# Inhibitors of BMP-1/tolloid-like proteinases: efficacy, selectivity and cellular toxicity

Maya Talantikite, Pascaline Lecorché, Fabrice Beau, Odile Damour, Christoph Becker-Pauly, Wen-Bin Ho, Vincent Dive, Sandrine Vadon-Le Goff, Catherine Moali

► To cite this version:

Maya Talantikite, Pascaline Lecorché, Fabrice Beau, Odile Damour, Christoph Becker-Pauly, et al. Inhibitors of BMP-1/tolloid-like proteinases: efficacy, selectivity and cellular toxicity. FEBS Open Bio, 2018, 8 (12), pp.2011-2021. 10.1002/2211-5463.12540. hal-02342437

HAL Id: hal-02342437

https://hal.science/hal-02342437v1

Submitted on 3 Mar 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# Inhibitors of BMP-1/tolloid-like proteinases: efficacy, selectivity and cellular toxicity

Maya Talantikite¹, Pascaline Lécorché², Fabrice Beau², Odile Damour¹,³, Christoph Becker-Pauly⁴, Wen-Bin Ho⁵, Vincent Dive², Sandrine Vadon-Le Goff¹ ID and Catherine Moali¹

1 Tissue Biology and Therapeutic Engineering Unit (LBTI), UMR5305, CNRS, Univ Lyon, Université Claude Bernard Lyon1, France

2 CEA Saclay, Institut Frédéric Joliot, Direction de la recherche fondamentale, SIMOPRO, Gif-sur-Yvette, France

3 Banque de Tissus et Cellules, Hospices Civils de Lyon, France

4 Institute of Biochemistry, Unit for Degradomics of the Protease Web, Christian-Albrechts-University, Kiel, Germany

5 FibroGen Inc, San Francisco, CA, USA

**Keywords**

astacin; cornea; extracellular matrix; fibrotic disorders; metalloproteinase; protease inhibition

**Correspondence**

S. Vadon-Le Goff, Tissue Biology and Therapeutic Engineering Unit (LBTI), Univ Lyon, Université Claude Bernard Lyon1, CNRS, UMR5305, 69367 Lyon, France
E-mail: sandrine.legoff@ibcp.fr

(Received 9 July 2018, revised 8 October 2018, accepted 8 October 2018)

doi:10.1002/2211-5463.12540

BMP-1/tolloid-like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well-characterized inhibitors and the questions that remain regarding their selectivity and *in vivo* efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG-2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP-1001) and broad-spectrum protease inhibitors (GM6001, actinonin). Their efficacy *in vitro*, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG-2575 and the protein sizzled being very powerful and selective inhibitors of BMP-1, whereas phosphinic peptide RXP-1001 behaves as a broad-spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation.

Bone morphogenetic protein-1 (BMP-1) belongs to a small family of zinc-dependent metalloproteinases, known as the BMP-1/tolloid-like proteinases (BTPs), which also includes in humans mammalian tolloid (mTLD, a splice variant of the *Bmp1* gene) and two homologous proteins, mammalian tolloid-like 1 and 2 (mTLL-1, mTLL-2) [1]. BTPs are composed of an astacin-like catalytic domain followed by several complement C1r/C1s, Uegf, BMP-1 (CUB) and epidermal growth factor (EGF) domains. They are members of

the astacin metalloproteinase subgroup, which also comprises meprin α, meprin β and ovastacin [2]. BMP-1 (also known as procollagen C-proteinase) and related tolloid proteinases were primarily identified as the main enzymes responsible for collagen maturation by excising the C-propeptide of procollagens I-III [3]. Today, around 30 substrates have been described for BTPs [1], turning them into key players in regulating processes such as morphogenesis, tissue repair or tumour progression. These substrates include several fibrillar

**Abbreviations**

bFGF, basic fibroblast growth factor; BMP-1, bone morphogenetic protein-1; BTP, BMP-1/tolloid-like proteinase; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; HRMS, high-resolution mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
procollagens (types I, II, III, V, XI), small leucine-rich proteoglycans (decorin, biglycan, osteoglycin), basement membrane components (laminin 332, procollagen VII, perlecan), lysyl oxidases (lysyl oxidase, lysyl oxidase-like) and mineralization factors (dentin matrix protein-1, dentin sialophosphoprotein). BTPs also activate numerous growth factors of the TGF-β superfamily, either by direct cleavage of their prodomain (GDF-8, GDF-11), or by processing of antagonist molecules (LTBP, betaglycan or CD109 for TGF-β1, chordin for BMP-2/4, etc.) [1]. Importantly, all BTPs seem to have the potential to cleave the same substrates, but their tissue distribution and catalytic efficiencies can differ significantly [4,5]. The catalytic domains of BTPs are very similar [6], and previous studies have shown that comparable results were found when testing inhibitors on several isoforms [7,8]. As the most active and widely distributed isoform, BMP-1 was therefore used in the present study to represent the BTP family.

Fibrotic disorders are forms of defective tissue repair characterized by increased cell proliferation and deposition of extracellular matrix (ECM), particularly fibrillar collagens. They can affect numerous organs such as heart (cardiac fibrosis), liver (liver fibrosis further evolving to cirrhosis), kidneys (renal fibrosis), skin (hypertrophic scars, keloids) and cornea (corneal scarring) and are critical factors in progressive organ dysfunction, altogether contributing to 45% of all-cause mortality worldwide [9]. Through the maturation of ECM components and the activation of growth factors, BTPs play major roles during tissue remodelling. As such, they are recognized as attractive therapeutic targets to prevent or treat various fibrotic pathologies [10–12].

Inhibitors of zinc metalloproteinases are classically designed as small molecules bearing zinc-binding groups which bind in the catalytic site and behave as competitive inhibitors. Following this strategy, a number of synthetic inhibitors of BTPs have been developed, most of them being hydroxamates [10,13–16]. Some have shown promising activity in cell-based [15,17] or animal [18] models of fibrosis; however, none of them have entered the clinic. Hydroxamates can actually suffer from a set of drawbacks linked to *in vivo* instability and poor specificity due to their high affinity for metal ions [19]. Other available inhibitors of BTPs include the *Xenopus* protein sizzled, which was previously found to be very potent and selective [7], and a newly developed phosphinic peptide inhibitor which is a broad-spectrum compound targeting both BTPs and meprins. Phosphinic peptide inhibitors have the advantage of showing remarkable *in vivo* stability and have been successfully used in various animal models to selectively block their targets [20,21].

Though some of these molecules have been designed to spare the activity of some important matrix metalloproteinases (MMPs), their inhibitory activity on other astacins is unknown. This specificity issue is crucial since it has been reported that BTPs and meprins have in common a striking specificity for acidic amino acids in the P₁' position (first residue C-terminal to the scissile bond) [22] and that they share a number of common substrates and a high degree of conservation in their catalytic domains [23]. Importantly, it was previously shown that, similarly to BTPs, meprin α and meprin β are able to perform the C-terminal maturation of procollagens I and III [24,25], making them further potential targets in collagen-related diseases. However, meprins also have distinct functions during various (patho)physiological conditions and have been implicated in inflammation, cancer progression or Alzheimer's disease [23].

Here, we provide the first systematic comparison of three hydroxamates previously reported as powerful inhibitors of BMP-1 [10,13,16,26], the newly developed phosphinic peptide and sizzled. The broad-spectrum metalloprotease inhibitor GM6001 (also known as galardin or ilomastat), and actinonin, a naturally occurring hydroxamate and effective meprin inhibitor, were also included in the study. We have characterized their cellular toxicity and inhibitory efficacy, both *in vitro* and in cultures of HT1080 cells and human primary corneal fibroblasts, and have determined their selectivity towards BTPs and related meprins. Altogether, this study will be very useful to guide the choice of suitable tools for the inhibition of BTPs in future studies.

# Materials and methods

## Proteinases

Recombinant Flag-tagged human BMP-1 (EC 3.4.24.19) was produced in 293-EBNA cells and purified as previously described [27]. Meprin α (EC 3.4.24.18) and meprin β (EC 3.4.24.63) were produced in insect cells, purified and activated according to the protocols described in [28,29].

## Inhibitors (Table 1)

Recombinant His-tagged sizzled (from *Xenopus laevis*) was produced in 293-EBNA cells and purified as described previously [7] (purity > 95% (SDS/PAGE), ESI-MS m/z calcd 35481.8 found 35483.2). S33A was synthesized as described in [13] (purity > 97% (HPLC); HRMS (high-resolution mass spectrometry; ESI (electrospray ionisation)) m/z: [MH+] calcd for C₂₇H₃₃N₄O₆S 541.2120; found 541.2119). FG-2575 was designed and prepared by FibroGen, Inc.
<table><caption>**Table 1.** Protease inhibitors profiled in this study.</caption><thead><tr><th>Name</th><th>Type</th><th>Target</th><th>Formula</th></tr></thead><tbody><tr><td>FG-2575</td><td>Hydroxamate</td><td>BMP-1</td><td rowspan="2">Undisclosed formula</td></tr><tr><td>UK383,367</td><td>Hydroxamate</td><td>BMP-1</td></tr><tr><td>S33A</td><td>Hydroxamate</td><td>BMP-1</td><td></td></tr><tr><td>Sizzled</td><td>Xenopus protein</td><td>BMP-1</td><td>UniProt O73821</td></tr><tr><td>RXP-1001</td><td>Phosphinic peptide</td><td>BMP-1</td><td>Fmoc-A-Y-Q-Ala(PO<sub>2</sub>CH<sub>2</sub>)hPhe-E-P-NH<sub>2</sub></td></tr><tr><td>GM6001</td><td>Hydroxamate</td><td>MMP</td><td></td></tr><tr><td>Actinonin</td><td>Hydroxamate</td><td>Meprin α</td><td></td></tr></tbody></table>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

(purity > 95% (HPLC); MW = 503.6251; HRMS (ESI) m/z: [MH+] within 1.6 ppm of calcd value). The synthesis of the phosphinic peptide RXP-1001 will be described elsewhere (Lecorché *et al.*, in preparation; purity > 95% (HPLC); MS (matrix-assisted laser desorption ionization)

m/z: [MH+] calcd for C<sub>55</sub>H<sub>67</sub>N<sub>8</sub>O<sub>14</sub>P 1095.46; found 1095.57). UK383,367 and actinonin were purchased from Sigma-Aldrich (L'isle d'abeau, France) and GM6001 from Enzo Life Sciences (Villeurbanne, France). All commercial compounds have purity > 98% (HPLC or TLC). Stock
solutions of the inhibitors were prepared at 10 mM in DMSO, except for RXP-1001 (3 mM in EtOH 50%) and sizzled (120 µM in HEPES 20 mM NaCl 0.5M pH 7.4).

## Cell culture

Human primary keratocytes were obtained from human corneas (collected from three donors aged around 70), harvested at the 'Banque de Tissus et Cellules' of the 'Hospices Civils de Lyon' in agreement with French ethical regulations. Informed consent was obtained from the donors' families, and the study conformed to the standards set by the Declaration of Helsinki. Keratocytes were grown in a medium without ascorbate shown to be optimal [30] to maintain their proliferation capacity and prevent their differentiation into myofibroblasts [Dulbecco's modified Eagle's medium (DMEM)/HamF12 1:1 (GE Healthcare, Velizy, France), 10% iron-supplemented newborn bovine serum (Hyclone, Fisher scientific, Illkirch, France), 1% antibiotic/antimycotic solution (AAS, Sigma-Aldrich) and 5 ng·mL⁻¹ basic fibroblast growth factor (bFGF) (Sigma-Aldrich)]. These culture conditions enabled easy detection of both unprocessed procollagen I and released C-propeptide. Cells were used between passage 6 and passage 9.

HT1080 cells (ATCC CCL-121) were grown in DMEM with 10% foetal bovine serum (FBS) (GE Healthcare) and 1% AAS.

Bone morphogenetic protein-1-overexpressing HT1080 cells were obtained as previously described [31] and grown in DMEM with 10% FBS and 500 µg·mL⁻¹ G418 sulphate (GE Healthcare). For inhibitor evaluation, 20 000 cells·well⁻¹ (in 12-well plates) were treated with 2.5 µM of synthetic inhibitors, 0.15 µM of sizzled or with an equal volume of vehicle starting 4 h after seeding. After 2 days, confluent cells were washed with PBS and maintained in serum-free medium (supplemented in inhibitor) for another 24–48 h. Modification of the cell morphology was visualized on a Nikon Eclipse TE300 Inverted Microscope.

## Inhibition assay

To determine inhibitor potencies, the fluorogenic peptide substrate Mca-Y-V-A-D-A-P-K-Dnp (20 µM; Enzo Life Sciences), which can be cleaved by both BTPs and meprins [7], was used as a substrate ($K_m$ = 7.2, 15 and 9 µM for BMP-1, meprin α and meprin β, respectively). It was incubated with 12 nM BMP-1, 1 nM meprin α or 0.5 nM meprin β, in the presence of increasing concentrations of the inhibitors (0.01–100 µM) in 50 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl₂, 0.02% octyl β-D-glucopyranoside. RXP-1001 was also assayed at pH 6.8. The cleavage assays were performed in 96-well nonbinding, flat-bottomed black plates (Corning, Amsterdam, The Netherlands) in 100 µL total reaction volume at 37 °C, and linear increase in fluorescence was monitored during 20 min using an Infinite M1000 Tecan reader

(excitation wavelength: 320 nm; emission wavelength: 405 nm). IC₅₀ values were determined by plotting the ratio of the inhibited versus uninhibited enzyme activities against the inhibitor concentration. Nonlinear regression analysis and calculations were performed using PRISM 5 (GraphPad Software, La Jolla, CA, USA) (n = 3).

## MTT Assay

HT1080 cells and keratocytes were seeded into 96-well culture plates (Corning) at a density of 1500 and 4000 cells·well⁻¹, respectively. After 48 h, cells were treated with various concentrations of the inhibitors, or with the corresponding vehicle, in serum-free medium (with AAS and bFGF for keratocytes), for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich) was then added to each well and incubated with the cells at 37 °C for 3 h. The supernatant was removed and a DMSO:ethanol (1/1) solution added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm using the Infinite reader. 100% values were obtained with cells treated with vehicle only.

## Procollagen cleavage assay

Keratocytes were seeded in 24-well plates (Corning, 20 000 cells·well⁻¹) and grown for 2 days (until confluence reached 90%). They were then washed three times with PBS followed by addition of serum-free, phenol red-free DMEM supplemented with AAS and bFGF and containing different concentrations of the inhibitors. After 48 h, the conditioned medium of each well was collected, clarified by centrifugation and boiled 5 min in SDS/PAGE sample buffer containing DTT. Equal volumes of each sample were loaded on 4–20% polyacrylamide gradient gels (Bio-Rad, Marnes La Coquette, France), followed by transfer to PVDF membranes (Millipore, St Quentin en Yvelines, France) and analysis by western blotting using the LF-41 rabbit antibody directed against the procollagen I C-propeptide (kind gift of L. Fisher, NIH Bethesda, USA) as primary antibody and goat HRP-linked anti-rabbit antibody (Cell Signaling, Leiden, The Netherlands) as secondary antibody. Protein bands were detected using the Clarity western solution (Bio-Rad) and a Fusion camera (Vilber-Lourmat, Marne-la-Vallée, France) then quantified by densitometry using ImageQuant TL (GE Healthcare).

## Results

The molecules selected for this study are as follows (Table 1): hydroxamates FG-2575 (sometimes referred to as BI-1 [8,26]), UK383,367 [16] and S33A [13], the sizzled protein [7] and the recently developed phosphinic peptide RXP-1001. GM6001 and actinonin, whose activity towards BTPs had never been documented, were also included.
## In vitro efficacy

The inhibitors were first profiled at pH 7.4 for their ability to block the cleavage of a fluorogenic peptide substrate by the three major human astacins (BMP-1, meprin α and meprin β) [7] and ranked according to their half-maximal inhibitory concentration (IC₅₀, Table 2). For BMP-1, these potencies can be ordered as follows: FG-2575 > S33A ≈ sizzled > UK383,367 > RXP-1001 >> actinin > GM6001. Thus, the results clearly show that, in these conditions, FG-2575 is the most powerful inhibitor of BMP-1 (IC₅₀ = 2.9 ± 0.3 nM). Together with the Xenopus protein sizzled (IC₅₀ = 8.5 ± 0.5 nM), it also appears to be highly specific for BMP-1 as their IC₅₀ values for meprins are more than 1000-fold higher. With 5- to 20-fold higher IC₅₀s, the other hydroxamates remain good inhibitors of BMP-1, but do not demonstrate the same level of specificity as they also inhibit meprin α with an IC₅₀ around 300 nM. As it was previously demonstrated that inhibition by phosphinic peptides, in contrast to hydroxamates, involves a proton uptake by a glutamate which is conserved in all metzincins [32,33], RXP-1001 was also tested at pH 6.8. This leads to significantly increased

potencies for all 3 enzymes (by a factor between 2.7 and 6.9). Interestingly, RXP-1001 was also the only molecule that showed a high degree of potency against all three enzymes (around micromolar or below at the two tested pH), and thus, it can be viewed as a broad-spectrum inhibitor of human astacins. GM6001 and actinin were inactive on BMP-1 (IC₅₀ > 10 µM), but both appeared as good inhibitors of meprin α, as already reported [34]. Actinin also displayed some inhibitory activity towards meprin β.

## Cellular toxicity of the inhibitors

In order to carefully choose the concentrations to be used in cell-based assays, evaluation of the toxicity of the inhibitors was needed. Viability of human primary keratocytes (corneal stromal cells) and HT1080 cells (fibrosarcoma-derived cancer cells) treated with increasing concentrations of the seven compounds was assessed using the MTT viability assay in serum-free conditions. The two cell types are very different in terms of origin, metabolism and nature of cell-matrix interactions (e.g. no fibrillar collagens are secreted by HT1080 cells) and, as can be seen in Fig. 1, responded

<table><caption>**Table 2.** Half-maximal inhibitory concentrations of the inhibitors for meprin α, meprin β and BMP-1 at pH 7.4 or (\*)6.8. A representative example of data determination (FG-2575 on BMP-1) is given in the Fig. S1.</caption><thead><tr><th rowspan="2">Compound</th><th colspan="3">IC<sub>50</sub> (nM)<sup>a</sup></th></tr><tr><th>Meprin α</th><th>Meprin β</th><th>BMP-1</th></tr></thead><tbody><tr><td>FG-2575</td><td>> 1000</td><td>> 10 000</td><td>2.9 ± 0.3</td></tr><tr><td>UK383,367</td><td>249 ± 17</td><td>> 3000</td><td>55 ± 15</td></tr><tr><td>S33A</td><td>393 ± 82</td><td>> 10 000</td><td>7.9 ± 0.6</td></tr><tr><td>Sizzled</td><td>> 1000</td><td>> 10 000</td><td>8.5 ± 0.3</td></tr><tr><td>RXP-1001</td><td>245 ± 40 (42 ± 9*)</td><td>1300 ± 100 (484 ± 33*)</td><td>359 ± 20 (52 ± 3*)</td></tr><tr><td>GM6001</td><td>185 ± 12</td><td>> 10 000</td><td>> 100 000</td></tr><tr><td>Actinin</td><td>55 ± 11</td><td>744 ± 29</td><td>> 10 000</td></tr></tbody></table>

<figure><img src="image_9.png" /><figcaption>**Fig. 1.** Evaluation of the cellular toxicity of the inhibitors. Viability of human keratocytes (A) or HT1080 cells (B) treated with increasing concentrations of inhibitors was measured using the MTT assay. Cells were seeded and grown for 2 days, washed with PBS and treated with inhibitors in serum-free medium for 48 (keratocytes) or 24 (HT1080) h. 100%: vehicle only. Mean ± SD, n = 3.</figcaption></figure>

<footnote><sup>a</sup>Mean values ± SD of three independent experiments performed in duplicate.</footnote>
differently to the addition of the inhibitors. Keratocytes were found to be very resistant, and even when 30 µM of the compounds was added to the cells, their viability was not significantly reduced (Fig. 1A). HT1080 cells were more sensitive to the cytotoxic effects of the molecules (Fig. 1B), at least in the serum-free conditions of the assay. For this reason, viability of HT1080 was measured after 24 h only, and DMSO concentration was kept below 0.3%, since it started to show some toxicity at around 1%. Whereas actininin and GM6001 were essentially harmless to HT1080 cells, UK383,367 and S33A were strongly cytotoxic at concentrations above 10 µM. FG-2575 was better tolerated, with a 30% drop in viability above 20 µM, which is a concentration 4 orders of magnitude above its active concentration. RXP-1001 was the least toxic compound since even at 30 µM it did not significantly modify HT1080 viability. Sizzled could not be evaluated at concentrations above 15 µM, but even at this relatively high concentration for a protein, it had little influence on HT1080 viability.

## Effect on BMP-1 activity in human keratocytes

To evaluate the inhibitors in a primary cell culture assay, we selected human keratocytes which were previously found to represent a good cellular system to study substrate processing by physiological levels of BMP-1 [31] and which are the main cell type involved in corneal scarring, a condition where BMP-1 is of

therapeutic interest [12]. Adult keratocytes are also known to deposit large amounts of collagen I, and they were used in our study to monitor the effect of the selected molecules on the BMP-1-dependent maturation of procollagen I into pN-collagen and C-propeptides. Subconfluent keratocytes were treated at the single concentration of 2.5 µM (synthetic inhibitors) or 0.25 µM (sizzled) for 48 h in serum-free medium and compared to untreated cells. Media were harvested and assayed for procollagen processing using western blotting and an antibody raised against the C-terminal propeptide of type I procollagen (Fig. 2A). In the absence of inhibitor or in the presence of actininin or GM6001, C-propeptide release was easily detected. In agreement with the *in vitro* data, other compounds were found to efficiently block this processing, as indicated by the decreased amounts of C-propeptide seen in Fig. 2A. Despite the difference in initial concentrations, the same level of inhibition was obtained with sizzled and the hydroxamates (around 90%, as quantified by western blotting, Fig. 2B). In contrast, RXP-1001, which efficiently inhibited recombinant BMP-1, required a concentration of 12 µM to reach the same efficiency (Fig. 2C).

## Effect on HT1080 cells overexpressing BMP-1

HT1080 cells do not express fibrillar collagens but were previously shown to secrete several established or putative BMP-1 substrates [31]. Also, when BMP-1

<figure><img src="image_10.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_12.png" /></figure>

**Fig. 2.** Effects of BTP inhibitors on the BMP-1-dependent maturation of procollagen I. (A–C) Subconfluent human keratocytes were treated with inhibitors or vehicle for 48 h. Medium was harvested, assayed by western blotting and probed with an antibody raised against the C-terminal propeptide of procollagen type I. (A) Treatment with 2.5 µM of inhibitors except sizzled which was assayed at 0.25 µM. (B) Quantification of band intensities in A. (C) Assay of RXP-1001 at two higher concentrations. One representative experiment out of 3 independent experiments is shown in all panels.
**Fig. 3.** Effects of BTP inhibitors on BMP-1-induced HT1080 change of cell morphology: Phase-contrast images of HT1080 cells stably overexpressing BMP-1, grown in the presence of vehicle or inhibitors, 24 h after serum deprivation. Cells treated with BMP-1 inhibitors at 2.5 µM (FG-2575, UK383,367, S33A and sizzled) show normal HT1080 morphology. On the contrary, untreated cells (DMSO panel) or cells treated with molecules that do not inhibit BMP-1 (GM6001, actininon) exhibit changes in morphology and start to detach from culture plates, indicative of the presence of active BMP-1. RXP-1001 at 2.5 µM has an intermediate effect, with only partial reversal of the phenotype. The 0.25% DMSO condition is shown as a representative control of all vehicle conditions. Scale bar: 100 µm.

<figure><img src="image_13.png" /></figure>

is transfected in HT1080 cells, a striking phenotype is induced, with cells losing their rounded shape and starting to detach from the culture plate when reaching high density (Fig. 3, DMSO panel). This change in cell morphology is due to the catalytic activity of BMP-1 since it is not present when a similar construct encoding an inactive BMP-1 mutant (E94A, Anastasi *et al.*, under revision) is used. We took advantage of this marked phenotype, which is a direct readout of BMP-1 activity, to assess the activity of the selected molecules in another type of cell-based assay.

As can be seen in Fig. 3, actininon and GM6001, which do not inhibit BMP-1 *in vitro*, had no impact on the phenotype, with cells behaving like untreated (DMSO) cells. In contrast, established inhibitors of BMP-1 had a remarkable effect, leading to the reversal of the adhesion phenotype when cells were grown in

the presence of these compounds. Similar to the results obtained with keratocyte cultures, sizzled was efficient at a concentration as low as 0.15 µM, with cells maintaining their normal aspect, while the hydroxamates and RXP-1001 required higher concentrations. Phenotype reversal was observed for hydroxamates at 2.5 µM, whereas, like for keratocytes, RXP-1001 was less efficient at this concentration.

## Discussion

This work was initiated in the course of our study on the role of BMP-1 in corneal scarring [12], for which we needed to select a potent, selective and nontoxic BMP-1 inhibitor. We wanted to be sure that only BTPs would be targeted by the selected molecule but realized that, except for sizzled, there was no information about the selectivity of BTP inhibitors towards
meprins. This is of utmost importance since BTPs and meprins have both common and specific substrates and, even for common substrates, the cleavage pattern can be significantly different leading to distinct physiologal effects [24,25]. Also, previous studies on other metalloproteases such as MMPs have demonstrated that the lack of selectivity of metalloproteinase inhibi tors can lead to erroneous conclusions and to dramatic side effects in clinical trials [35]. Noteworthy, FG- 2575, S33A, UK383,367, RXP-1001 and sizzled have been tested on representative panels of MMPs and found to have no significant effects [7,13,16]. In con trast, the natural antibiotic actinonin was previously shown to inhibit aminopeptidases or peptide deformy lases [36] and GM6001 is a broad-spectrum inhibitor of several metalloproteases [37].

The five established BTP inhibitors showed very dif-
ferent inhibition profiles. One major result of our
study is that sizzled and FG-2575 clearly stand out as
the most potent and selective compounds with low
nanomolar activities on BMP-1 and more than 1000-
fold higher IC50s for meprins. Sizzled has previously
been shown to efficiently inhibit the cleavage of other
BMP-1 substrates such as procollagen III, procollagen
V and pro-lysyl oxidase [7]. FG-2575 was also used to
inhibit the cleavage of laminin-332 both in vitro and
in cultures of human keratinocytes and to prevent
pro-lysyl oxidase maturation in 293-EBNA cells over-
expressing the protein [8,17]. These two compounds
actually behaved very well in our cell-based assays
combining efficiency and low cytotoxicity and appear
to be the most attractive tools to modulate BTP activ-
ity both in vitro and in cellulo. BTPs being known to
be important for matrix assembly, their inhibition
could interfere with the deposition of the provisional
matrix needed to support cell adhesion. It is worth
noting that we did not observe any detrimental effect
of the addition of the inhibitors in our culture condi-
tions, 4 (HT-1080) or 48 h (MTT assay) after seeding.
However, further studies will be needed to obtain
information about the usability and pharmacokinetic/
biodistribution properties of sizzled and FG-2575 in
whole-organism experiments.

In our cell-based assays, we found that the inhibi-
tory capacity of sizzled towards BMP-1 is stronger
than that of FG-2575. However, this advantage might
be counterbalanced by the increased production cost
of sizzled compared to a small chemical compound, by
the size of the protein (30 kDa) which may hamper its
diffusion within target tissues and by the possible
immunogenicity of a protein originating from Xenopus.
These various parameters will have to be carefully
evaluated in the future.

Though slightly less active on BMP-1, RXP-1001 is
the only compound tested here to be a broad-spec-
trum inhibitor of BTPs and meprins, which makes it
a very valuable tool to explore redundancy and cross-
reactivity of astacins. In particular, as our previous
results show that meprins can directly contribute to
collagen deposition in tissues [24,25], it could be very
informative to evaluate the impact of blocking all
procollagen C-proteinase activities simultaneously in
fibrosis models. However, RXP-1001 seems somewhat
less potent in cell cultures than other synthetic com-
pounds. This can be compensated by the use of
higher RXP-1001 concentrations without any obvious
toxicity for the two cell types tested here. This appar-
ent loss of inhibitory potency may be explained by its
relatively high pH sensitivity around physiological pH
or the possible sequestration of RXP-1001 by residual
serum proteins present in the medium. As already
observed for this type of compounds [38], this seques-
tration mechanism can be favoured by the hydropho-
bic residues and negative charges born by the
phosphinic peptide. This property can be a real bene-
fit in vivo, since it will hamper rapid clearance and
help the transport of the molecule to pathological
sites. Also, a previous report by Fish et al. [15] sug-
gested that ionized BTP inhibitors may have a lower
efficacy in cellulo compared to neutral compounds,
possibly because of repulsive interactions with the
biomolecules present in the specific microenviron-
ments where BMP-1 is located.

This study also shows that actinonin can be used
to modulate meprin activity with no impact on BTPs,
whereas S33A and UK383,367 target both BMP-1
and meprin α, with little effect on meprin β. Finally,
the widely used MMP inhibitor GM6001 does not
affect BMP-1 and meprin β in the tested concentra-
tion range but a clear off-target activity of this mole-
cule on meprin α is observed. This is an illustrative
example of why careful comparison is necessary
before choosing an inhibitor to probe enzymatic
activity.

In summary, this study reveals striking differences in
the potency and selectivity of the inhibitors tested. It
demonstrates that some of the molecules sometimes
claimed to be selective BMP-1 inhibitors by commer-
cial suppliers (UK383,367) actually also inhibit meprin
α. Also, the marked differences in inhibition profiles
suggest that the full spectrum of activities (from very
selective of one family member to family-wide) can be
achieved and confirm that there are both common and
specific features in the catalytic sites of BTPs and
meprins, as previously shown by crystallography
[6,39]. The study also highlights for the first time the
advantages and drawbacks of a diverse range of inhibitors that can be picked as required to modulate either one, two or the three proteases. It should help to select the right tool for the right experiment and pave the way towards novel *in vivo* applications of BTP inhibitors, for which only few examples have been described to date [18].

## Acknowledgement

We thank Larry W. Fisher for the LF-41 antibody and acknowledge the contribution of the SFR Biosciences (UMS3444/CNRS, US8/INSERM, ENS de Lyon, UCBL) Protein Science Facility (PSF) for HPLC (Eric Diesis) and mass spectrometry (Frédéric Delolme) analyses and for the access to the Tecan reader. The work was supported by the Centre National de la Recherche Scientifique and the Université Claude Bernard Lyon 1, by the Région Rhône-Alpes ARC2 (to MT and CM), by the Agence Nationale de la Recherche (Project Tollreg ANR 2010 BLAN 1526 01 to CM and VD) and by the Deutsche Forschungsgemeinschaft (DFG) SFB 877 (Proteolysis as a Regulatory Event in Pathophysiology, project A9 to CBP).

## Author contributions

SVLG and CM designed and coordinated the study, MT and SVLG performed the experiments, SVLG, PR, FB, WBH, CBP and VD produced and purified the enzymes and/or inhibitors used in this study. OD provided the keratocytes. SVLG, MT and CM analysed and interpreted the data, and wrote the paper. All authors reviewed the results, critically revised the manuscript and approved the final version.

## Conflict of interest

WBH is an employee of FibroGen, Inc. The other authors report no conflict of interests.

## References

1. Vadon-Le Goff S, Hulmes DJS and Moali C (2015) BMP-1/tolloid-like proteinases synchronize matrix assembly with growth factor activation to promote morphogenesis and tissue remodeling. *Matrix Biol* **44**, 14–23.

2. Sterchi EE, Stöcker W and Bond JS (2008) Meprins, membrane-bound and secreted astacin metalloproteinases. *Mol Aspects Med* **29**, 309–328.

3. Kessler E, Takahara K, Biniaminov L, Brusel M and Greenspan DS (1996) Bone morphogenetic protein-1: the type I procollagen C proteinase. *Science* **271**, 360–362.

4. Muir AM, Massoudi D, Nguyen N, Keene DR, Lee SJ, Birk DE, Davidson JM, Marinkovich MP and Greenspan DS (2016) BMP1-like proteinases are essential to the structure and wound healing of skin. *Matrix Biol* **56**, 114–131.

5. Bayley CP, Ruiz Nivia HD, Dajani R, Jowitt TA, Collins RF, Rada H, Bird LE and Baldock C (2016) Diversity between mammalian tolloid proteinases: oligomerisation and non-catalytic domains influence activity and specificity. *Sci Rep* **6**, 21456.

6. Mac Sweeney A, Gil-Parrado S, Vinzenz D, Bernardi A, Hein A, Bodendorf U, Erbel P, Logel C and Gerhartz B (2008) Structural basis for the substrate specificity of Bone Morphogenetic Protein 1/tolloid-like metalloproteases. *J Mol Biol* **384**, 228–239.

7. Bijakowski C, Vadon-Le Goff S, Delolme F, Bourhis J-M, Lécorché P, Ruggiero F, Becker-Pauly C, Yiallouros I, Stöcker W, Dive V *et al.* (2012) Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases. *J Biol Chem* **287**, 33581–33593.

8. Veitch DP, Nokelainen P, McGowan KA, Nguyen TT, Nguyen NE, Stephenson R, Pappano WN, Keene DR, Spong SM, Greenspan DS *et al.* (2003) Mammalian Tolloid metalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin. *J Biol Chem* **278**, 15661–15668.

9. Nanthakumar CB, Hatley RJD, Lemma S, Gauldie J, Marshall RP and Macdonald SJF (2015) Dissecting fibrosis: therapeutic insights from the small-molecule toolbox. *Nat Rev Drug Discov* **14**, 693–720.

10. Turtle ED and Ho W-B (2004) Inhibition of procollagen C-proteinase: fibrosis and beyond. *Expert Opin Ther Pat* **14**, 1–13.

11. Grgurevic L, Macek B, Healy DR, Brault AL, Erjavec I, Cipcic A, Grgurevic I, Rogic D, Galesic K, Brkljacic J *et al.* (2011) Circulating bone morphogenetic protein 1-3 isoform increases renal fibrosis. *J Am Soc Nephrol* **22**, 681–692.

12. Malecaze F, Massoudi D, Fournié P, Tricoire C, Cassagne M, Malbouyres M, Hulmes DJS, Moali C and Galiacy SD (2014) Upregulation of bone morphogenetic protein-1/mammalian tolloid and procollagen C-proteinase enhancer-1 in corneal scarring. *Invest Ophthalmol Vis Sci* **55**, 6712–6721.

13. Turtle E, Chow N, Yang C, Sosa S, Bauer U, Brenner M, Solow-Cordero D and Ho W-B (2012) Design and
synthesis of procollagen C-proteinase inhibitors.

*Bioorganic Med Chem Lett* **22**, 7397-7401.

14 Dowdell SE, Eidam HS, Elban M, Fox RM, Hammond M, Hilfiker MA, Hoang TH, Kallander LS, Lawhorn BG, Manns S *et al.* (2015) Hydroxy formamide derivatives and their use. WO 2015/104684 A1.

15 Fish PV, Allan GA, Bailey S, Blagg J, Butt R, Collis MG, Greiling D, James K, Kendall J, McElroy A *et al.* (2007) Potent and selective nonpeptidic inhibitors of procollagen C-proteinase. *J Med Chem* **50**, 3442-3456.

16 Bailey S, Fish PV, Billotte S, Bordner J, Greiling D, James K, McElroy A, Mills JE, Reed C and Webster R (2008) Succinyl hydroxamates as potent and selective non-peptidic inhibitors of procollagen C-proteinase: design, synthesis, and evaluation as topically applied, dermal anti-scarring agents. *Bioorganic Med Chem Lett* **18**, 6562-6567.

17 Pischon N, Babakhanlou-Chase H, Darbois L, Bin Ho W, Brenner MC, Kessler E, Palamakumbura AH and Trackman PC (2005) A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures. *J Cell Physiol* **203**, 111-117.

18 Reid RR, Mogford JE, Butt R, DeGiorgio-Miller A and Mustoe TA (2006) Inhibition of procollagen C-proteinase reduces scar hypertrophy in a rabbit model of cutaneous scarring. *Wound Repair Regen* **14**, 138-141.

19 Yiotakis A and Dive V (2009) Synthetic active site-directed inhibitors of metzincins: achievement and perspectives. *Mol Aspects Med* **29**, 329-338.

20 Johnson JL, Devel L, Czarny B, George SJ, Jackson CL, Rogakos V, Beau F, Yiotakis A, Newby AC and Dive V (2011) A selective matrix metalloproteinase-12 inhibitor retards atherosclerotic plaque development in apolipoprotein E-knockout mice. *Arterioscler Thromb Vasc Biol* **31**, 528-535.

21 Marchant DJ, Bellac CL, Moraes TJ, Wadsworth SJ, Dufour A, Butler GS, Bilawchuk LM, Hendry RG, Robertson AG, Cheung CT *et al.* (2014) A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity. *Nat Med* **20**, 493-502.

22 Becker-Pauly C, Barré O, Schilling O, Auf dem Keller U, Ohler A, Broder C, Schütte A, Kappelhoff R, Stöcker W and Overall CM (2011) Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates. *Mol Cell Proteomics* **10**, M111.009233.

23 Prox J, Arnold P and Becker-Pauly C (2014) Meprin alpha and meprin beta: procollagen proteinases in health and disease. *Matrix Biol* **44-46**, 7-13.

24 Broder C, Arnold P, Vadon-Le Goff S, Konerding MA, Bahr K, Müller S, Overall CM, Bond JS, Koudelka T, Tholey A *et al.* (2013) Metalloproteases

meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength. *Proc Natl Acad Sci USA* **110**, 14219-14224.

25 Kronenberg D, Bruns BC, Moali C, Vadon-Le Goff S, Sterchi EE, Traupe H, Böhm M, Hulmes DJS, Stöcker W and Becker-Pauly C (2010) Processing of procollagen III by meprins: new players in extracellular matrix assembly? *J Invest Dermatol* **130**, 2727-2735.

26 Moali C, Font B, Ruggiero F, Eichenberger D, Rousselle P, François V, Oldberg Å, Bruckner-Tuderman L and Hulmes DJS (2005) Substrate-specific modulation of a multisubstrate proteinase: C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1. *J Biol Chem* **280**, 24188-24194.

27 Blanc G, Font B, Eichenberger D, Moreau C, Ricard-Blum S, Hulmes DJS and Moali C (2007) Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity. *J Biol Chem* **282**, 16924-16933.

28 Becker C, Kruse M-N, Slotty KA, Köhler D, Harris JR, Rösmann S, Sterchi EE and Stöcker W (2003) Differences in the activation mechanism between the alpha and beta subunits of human meprin. *Biol Chem* **384**, 825-831.

29 Becker-Pauly C, Höwel M, Walker T, Vlad A, Aufvenne K, Oji V, Lottaz D, Sterchi EE, Debela M, Magdolen V *et al.* (2007) The α and β subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation. *J Invest Dermatol* **127**, 1115-1125.

30 Builles N, Bechetoille N, Justin V, Ducerf A, Auxenfans C, Burillon C, Sergent M and Damour O (2006) Development of an optimised culture medium for keratocytes in monolayer. *Biomed Mater Eng* **16**, S95-S104.

31 Delolme F, Anastasi C, Alcaraz LB, Mendoza V, Vadon-Le Goff S, Talantikite M, Capomaccio R, Mevaere J, Fortin L, Mazzocut D *et al.* (2015) Proteolytic control of TGF-β co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics. *Cell Mol Life Sci* **72**, 1009-1027.

32 Czarny B, Stura EA, Devel L, Vera L, Cassar-Lajeunesse E, Beau F, Calderone V, Fragai M, Luchinat C and Dive V (2013) Molecular determinants of a selective matrix metalloprotease-12 inhibitor: insights from crystallography and thermodynamic studies. *J Med Chem* **56**, 1149-1159.

33 Rouanet-Mehouas C, Czarny B, Beau F, Cassar-Lajeunesse E, Stura EA, Dive V and Devel L (2017) Zinc-metalloproteinase inhibitors: evaluation of the
complex role played by the zinc-binding group on
potency and selectivity. *J Med Chem* **60**, 403–414.

34 Kruse M-N, Becker C, Lottaz D, Köhler D, Yiallouros I, Krell H-W, Sterchi EE and Stöcker W (2004) Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors. *Biochem J* **378**, 383–389.

35 Coussens LM, Fingleton B and Matrisian LM (2002) Matrix Metalloproteinase inhibitors and cancer-Trials and tribulations. *Science* **295**, 2387–2392.

36 Ganji RJ, Reddi R, Gumpena R, Marapaka AK, Arya T, Sankoju P, Bhukya S and Addlagatta A (2015) Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: crystal structure in complex with E. coli aminopeptidase N. *Protein Sci* **24**, 823–831.

37 Saghatelian A, Jessani N, Joseph A, Humphrey M and Cravatt BF (2004) Activity-based probes for the proteomic profiling of metalloproteases. *Proc Natl Acad Sci USA* **101**, 10000–10005.

38 Bordenave T, Helle M, Beau F, Georgiadis D, Tepshi L, Barnes M, Ye Y, Levenez L, Poquet E, Nozach H *et al.* (2016) Synthesis and *in vitro* and *in vivo* evaluation of MMP-12 selective optical probes. *Bioconjug Chem* **27**, 2407–2417.

39 Arolas JL, Broder C, Jefferson T, Guevara T, Sterchi EE, Bode W, Stocker W, Becker-Pauly C and Gomis-

Ruth FX (2012) Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane. *Proc Natl Acad Sci USA* **109**, 16131–16136.

**Supporting information**

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Fig. S1.** Representative example of inhibition assay and IC₅₀ determination. (A) Inhibition of BMP-1 (12 nM) by increasing concentrations of FG-2575 (0–100 nM, each concentration in duplicate). Linear increase in fluorescence was monitored during 20 min (substrate concentration 20 µM, excitation wavelength 320 nm, emission wavelength 405 nm). Enzyme activity is given by the slope of the curve, determined by linear regression using Excel. One representative experiment out of 3 independent experiments is shown. (B) IC₅₀ determination of FG-2575 on BMP-1. Plot of % of residual activity (ratio of inhibited versus uninhibited enzyme activities) against FG-2575 concentration. Mean ± SD of 3 independent experiments performed in duplicate. IC₅₀ is determined by nonlinear regression using GraphPad Prism 5.