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Eur J Pharmacol. Author manuscript; available in PMC 2018 September 15.
Published in final edited form as:
Eur J Pharmacol. 2017 September 15; 811: 87–92. doi:10.1016/j.ejphar.2017.05.050.
Phenylpyrrolidine structural mimics of pirfenidone lacking
antifibrotic activity: a new tool for mechanism of action studies
Andrew J. Haak1, Megan A. Girtman3, Mohamed F. Ali2, Eva M. Carmona2,3, Andrew H.
Limper2,3, and Daniel J. Tschumperlin1
1Department
of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
55905
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2Thoracic
Diseases Research Unit, Department of Medicine, Mayo Clinic and Foundation,
Rochester, MN 55905
3Division
of Pulmonary Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic
and Foundation, Rochester, MN 55905
Abstract
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Pirfenidone recently received FDA approval as one of the first two drugs designed to treat
idiopathic pulmonary fibrosis. While the clinical data continues to support the efficacy of
pirfenidone, the specific molecular mechanism of action of this drug has not been fully defined.
From a chemical perspective the comparatively simple and lipophilic structure of pirfenidone
combined with its administration at high doses, both experimentally and clinically, complicates
some of the basic tenants of drug action and drug design. Our objective here was to identify a
commercially available structural mimic of pirfenidone which retains key aspects of its physical
chemical properties but does not display any of its antifibrotic effects. We tested these molecules
using lung fibroblasts derived from patients with idiopathic pulmonary fibrosis and found
phenylpyrrolidine based analogs of pirfenidone that were non-toxic and lacked antifibrotic activity
even when applied at millimolar concentrations. Based on our findings, these molecules represent
pharmacological tools for future studies delineating pirfenidone’s mechanism of action.
Keywords
Pirfenidone; Fibrosis; Antifibrotic; IPF; Idiopathic Pulmonary Fibrosis; Interstitial Lung Disease;
p38
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Corresponding Author: D.J. Tschumperlin, Mayo Clinic College of Medicine, 200 1st St SW, Rochester MN 55906, 507-255-8475
(Tschumperlin.daniel@mayo.edu).
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CONFLICT OF INTEREST:
There is no conflict of interest
Haak et al.
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1. INTRODUCTION
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The molecular mechanism of action of pirfenidone is still not well defined. In pulmonary
fibrosis, activated resident fibroblasts are thought to be the main cellular contributor to
matrix deposition and scarring (Barkauskas and Noble, 2014). In an effort to identify the
mechanism of action of pirfenidone, its in vitro antifibrotic efficacy was established in
isolated fibroblasts (Conte et al., 2014; Hewitson et al., 2001; Lehtonen et al., 2016a). A
major limitation of pirfenidone is the compound’s potency. In order to produce antifibrotic
activity in vitro, concentrations between 1–5mM are required (Conte et al., 2014; Lehtonen
et al., 2016b; Nakayama et al., 2008). Pirfenidone’s lack of potency, lipophilic nature, and
minimal number of heteroatoms (Fig. 1) has led to debate about the potential for pirfenidone
to bind to a selective target or pocket, and has stimulated an alternative hypothesis that
pirfenidone simply works as a free radical scavenging antioxidant (Mitani et al., 2008;
Salazar-Montes et al., 2008). Some very recent work has suggested pirfenidone is an
inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway (Li et al., 2016;
Ma et al., 2014; Neri et al., 2016; Yin et al., 2016). A major barrier to further delineating
pirfenidone’s mechanism of action is the absence of structurally similar compounds which
lack antifibrotic efficacy. Inactive structural analogs are extremely valuable tools when
attempting to further define the mechanism of action or molecular target of small molecules
(Dancy et al., 2012; Fu et al., 2008; Lim et al., 2004).
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Idiopathic pulmonary fibrosis (IPF) is a chronic disease characteristically defined by
excessive deposition of extracellular matrix and destruction of the lung’s normal
architecture. Over time this progressive scarring and alteration in tissue leads to declining
lung function and often death. Despite the scope and severity of the disease burden
associated with IPF, it has only recently become the target of clinically approved
therapeutics, with the approval of nintedanib and pirfenidone. Interest in the latter began
with the finding that phenylamine derivative substituted pyridones produce analgesic
properties (Scudi et al., 1960), leading to the synthesis of 5-Methyl-1-phenyl-2-(1H)pyridone (initially referred to as AMR-69, later referred to as pirfenidone) and description of
its analgesic, antipyretic, and anti-inflammatory effects (Shreekrishna Manmohan Gadekar,
1972). Decade’s later the antifibrotic properties of pirfenidone began to emerge (Iyer et al.,
1995; Kehrer and Margolin, 1997; Schelegle et al., 1997).
We set out to identify a chemical mimic of pirfenidone which retains its low molecular
weight and lipophilic nature but does not display any antifibrotic activity against pulmonary
fibroblasts. Our criteria for an inactive mimic thus included commercial availability and nontoxicity at the very high doses (millimolar) at which pirfenidone shows antifibrotic activity.
For our initial testing we identified a diverse pilot set of compounds with physical chemical
properties consistent with pirfenidone (To ease communication the compounds are
abbreviated MC-2 through MC-7) (Fig. 1). We tested these compounds in primary human
pulmonary fibroblasts obtained from patients with IPF. As a proof of concept demonstrating
the utility of these compounds, we used the inactive analogs identified here to address the
role of p38 MAPK in mediating antifibrotic effects of pirfenidone in cultured lung
fibroblasts.
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2. MATERIALS AND METHODS
2.1. Compounds
Pirfenidone (PFD) (PubChem CID:40632) was purchased from Sigma Aldrich, St. Louis,
MO. 4-amino-1-phenyl-1,2-dihydropyridin-2-one (MC-2) (PubChem CID:664174); 4,6dimethyl-1-phenyl-1,2-dihydropyridin-2-one (MC-3) (PubChem CID:N/A); 2-phenylphenol
(MC-4) (PubChem CID: 24938931); 1-phenylpyrrolidin-2-one (MC-5) (PubChem CID:
78375); 1-(3-methylphenyl)pyrrolidin-2-one (MC-6) (PubChem CID: 314213); and 1-(3,5dimethylphenyl)pyrrolidin-2-one (MC-7) (PubChem CID: 847897) were purchased from
MolPort, Riga, Latvia. All compounds were dissolved in DMSO for a stock concentration of
600mM. TPSA and cLogP were calculated using the OSIRIS Property Explorer available
through the Organic Chemistry Portal: http://www.organic-chemistry.org/prog/peo/.
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2.2. Cell Culture
Primary human lung fibroblasts (generously provided by Peter Bitterman and Craig Henke
at the University of Minnesota) were isolated by explant culture from the lungs of subjects
diagnosed with IPF who underwent lung transplantation, under a protocol approved by the
University of Minnesota Institutional Review Board. Primary fibroblasts were maintained in
EMEM (ATCC, Manassas, VA) containing 10% FBS, unless otherwise noted. All primary
cell culture experiments were performed with cells at passage six or less.
2.3. Viability Assay
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Lung fibroblasts were plated into 96-well plates in EMEM containing 10% FBS and allowed
to attach for 6 h. Media was then exchanged with EMEM containing 0.1% FBS + the
indicated compound concentration. All wells were treated with a final concentration of 0.5%
DMSO (We have previously found these cells tolerate up to 1% DMSO without any effects
on viability). After 24 h the cellular viability/toxicity was analyzed using the WST-1 reagent
(Sigma Aldrich) following the manufacturer’s protocol. Results are expressed as the
absorbance at 450nm relative to DMSO control.
2.4. Immuno-ECM Assay
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Adapting from previously published methods (Jones et al., 2010; Vogel et al., 2014), lung
fibroblasts were plated to confluence in clear-bottom 96-well plates in EMEM containing
10% FBS and allowed to attach for 6 h. Media was then exchanged with EMEM containing
0.1% FBS plus the indicated compound concentration. All wells were treated with a final
concentration of 0.5% DMSO. After 72 h cells were fixed with 4% formalin, then treated
with 0.25% Triton X-100 and blocked with Li-Cor Odyssey Blocking Buffer (Li-Cor
Biosciences, Lincoln, NE) before overnight incubation in a polyclonal rabbit antibody for
collagen I (Novus NB600-408) diluted 1:150 in blocking buffer. Wells where then incubated
with IR-dye-conjugated secondary antibody (Li-Cor #926-32211) diluted 1:500. Plates were
imaged via a Li-Cor OdysseyXL system with quantification performed via densitometry.
Results are represented as collagen I signal intensity relative to DMSO control.
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2.5. qPCR Analysis
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Cells were plated and treated as indicated prior to RNA isolation using RNeasy Plus Mini
Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Isolated RNA
(250ng) was then used to synthesize cDNA using SuperScript VILO (Invitrogen, Carlsbad,
CA). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche
Applied Science, Penzberg, Germany) and analyzed using a LightCycler 96 (Roche Applied
Science). Results are expressed as a fold change by ΔΔCt relative to glyceraldehyde-3phosphate dehydrogenase (GAPDH). Primers: GAPDH; F:
GGAAGGGCTCATGACCACAG, R: ACAGTCTTCTGGGTGGCAGTG. ACTA2; F:
GTGAAGAAGAGGACAGCACTG, R: CCCATTCCCACCATC ACC. COL1A1; F:
AAGGGACACAGAGGTTTCAGTGG, R: CAGCACCAGTAGCACCATCATTTC.
COL1A2; F: CTTGCAGTAACCTTATGCCTAGCA,
R:CCCATCTAACCTCTCTACCCAGTCT
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2.6. Western Blot Analysis
Cells were plated in EMEM containing 10% FBS and allowed to attach for 6 h. Cells were
then starved for 24 h in EMEM containing 0.1% FBS prior to stimulation with 2ng/mL
TGFβ with or without the indicated concentration of compound for 30 min. Total protein
was isolated using RIPA buffer (Thermo Scientific, Rockford, IL) and quantified with a
BCA protein assay kit (Thermo Scientific, Rockford, IL) according to manufacturer’s
instructions. Membranes were probed for phospho-p38 (Cell Signaling, Danvers, MA
#9211) and total p38 (Cell Signaling, Danvers, MA #9212) followed by IR conjugated
secondary antibodies (Li-Cor Biosciences, Lincoln, NE). Bands were visualized and
quantified using a Li-Cor OdysseyXL.
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2.7. Statistical Analysis
Groups were compared by one-way ANOVA with Tukey’s multiple comparison’s test. All
statistical tests were carried out using GraphPad Prism 6 with statistical significance defined
as p < 0.05. Results are expressed throughout as the mean ± standard error of the mean
(S.E.M.).
3. RESULTS
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We began this campaign through molport.com searching for compounds which have a
similarity of 0.65 or greater (Tanimoto metrics) to PFD. (Fig. 1). Compounds MC-2 and
MC-3 retain the phenylpyridine core structure of PFD with moderate substitutions, and the
phenylphenol, MC-4 and the phenylpyrrolidine MC-5 offer alternative backbones but retain
the basic size and atom representation of PFD. Previous work has shown that pirfenidone
can be given at millimolar concentrations in vitro without causing any cellular toxicity
(Conte et al., 2014; Nakayama et al., 2008). To screen out any toxic compounds we treated
IPF lung fibroblasts with 0.3 and 3.0mM concentrations of each compound for 24 h and then
measured cellular viability with WST-1 reagent, a tetrazolium salt that is cleaved into a
formazan dye in metabolically active, viable cells (Fig. 2A). Consistent with prior
observations, PFD did not reduce cellular viability. However 3.0mM treatment with MC-2
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and MC-4 was toxic to the cells. At 3.0mM MC-4 became insoluable and fell out of
solution, likely due to the lipopholic nature of this compound (cLogP = 2.97).
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In previous in vitro work (Conte et al., 2014) with pulmonary fibroblasts PFD has been
shown to reduce gene expression of COL1A1 (which codes for collagen 1α1 protein, a
major fibrillar collagen component in the extracellular matrix). To translate this readout into
a higher-througput assay we measured collagen I protein levels using an in-cell Western blot
technique. In this experiment we plated IPF lung fibroblasts at confluence in a 96-well plate
and allowed them to synthesize and deposit matrix for 72 h in the presense of PFD, MC-3,
MC-5 (non-toxic from Fig. 2A) or DMSO control. We then immunostained the whole well
for collagen I protein and visualized and quantitatively analyzed using infrared-emitting
secondary antibody. PFD reduced collagen protein levels at the higher concentration, and
interestingly MC-3 appeared to perform as an active structural mimic of PFD, as it was both
non-toxic to the fibroblasts and reproduced the antifibrotic phenotype of reducing collagen
deposition. The phenylpyrrolidine-based backbone MC-5 appeared to be a good framework
to move forward as an inactive structural mimic of pirfenidone based on the combination of
low toxicity and absence of antifibrotic effect on collagen 1 expression.
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Based on the results from Fig. 2 we identified a second round of commercially availible
pirfenidone mimics based off of the phenylpyrrolidine backbone. Aside from the number of
hydrogens; MC-5 and PFD only differed by two carbon atoms so we searched for
phenylpyrridone anologs with aditional carbon substitutions. We also reasoned that
inactivity would be associated with compounds which were substituted on the phenyl ring
rather than the pyridine as in PFD. Methyl, MC-6 and dimethyl, MC-7 derivatives of PFD
were thus obtained and tested in the same assays as in Fig. 2. Interstingly, the dimethyl
substituted analog, MC-7 was toxic to the cells (and also reduced collagen deposition),
whereas MC-6, with a single methyl substitution onto MC-5, was both non-toxic and
inactive in the collagen deposition assay (Fig. 3).
For our final analysis testing MC-5 and MC-6 against pirfenidone we measured expression
of three fibrosis-associated genes: ACTA2 (coding for α-smooth muscle actin, a hallmark of
activated and contractile myofibroblasts), and COL1A1 and COL1A2 (both genes required
for mature collagen I protein). IPF lung fibroblasts were treated for 48 h with compounds or
DMSO control and gene expression was determined by qPCR relative to the housekeeping
gene GAPDH. As predicted, pirfenidone reduced expression of all three profibrotic genes at
the higher concetration; in contrast, MC-5 and MC-6 exhibited no effect in this highly
sensitive assay (Fig. 4A).
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In response to recent evidence suggesting p38 MAPK pathway inhibition is the mechanism
of action of pirfenidone (Li et al., 2016; Ma et al., 2014; Neri et al., 2016), we stimulated
IPF lung fibroblasts with TGFβ for 30 min in the presence of pirfenidone and our inactive
mimics (Fig. 4B). TGFβ has been shown to induce p38 phosphorylation through multiple
pathways (Yu et al., 2002; Zhang, 2009), and consistent with these prior publications we
observed a 2-fold induction in phospho-p38 under these conditions. As previosuly reported,
pirfenidone was able to significantly inhibit this TGFβ-induced p38 activation. However,
while one of the inactive structural mimics, MC-5 showed no effect on inhibitng p38
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phosphorylation, the other MC-6 did significantly inhibit p38 at a concentration which
showed no antifibrotic activity (Fig. 3 and Fig. 4), consistent with pirfenidone acting through
additional or alternative mechanisms beyond p38 in mediating its antifibrotic effects in
fibroblasts.
4. DISCUSSION
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Approved in 2014 for the treatment of idiopathic pulmonary fibrosis, pirfenidone became
one of the first in a class of new “antifibrotic drugs” which will likely expand in the future.
Multiple groups are currently attempting to understand the mechanism of action that leads to
the antifibrotic efficacy of pirfenidone. One of the basic challenges in interpreting
pirfenidone’s clinical efficacy is the dosage of the drug required to produce any biological
activity. We set out to identify one or several structural mimics of pirfenidone that can be
used as a negative control which accounts for the amount of lipophilic compound delivered
to the cells. Taken together, our data demonstrate that phenylpyrrolidine backbones produce
adequate structural mimics of pirfenidone. Our results are also consistent with the concept
that pirfenidone binds its antifibrotic target(s) in a specific manner that is highly dependent
on the 3-dimensional planar shape of pirfenidone or potentially the 5-methyl substitution on
the pyridine ring, not present in MC-5 and MC-6. Interestingly, F-351, a hydroxyl
substituted analog of pirfenidone, is currently in clinical trials for liver and kidney fibrosis
(Nanthakumar et al., 2015). This modification results in more “drug like” physical chemical
properties than pirfenidone and retains the antifibrotic nature of the drug.
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MC-5 and MC-6 are available from molport.com (MolPort-001-012-470 and
MolPort-001-028-037) through multiple venders for less than $10/mg, making them very
affordable, and based on their structural similarity to pirfenidone (Fig. 1) and inactivity in
cellular assays of fibrosis (Fig. 2–4), should be considered as potentially informative inactive
structural mimics for pirfenidone. The use of phenotype-based assays in drug discovery is
continuing to expand, leading to the identification of promising drug candidates with no
known mechanism of action or molecular target (Etzion and Muslin, 2009; Lee and Berg,
2013; Moffat et al., 2014). One of the common approaches to identifying or validating the
molecular target of a small molecule is to design linkable chemical analogs to perform pulldown assays followed by proteomics analysis (Leslie and Hergenrother, 2008). In these
types of assays a pull-down experiment can be performed using both the active and inactive
compounds. Inevitably, highly abundant or adherent proteins will be observed in both cases,
and only by comparing which protein(s) are exclusively associated with the active molecule
can specific targets be identified (Bell et al., 2013; Colca et al., 2003). Even with very potent
drugs nonspecific binding is always a concern when interpreting the effects of adding a
compound to a biological system. In the case of pirfenidone where millimolar concentrations
are required, these concerns are amplified. In recent years the growth of biologics based
therapeutics has created a new standard for preclinical drug discovery where the use of
inactive control molecules is customary. Antibody based therapies are compared to an
isotype control, siRNA treatments are compared with scrambled nontargeting sequences and
peptides are compared to an inactive derivative sequence. Although less exciting in the short
term, inactive analogs play an important role in small molecule based drug discovery.
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It is important to note that although we have shown MC-5 and MC-6 are inactive in fibrosis
assays relative to pirfenidone, no small molecule is absolutely inactive. MC-5 and MC-6 are
both in the PubChem database but only MC-5 has been tested in biological assays;
reassuringly this compound showed no activity in these screens. The interesting results we
observed in the p38 MAPK phosphorylation experiments serves as proof of concept of their
utility, and suggest that further work is needed to define the role of p38 MAPK as a potential
mechanism of action for pirfenidone antifibrotic effects in fibroblasts. The identification and
characterization of these compounds should help facilitate further delineation of the
molecular mechanism of action of pirfenidone, and ultimately lead to more potent and more
efficacious antifibrotic drugs in the future.
Acknowledgments
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This work was supported by NIH HL092961 and HL113796 (DJT), American Lung Association Senior Research
Training Fellowship (AJH), and funds from the Caerus Foundation.
References
Author Manuscript
Author Manuscript
Barkauskas CE, Noble PW. Cellular Mechanisms of Tissue Fibrosis. 7. New insights into the cellular
mechanisms of pulmonary fibrosis. Am J Physiol-Cell Ph. 2014; 306:C987–C996.
Bell JL, Haak AJ, Wade SM, Sun YH, Neubig RR, Larsen SD. Design and synthesis of tag-free
photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the
Rho/MKL1/SRF signaling pathway. Beilstein J Org Chem. 2013; 9:966–973. [PubMed: 23766813]
Colca JR, McDonald WG, Waldon DJ, Thomasco LM, Gadwood RC, Lund ET, Cavey GS, Mathews
WR, Adams LD, Cecil ET, Pearson JD, Bock JH, Mott JE, Shinabarger DL, Xiong LQ, Mankin AS.
Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics. J Biol Chem.
2003; 278:21972–21979. [PubMed: 12690106]
Conte E, Gili E, Fagone E, Fruciano M, Iemmolo M, Vancheri C. Effect of pirfenidone on
proliferation, TGF-beta-induced myofibroblast differentiation and fibrogenic activity of primary
human lung fibroblasts. Eur J Pharm Sci. 2014; 58:13–19. [PubMed: 24613900]
Dancy BM, Crump NT, Peterson DJ, Mukherjee C, Bowers EM, Ahn YH, Yoshida M, Zhang J,
Mahadevan LC, Meyers DJ, Boeke JD, Cole PA. Live-Cell Studies of p300/CBP Histone
Acetyltransferase Activity and Inhibition. Chembiochem. 2012; 13:2113–2121. [PubMed:
22961914]
Etzion Y, Muslin AJ. The Application of Phenotypic High-Throughput Screening Techniques to
Cardiovascular Research. Trends Cardiovas Med. 2009; 19:207–212.
Fu Y, Han J, Ishola T, Scerbo M, Adwanikar H, Ramsey C, Neugebauer V. PKA and ERK, but not
PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Mol Pain. 2008; 4
Hewitson TD, Kelynack KJ, Tait MG, Martic M, Jones CL, Margolin SB, Becker GJ. Pirfenidone
reduces in vitro rat renal fibroblast activation and mitogenesis. J Nephrol. 2001; 14:453–460.
[PubMed: 11783601]
Iyer SN, Wild JS, Schiedt MJ, Hyde DM, Margolin SB, Giri SN. Dietary intake of pirfenidone
ameliorates bleomycin-induced lung fibrosis in hamsters. J Lab Clin Med. 1995; 125:779–785.
[PubMed: 7539478]
Jones B, Bucks C, Wilkinson P, Pratta M, Farrell F, Sivakumar P. Development of cellbased
immunoassays to measure type I collagen in cultured fibroblasts. Int J Biochem Cell Biol. 2010;
42:1808–1815. [PubMed: 20656053]
Kehrer JP, Margolin SB. Pirfenidone diminishes cyclophosphamide-induced lung fibrosis in mice.
Toxicol Lett. 1997; 90:125–132. [PubMed: 9067480]
Lee JA, Berg EL. Neoclassic Drug Discovery: The Case for Lead Generation Using Phenotypic and
Functional Approaches. J Biomol Screen. 2013; 18:1143–1155. [PubMed: 24080259]
Eur J Pharmacol. Author manuscript; available in PMC 2018 September 15.
Haak et al.
Page 8
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Lehtonen ST, Veijola A, Karvonen H, Lappi-Blanco E, Sormunen R, Korpela S, Zagai U, Skold MC,
Kaarteenaho R. Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts
in idiopathic pulmonary fibrosis. Respir Res. 2016a; 17
Lehtonen ST, Veijola A, Karvonen H, Lappi-Blanco E, Sormunen R, Korpela S, Zagai U, Skold MC,
Kaarteenaho R. Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts
in idiopathic pulmonary fibrosis. Respir Res. 2016b; 17:14. [PubMed: 26846335]
Leslie BJ, Hergenrother PJ. Identification of the cellular targets of bioactive small organic molecules
using affinity reagents. Chem Soc Rev. 2008; 37:1347–1360. [PubMed: 18568161]
Li Z, Liu X, Wang B, Nie Y, Wen J, Wang Q, Gu C. Pirfenidone suppresses MAPK signaling pathway
to reverse epithelial-mesenchymal transition and renal fibrosis. Nephrology (Carlton). 2016
Lim S, Kang KW, Park SY, Kim SI, Choi YS, Kim ND, Lee KU, Lee HK, Pak YK. Inhibition of
lipopolysaccharide-induced inducible nitric oxide synthase expression by a novel compound,
mercaptopyrazine, through suppression of nuclear factor-kappaB binding to DNA. Biochem
Pharmacol. 2004; 68:719–728. [PubMed: 15276079]
Ma Z, Pan YL, Huang WH, Yang YW, Wang ZY, Li Q, Zhao Y, Zhang XY, Shen ZR. Synthesis and
biological evaluation of the pirfenidone derivatives as antifibrotic agents. Bioorg Med Chem Lett.
2014; 24:220–223. [PubMed: 24332090]
Mitani Y, Sato K, Muramoto Y, Karakawa T, Kitamado M, Iwanaga T, Nabeshima T, Maruyama K,
Nakagawa K, Ishida K, Sasamoto K. Superoxide scavenging activity of pirfenidone-iron complex.
Biochem Biophys Res Commun. 2008; 372:19–23. [PubMed: 18468515]
Moffat JG, Rudolph J, Bailey D. Phenotypic screening in cancer drug discovery - past, present and
future. Nature Reviews Drug Discovery. 2014; 13:588–602. [PubMed: 25033736]
Nakayama S, Mukae H, Sakamoto N, Kakugawa T, Yoshioka S, Soda H, Oku H, Urata Y, Kondo T,
Kubota H, Nagata K, Kohno S. Pirfenidone inhibits the expression of HSP47 in TGF-beta1stimulated human lung fibroblasts. Life Sci. 2008; 82:210–217. [PubMed: 18093617]
Nanthakumar CB, Hatley RJD, Lemma S, Gauldie J, Marshall RP, Macdonald SJF. Dissecting fibrosis:
therapeutic insights from the small-molecule toolbox. Nature Reviews Drug Discovery. 2015;
14:693–720. [PubMed: 26338155]
Neri T, Lombardi S, Faita F, Petrini S, Balia C, Scalise V, Pedrinelli R, Paggiaro P, Celi A. Pirfenidone
inhibits p38-mediated generation of procoagulant microparticles by human alveolar epithelial
cells. Pulm Pharmacol Ther. 2016; 39:1–6. [PubMed: 27237042]
Salazar-Montes A, Ruiz-Corro L, Lopez-Reyes A, Castrejon-Gomez E, Armendariz-Borunda J. Potent
antioxidant role of pirfenidone in experimental cirrhosis. Eur J Pharmacol. 2008; 595:69–77.
[PubMed: 18652820]
Schelegle ES, Mansoor JK, Giri S. Pirfenidone attenuates bleomycin-induced changes in pulmonary
functions in hamsters. P Soc Exp Biol Med. 1997; 216:392–397.
Scudi, JV., Reisner, DB., Childress, SJ., Walter, LA. Substituted 1-m-aminophenyl-2-pyridones. Office,
USP., editor. WALLACE & TIERNAN INC; United States: 1960.
Shreekrishna Manmohan Gadekar. N-SUBSTITUTED PYRIDONE AND GENERAL METHOD FOR
PREPARING PYRIDONES. Office, USP., editor. United States: 1972.
Vogel ER, VanOosten SK, Holman MA, Hohbein DD, Thompson MA, Vassallo R, Pandya HC,
Prakash YS, Pabelick CM. Cigarette smoke enhances proliferation and extracellular matrix
deposition by human fetal airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2014;
307:L978–986. [PubMed: 25344066]
Yin N, Qi X, Tsai S, Lu Y, Basir Z, Oshima K, Thomas JP, Myers CR, Stoner G, Chen G. p38 gamma
MAPK is required for inflammation-associated colon tumorigenesis. Oncogene. 2016; 35:1039–
1048. [PubMed: 25961922]
Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smadindependent TGF-beta responses. Embo J. 2002; 21:3749–3759. [PubMed: 12110587]
Zhang YE. Non-Smad pathways in TGF-beta signaling. Cell Res. 2009; 19:128–139. [PubMed:
19114990]
Eur J Pharmacol. Author manuscript; available in PMC 2018 September 15.
Haak et al.
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Fig. 1.
Pirfenidone (PFD) and structural mimics: physical chemical properties. Molecular weight
(Mol. Weight), calculated logarithm of the partition coefficient (cLogP), topological polar
surface area (tPSA), and the non-carbon heteroatoms are compared. Based on the data
presented here the compounds are color-coded to summarize whether they were antifibrotic
(black), acutely toxic (red) or inactive structural mimics of pirfenidone (green).
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Fig. 2.
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Effects of high concentration pirfenidone and initial test compounds on cellular viability and
collagen I protein deposition. A. Toxicity. Pulmonary fibroblasts derived from patients with
IPF were treated for 24 h at 0.3 and 3.0mM to initially rule out any acutely toxic
compounds. Data represent the mean (±S.E.M.) from three patient samples each run in
duplicate (**** P < 0.0001 vs. DMSO control). B. Collagen I Deposition. One of the
hallmarks of pulmonary fibrosis is activated fibroblasts which synthesize excessive
extracellular matrix proteins; principally collagen I. Pulmonary fibroblasts derived from
patients with IPF were treated for 72 h with PFD, MC-3, and MC-5 at 0.3 and 3.0mM to
measure the effect on collagen deposition. Shown on the left is a representative image from
one patient sample run in triplicate for each compound at 3.0mM. Data represent the mean
(±S.E.M.) from three patient samples each run in triplicate (*** P < 0.001, **** P < 0.0001
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vs. DMSO control). Pirfenidone was not acutely toxic to the cells but did reduce matrix
deposition; in contrast MC-5 did not affect the cells in either assay.
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Fig. 3.
Methyl substituted phenylpyrrolidines effect on cellular viability and collagen I protein
deposition. A. Toxicity. Pulmonary fibroblasts derived from patients with IPF were treated
for 24 h with MC-6 and MC-7 at 0.3 and 3.0mM to measure acute toxicity. Data represent
the mean (±S.E.M.) from three patient samples each run in duplicate (**** P < 0.0001 vs.
DMSO control). B. Collagen I Deposition. Pulmonary fibroblasts derived from patients with
IPF were treated for 72 h with MC-6 and MC-7 at 0.3 and 3.0mM to measure the effect on
collagen deposition. Shown on the left is a representative image from one patient sample run
in triplicate for each compound at 3.0mM. Data represent the mean (±S.E.M.) from three
patient samples each run in triplicate (**** P < 0.0001 vs. DMSO control). Similar to MC-5
in Fig. 2, MC-6 is nontoxic and has no effect on collagen deposition.
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Fig. 4.
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Pirfenidone and phenylpyrrolidine mimics effects on profibrotic gene expression and
inhibition of p38 phosphorylation. A. IPF lung fibroblasts were treated for 48 h with PFD,
MC-5, and MC-6 at 0.3 and 3.0 mM, followed by RNA isolation and qPCR analysis. Data
represent the mean (±S.E.M.) from three patient samples (* P < 0.05, ** P < 0.01 vs. DMSO
control). The approved antifibrotic drug pirfenidone reduced expression of all three genes
but the inactive structural mimics had no effect. B. IPF lung fibroblasts were starved for 24 h
in media containing 0.1% FBS and then stimulated with 2ng/mL TGFβ +/− PFD, MC-5,
and MC-6 for 30 min. A representative blot from a single patient sample is shown on the
left, while the plot on the right represents the mean (±S.E.M.) from three patient samples
each run in triplicate (#### P < 0.0001 vs. DMSO control –TGFβ, *** P < 0.001, **** P <
0.0001 vs. DMSO control +TGFβ).
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Eur J Pharmacol. Author manuscript; available in PMC 2018 September 15.