
Therapeutics 303: 52-57.
Oyadomari S, Takeda K, Takiguchi M, Gotoh T, Matsumoto M, Wada I, et al. (2001). Nitric oxide-
induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway.
Proc Natl Acad Sci U S A 98: 10845-10850.
This article is protected by copyright. All rights reserved.

Pan J, Burgher KL, Szczepanik AM, Ringheim GE (1996). Tyrosine phosphorylation of inducible nitric
oxide synthase: implications for potential post-translational regulation. Biochemical Journal 314: 889-
894.
Papaccio G, Graziano A, Valiante S, D'Aquino R, Travali S, Nicoletti F (2005). Interleukin (IL)-1β toxicity
to islet β cells: Efaroxan exerts a complete protection. Journal of Cellular Physiology 203: 94-102.
Philip D, Janine S, Stefan P (2008). High-Content Analysis in Preclinical Drug Discovery. Combinatorial
Chemistry & High Throughput Screening 11: 216-230.
Pop C, Timmer J, Sperandio S, Salvesen GS The Apoptosome Activates Caspase-9 by Dimerization.
Molecular Cell 22: 269-275.
Qader SS, Ekelund M, Andersson R, Obermuller S, Salehi A (2003). Acute pancreatitis, expression of
inducible nitric oxide synthase and defective insulin secretion. Cell and Tissue Research 313: 271-279.
Reilly SM, Chiang S-H, Decker SJ, Chang L, Uhm M, Larsen MJ, et al. (2013). An inhibitor of the protein
kinases TBK1 and IKK-[epsiv] improves obesity-related metabolic dysfunctions in mice. Nat Med 19:
313-321.
Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS (2001). Dimer formation drives the
activation of the cell death protease caspase 9. Proceedings of the National Academy of Sciences 98:
14250-14255.
Rissanen A, Howard CP, Botha J, Thuren T, Global I (2012). Effect of anti-IL-1beta antibody
(canakinumab) on insulin secretion rates in impaired glucose tolerance or type 2 diabetes: results of a
randomized, placebo-controlled trial. Diabetes Obes Metab 14: 1088-1096.
Sennequier N, Wolan D, Stuehr DJ (1999). Antifungal Imidazoles Block Assembly of Inducible NO
Synthase into an Active Dimer. Journal of Biological Chemistry 274: 930-938.
Shimabukuro M, Ohneda M, Lee Y, Unger RH Role of nitric oxide in obesity-induced beta cell disease.
The Journal of Clinical Investigation 100: 290-295.
Sloan-Lancaster J, Abu-Raddad E, Polzer J, Miller JW, Scherer JC, De Gaetano A, et al. (2013). Double-
blind, randomized study evaluating the glycemic and anti-inflammatory effects of subcutaneous
LY2189102, a neutralizing IL-1beta antibody, in patients with type 2 diabetes. Diabetes Care 36: 2239-
2246.
Sugita H, Fujimoto M, Yasukawa T, Shimizu N, Sugita M, Yasuhara S, et al. (2005). Inducible Nitric-oxide
Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells.
Journal of Biological Chemistry 280: 14203-14211.
This article is protected by copyright. All rights reserved.

Symons KT, Nguyen PM, Massari ME, Anzola JV, Staszewski LM, Wang L, et al. (2011). Pharmacological
Characterization of KLYP961, a Dual Inhibitor of Inducible and Neuronal Nitric-Oxide Synthases.
Journal of Pharmacology and Experimental Therapeutics 336: 468-478.
Torres S, De Sanctis J, de Briceno LM, Hernandez N, Finol H (2004). Inflammation and nitric oxide
production in skeletal muscle of type 2 diabetic patients. Journal of Endocrinology 181: 419-427.
van Asseldonk EJ, van Poppel PC, Ballak DB, Stienstra R, Netea MG, Tack CJ (2015). One week
treatment with the IL-1 receptor antagonist anakinra leads to a sustained improvement in insulin
sensitivity in insulin resistant patients with type 1 diabetes mellitus. Clin Immunol 160: 155-162.
Víteček J, Lojek A, Valacchi G, Kubala L (2012). Arginine-Based Inhibitors of Nitric Oxide Synthase:
Therapeutic Potential and Challenges. Mediators of Inflammation 2012: 22.
Waelchli R, Bollbuck B, Bruns C, Buhl T, Eder J, Feifel R, et al. (2006). Design and preparation of 2-
benzamido-pyrimidines as inhibitors of IKK. Bioorganic & Medicinal Chemistry Letters 16: 108-112.
Wei C-C, Wang Z-Q, Tejero J, Yang Y-P, Hemann C, Hille R, et al. (2008). Catalytic Reduction of a
Tetrahydrobiopterin Radical within Nitric-oxide Synthase. Journal of Biological Chemistry 283: 11734-
11742.
Welsh N, Eizirik DL, Bendtzen K, Sandler S (1991). Interleukin-1 beta-induced nitric oxide production
in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs
cycle enzyme aconitase. Endocrinology 129: 3167-3173.
Xie QW, Kashiwabara Y, Nathan C (1994). Role of transcription factor NF-kappa B/Rel in induction of
nitric oxide synthase. Journal of Biological Chemistry 269: 4705-4708.
Xu C, Ma H, Inesi G, Al-Shawi MK, Toyoshima C (2004). Specific structural requirements for the
inhibitory effect of thapsigargin on the Ca2+ ATPase SERCA. J Biol Chem 279: 17973-17979.
Yang YH, Vilin YY, Roberge M, Kurata HT, Johnson JD (2014). Multiparameter screening reveals a role
for Na+ channels in cytokine-induced beta-cell death. Mol Endocrinol 28: 406-417.
This article is protected by copyright. All rights reserved.

Figure 1 High throughput screening for small molecule inhibitors mitochondrial
apoptosis. A) A schematic description of the intrinsic (mitochondrial) pathway of apoptosis.
Cytokines induce intracellular stress and ultimately apoptosis. A high-content image-based
phenotypic assay was designed to identify small molecules that protect β-cells from cytokine-
This article is protected by copyright. All rights reserved.

induced activation of mitochondrial apoptosis by monitoring cleaved caspase-9 levels. B)
Representative screening setup from a subset of Life Chemicals Library (30,000 compounds)
screen which identified the hit ATV399. Rat insulin-producing INS1E cells were pretreated
with compounds for 24 h. Cytokines (2.5 ng/mL IL-1β and 100 ng/mL IFNγ) were then
supplemented in the presence of high glucose (33 mM) to induce intracellular stress and
activate cleaved caspase-9. Compounds reducing > 50% of cleaved caspase-9 in the presence
of cytokines were considered as hits. Z’ > 0.7 was calculated based on DMSO versus cytokine
treated wells, indicating the assay was robust and suitable for high throughput screening.
ATV399 was identified as one of the most potent hit compounds. C) Schematic workflow of
the high-throughput screen. D) Chemical structures of the selected hit compounds. Everolimus,
IKK-16, baricitinib, and flumazenil were identified as hit compounds with known mechanisms
of action.
This article is protected by copyright. All rights reserved.

Figure 2 ATV399 protected rat insulin-producing INS1E cells in vitro from cytokine
stress. A) Phenotypic activities of ATV399. Left, dose response reduction of cleaved caspase-
9 levels as determined from the screening assay. Middle, dose response reduction of rat insulin-
producing INS1E cell viability as measured by CTG assay (n = 6). Right, dose response rescue
of secreted insulin in culture medium as determined by HTRF assay (n=6). Rat insulin-
producing INS1E cells were pretreated with compounds for 24 h, followed by co-treatment
with pro-inflammatory cytokines (10 ng/mL IL-1β, 100 ng/mL IFNγ) for an additional 24 h
and analyzed accordingly. B) ATV399 attenuated ER stress-related genes induced by pro-
inflammatory cytokines (n=3). C) ATV399 reduced downstream apoptotic proteins, such as
CHOP and cleaved caspase-3, which were induced cytokines. In both B) & C), cells were
pretreated with compounds for 8 h, followed by co-treatment with cytokines (10 ng/mL IL-1β,
100 ng/mL IFNγ) for an additional 8 h and gene expression and protein levels were analyzed
by qPCR and western blot, respectively. Data are represented as Mean ± SD. ** P < 0.01.
This article is protected by copyright. All rights reserved.

Figure 3. Pre- and post-treatment of CAT639 protect INS-1e βcells. A, C) Pre-treatment of CAT639
for 6 hrs decreased caspase 9 activity induced by cytokines (upper panels) and rescued cell viability
impaired by cytokines (bottom panels). INS-1e βcells were pretreated with compounds for 6 h,
followed by co-treatment with cytokines (10 ng/mL IL-1β, 100 ng/mL IFNγ) for an additional
24 h and caspase-9 and cell viability were measured by Caspase Glo-9 or Cell Titer Glo,
according to the manufacturer’s manual (n = 6). B, D) Post-treatment of CAT639 6 hrs after
cytokine treatment also partially decreased caspase 9 activity induced by cytokines (upper panels) and
rescued cell viability impaired by cytokines (bottom panels). INS-1e βcells were plated overnight and
treated with cytokines (10 ng/mL IL-1β, 100 ng/mL IFNγ) for 6 hrs followed by compound
treatment for 24 h, and caspase-9 and cell viability were measured by Caspase Glo-9 or Cell
Titer Glo, according to the manufacturer’s manual (n = 6). Data are represented as Mean ± SD.
** P < 0.01
This article is protected by copyright. All rights reserved.

Figure 4: CAT639 protected dispersed rat primary islets cells from pro-inflammatory
cytokine stress. A) CAT639 rescued cell apoptosis and B) insulin secretion in the presence of
cytokine stress (n = 6). Dissociated rat primary islets (5000 cells/well) were seeded in 96-well
plates and treated with a cocktail of pro-inflammatory cytokines. The concentrations of
cytokines for cell viability assay was 5 ng/ml IL-1β and 100 ng/ml IFN-γ, and the
concentrations of cytokines for insulin secretion was 0.3 ng/ml IL-1 and 25 ng/ml IFN-γ. For
GSIS assay, cells were pre-incubated in 2.8 mM glucose for 1 hr then incubated in 2.8
mM/20mM glucose for 1 hr. Secreted insulin in the medium was measured by ELISA. Data
are represented as Mean ± SD. ** P < 0.01.
This article is protected by copyright. All rights reserved.

Figure 5: Activity of CAT639 in rat insulin-producing INS1E cells treated with pro-
inflammatory cytokines. A) CAT639 decreased caspase-9 activity induced by IL-1 alone,
increased cell viability and insulin secretion impaired by pro-inflammatory cytokine (n=6).
B&C) qRT-PCR analysis of gene expression of rat insulin-producing INS1E cells. Cells were
pre-treated with CAT639 for 8 h prior to the addition of cytokines for 8 h (n = 3). D) CAT639
decreased the percentage of cells exhibiting loss of mitochondrial membrane potential. Cells
were pre-treated with CAT639 for 4 h prior to the addition of cytokines (10 ng/ml IL-1) for 24
h (n=3). JC1 was used as an indicator of mitochondrial membrane potential and analyzed by
FACS. Data are represented as Mean ± SD. ** P < 0.01.
This article is protected by copyright. All rights reserved.

Figure 6: CAT639 inhibited dimerization of iNOS. A) CAT639 slightly inhibited iNOS
mRNA expression (n=3). B) CAT639 inhibited nitric oxide production induced by IL-1 alone
or IL-1β +IFN-γ. NO was measured using a nitrate/nitrite colorimetric assay kit (n=3). C)
CAT639 had no effect on apoptosis induced by SNAP (n=3). D) CAT639 destabilized iNOS
dimer in rat insulin-producing INS1E cells. Dimeric proteins were determined by performing
low temperature SDS-PAGE gel. Samples lysed in sample buffer with β -ME and boiled for 5
min were used as a positive control of total iNOS. Data are represented as Mean ± SD.
This article is protected by copyright. All rights reserved.

Table 1. SAR of ATV399 led to compounds with increased potency
EC50
(µM)
EC50
(µM)
Entry Compound
Structure
Entry Compound
Structure
ATV399
CAT628
CAT640
2.90
CAT639
CAT636
CBD498
0.48
3.10
1.2
1
2
3
8
9
>20
>20
10
CAT635
CAT648
CAT646
CAT634
8.75
9.54
>20
>20
CBD504
CBD505
CBD501
CBE173
0.098
0.12
0.91
8.8
4
5
6
7
11
12
13
14
Rat insulin-producing INS1E cells were pretreated with compounds in dose response (n =6)
for 24 h, followed by co-treatment with pro-inflammatory cytokines (10 ng/mL IL-1β, 100
ng/mL IFNγ) for an additional 24 h and cleaved caspase-9 levels were determined using
Caspase-9 Glo and the evolved luminescence signal was read used to calculate EC50 values.
This article is protected by copyright. All rights reserved.