Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes

Article

Discovery and Development of TMPRSS6 Inhibitors

Modulating Hepcidin Levels in Human Hepatocytes

Graphical Abstract

Authors

´

Franc¸ ois Beliveau, Aarti Tarkar,

´

Sebastien P. Dion, ...,

Michael Ouellette, Andrew J. Pope,

Richard Leduc

Correspondence

richard.leduc@usherbrooke.ca

In Brief

TMPRSS6 is a human liver enzyme

considered as a therapeutic target for iron

overload-related diseases, such as

hereditary hemochromatosis and

´

b-thalassemia. In this study, Beliveau

et al. discovered and characterized three

different classes of TMPRSS6 inhibitors

that increase production of hepcidin, the

major iron regulatory hormone, in human

hepatocytes.

Highlights

d

Discovery of TMPRSS6 inhibitors from high-throughput

screening

d

In cellulo characterization of peptidomimetic and non-

peptidic inhibitors of TMPRSS6

d

Inhibitors abolish TMPRSS6-mediated HJV cleavage

Inhibitors upregulate hepcidin production in human

hepatocytes

´

Beliveau et al., 2019, Cell Chemical Biology 26, 1–14

November 21, 2019 ª 2019 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2019.09.004

´

Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

Cell Chemical Biology

Article

Discovery and Development of TMPRSS6 Inhibitors

Modulating Hepcidin Levels in Human Hepatocytes

1 ,2

3

1 ,2

Franc¸ ois Beliveau, Aarti Tarkar, Sebastien P. Dion, Antoine Desilets,^1,2Mariana Gabriela Ghinet,^1,2

´

Pierre-Luc Boudreault,^1,2Catherine St-Georges,^1,2Eric Marsault,^1,2Daniel Paone,³Jon Collins,³Colin H. Macphee,³

Nino Campobasso,⁴Arthur Groy,⁴Josh Cottom,⁴Michael Ouellette,⁴Andrew J. Pope,³and Richard Leduc^1,2,5,

´

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, 3001, 12 Avenue Nord,

Sherbrooke, QC J1H 5N4, Canada

Institut de Pharmacologie de Sherbrooke, Universite de Sherbrooke, Sherbrooke, QC, Canada

³Discovery Partnerships with Academia, GlaxoSmithKline, Collegeville, Philadelphia, PA 19426-0989, USA

⁴Platform Technology & Science, GlaxoSmithKline, Collegeville, Philadelphia, PA 19426-0989, USA

⁵Lead Contact

´

*

1

e

2

´

*Correspondence: richard.leduc@usherbrooke.ca

https://doi.org/10.1016/j.chembiol.2019.09.004

SUMMARY

The role of TMPRSS6 in iron regulation was ﬁrst revealed by

association of TMPRSS6 genetic mutations found in patients

Iron overload disorders are characterized by the suffering from iron-refractory iron deﬁciency anemia (IRIDA), a

condition associated with low circulating iron, elevated hepcidin

levels and unresponsiveness to oral iron therapy (Finberg et al.,

body’s inability to regulate iron absorption and its

storage which can lead to organ failures. Accumu-

2008). Concomitantly, a chemically induced mutant mouse

model (mask) exhibiting microcytic anemia where a splicing

defect led to the production of a catalytically inactive TMPRSS6

lated evidence has revealed that hepcidin, the

master regulator of iron homeostasis, is negatively

modulated by TMPRSS6 (matriptase-2), a liver-

speciﬁc type II transmembrane serine protease

(TTSP). Here, we report that treatment with a pep-

tidomimetic inhibitor affecting TMPRSS6 activity

protein was described (Du et al., 2008). A Tmprss6^ꢀ/ꢀmice

model also conﬁrmed that absence of the protease causes

anemia associated with elevated hepcidin levels (Folgueras

et al., 2008). In these models, loss of enzymatic function was

increases hepcidin production in hepatic cells.

Moreover, similar effects were observed when us-

associated with an increase in the serum levels of hepcidin, a

secreted hepatic hormone that decreases the body’s iron con-

ing non-peptidic inhibitors obtained through opti- tent through ferroportin (FPN) occlusion and degradation, result-

ing in IRIDA (Aschemeyer et al., 2018; Finberg et al., 2008).

At the cell surface, TMPRSS6 is a negative regulator of hepci-

mization of hits from high-throughput screening.

Using HepG2 cells and human primary hepato-

cytes, we show that TMPRSS6 inhibitors block

TMPRSS6-dependent hemojuvelin cleavage and

din production by acting at the apex of a bone morphogenetic

protein/SMAD signaling pathway modulating expression of

HAMP, the gene coding for hepcidin (Du et al., 2008; Nemeth

increase HAMP expression and levels of secreted

et al., 2004). TMPRSS6 exerts its effect via cleavage of hemoju-

hepcidin.

velin (HJV), a co-receptor for the BMP family, which is an essen-

tial element, along with BMP receptors, transferrin receptor-2,

and homeostatic iron regulator protein (Finberg et al., 2010; Sil-

INTRODUCTION

vestri et al., 2008a; Wahedi et al., 2017) contributing to iron ho-

meostasis. Therefore, it can be postulated that inhibition of

TMPRSS6 may result in increased hepcidin levels, making it a

potentially attractive pharmacological target for diseases char-

Type II transmembrane serine proteases (TTSPs) are proteolytic

enzymes localized at the plasma membrane that share

conserved domains. They possess an amino-terminal cyto- acterized by iron overload such as thalassemias and hemochro-

matosis (Beckmann et al., 2016a; Wang et al., 2014). Proof-of-

concept for this idea has been demonstrated in pre-clinical

plasmic region, a single-pass transmembrane domain, and an

extracellular domain composed of various regions ultimately

ending with a C-terminal serine protease catalytic domain (Ant- model systems by knockout of the TMPRSS6 gene or reduction

of its mRNA expression using antisense oligonucleotides

(ASOs). These approaches led to reduced iron levels and related

alis et al., 2011; Hooper et al., 2001). According to the MEROPS

peptidase database (Rawlings et al., 2018), TTSPs belong to the

chymotrypsin-fold PA clan of proteases and are part of the S1 symptoms (e.g., siderosis, quality of erythropoiesis) in mouse

subfamily. TMPRSS6, also known as matriptase-2, is a TTSP models of hemochromatosis and b-thalassemia (Casu et al.,

2016; Finberg et al., 2011; Guo et al., 2013; Nai et al., 2012,

almost exclusively expressed at the surface of hepatocytes in

the liver (Dion et al., 2018a; Ramsay et al., 2008) and plays a 2014; Schmidt et al., 2013). Recently, small molecules (thiazoli-

key role in iron homeostasis via modulation of hepcidin expres- dinones) have been shown to reduce iron overload in mice

models of hereditary hemochromatosis and b-thalassemia

through hepcidin upregulation, by a mechanism involving

sion (Du et al., 2008; Finberg et al., 2008; Folgueras et al.,

2008; Silvestri et al., 2008a).

Cell Chemical Biology 26, 1–14, November 21, 2019 ª 2019 Elsevier Ltd.

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Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

TMPRSS6 reduction at both the mRNA and protein levels (Liu S762A; Figure 1B). Inhibitors (100 nM) or vehicle (DMSO) were

et al., 2019). While these compounds are not direct protease in- added 24 h post transfection and cell media were collected after

hibitors, it warrants the use of small molecules targeting a 24-h incubation. Proteolytic activity was evaluated using a

TMPRSS6 in a therapeutic context.

TMPRSS6 ﬂuorogenic peptide substrate. Of the 12 compounds

Like other serine proteases, TTSPs have similar substrate tested, three; compounds 2 ((H)Y-Y-V-R-kbt), 8 ((H)F-(thiazol-4-

speciﬁcities, each protease possessing distinct structural deter- yl)alanine-V-R-kbt), and 29 ((H)F-hF-(Allo)T-R-kbt) (Cpd-2, -8,

´

minants (Barre et al., 2014; Beliveau et al., 2009). Because and -29) (chemical structures in Figure S1) showed signiﬁcantly

closely related TTSPs play essential physiological roles, for lower residual TMPRSS6 proteolytic activity compared with

example matriptase regulates epithelial barrier formation and untreated cells and comparable activity with cells transfected

permeability in the intestine (Buzza et al., 2010), a highly selective with the catalytically inactive mutant (S762A). Noteworthy,

TMPRSS6 inhibitor would be required to avoid potential side Cpd-1 (Y-Y-V-R-kbt), the only non-desamino compound tested,

effects. Several groups including ours have reported on the did not exhibit TMPRSS6 inhibition in transfected cells. This

development of different classes of TMPRSS6 inhibitors (Beck- compound was shown to be unstable in mouse plasma (St-

ˆ

mann et al., 2016a, 2016b; Dosa et al., 2012; Duchene et al., Georges et al., 2017), which may explain the absence of inhibi-

€

2014; Gitlin et al., 2015; Haußler et al., 2016; Maurer et al., tion in treated cells.

2013; Roydeva et al., 2016; Sisay et al., 2010; St-Georges

We further evaluated the capacity of these three compounds

et al., 2017). However, many of these studies did not exhaus- (Cpd-2, -8, -29) to prevent TMPRSS6 shedding, an autocata-

tively address the compounds’ selectivity for TMPRSS6 over lytic event (Stirnberg et al., 2010), in the same conditions

other TTSPs or serine proteases in general. Highly potent (low (Figure 1C). Cpd-1 and the catalytically inactive mutant S762A

nM K_i) and selective (>60-fold selectivity for TMPRSS6 over were used as controls. In the cell media of transfected and

matriptase) peptidomimetic inhibitors using unnatural amino compound-treated cells, a signiﬁcant decrease of the shed

acids and a ketobenzothiazole serine trap (St-Georges et al., forms of TMPRSS6 (zymogen shed form, 110 kDa; active

2017) currently exist but those compounds have only been char- shed form, 32 kDa) was observed in cells treated with Cpd-2

acterized in vitro.

and -8 and to a lesser extent with Cpd-29. Cpd-1 did not

Here, we report that a previously developed peptidomimetic have an effect and no cell surface shedding of TMPRSS6 was

compound with low nanomolar K_iand >10-fold selectivity for detected in cells transfected with the catalytically inactive

TMPRSS6 over matriptase inhibits TMPRSS6 proteolytic activity mutant (Figure 1C).

in transfected cells and modulates the BMP6-hepcidin pathway

To compare the potencies of the best compounds in cellulo

in cellular models. In addition, we performed high-throughput toward TMPRSS6, we calculated half maximal inhibitory con-

screening campaigns to identify non-peptidomimetic com- centration (IC₅₀) values for Cpd-2, -8, and -29 in TMPRSS6-

pounds. This led to the identiﬁcation and characterization of transfected HEK293 cells (Figure 1D). All three compounds dis-

two distinct additional chemical classes of TMPRSS6 inhibitors, played low nanomolar IC_50s, Cpd-2 (4.75 1.47 nM) and Cpd-

i.e., pyrrolidinones and boronic acids. Using the immortalized 8 (7.65 1.86 nM) being the most potent, followed by Cpd-29

hepatocellular carcinoma cell line HepG2 as well as human pri- (43.1 17.7 nM). No cytotoxicity was observable at the highest

mary hepatocytes, we demonstrate that exemplar compounds compound concentration used (10 mM) (Figure S2A), and com-

from all three classes signiﬁcantly reduce TMPRSS6-dependent pounds did not negatively affect TMPRSS6 plasma membrane

HJV cleavage as well as increase HAMP expression and hepci- localization (Figure S3A), suggesting that reduction in activity

din levels.

was due to direct protease inhibition.

To gain information on compound selectivity, we calculated

their IC₅₀values using various recombinant proteases in vitro.

We ﬁrst compared inhibitory activity toward TMPRSS6 and

two closely related TTSPs (matriptase and hepsin). Matriptase

is a potential off-target due to high similarity although it is not

RESULTS

Benzothiazole-Based Peptidomimetics Compounds

Inhibit TMPRSS6 In Cellulo

We previously developed a series of peptidomimetic inhibitors expressed in the liver according to the Human Protein Atlas

´

of TMPRSS6 with a generic scaffold composed of a ketobenzo- (Uhlen et al., 2015), while hepsin is highly expressed in the liver

thiazole serine trap at the C terminus ﬂanked by an arginine in P1, (Table S2). We also tested the compounds against two serine

and in which positions P4, P3, and P2 were varied using unnat- proteases of the same protease clan and family (PA, S1) ex-

ural amino acids (St-Georges et al., 2017) (Figure 1A). These pressed in the liver (thrombin and factor Xa; Table S2), and furin,

compounds were characterized in vitro using recombinant a serine protease known to be involved in HJV and hepcidin pro-

human TMPRSS6 and matriptase to determine K_ivalues and cessing (Silvestri et al., 2008b; Valore and Ganz, 2008). As ex-

selectivity, but their effects in cellular assays were never evalu- pected, all three compounds exhibit low nanomolar IC₅₀values

ated. To assess their ability at inhibiting TMPRSS6 activity in cel- for TMPRSS6 with values of 11 nM for Cpd-2, 8.1 nM for Cpd-

lulo, we selected the most potent and speciﬁc TMPRSS6 com- 8, and 25 nM for Cpd-29 (Figure 1E for heatmap representation

pounds. Those with K_i< 5 nM and a 10-fold selectivity over supported by Table S3). When compared with other TTSPs (ma-

matriptase were chosen. A total of 12 compounds respecting triptase and hepsin), Cpd-2 and -8 were not as potent, with IC₅₀

those conditions were initially selected (Table S1).

values toward matriptase of 158 and 52 nM, and toward hepsin

To evaluate the inhibitory effects of these compounds on of 74 and 16 nM, respectively. Cpd-29 is a weaker inhibitor of

TMPRSS6 activity in cellulo, we transfected HEK293 cells with both matriptase (IC₅₀= 1.5 mM) and hepsin (IC₅₀= 0.4 mM). Inter-

TMPRSS6 and its catalytically inactive counterpart (TMPRSS6 estingly, all three compounds were shown to be weak inhibitors

2

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Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

Figure 1. Inhibition by Benzothiazole-Based Compounds

(A) Generic scaffold of ketobenzothiazole serine trap peptidomimetic inhibitors. Positions P4, P3, and P2 (red) were varied using natural and unnatural amino

acids when position P1 was ﬁxed to Arg. A ketobenzothiazole serine trap is present in the C-terminal position (orange).

(B) HEK293 cells were transfected with TMPRSS6-V5 and cell media were replaced after 24 h with serum-free media with or without inhibitor. Activity in cell media

treated or not with 100 nM inhibitor was evaluated 24 h after media change with Boc-QAR-AMC ﬂuorescent substrate and reported as percent residual activity. Boxes

show quartiles and whiskers show the last data within 1.5 interquartile range between lower and upper quartile (Tukey boxplot) (n R 5). Statistical analyses were

determinedusingKruskal-Wallistest. Asterisks indicatesigniﬁcantdifferencesfromTMPRSS6-V5controlstreatedwithvehicle(0.1%DMSO). *p<0.05, ****p< 0.0001.

(C) Immunoblotting of TMPRSS6-V5 in cell lysate (CL) and cell media with reference compound (Cpd-1) and inhibitors (100 nM) exhibiting the highest level of

inhibition of proteolytic activity (compounds 2, 8, and 29). Expression was detected by western blotting with anti-V5 antibody. An equal amount of CL was loaded

on an SDS-polyacrylamide gel (upper panels). The cell medium (CM) was concentrated and loaded on a 12% SDS-polyacrylamide gel (lower panel). CL GAPDH

was blotted as a loading control (n = 3) (middle panel).

(D) Mean IC₅₀values were determined by monitoring proteolytic activity in cell media of HEK293 transfected cells treated with inhibitors signiﬁcantly decreasing

TMPRSS6’s activity. Relative activity is expressed as milliﬂuorecence units per minutes (mF.U./min). Mean values SD are presented alongside a representative

IC₅₀curve.

(E) Speciﬁcity of benzothiazole-based compounds toward other serine proteases. Data are represented as a heat map using log(IC₅₀) mean values, N R 3.

See also Figures S1–S3 and Tables S1–S3.

of thrombin and factor Xa and had no inhibitory activity inhibitor both in vitro and in cellulo and is the most stable in

toward furin. mouse plasma compared with Cpd-2 (St-Georges et al., 2017),

Overall, compounds showed speciﬁcity for TTSPs with a pref- it was selected for further characterization as a tool compound

erence for TMPRSS6. Because Cpd-8 is a potent TMPRSS6 for its potential effect on BMP6-HJV-hepcidin axis modulation.

Cell Chemical Biology 26, 1–14, November 21, 2019

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Figure 2. Interactions of Cpd-8 with Matriptase and TMPRSS6

(A) Chemical structure of benzothiazole inhibitor Cpd-8. The compound is composed of a desaminophenylalanine residue (P4), (thiazol-4-yl)alanine (P3), valine

(P2), arginine (P1), and a ketobenzothiazole (kbt) at the C terminus.

(legend continued on next page)

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

Cpd-8 Forms a Reversible Covalent Bond with TMPRSS6

A closer look at the models reveal key interactions inside the

Cpd-8 is composed of a desamino phenylalanine residue in P4, binding pocket between Cpd-8 and matriptase (crystal structure)

(thiazol-4-yl)alanine in P3, valine in P2, arginine in P1, and a ke- as well as with TMPRSS6 (homology model) (Figures 2E and 2F).

tobenzothiazole at the C terminus (Figure 2A). To understand A strong hydrogen bond network is observed between the guani-

how this compound achieves its high inhibitory potential, we ob- dinium group (-C-(NH₂)²⁺) of Cpd-8 Arg (P1) and Asp799 in ma-

tained a crystal structure of this compound bound to matriptase triptase (Figure 2E) or the equivalent Asp756 position in TMPRSS6

(PDB: 6N4T; Figure 2B). A homology model of TMPRSS6 was (Figure 2F). The guanidinium group was found deep in the cata-

then built using the crystal structure of matriptase (Figure 2C). lytic pocket, which is common for this type of enzyme (Czapinska

Alignment to the residues of the catalytic domain of matriptase and Otlewski, 1999). Gly829 (matriptase) and Gly786 (TMPRSS6)

resulted in 45% and 62% sequence identity and similarity, are the backbone hydrogen bond acceptor in both cases. Another

respectively, allowing to build a high-quality model. We had pre- interesting interaction is an arene-H between a proton of the

viously published a homology model of TMPRSS6 (St-Georges Cpd-8 Val (P2) side chain and His656 (matriptase) or His617

et al., 2017) based on a different matriptase crystal (PDB: (TMPRSS6), which is part of the catalytic triad. Other similarities

3NCL; Brown et al., 2011). Comparison of those TMPRSS6 ho- include hydrogen bond acceptors and donors of the (thiazol-4-

mology models revealed only two minor differences, mostly yl)alanine (P3) backbone with Gly827 (matriptase) and Gly784

due to the shape and the conformation of their corresponding (TMPRSS6), a hydrogen bond between the arginine (P1) amide

ligand. First, to accommodate the P2 Val residue (PDB: 6N4T), and the Ser825 (matriptase) or Ser782 (TMPRSS6) backbone,

Phe708 side chain must rotate 90^ꢁ. Finally, translation and rota- and a hydrogen bond network with Gly803 and Ser805 (matrip-

tion of Gln801 allows the formation of a hydrogen bond with the tase) or Gly760 and Ser762 (TMPRSS6), which presumably plays

ﬁrst amide bond of Cpd-8, which was not possible with benzami- a critical role in the hemiketal formation and stability. Ligand expo-

dine phosphate inhibitors as shown in Figure S4A.

Superimposition of the previously proposed model (docked desamino phenylalanine, and (thiazol-4-yl)alanine.

with inhibitor Y-Y-V-R-Kbt) with the newly obtained crystal struc- Conversely, differences were also observed. Matriptase

sures are also identical and include a benzothiazole aromatic ring,

ture of matriptase with Cpd-8 (PDB: 6N4T) shows a good overlay Tyr755 is replaced in TMPRSS6 with a smaller residue (Glu712)

of the peptidomimetic backbone of both inhibitors (see Fig- leading to a conformation change of the ligand to accommodate

ure S4B). Moreover, no major difference can be observed around the (thiazol-4-yl)alanine in the crystal structure of matriptase.

the binding pocket of matriptase and TMPRSS6 regarding the Sulfur forms a hydrogen bond with the phenol group of the tyro-

orientation of the amino acids for each enzyme. In other words, sine. However, Glu712 in the TMPRSS6 model plays a similar

the new crystal structure did not provide major new insights role and is a side chain hydrogen bond acceptor with the (thia-

into the possible future design of molecules or the interpretation zol-4-yl)alanine amine instead.

of the selectivity of peptides. Comparison of the binding pocket

of matriptase and TMPRSS6’s homology model revealed only Inhibition of TMPRSS6 by Non-peptidic Compounds

seven differences (Figure 2C). At the P2 position, matriptase Use of peptidic compounds as drugs is limited by their rapid pro-

His665 is replaced by Phe708 in TMPRSS6, another aromatic res- teolysis and generally poor membrane permeability and oral

idue. There are also three major differences around (thiazol-4-yl) bioavailability (Qvit et al., 2017). In an attempt to identify more

alanine in P3, which can eventually be used to optimize selectivity: druggable compounds, an integrated screening approach was

Asp728 / Leu785, Gln838 / Arg789, and Tyr755 / Glu712. used to identify TMPRSS6 inhibitors (Leveridge et al., 2018) in

Along these lines, at the P4 extension, aromatic residue Phe706 which both a conventional HTS campaign and a DNA-encoded

is replaced by an acidic and polar Asp663. Another variation is library screen ELT (Arico-Muendel, 2016) were performed. HTS

located in the ketobenzothiazole environment while the polar hy- was conducted against GSK’s collection of around 2 million

drophilic Gln620 replaces the hydrophobic Ile559. Another impor- compounds, with each compound tested at 10 mM using a ﬂuo-

tant difference is a deletion of three residues observed for rescence-based assay for TMPRSS6 activity. The screen output

TMPRSS6 leading to a conformational change and a complete was triaged by re-testing putative hits in dose-response tests

ﬂip of Glu621 which replaces Asp660 in matriptase.

conducted under a range of conditions to give preliminary infor-

A striking observation is that Cpd-8 forms a covalent bond mation on potency, speciﬁcity (versus matriptase) and inhibition

with the catalytic triad residue Ser762 of TMPRSS6 (His617 mechanism (e.g., time-dependence of inhibition). Potentially

and Asp668) as shown in Figure 2D, which is also found in the useful chemotypes were then conﬁrmed by re-synthesis of

crystal structure of matriptase (Figure 2B). To the best of our exemplars and re-testing against TMPRSS6. In the case of

knowledge, this constitutes the ﬁrst observation of the covalent ELT, selections were performed against puriﬁed TMPRSS6 using

bond formation of ketobenzothiazoles with TTSPs. This observa- methods described previously (Arico-Muendel, 2016; Belyan-

tion strongly supports the designed reversible tight-binding skaya et al., 2017), and putative hits were re-synthesized and

mode of inhibition.

tested as described above.

(B) Cpd-8 forms a covalent bond with matriptase catalytic triad residue Ser7805. Residues of the catalytic triad are shown in cyan and Cpd-8 in green.

(C) Comparison of the binding pocket of matriptase crystal structure (PDB: 6N4T) (orange) and TMPRSS6 homology model (purple). Matriptase-associated

Cpd-8 is blue and green for TMPRSS6.

(D) Cpd-8 forms a covalent bond with catalytic triad residue Ser762. Residues of the catalytic triad are shown in cyan and Cpd-8 in green.

(E and F) Interactions of Cpd-8 with (E) matriptase and (F) TMPRSS6. Orange highlights inhibitor side chains.

See also Figures S2, S5, and S6.

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Figure 6. TMPRSS6 Inhibition Increases HAMP Expression in Human Primary Hepatocytes

Human primary hepatocytes from four different donors (1797, 1836, 8148, and 8279) were treated with either DMSO (0.1%) or increasing concentrations

(0.1, 1, 5, and 10 mM) of (A) Cpd-8, (B) Cpd-A, or (C) Cpd-B at 30 ng/mL hBMP6. Cells were plated in media with fetal bovine serum for 24 h then media

(legend continued on next page)

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Figure 7. TMPRSS6 Inhibition Increases Hepcidin Levels in Human Primary Hepatocytes

Human primary hepatocytes from donor 8279 were treated with either DMSO (0.1%) or increasing concentrations of (A) Cpd-8, (B) Cpd-A, or (C) Cpd-B (0.1, 1, 5,

and 10 mM) at 5 ng/mL hBMP6. Cells were plated in media with fetal bovine serum for 24 h then media were replaced by serum-free media containing inhibitors for

6 h. hBMP6 was then added for 24 h and hepcidin-25 in cell media was quantiﬁed by ELISA. Results are expressed as hepcidin fold change over vehicle-treated

cells (n = 3). All statistical analyses were determined using one-way ANOVA. Asterisks indicate signiﬁcant differences compared with vehicle (DMSO 0.1%)

treated cells.*p < 0.05, ***p < 0.001. See also Figure S2.

activity have not been reported to efﬁciently modulate hepci- and pyrrolidinones) constitute a potential strategy for future

din expression in cellular models. studies aimed at developing molecules for therapeutic

Data presented herein demonstrate that three different purposes.

compound classes inhibit TMPRSS6 proteolytic activity in

Other groups have developed TMPRSS6 proteolytic inhibi-

a cellular context. We show that TMPRSS6 inhibition abol- tors using different strategies based on benzylamide (Sisay

ishes HJV cleavage, a TMPRSS6 substrate and an upstream et al., 2010), bisbenzamidines (Beckmann et al., 2016b), sun-

element of the BMP/SMAD pathway, leading to the produc- ﬂower trypsin inhibitor-1 (Gitlin et al., 2015; Gitlin-Domagalska

tion of hepcidin. These compounds exhibited a dose-depen- et al., 2017), or Kunitz-type inhibitors such as HAI-1 and

dent increase in HAMP expression and hepcidin levels in HAI-2 (Beckmann et al., 2016a). Although some of these exhibit

liver-related models such as HepG2 cells and human primary potency and selectivity for TMPRSS6, their use in a cellular

hepatocytes. Although we cannot rule out the possibility that context has not been reported. In this study, we demonstrate

the effect observed on HAMP and hepcidin levels in primary for the ﬁrst time that targeting TMPRSS6 proteolytic activity

hepatocytes is due to inhibition of off-target proteases, we with protease inhibitors can be used to increase hepcidin

believe it is likely to be the consequence of TMPRSS6 expression in liver cellular models, constituting a potential

inhibition. Compounds tested in this study are also potent therapeutic approach aimed at treating patients with iron over-

against matriptase and hepsin (Figures 1E and 3E), two TTSPs load conditions.

with high similarity regarding their catalytic domain when

compared with TMPRSS6. However, while matriptase is SIGNIFICANCE

virtually not expressed in the liver (Table S2), no reported

data support the involvement of the liver-expressed hepsin TMPRSS6 is a key modulator of the iron homeostasis

in hepcidin regulation. Reﬁnement of inhibitor selectivity using pathway. Previous studies have shown that downregulation

structure-activity relationship strategy and the use of of the protease increases hepcidin levels thus reducing iron

TMPRSS6 and hepsin knockout hepatocytes would be useful import. Those studies have also shown that, in models of

to determine precisely the mechanism of action of these iron overload diseases, such as hemochromatosis and

molecules and avoid potential off-target effects. These results b-thalassemia, knockout of the enzyme diminishes related

could also be very important for further characterization of effects. However, inhibition of the catalytic activity of the

in vivo mouse models of b-thalassemia and hemochromatosis. enzyme by small molecules resulting in similar outcomes

Overall, compound optimization for the three classes used has not been demonstrated. Here we show that three

here (peptidomimetic benzothiazole-based, boronic acids, different classes of proteolytic inhibitors targeting

were replaced by serum-free media containing inhibitors for 6 h. hBMP6 was then added for 24 h. HAMP expression was quantiﬁed by RT-qPCR and

expressed as HAMP fold change over vehicle-treated cells (DMSO 0.1%) (n R 3). All statistical analyses were determined using one-way ANOVA. As-

terisks indicate signiﬁcant differences compared with vehicle (DMSO 0.1%)-treated cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also

Figures S2 and S7.

Cell Chemical Biology 26, 1–14, November 21, 2019 11

´

Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

DECLARATION OF INTERESTS

TMPRSS6 catalytic activity increase hepcidin production in

a liver cellular model and in primary human hepatocytes.

R.L. and E.M. hold patent WO2012162828A1 related to peptidomimetic serine

protease inhibitors.

STAR+METHODS

Received: November 29, 2018

Revised: June 6, 2019

Detailed methods are provided in the online version of this paper

and include the following:

Accepted: September 3, 2019

Published: September 19, 2019

d KEY RESOURCES TABLE

REFERENCES

d LEAD CONTACT AND MATERIALS AVAILABILITY

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Chemical Synthesis of Compounds

B Cell Culture

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B Screening of Inhibitory Potency in Cells

B Compounds IC₅₀on TMRSS6 Activity In Cellulo

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B Matriptase/Cpd-8 Crystal Structure

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B Hemojuvelin Cleavage Assay

B TMPRSS6 Western Blot

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B Statistical Analysis

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d DATA AND CODE AVAILABILITY

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SUPPLEMENTAL INFORMATION

Evaluation of bisbenzamidines as inhibitors for matriptase-2. Bioorg. Med.

Supplemental Information can be found online at https://doi.org/10.1016/j.

chembiol.2019.09.004.

Chem. Lett. 26 , 3741–3745.

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ACKNOWLEDGMENTS

B e ´ liveau, F., Brul e ´ , C., D e ´ silets, A., Zimmerman, B., Laporte, S.A., Lavoie, C.L.,

and Leduc, R. (2011). Essential role of endocytosis of the type II transmem-

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This work was supported by the Canadian Institutes of Health Research (CIHR)

(grant number MOP 133400) to R.L. and E.M. and by the Discovery Partner-

ships with Academia (DPAC) group, part of GlaxoSmithKline Research and

Development. F.B. has received a MITACS fellowship in partnership with Glax-

´

oSmithKline. S.P.D. is a Ph.D. fellow of the Fonds de Recherche du Quebec –

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Discovering drugs with DNA-encoded library technology: from concept to

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´

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Brosseau, J.-P., Lucier, J.-F., Lapointe, E., Durand, M., Gendron, D., Gervais-

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zothiazole-based compounds screening, IC₅₀determination, cell surface

localization assays in HEK293 cells, and cytotoxic assays in all cellular models.

A.D. performed the compounds IC₅₀determination in vitro. N.C. determined

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modeling of Cpd-8 in the TMPRSS6 homology model based on matriptase.

M.G.G. performed the HJV cleavage assay in HepG2 cells. A.T. performed

BacMam assay in HepG2 cells. A.T. and A.G. performed HAMP RT-qPCR

assay in human primary hepatocytes. F.B. performed hepcidin ELISA assay

in human primary hepatocytes and cell surface localization assays in HepG2

cells. F.B., S.P.D., A.D., P.-L.B., A.J.P., and R.L. wrote the manuscript. R.L.

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Mouse Anti-V5 Monoclonal Antibody

Mouse Anti-V5 Monoclonal Antibody, HRP Conjugated

Thermo Fisher Scientiﬁc

Cell Signaling Technology

Cat# R960-25; RRID: AB_2556564

Cat# R961-25; RRID: AB_2556565

Cat# 3683; RRID: AB_1642205

Rabbit Anti-GAPDH Monoclonal Antibody (14C10),

HRP Conjugated

Goat Anti-Hemojuvelin Polyclonal Antibody

Rabbit Anti-Goat IgG (H+L) Secondary Antibody, HRP

Horse Anti-Mouse IgG, HRP Conjugated

R & D Systems

Cat# AF3720; RRID: AB_2264104

Cat# A16136; RRID: AB_2534807

Thermo Fisher Scientiﬁc

Cell Signaling Technology

Cat# 7076;

RRID: AB_330924

Mouse anti-GAPDH monolconal Antibody

Rabbit anti-Matriptase2 Antibody

Goat Anti-Mouse 680LT

Sigma-Aldrich

Abcam

Cat# G8795; RRID: AB_1078991

Cat# ab28287; RRID: AB_2203535

Cat# 926-68020; RRID: AB_10706161

Cat# 926-32211; RRID: AB_621843

LICOR

Goat Anti-Rabbit 800 CW

LICOR

Bacterial and Virus Strains

Bacmam/TMPRSS6-WT isoform 2

Bacmam/TMPRSS6-S762A isoform 2

Biological Samples

This paper

N/A

Human Plateable Hepatocytes, Induction Qualiﬁed

ThermoFisher Scientiﬁc

Cat# HMCPIS, Lot# Hu1797, Hu1836,

Hu1880, Hu8148, Hu8279

Chemicals, Peptides, and Recombinant Proteins

Boc-Gln-Ala-Arg-AMC

R & D Systems

Bachem

Cat# ES014

Boc-Ile-Glu-Gly-Arg-AMC acetate salt

Boc-Arg-Val-Arg-Arg-AMC acetate salt

Cat# 4004961

Cat# 4018735

Cat# sc-210120

Bachem

4-Methylumbelliferyl 4-guanidinobenzoate

Hydrochloride (MUGB-HCl)

Santa Cruz Biotechnology

Compound-2

(St-Georges et al., 2017)

This paper

N/A

Compound-8

N/A

Compound-29

N/A

Compound-A

N/A

Compound-B

This paper

N/A

William’s E Medium, no phenol red

Primary Hepatocyte Thawing and Plating Supplements

Primary Hepatocyte Maintenance Supplements

Cryopreserved Hepatocyte Recovery Medium (CHRM)

ThermoFisher Scientiﬁc

WISENT Bioproducts

Cat# A1217601

Cat# CM3000

Cat# CM4000

Cat# CM7000

Cat# 319-005-CL

High glucose Dulbecco’s Modiﬁed Eagle’s

Medium (DMEM)

Eagle’s Minimum Essential Medium (EMEM)

HCell-100

WISENT Bioproducts

ThermoFisher Scientiﬁc

Sigma-Aldrich

Cat# 320-005-CL

Cat# 001-035-CL

Cat# 080-150

Fetal bovine serum (FBS)

Penicillin-Streptomycin L-Glutamine

Lipofectamine 3000 Transfection Reagent

Complete Protease Inhibitor Cocktail

Recombinant Human BMP-6 Protein

Clarity Western ECL Substrate

Basal Medium Eagle (BME)

Cat# 450-202-EL

Cat# L3000015

Cat# 11836145001

Cat# 507-BP-020

Cat# 1705060

R & D Systems

Bio-Rad

Thermo Fisher Scientiﬁc

Cat#21010046

Insulin-Transferrin-Selenium (ITS-G) 100X

Cat# 41400045

(Continued on next page)

Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e1

´

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

Continued

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Recombinant Human BMP6 (Carrier-free)

Recombinant Human TMPRSS6

TONBO Biosciences

GlaxoSmithKline

R & D Systems

Cat# 21-7004-U100

Recombinant Human Coagulation Factor Xa

Protein, CF

Cat# 1063-SE-010

Thrombin from Human Plasma, High Activity

Recombinant Human Furin Protein, CF

Restore^TMPLUS Western Blot Stripping Buffer

TMB One Component Substrate

Millipore Sigma

R & D Systems

Thermo Fisher Scientiﬁc

Bethyl

Cat# 605195

Cat# 1503-SE-010

Cat# 46430

Cat# E102

3,3⁰,5,5⁰-Tetramethylbenzidine Liquid Substrate

Transcriptor First Strand cDNA Synthesis Kit

RNaseOUT Recombinant Ribonuclease Inhibitor

iTaq Universal SYBRꢀ Green Supermix

TRIzol Reagent

Sigma

Cat# T4319

Roche

Cat# 04897030001

Cat# 10777019

Cat# 1725120

Cat# 15596018

Cat# 89900

Thermo Fisher Scientiﬁc

Bio-Rad

Thermo Fisher Scientiﬁc

LICOR

RIPA Lysis Buffer

Odysseyꢀ One-Color Protein Molecular Weight

Marker

Cat# 928-40000

Odysseyꢀ Blocking Buffer in TBS

Critical Commercial Assays

LICOR

Cat# 927-50000

Hepcidin-25 (Human) EIA kit

Peninsula Laboratories

Thermo Fisher Scientiﬁc

Promega

Cat# S-1337

Cat# A25602

Cat# G7572

Cells-to-CT 1-Step TaqMan Kit

CellTiter-Glo Luminescent Cell Viability Assay

Cytotoxicity Detection Kit^PLUS(LDH)

Pierce Cell Surface Protein Isolation Kit

Pierce BCA Protein Assay Kit

Deposited Data

Roche

Cat# 04744926001

Cat# 89881

Thermo Fisher Scientiﬁc

Cat# 23227

Matriptase/Compound-8 structure

This study

PDB: 6N4T

PDB: 3NCL

Crystal Structure of MT-SP1 bound to Benzamidine

Phosphonate Inhibitor

(Brown et al., 2011)

Crystal Structure of Matriptase in complex with

Inhibitor

(Goswami et al., 2013)

PDB: 4JYT

Experimental Models: Cell Lines

HEK293

ATCC

Cat# CRL-1573; RRID: CVCL_0045

Cat# HB-8065; RRID: CVCL_002

HepG2

Oligonucleotides

HAMP TaqMan Assay (Human)

B2M TaqMan Assay (Human)

PPIA TaqMan Assay (Human)

Additional oligonucleotides are described in Table S5

Recombinant DNA

Thermo Fisher Scientiﬁc

Cat# 4331182/Hs00221783_m1

Cat# 4331182/Hs99999907_m1

Cat# 4331182/Hs99999904_m1

´

(Beliveau et al., 2011)

pcDNA6/V5

N/A

pcDNA6/TMPRSS6-WT-V5 isoform 1/V5

pcDNA6/TMPRSS6-S762A-V5 isoform 1/V5

pcDNA6/TMPRSS6-WT-V5 isoform 2/V5

pcDNA6/TMPRSS6-S762A-V5 isoform 2/V5

pCMV6-Entry/HJV variant a

pET28a/ST14 (aa 615-855)

Software and Algorithms

(B e ´ liveau et al., 2011)

N/A

´

(Beliveau et al., 2011)

N/A

(Dion et al., 2018a)

Origene

N/A

Cat# RC217954

N/A

This study

GraphPad Prism 7

GraphPad

Version 7.04 for Windows

Version 8.0.2 for Windows

GraphPad Prism 8

(Continued on next page)

e2 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

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Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

Continued

REAGENT or RESOURCE

Molecular Operating Environment (MOE)

Bio-1D

SOURCE

IDENTIFIER

Version 2013.08

Version 15.05

N/A

Chemical Computing Group

Vilber Lourmat

IDBS

IDBS ActivityBase software

Other

Amicon Ultra-0.5 Centrigugal Filter Unit

(10,000 NMWL)

Millipore Sigma

Qiagen

Cat# UFC501096

Cat# 74106

RNeasy Mini Kit

LEAD CONTACT AND MATERIALS AVAILABILITY

e

´

Please contact Richard Leduc, Universite de Sherbrooke. 3001, 12 Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4. e-mail:

Richard.Leduc@USherbrooke.ca, phone: +1 (819) 821-8000 ext. 75413, Fax: +1 (819) 564-5400 for reagents and resources gener-

ated in this study.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Chemical Synthesis of Compounds

Synthesis of Cpd-A

Synthesis of (S)-tert-butyl ((4’-(3-((5-(((tert-butoxycarbonyl)amino)methyl)-2- methylphenyl) sulfonamido)-2-oxopyrrolidin-1-yl)-2’,6’-

dimethyl-[1,1’-biphenyl]-2- yl)methyl)carbamate (23). Tert-butyl 3-(chlorosulfonyl)-4-methylbenzylcarbamate (B) (330 mg,

1.03 mmol) was dissolved in acetonitrile (21 mL), then pyridine (2.2 mL) was added followed by (S)-tert-butyl ((4’-(3-amino-2- oxo-

pyrrolidin-1-yl)-2’,6’-dimethyl-[1,1’-biphenyl]-2-yl)methyl)carbamate (21) (384 mg, 0.94 mmol). After stirring at room temperature

for 1 h, the mixture was poured in water (300 mL), extracted with ethyl acetate (3 x 200 mL), dried over Na₂SO₄, ﬁltered, and concen-

trated in vacuum. The residue (700 mg, yellow solid) was puriﬁed by thin layer chromatography, developed with petroleum:ethyl ac-

etate=3:2. This afforded (S)-tert-butyl ((4’-(3-((5-(((tert butoxycarbonyl)amino) methyl)-2-methylphenyl)sulfonamido)-2-oxopyrrolidin-

1-yl)-2’,6’ dimethyl-[1,1’-biphenyl]-2-yl) methyl)carbamate (23) (610 mg, 94%, purity: 97%) as a white solid. ¹HNMR (400 MHz,

CDCl₃): d 7.98 (s, 1H), 7.47-7.43 (m, 2H), 7.36-7.30 (m, 5H), 7.00-6.98 (m, 1H), 4.98 (br, 1H), 4.57 (br, 1H), 4.36 (br, 2H), 3.93-3.90

(m, 2H), 3.85-3.77 (m, 3H), 2.77 (s, 3H), 2.71 2.64 (m, 1H), 2.18-2.10 (m, 1H), 1.96 (s, 6H), 1.49 (s, 9H), 1.42 (s, 9H). LCMS (ESI):

m/z 715 [M+H]⁺.

Synthesis of (S)-5-(aminomethyl)-N-(1-(2’-(aminomethyl)-2,6-dimethyl-[1,1’-biphenyl]-4-yl)-2-oxopyrrolidin-3-yl)-2-methylbenzenesul-

fonamide (Cpd-A). (S)-Tert-butyl ((4’-(3-((5-(((tert-butoxycarbonyl)amino)methyl)-2-methylphenyl)sulfonamido)-2-oxopyrrolidin-1-yl)-

2’,6’-dimethyl-[1,1’-biphenyl]-2-yl)methyl)carbamate (23) (600 mg, 0.87 mmol) was dissolved in HCl/dioxane (4 M, 10 mL) and the

mixture was stirred for 1 h at room temperature after which the solvent was removed in vacuum, and the residue (800 mg, white solid)

was dissolved in MeOH (5 mL). The mixture was puriﬁed with the following conditions: Column, XBridge Prep C18 OBD Column

19x150 mm, 5 mm; Mobile Phase A: water (0.05% TFA), Mobile Phase B: ACN; Flow Rate: 25 mL/min; Gradient: 5% B to 30% B in

Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e3

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

10 min; 254 nm; Rt: 8.25 min. This afforded (S)-5-(aminomethyl)-N-(1-(2’-(aminomethyl)-2,6-dimethyl-[1,1’-biphenyl]-4-yl)-2-oxopyrro-

lidin-3-yl)-2-methylbenzenesulfonamide (Cpd-A, bis-TFA salt) (160 mg, 26% yield, purity: 99%, %ee: 100) as a white solid. ¹HNMR (400

MHz, CD₃OD): d 8.23 (d, J = 2.0 Hz, 1H), 7.62-7.50 (m, 5H), 7.41-7.40 (m, 2H), 7.15-7.12 (m, 1H), 4.26-4.19 (m, 3H), 3.84-3.80 (m, 2H),

3.76 (s, 2H), 2.78 (s, 3H), 2.67-2.47 (m, 1H), 2.11-2.00 (m, 1H), 1.97 (s, 6 H). LCMS (ESI): m/z 493 [M+H]⁺. ¹⁹FNMR (300 MHz, CD₃OD): d:

-76.93, -76.90.

Synthesis of Cpd-B

Synthesis of tert-butyl (3-((4-bromophenyl)amino)-3-oxopropyl)carbamate (30). To a solution of 4-bromoaniline (12 g, 70.6 mmol) in

dichloromethane (120 mL) at room temperature was added 3-((tert-butoxycarbonyl) amino) propanoic acid (16 g, 84.7 mmol), HATU

(40 g, 105 mmol), and diisopropylethylamine (45 g, 349 mmol). After 2 h water (100 mL) was added, and the aqueous phase was ex-

tracted with dichloromethane (2 3 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, ﬁltered, and

concentrated. Puriﬁcation by silica gel chromatography (ethyl acetate: petroleum ether= 1:1) afforded tert-butyl (3-((4-bromophenyl)

amino)-3- oxopropyl)carbamate (30) (22 g, 91% yield, purity: 100%) as a yellow solid. ¹HNMR (300 MHz, DMSO-d6): d 10.06 (s, 1H),

7.59-7.55 (m, 2H), 7.49-7.45 (m, 2H), 6.88 (t, J = 5.6 Hz, 1H), 3.24-3.18 (m, 2H), 2.47- 2.44 (m, 2H), 1.38 (s, 9H). LCMS (ESI): m/z

343 [M+H]⁺.

Synthesis of 3-amino-N-(4-bromophenyl)propanamide (31). 4M HCl in 1,4-dioxane (120 mL) was added to tert-butyl (3-((4-bromo-

phenyl)amino)-3-oxopropyl)carbamate (30) (12 g, 35.1 mmol) at room temperature. After 2 h, the solvent was removed under reduced

pressure to afford 3-amino-N-(4-bromophenyl)propenamide, HCl salt (31) (9.2 g, 94% yield, purity: 97%). ¹HNMR (400 MHz,

DMSO-d₆): d 10.59 (br, 1H), 8.07 (br, 3H), 7.64-7.60 (m, 2H), 7.51-7.47 (m, 2H), 3.09-3.04 (m, 2H), 2.81-2.75 (m, 2H). LCMS (ESI):

m/z 242 [M+H]⁺.

Synthesis of N-(4-bromophenyl)-3-((6-chloro-5-nitropyrimidin-4-yl)amino)propanamide (32). To a solution of 3-amino-N-(4-bromo-

phenyl)propenamide, HCl salt (31) (5.5 g, 19.8 mmol) and diisopropylethylamine (12.8 g, 99.2 mmol) in THF (150 mL) at 0^ꢁC was

added a solution of 4,6-dichloro-5-nitropyrimidine (4.6 g, 23.8 mmol) in THF (100 mL) dropwise. After 1 h at 0^ꢁC the solvent was

removed under reduced pressure. The residue was then diluted with water (100 mL), extracted with ethyl acetate (100 mL 3 3),

and the combined organic extracts were dried over anhydrous sodium sulfate, ﬁltered, and concentrated. Puriﬁcation by silica gel

chromatography (ethyl acetate: petroleum ether= 1:1) afforded N-(4-bromophenyl)-3-((6-chloro-5-nitropyrimidin-4-yl)amino)propa-

namide (32) (5.8 g, 73% yield, purity: 96%) as a yellow solid. ¹HNMR (400 MHz, DMSO-d₆): d 10.23 (s, 1H), 9.73 (t, J = 5.9 Hz, 1H), 8.12

(s, 1H), 7.58-7.56 (m, 2H), 7.49-7.47 (m, 2H), 3.90-3.85 (m, 2H), 2.72-2.65 (m, 2H). LCMS (ESI): m/z 399 [M+H]⁺.

Synthesis of 3-((5-amino-6-chloropyrimidin-4-yl)amino)-N-(4-bromophenyl)propanamide (33). To a solution of N-(4-bromophenyl)-

3-((6-chloro-5-nitropyrimidin-4-yl)amino)propanamide (32) (1.5 g, 3.76 mmol) in ethanol (60 mL) and water (15 mL) was added iron(0)

(2.1 g, 37.5 mmol) and NH₄Cl (994 mg, 18.6 mmol) and the reaction heated to 70^ꢁC. After 1 h the mixture was allowed to cool to room

temperature, ﬁltered, and concentrated. The residue was diluted with water (100 mL), extracted with ethyl acetate (100 mL33), and

e4 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

the combined organic extracts were dried over anhydrous sodium sulfate, ﬁltered, and concentrated. Puriﬁcation by silica gel

chromatography (methanol: dichloromethane= 1:10) afforded 3-((5-amino-6-chloropyrimidin-4-yl)amino)-N-(4-bromophenyl)propa-

namide (33) (640 mg, 46% yield, purity: 98%) as a yellow solid. ¹HNMR (400 MHz, DMSO-d₆): d 10.08 (s, 1H), 7.76 (s, 1H), 7.60-7.56

(m, 2H), 7.49-7.45 (m, 2H), 6.99 (t, J = 5.5 Hz, 1H), 5.05 (s, 2H), 3.69-3.65 (m, 2H), 2.65 (t, J = 6.6 Hz, 2H). LCMS (ESI): m/z 370 [M+H]+.

Synthesis of (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-6-chloro-8,9-dihydro-7H-purin-8-yl)-3,5-diﬂuorophenyl)boronic acid

(34). To a solution of 3-((5-amino-6-chloropyrimidin-4-yl)amino)-N-(4-bromophenyl)propanamide (33) (600 mg, 1.63 mmol) in

NMP (12 mL) was added 3,5-diﬂuoro-4-formylphenyl)boronic acid (361 mg, 1.94 mmol) and TMSCl (875 mg, 8.1 mmol) and the re-

action was heated to 50^ꢁC. After 16 h, the mixture was diluted with water (100 mL), extracted with ethyl acetate (100 mL 3 3), and the

combined organic extracts were washed with saturated brine (150 mL 3 3), dried over anhydrous sodium sulfate, ﬁltered and

concentrated to afford (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-6-chloro-8,9-dihydro-7H-purin-8-yl)-3,5-diﬂuorophenyl)

boronic acid (34) as a yellow solid (760 mg, 88% yield, purity: 71%). ¹HNMR (300 MHz, DMSO-d₆): d 10.11-10.07 (m, 1H), 8.47 (s,

1H), 7.70 (s, 1H), 7.40-7.33 (m, 4H), 6.75-3.78 (m, 2H), 4.05-3.98 (m, 1H), 3.71-3.63 (m, 1H), 3.26-3.17 (m, 1H), 2.74-2.64 (m, 1H),

2.55-2.52 (m, 1H). LCMS (ESI): m/z 538 [M+H]⁺.

Synthesis of (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-6-chloro-9H-purin-8-yl)-3,5-diﬂuorophenyl)boronic acid (35). To a solu-

tion of (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-6-chloro-8,9-dihydro-7H-purin-8-yl)-3,5-diﬂuorophenyl)boronic acid (34)

(760 mg, 1.4 mmol) in THF (12 mL) was added DDQ (636 mg, 2.8 mmol). After stirring at room temperature for 2 h, the solvent

was removed under reduced pressure. The residue was then dissolved in DMF (6 mL) and puriﬁed by reversed phase HPLC with

the following conditions: Column: C18 silica gel 80 g, 20-35 mm; Mobile Phase A: Water (0. 05% TFA), Mobile Phase B: ACN;

Flow rate: 50 mL/min; Gradient: 5% B to 55% B in 20 min; 254 nm. This afforded (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-

6-chloro-9H-purin-8-yl)-3,5-diﬂuorophenyl)boronic acid (35) (380 mg, 51% yield, purity: 100%) as a yellow solid. ¹HNMR (300

MHz, DMSO-d₆): d 10.06-10.04 (m, 1H), 8.90-8.89 (m, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.60 (dd, J = 17.1, 8.7 Hz, 1H), 7.42-7.34

(m, 4H), 4.54-4.46 (m, 2H), 4.10 (s, 2H), 2.96-2.88 (m, 2H). LCMS (ESI): m/z 536 [M+H]⁺.

Synthesis of (4-(6-((3-(aminomethyl)benzyl)amino)-9-(3-((4-bromophenyl)amino)-3-oxopropyl) -9H-purin-8-yl)-3,5-diﬂuorophenyl)

boronic acid (Cpd-B). To a solution of (4-(9-(3-((4-bromophenyl)amino)-3-oxopropyl)-6-chloro-9H-purin-8-yl)-3,5-diﬂuorophenyl)

boronic acid (35) (280 mg, 0.52 mmol) in DMF (9 mL) was added 1,3-phenylenedimethanamine (354 mg, 2.6 mmol) and K₂CO₃

(359 mg, 2.6 mmol) and the mixture was heated to 90^ꢁC. After 2 h the mixture was allowed to cool to room temperature, ﬁltered

and submitted to HPLC puriﬁcation with the following condtions: Column: XBridge Prep C18 OBD Column 193150 mm; 5 mm; Mobile

Phase A: Water (0. 05% TFA), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 17% B to 37% B in 8 min; 254 nm; Rt: 6.55min.

This afforded (4-(6-((3-(aminomethyl)benzyl)amino)-9-(3-((4-bromophenyl)amino)-3-oxopropyl)-9H-purin-8-yl)-3,5 -diﬂuorophenyl)

boronic acid (Cpd-B, TFA salt) (102 mg, 26% yield, purity: 100%) as a white solid. ¹HNMR (300 MHz, DMSO-d₆): d 8.37 (s,1H),

7.50-7.43 (m, 3H), 7.39-7.31 (m, 7H), 4.86 (s,2H), 4.53 (t, J = 4.0 Hz, 2H), 4.09 (s,2H), 2.87 (t, J = 4.2 Hz, 2H). LCMS (ESI): m/z 636

[M+H]⁺. ¹⁹FNMR (300 MHz, DMSO-d₆): d: -113.30, -77.23.

Cell Culture

HEK293 cells were grown in High glucose Dulbecco’s Modiﬁed Eagle’s Medium (DMEM), HepG2 were grown in Eagle’s Minimum

Essential Medium (EMEM), both media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium

pyruvate, 100 IU/mL penicillin and 100 mg/mL streptomycin.

HepG2 cells for qPCR analysis were grown in Basal Eagle’s Medium (BME) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM

sodium pyruvate and 1X ITS. After incubation overnight at 37^ꢁC for cell attachment, cell medium was replaced with the same media

without serum.

Cryopreserved human primary hepatocytes are from different donors: Hu1797 (Caucasian male, 77-year-old), Hu1836 (Caucasian

female, 66-year-old), Hu8148 (Caucasian female, 55-year-old) and Hu8279 (Caucasian female, 53-year-old). Cells were thaw in a

37^ꢁC water bath for 2 min, transferred in pre-warmed Cryopreserved Hepatocyte Recovery Medium (CHRM) and centrifuged at

100 g for 10 min at room temperature. Pelleted cells were resuspended in Plating Medium (William’s E Medium without phenol

red supplemented with Primary Hepatocyte Thawing and Plating Supplements). After overnight incubation at 37^ꢁC for cell attach-

ment, cell medium was replaced with Maintenance Medium (William’s E Medium without phenol red supplemented with Primary He-

patocyte Maintenance Supplements).

The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed con-

sents under an IRB/EC approved protocol.

METHOD DETAILS

Screening of Inhibitory Potency in Cells

HEK293 cells were transfected with mock (pcDNA6/V5), TMPRSS6 WT (pcDNA6/TMPRSS6-WT-V5 isoform 1/V5) (UniProt

Q8IU80-4) or TMPRSS6 S762A (pcDNA6/TMPRSS6-S762A-V5 isoform 1/V5) cDNA using Lipofectamine 3000 in 6-well plates. After

24h transfection, cell media were replaced with HCell-100 media containing vehicle (DMSO 0.1%) or 100 nM compound (Table S1)

for 24h. Cell media was collected, and cells were lysed in lysis buffer (1% Triton, 50 mM Tris, 150 mM NaCl, 5 mM EDTA) supple-

mented with Protease Inhibitor Cocktail.

Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e5

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tocytes, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.09.004

For proteolytic activity measurements, release of ﬂuorescence (excitation, 360 nm; emission 460 nm) with 200 mM Boc-QAR-AMC

was measured for 1h at room temperature in an FLx800 TBE microplate reader (Bio-Tek Instruments) in collected cell media. Pro-

teolytic activities (mF.U/min) were normalized to protein quantity in cell lysate (mF.U/min per mg of protein) and reported on

vehicle-treated TMPRSS6-transfected cells activity and expressed as residual activity (%).

For TMPRSS6 immunodetection in cell media, 1 mL of media was concentrated using centrifugal ﬁlters (10,000 NMWL, 21,100 g,

15 min, 4^ꢁC). Concentrated samples were loaded on 12% SDS-polyacrylamide gels under denaturing conditions. As loading and

TMPRSS6 expression controls, 30 mg of cell lysates were loaded on 12% SDS-polyacrylamide gels. Migrated proteins were elec-

tro-transferred onto PVDF membranes. Nonspeciﬁc protein binding was blocked using a solution of 10% skimmed milk in TBS

0.1% Tween-20. Primary antibodies, anti-V5-HRP for concentrated cell media and cell lysate; and anti-GAPDH-HRP for cell lysate,

were diluted in 5% skimmed milk in TBS 0.1 % Tween-20 and incubated overnight at 4^ꢁC. After three sequential washing steps (5 min

each) with TBS 0.1% Tween-20, Clarity Western ECL substrate (Bio-Rad) was put on membranes and blot images were acquired

using a Fusion Pulse system (Vilber Lourmat).

Compounds IC₅₀on TMRSS6 Activity In Cellulo

HEK293 cells were reverse transfected with mock (pcDNA6/V5) or previously described TMPRSS6 WT (pcDNA6/TMPRSS6-WT-V5

isoform 2/V5) (UniProt Q8IU80-2) cDNA (Dion et al., 2018a) using Lipofectamine 3000 in 96-well plates. After 24h transfection, cell

media were washed twice with PBS and HCell-100 media containing vehicle (DMSO 0.1%) or compound was added. For all com-

pounds, 10 tree-fold serial dilutions were tested. The highest doses were 900 nM for Cpd-2 and Cpd-29, 300 nM for Cpd-8 and 30 mM

for Cpd-A and Cpd-B. After 24h, cell media was collected and proteolytic activities were measured in cell media using Boc-QAR-

AMC (mF.U/min). IC₅₀values were derived by a sigmoidal dose-response (variable slope) curve using GraphPad Prism 8 software

from a log([Compound]) versus cell media proteolytic activity plot. GraphPad was used to identify and eliminate outliers (Q=1) and

to assess the goodness of the ﬁts. Only curves presenting a R squared R 0.8 were used for the mean IC50 calculation. All assays

were performed at least three times and data are presented as mean standard deviation (SD).

In Vitro IC₅₀Determination

Recombinant human TMPRSS6, matriptase and hepsin were expressed and puriﬁed as described previously (Beliveau et al., 2009).

´

Recombinant human factor Xa, and furin (R & D Systems) as well as human thrombin from plasma (Millipore Sigma) were obtained

from commercial sources. Enzymatic assays were all performed at room temperature in an assay buffer containing 50 mM HEPES pH

7.4, 1 mM b-mercaptoethanol, 1 mM CaCl₂and 500mg/ml BSA for furin and 50 mM HEPES pH 7.4, 150 mM NaCl and 500 mg/ml BSA

for all other proteases. All assays were performed using a ﬂuorogenic substrate (Boc-RVRR-AMC for furin, Boc-IEGR-AMC for

factor Xa and Boc-QAR-AMC for other proteases) at a concentration equivalent to the Michaelis constant K_M. To determine IC₅₀

,

1 nM (TMPRSS6, matriptase, hepsin, thrombin) or 2 nM (factor Xa, furin) was added to a reaction buffer containing serial-diluted in-

hibitor and the ﬂuorogenic substrate. Proteolytic activity was then monitored by measuring the release of ﬂuorescence (excitation,

360 nm; emission, 441 nm) in a FLX800 TBE microplate reader (BioTek Instruments) for 1 h. All velocities were retrieved, and data

were ﬁtted by non-linear regression analysis to an inhibitory dose-response curve according to the variable slope model (four-param-

eter, 4PL). All assays were performed at least three times and data are presented as mean standard deviation (SD). Nonlinear

regression and statistical analysis were performed using GraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla

California USA, www.graphpad.com).

Compound Toxicity In Cellulo

HEK293, HepG2 and human primary hepatocytes (Hu1880) were seeded in 96 well-plates. After 24h plating, cells were washed with

PBS and media containing vehicle or compound was added to cells. For HEK293 cells, Cpd-2, -8 and -29 were tested at 10 mM and

Cpd-A and -B were tested at 30 mM. HepG2 cells and human primary hepatocytes were tested with Cpd-8, -A and -B at 10 mM. After

24h exposure to compounds, lactate dehydrogenase (LDH) release was measured at 492 nm using Roche’s Cytotoxicity Detection

Kit^PLUS(LDH) as recommended by the manufacturer. For each cell lines, assays were performed in triplicate, three times. Results are

background corrected and presented as the mean Cytotoxicity (%) compared to lysed cells standard deviation (SD).

Compound Effect on Cell Surface Expression in HEK293 Cells

HEK293 cells were seeded on collagen-coated 24 well plates. 24h post-plating, cells were washed with PBS and transfected with

mock (pcDNA6/V5) or TMPRSS6 WT (pcDNA6/TMPRSS6-WT-V5 isoform 2/V5) (UniProt Q8IU80-2) cDNA using Lipofectamine

3000. After 24h transfection, cells were washed twice with PBS and incubated with HCell media containing compound or vehicle.

Concentrations tested were 10 mM for Cpd-2, -8 and -29 and 30 mM for Cpd-A and -B. After 24h treatment, cell media were removed,

cells ﬁxed in 3.7% formaldehyde, diluted in PBS, for 5 min at room temperature (RT). Cells were washed with PBS prior to blocking in

PBS-10% skimmed milk for 1h at RT. Blocking solution was removed and cells incubated with 500 mL of anti-V5 primary antibody

(1:500) diluted in PBS-5% skimmed milk for 1h at RT. The primary antibody containing solution was removed and cells washed twice

with PBS prior incubation with horse anti-mouse HRP conjugated antibody (1:2000) diluted in PBS-5% skimmed milk for 1h at RT.

Cells were washed twice with PBS and incubated with 3,3’,5,5’-tetramethylbenzidine (TMB) for 5 min prior to stopping the reaction

with 500 mL of HCl 2N. Absorbance was measured at 450 nm. Results are background corrected and presented as cell surface

expression fold over vehicle treated cells standard deviation (SD).

e6 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

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Compound Effect on Cell Surface Expression in HepG2 Cells

HepG2 cells were transfected with 5 mg of TMPRSS6 WT (pcDNA6/TMPRSS6-WT-V5 isoform 2/V5) (UniProt Q8IU80-2) and same

quantity of mock (pcDNA6/V5) or pCMV6-Entry/HJV variant a (UniProt Q6ZVN8-1) cDNA using Lipofectamine 3000. After a 24-h

transfection, biotinylation of HepG2 surface proteins was performed with Pierce cell-surface protein isolation kit. Equal quantities

of proteins from cell lysate were precipitated (biotinylated cell-surface proteins) or not (total proteins) with avidin and the resulting

samples were loaded on a 10% SDS-polyacrylamide gels under denaturing conditions. Migrated proteins were electro-transferred

onto nitrocellulose membranes. Nonspeciﬁc protein binding was blocked using a solution of 5% bovine serum albumin (HJV) or 10%

skim milk (V5) in TBS 0.1% Tween-20. Anti-HJV antibodies were diluted in a solution of 2.5% BSA in TBS 0.1% Tween-20, anti-V5

and anti-GAPDH-HRP antibodies were diluted in a solution of 5% skim milk in TBS 0.1% Tween-20. All membranes were incubated

1h at RT. For anti-HJV and anti-V5 antibodies, after three sequential washing steps (5 min each) with TBS 0.1% Tween-20, mem-

branes were incubated with horseradish peroxidase-conjugated secondary antibodies (Rabbit anti-Goat for anti-HJV or Horse

anti-mouse for anti-V5) for 1h at RT and washed three times (5 min) in TBS 0.1% Tween-20. Clarity Western ECL substrate was

used on membranes and blot images were acquired using a Fusion Pulse system. Band intensities were quantiﬁed by densitometry

analysis using Bio-1D software (Vilber Lourmat), normalized with GAPDH expression and reported on vehicle-treated cells

(0.1 % DMSO).

Matriptase/Cpd-8 Crystal Structure

Crystallization of matriptase (ST14) with Cpd-8 was performed at the Contract Research Organization (CRO), Aurigene Discovery

Technologies Limited using previously described methods (Goswami et al., 2013). In brief, matriptase (ST14) (GenBank:

AF057145, GenBank: NM_021978.3) residues 615 to 855 were cloned into pET28a. Over expression was performed by induction

of mid-log phase BL21 DE3 Escherichia coli cells with 1 mM isopropyl-1-thio-b-D-galactopyranoside for 4 hrs at 37^ꢁC. Cell pellets

were washed with lysis buffer (10 mM Tris pH 8.0, 150 mM NaCl and 2 mM DTT) and treated with 50 mg/mL lysozyme for 30 min. The

cells lysed by sonication and the cell debris cleared by centrifugation at 12 000 rpm for 20 min at 4^ꢁC. The inclusion bodies were

washed thoroughly with buffer (10 mM Tris pH 8.0, 150 mM NaCl). Pellets were then solubilized in a buffer containing 10 mM Tris

pH 8.0, 150 mM NaCl, 6 M Guanidinium chloride, 100 mM sodium phosphate pH 8 for 4 hrs by stirring at room temperature. Solu-

bilized protein was diluted in a buffer consisting of 50 mM Tris pH 8.0, 100 mM NaCl, 0.5 M Arginine, 0.2 mM GSSH (Oxidized gluta-

thione), 2 mM GSH (Reduced glutathione), 20 mM CaCl₂, 1 mM EDTA and 1 M Urea at 4^ꢁC for 72 hrs. Protein was dialyzed against 15

volumes of dialysis buffer (25 mM Tris pH 8, 50 mM NaCl and 1 M Urea) at 4^ꢁC. The dialyzed protein was subjected to anion-exchange

column chromatography (HiTrap Q FF, GE Healthcare) and the bound protein eluted by step gradient using buffer 25 mM Tris pH 8.0,

1 M NaCl using AKTA FPLC (GE Healthcare) system. Refolded protein was eluted between 50-150 mM of NaCl concentration was

later passed through superdex-75 column (GE Healthcare) using 50 mM Tris pH 8.0, 500 mM NaCl.

Crystals were grown by Aurigene. Protein was concentrated with benzamidine to 30 mg/mL and crystallization was setup under

conditions with reservoir solution (0.1 Tris, pH 8.2-8.7, PEG800 (18% - 22%) and 200 mM MgCl₂) using hanging drop vapor diffusion

at room temperature. Crystals grew overnight, were harvested and soaked into a solution with Cpd-8. The crystals were transferred

to a cryo-protectant and ﬂash frozen at 100 K. The crystals were sent to the Advanced Photon Source for X-ray data collection at

Beamline 22ID. The diffraction data were processed using XDS (Vonrhein et al., 2011). The crystal structure was determined via

the method of molecular replacement (McCoy et al., 2007) using coordinates from a previously determined crystal structure

(PDB: 4JYT) (Adams et al., 2002, 2010; Goswami et al., 2013). Coot (Emsley and Cowtan, 2004) was used for model building and

reﬁnement was done with the PHENIX software package (Adams et al., 2002). A summary of the data reduction and structure reﬁne-

ment statistics are provided in Table S4. Coordinates of the X-ray structure have been deposited in the Protein Data Bank

(PDB: 6N4T).

TMPRSS6 Homology Model Using MOE Modeling

A homology model of TMPRSS6 catalytic domain was built using the structure of matriptase (PDB: 6N4T) with the ‘‘Homology Model’’

module of the Molecular Operating Environment (MOE) from the Chemical Computing Group. Sequence alignment of catalytic do-

mains of matriptase (Uniprot: Q9Y5Y6) with TMPRSS6 (Uniprot: Q8IU80) using ‘‘Align Sequences Protein BLAST’’ resulted in 45%

and 62% sequence identity and similarity, respectively allowing to build a high-quality model. Ten models were created and the ﬁnal

model was selected using the best score obtained by the Generalized-Born Volume Integral/Weighted Surface area (GBVI/WSA)

scoring method (Corbeil et al., 2012). The ﬁnal model was reﬁned and minimized using Amber10: Extended Huckel Theory (EHT) force

ﬁeld.

Hemojuvelin Cleavage Assay

HepG2 cells were co-transfected in 6-well plates with pcDNA6/V5 or pcDNA6/TMPRSS6-WT-V5 isoform 2/V5 (UniProt Q8IU80-1)

and pCMV6-Entry/HJV variant a (UniProt Q6ZVN8-1) using Lipofectamine 3000. 24h after transfection, cell media were replaced

with HCell-100 media for another 24h. Cell media was collected, and cells lysed. Cell media was concentrated using centrifugal ﬁlters

(10,000 NMWL, 21,100 g, 15 min, 4^ꢁC). Concentrated samples were loaded on 12% SDS-polyacrylamide gels under denaturing

conditions. Migrated proteins were electro-transferred onto nitrocellulose membranes. Nonspeciﬁc protein binding was blocked

using a solution of 5% bovine serum albumin (BSA) in TBS 0.1% Tween-20. Anti-hemojuvelin antibodies were diluted in a solution

of 2.5% BSA in TBS 0.1% Tween-20 and incubated overnight at 4^ꢁC. After three sequential washing steps (5 min each) with TBS

Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e7

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Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

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0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Rabbit anti-Goat) for

1h at RT and washed three times (5 min) in TBS 0.1% Tween-20. Clarity Western ECL substrate was put on membranes and blot

images were acquired using a Fusion Pulse system. HJV cleavage fragments intensities were quantiﬁed by densitometry analysis

using Bio-1D software (Vilber Lourmat) and reported on vehicle-treated cells (0.1 % DMSO). Results are expressed as HJV cleavage

(%). IC₅₀values were derived by a sigmoidal dose-response (variable slope) curve using GraphPad Prism software from a log([Com-

pound]) versus HJV cleavage plot.

TMPRSS6 Western Blot

HepG2 cells were plated on a 96-well plate at a density of 10,000 cells/well in BME media containing serum and supplements. The

cells were incubated overnight to allow for cell attachment. Next day, complete media was changed to serum-free media. Depending

on the conditions, the cells were then treated with the wild-type or the S762A-mutated TMPRSS6 BacMam or no Bacmam. The cells

were incubated overnight. The media was removed from the cells and the cells were washed with PBS twice. The cells were then

treated with RIPA lysis buffer with proteinase inhibitor cocktail and incubated for 20 mins on ice with intermittent vortexing. The lysate

was collected in tubes and was further sonicated to ensure effective lysis. The lysate was then spun at 1200rpm for 10 mins at 4 de-

grees. The supernatant was transferred to a separate tube and BCA assay was performed to determine protein quantities. 40ug of

protein was loaded onto the gel for each of the conditions. The gel was run and the proteins were transferred on the nitrocellulose

membrane. The membrane was incubated overnight with anti-TMPRSS6 antibody to detect TMPRSS6 protein while anti-GAPDH

was used as an endogenous control. The blot was washed thrice with TBS-T buffer and then incubated in Licor secondary antibodies

for 2 hours and washed with TBS-T buffer. The blot was then scanned with Licor Odysser scanner.

HAMP RT-qPCR

HepG2 cells were plated on 96-well plate at a density of 10,000 cells/well in BME media containing serum and supplements. The cells

were incubated overnight to allow for cell attachment. Next day, complete media was changed to serum-free media. Depending on

the conditions, the cells were then treated with wild-type or S762A-mutated TMPRSS6 BacMam. The cells were treated with either

DMSO (0.1%) or TMPRSS6 inhibitor compounds (Cpd-8, Cpd-A and Cpd-B) for 6hr, following which the cells were stimulated

hBMP6 (3 ng/mL) for 24 hrs. The cell media was collected for Hepcidin-25 ELISA. Human primary hepatocytes, freshly thawed cells

from donors 1797, 1836, 8148 and 8279 were plated on a 96-well plate (15,000 cells/well) in Plating media containing FBS. After 24h,

Plating media were replaced by serum free Maintenance media. Cells were treated with either DMSO or 0.1, 1, 5 and 10 mM of Cpd-8,

Cpd-A and Cpd-B inhibitor for 6h. Cells were stimulated using hBMP6 (30 ng/mL) for 24 h. The cells (HepG2 or Hepatocytes) were

lysed using the lysis buffer from the Cells-to-CT kit for 7 min and the reaction was terminated by adding Stop Solution from the kit. RT-

qPCR was performed using the 1-Step TaqMan reagent provided in the kit using the human TaqMan assays for HAMP, B2M and

PPIA on the QuantStudio 6ﬂex. The raw data obtained was analyzed using Array Studio software with 2 housekeeper normalization.

Hepcidin Quantiﬁcation by ELISA

HepG2 cells were plated on 96-well plate at a density of 10,000 cells/well in BME media containing serum and supplements. The cells

were incubated ovenight to allow for cell attachment. Next day, complete media was changed to serum-free media with or without

TMPRSS6 BacMam. The cells were treated with either DMSO (0.1%) or TMPRSS6 inhibitor compounds (Cpd-8, Cpd-A and Cpd-B)

for 6hr, following which the cells were stimulated hBMP6 (3 ng/mL) for 24 hrs. The cell media was collected for Hepcidin-25

ELISA.Human primary hepatocytes, freshly thawed cells from donor 8279, were plated on a 96-well plate (85,000 cells/well) in Plating

media containing FBS. After 24h, Plating media were replaced by serum free Maintenance media. Cells were treated with either

DMSO or 0.1, 1, 5 and 10 mM of Cpd-8, Cpd-A and Cpd-B inhibitor for 6h. Cells were stimulated using hBMP6 (5 ng/mL) for

24 hr. Cell media were collected for Hepcidin-25 quantiﬁcation and number of viable cells was determined using CellTiter-Glo

Cell Viability Assay (CTG) (Promega) on wells attached cells according to the manufacturer protocol. Resulting luminescence was

measured using a TriStar²LB 942 (Berthold Technologies) microplate reader.

Hepcidin-25 was assessed in cell media by a competitive ELISA method using the human hepcidin-25 enzyme immunoassay kit

from Peninsula Laboratories following manufacturer protocol (protocol II, for increased sensitivity and signal). After the termination of

reactions, absorbance at 450 nm was read with a Tecan Inﬁnite M200 microplate reader. Absorbance of known hepcidin concentra-

tions (log[Hepcidin]) was plotted and a standard curve was determined with a sigmoidal dose-response (variable slope) curve using

GraphPad Prism software. Samples hepcidin concentrations was determined using the standard curve. Obtained concentrations

were corrected for cell quantity in well according to the number of viable cells determined previously by CTG assay.

ID1, SMAD7 and TMPRSS6 RT-qPCR

Total RNA extractions of human primary hepatocytes (Hu8279) total RNA extractions were performed from plated cells (24-well plate)

using TRIzol (Invitrogen) with chloroform as recommended by the manufacturer’s protocol. The aqueous layer was recovered, mixed

with 1 volume of 70% ethanol, and applied directly to a RNeasy Mini kit column (Qiagen). DNase treatment on the column and total

RNA recovery were performed according to the manufacturer’s protocol. RNA quality and the presence of contaminating genomic

DNA were veriﬁed as described previously (Brosseau et al., 2010). RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agi-

lent Technologies). Reverse transcription was performed on 1.1 mg of total RNA with Transcriptor reverse transcriptase, random hex-

amers, dNTPs (Roche Diagnostics), and 10 units of RNaseOUT (Invitrogen) following the manufacturer’s protocol in a total volume of

e8 Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019

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Please cite this article in press as: Beliveau et al., Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepa-

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10 mL. All forward and reverse primers were individually resuspended to 20–100 mM stock solutions in Tris-EDTA buffer and diluted as

a primer pair to 1 mM in RNase DNase-free water. Real-time qPCRs were performed in 10 mL in 96-well plates on a CFX-96 thermo-

cycler (Bio-Rad) with 5 ml of 2 x iTaq Universal SYBR Green Supermix (BioRad), 10 ng (3 mL) of cDNA, and 200 nM (ﬁnal concentration;

2 mL) primer pair solutions. The following cycling conditions were used: 3 min at 95^ꢁC and 50 cycles of 15 s at 95^ꢁC, 30 s at 60^ꢁC, and

30 s at 72^ꢁC. Relative expression levels were calculated using the qBASE framework (Hellemans et al., 2007) and the housekeeping

genes YWHAZ, PUM1, and MRPL19 for human cDNA. Primer design and validation were evaluated as described elsewhere (Bros-

seau et al., 2010). In every qPCR run, a no-template control was performed for each primer pair, and these were consistently

negative.

Statistical Analysis

Statistical values including the number of times the data was replicated (‘‘n’’) and statistical signiﬁcance are reported in the Fig-

ure Legends. Statistical analyses were conducted using GraphPad Prism version 7.0c or 8.0.2 (GraphPad Software, La Jolla, CA).

Outliers were removed using the ROUT method (Q = 1%). For activity, IC₅₀and hemojuvelin cleavage assays, data are represented

as the mean SD and signiﬁcant differences between groups were determined using non-parametric Kruskal-Wallis test. For RT-

qPCR and ELISA results, data are represented as the mean SEM and signiﬁcant differences between groups were determined

by one-way ANOVA test. For cell surface localization of TMPRSS6 and HJV in HepG2 treated cells, densitometry results are repre-

sented as the mean SEM and signiﬁcant differences between groups were determined using the one sample t test using 1 (vehicle

treated cells value) as hypothetical mean.

DATA AND CODE AVAILABILITY

The accession number for the matriptase/Cpd-8 structure reported in this paper is PDB: 6N4T.

Cell Chemical Biology 26, 1–14.e1–e9, November 21, 2019 e9

Article Doi

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes

DOI: 10.1016/j.chembiol.2019.09.004

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Article abstract of DOI:10.1016/j.chembiol.2019.09.004

Full text of DOI:10.1016/j.chembiol.2019.09.004

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