Discovery of Selective Matriptase and Hepsin Serine Protease Inhibitors

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Discovery of Selective Matriptase and Hepsin Serine Protease Inhibitors: Useful Chemical Tools for Cancer Cell Biology Vishnu C Damalanka, Zhenfu Han, Partha Karmakar, Anthony J. O'Donoghue, Florencia La Greca, Tommy Kim, Shishir Pant, Jonathan Helander, Juha Klefström, Charles S. Craik, and James W Janetka J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01536 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Journal of Medicinal Chemistry

Discovery of Selective Matriptase and Hepsin Serine Protease Inhibitors: Useful Chemical Tools for Cancer Cell Biology

Vishnu C. Damalankaa, Zhenfu Hana, Partha Karmakara, Anthony J. O’Donoghueb, #, Florencia La Grecab, Tommy Kima, Shishir M. Pantc, Jonathan Helandera, Juha Klefströmc, Charles S. Craikb, James W. Janetkaa, *

aDepartment

of Biochemistry and Molecular Biophysics, Washington University School of

Medicine, St. Louis, Missouri, 63110, USA # Skaggs

School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego,

California, 92093, USA bDepartment

of Pharmaceutical Chemistry, University of California, San Francisco, California,

94158, USA cCancer

Cell Circuitry Laboratory, Research Programs Unit / Translational Cancer Biology &

Medicum, University of Helsinki, P.O. Box 63 (Street address: Haartmaninkatu 8), 00014 University of Helsinki, Finland

ABSTRACT Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA) is associated with unregulated cell signaling and tumor progression through increased MET and

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RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL) which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.

INTRODUCTION Proteolytic regulation of growth factors, cytokines and cell-surface receptors are important processes in normal physiology but often becomes dysregulated in disease1-9. The aberrant activity of the trypsin-like S1 serine proteases matriptase10, hepsin11-12 and hepatocyte growth factor activator (HGFA)13 in cancer leads to increased proteolysis of the growth factors HGF and macrophage stimulating protein (MSP) among others. HGF is the activating ligand for the oncogene MET and MSP is the ligand for RON. MET and RON are structurally related receptor tyrosine kinases (RTKs) which when activated promote downstream effects including cell proliferation, survival, motility, and epithelial to mesenchymal transition (EMT). Increased HGF/MET14-19 and MSP/RON20-22 signaling, prompted by deregulated HGFA, matriptase and hepsin activity23 in the tumor microenvironment24, has been clearly associated with oncogenesis 25-26,

tumor progression27-31 and resistance to targeted therapy in cancer32-45. Loss of epithelial

integrity is a diagnostic hallmark of all advanced epithelial cancers46-47. In healthy epithelial tissue


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the integrity is maintained by complex network of cell-cell junctions, which include tight junctions, adhesion junctions and desmosomes as well as cell contacts to surrounding basement membrane48. The desmosomal localization of hepsin and disruption of desmosomal junction upon hepsin expression suggest important role for hepsin in regulation of epithelial integrity and tumor invasive processes49-52. Therefore, inhibitors of these proteases offer a novel therapeutic strategy53 for targeting these signaling pathways and associated cross-talk for the treatment of cancer and the prevention of tumor progression. The growth factors HGF and MSP are secreted as single-chain zymogens, proHGF and proMSP, which are incapable of activating their respective receptors, MET and RON. Matriptase, hepsin and HGFA are the three most efficient activators of HGF and MSP54. Proteolytic processing of proHGF and proMSP occurs by peptide bond hydrolysis between an Arg and Val residue. HN H 2N

Ac

H N

NH2

O

HN

NH2

NH

O N H

NH2

H N O

NH

N

O N H

NH2

O S

Ac

O

N

N H

HO

Ac-KQLR-kbt (1)

H N O

O

N N H

S O

Ac-SKLR-kbt (2)

Figure 1: Previously reported pro-HGF and pro-MSP substrate-based triplex ketobenzothiazole (kbt) inhibitors of HGFA, matriptase and hepsin.

Subsequent disulfide bond formation leads to an active two-chain protein which can bind to and activate the receptor. This processing is the rate-limiting step in MET and RON kinase signaling. Normally, the activity of these proteases is regulated by the endogenous polypeptide



inhibitors, HAI-1 and HAI-255-60 which are both selective for HGFA, matriptase and hepsin. In multiple tumor types, especially of advanced disease, HAI-1 and/or HAI-2 are either downregulated or silenced through transcription leaving protease activity unchecked and dysregulated61-63. We have recently reported on substrate-based64-65 covalent (Fig. 1) and small molecule benzamidine38,

66-68

triplex inhibitors of HGFA, matriptase and hepsin. We have

demonstrated these novel inhibitors display anticancer properties in breast65, prostate64, colon68, and lung38 cancer cells. In this present communication, we have discovered new and smaller molecular weight dipeptides which contain a P1 Arginine (R) ketobenzothiazole (kbt) warhead that are not triplex inhibitors of HGFA, matriptase and hepsin but rather selective inhibitors of each of the proteases matriptase and hepsin alone. These selective inhibitors were used as discriminating chemical tools to study the individual roles of HGFA, hepsin and matriptase in the regulation of epithelial cell membrane integrity in breast cancer cells.

RESULTS AND DISCUSSION Previously, we conducted several structure activity relationship (SAR) studies based on the tetrapeptide inhibitors, Ac-KQLR-kbt (1) and Ac-SKLR-kbt (2) shown in Figure 164. The significance of these two peptide sequences is that they correspond to the N-terminal portion of the pro-HGF and pro-MSP substrate cleavage sites for HGFA, matriptase and hepsin. To date, our SAR studies have shown that hepsin and HGFA prefer a Leu in the P2 site adjacent to the essential P1 Arg. Other groups have found a preference for a P2 is Leu in hepsin by screening combinatorial substrate libraries using positional scanning of substrate combinatorial libraries/PS-SCL69-70. Published reports on PS-SCL on matriptase substrates71-72 and synthetic studies of inhibitors73-74 have produced confusing results indicating that matriptase prefers small P2 hydrophobic


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sidechains, while others, like ours, indicated other sidechains were also tolerated. For example, the ketothiazole inhibitor Ac-KQFR-kt65 and ketobenzothiazole inhibitor RQYR-kbt73 were shown to have Ki* of 0.69 nM and 0.18 nM, respectively, for matriptase.

Figure 2: PS-SCL evaluation of HGFA substrate specificity.

A. Substrate Specificity of HGFA. To more fully understand the substrate specificity for HGFA and to enable the design of more selective inhibitors for not only HGFA but also of matriptase and hepsin, we performed positional scanning of substrate combinatorial libraries (PS-SCL) on a recombinant form of the HGFA serine protease domain. The PS-SCL method and the same libraries have been utilized extensively in the determination of the substrate specificities of other proteases including matriptase and hepsin. Four compound libraries (P1, P2, P3, P4) were screened where one of the 20 amino acids (Cys is substituted with norleucine) is held constant while the other three positions are an isokinetic mixture of the same 20 amino acids (see experimental for details). In this study, we found that HGFA clearly prefers Arg in the P1 position which is consistent with other S1 trypsin-like serine proteases. From the subsequent results of screening the P2, P3 and P4 positions, we found HGFA discriminates substrate preference mainly in the S2 pocket, strongly preferring Leu, then Met and norleucine (n), followed by Phe, Thr and Val. These data support the substrate cleavage of pro-HGF and pro-MSP by HGFA, as both pro-protein activation sites contain Leu in the P2 position. The P3 position of pro-HGF and pro-MSP are Gln



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and Lys respectively, however, HGFA hydrolyzes fluorescent substrates with P3-Lys 3-fold faster than substrates with P3-Gln. This might suggest HGFA is a more efficient activator of pro-MSP versus pro-HGF. Finally, it appears that HGFA is promiscuous in discerning selectivity at the P4 position, but it is apparent that acidic residues (Glu and Asp) are not preferred in the S4 pocket.

B. Synthesis and evaluation of a dipeptide library of P2-Arg-Kbt inhibitors. To validate the results from PS-SCL studies on all three proteases HGFA, matriptase and hepsin in the P2 position and solidify our SAR for this position of HGFA, matriptase and hepsin inhibitors, we set out to create a comprehensive synthetic inhibitor library of dipeptides consisting of an Arg-kbt warhead in the P1 position and all of the naturally occurring amino acids, except Cys in the P2 position. Shown in Scheme 1, we employed a simple, high-yielding 2-step synthetic strategy (Scheme 1B) to prepare the dipeptide inhibitor library (6) where the side chain protected Arg-kbt (4), synthesized as shown in

Scheme 1: Synthesis of matriptase, hepsin and HGFA inhibitor library of P1 Arg dipeptide kbts. A. O

OH

OMe N

O NH Boc

MeO S O2

H N

NH Boc

a Mtr

NH

HN

NH

b, c Mtr HN

3

HCl NH 4

NH

B.

H 2N

NH NH

4

+

O

H N

O O

d, e OH

P2

O

5

O

H N

O

ACS Paragon Plus Environment

S N H

P2 6

6

N NH2

NH NH

Boc-Arg(Mtr)-OH

S O

N O

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Reagents and conditions: a) HCl·HN(Me)OMe, HATU, iPr2NEt, DMF, RT; b) benzothiazole, nBuLi in hexane, THF, -78 °C, 1.5 h, then 3, -78 °C, 2 h; c) 4 M HCl in dioxane, RT; d) HATU, iPr

2NEt,

DMF, RT; e) TFA/thioanisole/H2O (95:2.5 :2.5 v/v/v), RT, 4 h.

Scheme 1A64, was reacted with the side chain protected Fmoc-amino acids (5) using standard coupling conditions in solution phase followed by global deprotection of the side chains and purification by HPLC. Using the fluorogenic protease substrates, Boc-QLR-AMC (HGFA) or Boc-QAR-AMC (matriptase and hepsin) in previously published kinetic enzyme assays64. Briefly, eleven different concentrations of compound were pre-incubated with protease followed by the addition of the substrate. Inhibition of substrate proteolysis derived fluorescence was monitored kinetically over a period of one hour. We experimentally determined the IC50 values and then calculated75 apparent inhibition constants (Ki *) of each dipeptide for their inhibition of HGFA, matriptase and hepsin. Interestingly, none of the Fmoc dipeptides showed good potency against HGFA (Table 1), with Fmoc-LR-kbt (6l), Fmoc-MR-kbt (6i) and Fmoc-FR-kt (6r) showing the highest activity with Ki*s of 468, 1208 and 963 nM, respectively. This observation is supported by our results from PS-SCL, molecular modeling and previous SAR that additional amino acid side chains at the P3 and P4 positions are necessary for effective HGFA binding and enzyme inhibition.

Table 1: Enzyme activity1,2 and selectivity data for inhibitors against HGA, matriptase and hepsin.

Compound

Matriptase

Hepsin

HGFA

Matriptase

Hepsin

Ki* (nM)

Ki* (nM)

Ki* (nM)

Selectivity

Selectivity

Structure



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1

Ac-KQLR-kbt

0.55

0.09

30

-

6.1

2

Ac-SKLR-kbt

3.1

0.16

33

-

19

6a

Fmoc-RR kbt

3.9

102

8634

26

-

6b

Fmoc-GR kbt

0.13

831

9375

6392

-

6c

Fmoc-KR kbt

0.22

104

>10,000

473

-

6d

Fmoc-AR kbt

0.36

240

7984

667

-

6e

Fmoc-QR kbt

0.65

116

6827

179

-

6f

Fmoc-ER kbt

7.2

372

>10,000

52

-

6g

Fmoc-HR kbt

11

226

6122

21

-

6h

Fmoc-PR kbt

0.50

391

>10,000

782

-

6i

Fmoc-MR kbt

5.4

227

1208

42

-

6j

Fmoc-SR kbt

4.9

555

>10,000

113

-

6k

Fmoc-NR kbt

106

1.7

4771

-

62

6l

Fmoc-LR kbt

253

67

468

-

3.8

6m

Fmoc-DR kbt

294

98

>10,000

-

3.0

6n

Fmoc-YR kbt

25

256

2961

10

6o

Fmoc-IR kbt

486

228

4303

-

2.1

6p

Fmoc-WR kbt

294

302

3197

1.0

1.0

6q

Fmoc-TR kbt

271

180

>10,000

-

1.5

6r

Fmoc-FR kbt

188

764

963

4.0

6s

Fmoc-VR-kbt

18

125

4634

6.9

1K * i

values were calculated using the Cheng and Prusoff equation75 (Ki= IC50/(1+[S]/Km).

2Values

are an average of three experiments with ±10% standard deviation. 8 ACS Paragon Plus Environment

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C. TTSP Selectivity and structure activity relationships (SAR). In contrast to HGFA, multiple members of this dipeptide library are potent inhibitors of matriptase and hepsin. The dipeptides 6a-6j and 6n display high potency for matriptase and >10-fold selectivity over hepsin. In contrast, the dipeptides 6k-6m and 6o are more potent and selective for hepsin over matriptase. In general the analogs with the highest potency against matriptase are more potent than the most potent hepsin inhibitors. For example, the most potent inhibitor of matriptase is Fmoc-GR-kbt (6b) with a Ki* of 0.13 nM while the most potent for hepsin is Fmoc-NR-kbt (6k) with a Ki* of 1.7 nM. To better understand the selectivity of these compounds against these three proteases, virtual docking was employed to identify key interactions. Generally, these compounds adopt similar conformations, with the expected P1 and P2 binding sites occupied by the conserved Arg and then the variable second sidechain, respectively. The Fmoc group was observed to form π-π interactions with the conserved Trp215 (chymotrypsin numbering) in the P4 pocket. First, we sought to understand the good affinity of compound 6a for matriptase. A saltbridge of the P2 Arg with Asp96 (chymotrypsin numbering; Asp101 in



Figure 3. Docking model and binding orientations of 6a to matriptase (A, PDB ID: 3NCL, protein in light blue, ligand in gold) and hepsin (C, PDB ID:1Z8G protein in tan, ligand in green) with respective ligand interaction diagrams (B; matriptase numbering, D; hepsin numbering). matriptase), in addition to the proximity of the Fmoc group by three aromatic residues (Figure 3A, B) provide favorable binding interactions in matriptase. Docking of 6a in hepsin indicates the guanidine of the P2 Arg sidechain can form hydrogen bonds (H-bonds) with the side chain of Asn99 and the backbone carbonyls of both Ser97 and Pro96 (Figure 3C, D; chymotrypsin numbering, 254, 251, 249 respectively for hepsin). However, this orientation requires Asn99 to occupy a space directly above the Trp215 residue, which disrupts hydrophobic π-π interactions 10 ACS Paragon Plus Environment

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with the Fmoc group thus, the hydrophobic interactions in the P4 pocket and hydrophilic interactions in the P2 pocket are both highly favorable for compound 6a in matriptase, but in hepsin require a tradeoff between these two pocket’s ability to simultaneously, and favorably, interact with this ligand, otherwise known as cooperativity. These interactions are likely mimicked with compound 6c which could potentially form a salt-bridge with Asp96 as well, but with the amine unable to form the extensive

Figure 4. Docking model of 6k bound to hepsin. A) The H-bond capabilities of 6k (cyan) with Asn99 of hepsin (tan, PDB ID: 1Z8G). B) The overlap of matriptase (PDB ID: 3NCL) Phe99 and the inability of HGFA (PDB ID: 2WUC) Ser99 indicate a greatly diminished ability to form favorable interactions in the S2 pocket when P2 is Asn. C) Surface view of hepsin-bound 6k with the P2 Asn residue fitting into the S2 pocket. This amide-amide interaction is likely weakened or lost in hepsin when introducing another carbon linker (Gln); see 6e and 6f. H-bonding network observed with the guanidyl group and the hepsin backbone, thus explaining the increased selectivity seen with the Lys sidechain (Table 1). In a more general analysis, the relationship of the inhibitor selectivity profile with the size, branching, charge and H-bond donor ability of the side chain of the P2 amino acid was investigated. When the P2 is changed from Glu (compound 6f) to Gln (compound 6e), the matriptase selectivity increased over 3-fold over hepsin, with a 10-fold increase in potency (Table



1). This increase in activity from carboxylic acid to amide is also observed in 6m to 6k. However, both 6m and 6k are selective for hepsin over matriptase. To understand this result, 6k was docked into hepsin (Figure 4A, C) which indicated a dual H-bond of the P2 Asn sidechain with Asn99 (chymotrypsin numbering, Asn254 in hepsin). This H-bonding capability is disrupted in matriptase, where the homologous Phe99 (chymotrypsin numbering, Phe104 in matriptase) overlaps with this required positioning of the P2 Asn amide (Figure 4B). Additionally, HGFA is unable to facilitate this H-bonding, due to the homologous Ser pointing away from the ligand. The smallest sidechain of Ala at the P2 position (6d) showed excellent selectivity for matriptase over hepsin. This preference increases to over 9-fold when the P2 sidechain is removed altogether with Gly, as in compound 6b, the most potent (Ki* 0.13 nM) and selective (6400-fold over hepsin) inhibitor we identified for matriptase. The preference for small amino acids in P2 is known for matriptase and here its selectivity over hepsin and HGFA can be explained by the diverse S2 amino acid residues. For example, the S2 pocket of matriptase is disrupted in the crystal structure of PDB ID: 3NCL by Phe99 (Figure 5A, C), potentially introducing steric clashes with large amino acids in the P2 position. However, hydrophobic interaction between the α-carbon of glycine and Phe99 would be present in this case. In contrast, hepsin has a larger pocket which is left unfilled with the simple Gly residue at this location (Figure 5B). Interestingly, 6h (Fmoc-PRkbt) is incredibly the second most selective inhibitor for matriptase also indicating small sidechains are preferred.


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Figure 5. Docking model of 6b to A) Matriptase (PDB ID: 3NCL, blue) surface view with 6b (purple) with the P2 position indicated with red circle, B) Hepsin (PDB ID: 1Z8G, tan) surface view with docked compo 6b (green) and P2 position with clear pocket indicated with red circle. C) Overlay of matriptase (blue ribbon, residues) and hepsin (tan ribbons, residues) showing the important Phe99 (chymotrypsin numbering) occupying the P2 pocket for matriptase, homologous residue Asn99 of Hepsin pointed away, to the left. D) Ligand interaction diagram of 6b with hepsin (residue numbering for hepsin). Compound 6d (Fmoc-AR-kbt) is the third most selective and potent inhibitor for matriptase and with a Ki* of 0.36 nM. It is noteworthy that another group74 previously identified AR-kbt 13 ACS Paragon Plus Environment


without the Fmoc group as a potent inhibitor of matriptase with a Ki* of 1.4 nM. Compound 6l with Leu at P2 (Fmoc-LR-kbt) is the second most potent hepsin inhibitor we found (Ki* of 67 nM). This result is consistent with that reported by another group76 who showed Ac-LR-kbt was 60-fold more selective for hepsin over matriptase. The effect of branching at the C3 carbon of the side chain in selectivity was also demonstrated since when the P2 amino acid was changed from Ser (compound 6j) to Thr (compound 6q), the selectivity was altered considerably (Table 1) going from 113-fold selectivity for matriptase to almost a 2-fold preference for hepsin. Overall, the selectivity strongly favoring hepsin and matriptase over HGFA is difficult to interpret using our models since several analogs dock well into the active site and subpockets of HGFA. Since our previously reported tetrapeptides (compounds 1 and 2) show excellent activity against HGFA, it may be indicative of the inability for HGFA to adopt a favorable conformation in the P4 pocket to allow the Fmoc favorable interactions (e.g. with the conserved Trp215). In our development of non-peptide benzamidine inhibitors of matriptase, hepsin and HGFA66, we also observe a similar trend where we can achieve excellent matriptase and hepsin potency and selectivity but not for HGFA. Further biochemical and biophysical studies are in progress to help understand these surprising results but will be reported elsewhere.

D. Selective hepsin inhibitor 6k inhibits cellular proteolytic activity of hepsin and prevents hepsin-mediated loss of desmosomal junctions in breast cancer cells. Loss of epithelial integrity is a defining feature during tumor progression and recently it has been realized that the endogenous inhibitors of HGFA, matriptase and hepsin, HAI-1 and HAI-2 may play a vital role55. It is known that hepsin co-localizes with desmosomes in epithelial cells and elevated oncogenic levels of hepsin disrupt the desmosomal junction52, 77. Herein, we asked if selective hepsin inhibitor


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6k could intervene the desmosome degrading action of hepsin overexpression. We first show that our potent and selective hepsin inhibitor 6k inhibits the cellular-mediated proteolysis of the fluorogenic hepsin substrate Boc-QRR-AMC78 (Km is >3-fold lower than Boc-QAR-AMC) in modified doxycycline inducible MCF10A mammary epithelial cells (MCF10A-Indu20-hepsin) with an IC50 of 92 nM (Figure 6A) while matriptase selective inhibitor 6b has an IC50 >10 µM. In this assay hepsin is membrane-bound on the extracellular surface of MCF10A cells in contrast to the biochemical enzyme assay described earlier which uses a soluble recombinant form of hepsin. It is important to note that hepsin, the substrate and inhibitors are all functioning outside the cell and not expected to have any cellular permeability. Next, we demonstrated that the hepsin selective inhibitor 6k was able to rescue pericellular expression of desmoglein 2, thus indicating inhibition of desmosome degradation in these cells at a concentration of 1 µM (Figure 6B). In contrast, the selective matriptase inhibitor 6b had no effect in

this assay. This significant finding shows that epithelial integrity damaging actions of oncogenic hepsin can be inhibited by selective hepsin inhibitor but not by matriptase inhibitor. Taken together, these results suggest a critical role for hepsin alone and not matriptase or HGFA in epithelial integrity damage and interventional strategy to inhibit transition of in situ tumors to invasive cancer.

A.



B.

C.

Figure 6. Inhibition of cell-based hepsin activity by 6k and 6b. A) Inhibition of Boc-QRR-AMC cleavage in MCF10A cells with doxycycline inducible hepsin (MCF10A-Indu20-hepsin) by matriptase inhibitor 6b and hepsin inhibitor 6k. B) Inhibition of hepsin-mediated loss of desmosomal cadherin desmoglein 2 by 6b and 6k. Representative confocal immunofluorescence microscopy images of MCF10Aindu20-hepsin cells without (-DOX) or with (+DOX) induction of hepsin and pre-treated with indicated inhibitors. Asterisks denote cells with at least two sides bordering the neighboring cells staining positive for desmoglein 2. Scale bar is 10 μm. C) Quantitation of desmosomal integrity. Cells expressing pericellular desmoglein 2 staining in at least two sides were counted positive from the immunofluorescent 16 ACS Paragon Plus Environment

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images. Cells were counted from 10 field of view per treatment from three biological repeats and shown as percentage. Student’s t test was used for statistical analysis.

CONCLUSION We have developed a small library of small but selective and potent dipeptide inhibitors of hepsin and matriptase based on PS-SCL screening of HGFA combined with the previously reported data for matriptase and hepsin. The SAR analysis validates these PS-SCL data and indicates that selectivity is highly dependent on the size, branching, charge and H-bond donor ability of the side chain at P2 position of the dipeptides. When combined with computational docking results, we show that the S2 pocket encompasses differential residue interactions that could be further targeted for further optimization. Small residues such as Gly, Pro and Ala as well as basic residues produce selective and potent ligands for matriptase, while the intermediate size residues such as Asn are the most potent for hepsin. We hypothesize from our models that the Fmoc group introduces steric bulk that cannot be accommodated by the S4 pocket of HGFA. Finally, we demonstrated the selective matriptase and hepsin inhibitors, 6b and 6k, respectively, are useful tools for chemical biology studies. To that end, we have provided strong evidence that hepsin alone and not matriptase or HGFA, plays a key role in diminishing epithelial cell membrane integrity through degradation of desmogelin-2 in breast cancer cells. The low molecular weight, high potency and selectivity, as well as the facile synthesis enable these new inhibitors as exciting leads for further optimization as matriptase and hepsin TTSP serine protease small molecule inhibitor-based therapeutics and chemical tools.

Experimental.



General synthesis, purification, and analytical chemistry procedures. Starting materials, reagents, and solvents were purchased from commercial vendors unless otherwise noted. 1H NMR spectra were measured on a Varian 400 MHz NMR instrument. The chemical shifts were reported as δ ppm relative to TMS using residual solvent peak as the reference unless otherwise noted. The following abbreviations were used to express the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad. High-performed liquid chromatography (HPLC) was carried out on GILSON GX-281 using Waters C18 5μM, 4.6*50mm and Waters Prep C18 5μM, 19*150mm reverse phase columns, eluted with a gradient system of 5:95 to 95:5 acetonitrile:water with a buffer consisting of 0.05% TFA. Mass spectra (MS) were performed on HPLC/MSD using electrospray ionization (ESI) for detection. All reactions were monitored by thin layer chromatography (TLC) carried out on Merck silica gel plates (0.25 mm thick, 60F254), visualized by using UV (254 nm) or dyes such as KMnO4, p-Anisaldehyde and CAMA (Cerium Ammonium Molybdate or Hanessian's Stain). Silica gel chromatography was carried out on a Teledyne ISCO CombiFlash purification system using pre-packed silica gel columns (12g~330g sizes). All compounds used for biological assays are greater than 95% purity based on NMR and HPLC by absorbance at 220 nm and 254 nm wavelengths.

Substrate specificity profiling of HGFA: PS-SCL is a combinatorial peptide library used to uncover the non-prime substrate specificity of proteases. It consists of four sublibraries of fluorogenic substrates with the general structure acetyl-P4-P3-P2-P1-ACC where ACC corresponds to amino-4-carbamoyl-methylcoumarin. In each sublibrary, one position is fixed, whereas the remaining positions contain an isokinetic mixture of 20 amino acids with cysteine omitted and norleucine included. Synthesis of this library has been described previously79. When


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protease mediated cleavage occurs between the P1 amino acid and ACC, a quantitative increase in fluorescence takes place. HGFA (500 nM) was assayed with each sublibrary in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM CaCl2, 0.01% Triton X-100 for 1 h at 25°C. All reactions were run in triplicate in Costar black 96-well round bottom plates (Corning, NY) using a BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader with λex = 380 nm and λem = 460 nm and the photomultiplier tube set to a gain of 63. Initial velocity in relative fluorescence units per second (RFU/s) was calculated using a linear fit of the progress curves with Gen5 software v.2.03.

General docking protocol of dipeptides into hepsin, HGFA, and matriptase: Receptor grid files were prepared using Schrödinger’s (Schrödinger Release 2018-1: Glide, Schrödinger, LLC, New

York,

NY,

2018.)

protein

preparation

wizard

and

the

glide

module

(ref:

https://pubs.acs.org/doi/abs/10.1021/jm051256o). For all grid files, two constraints were applied; an H-bond requirement to the conserved Asp189 (chymotrypsin numbering), and a sulfur atom (as in the benzothiazole ring) within 4 Å radius of the His57 and Ser195 (chymotrypsin numbering). Ligands were prepared using Schrödinger’s ligprep module and docking using glide in SP mode with enhanced conformational sampling. Protonation states of the acidic and basic amino acid sidechain residues are those which exist at pH 7.4. Subsequent ligand interactions were generated in the Maestro UI of Schrödinger. Surface and ribbon views were generated using UCSF Chimera (ref: https://onlinelibrary.wiley.com/doi/abs/10.1002/jcc.20084)

General procedure for the preparation of dipeptides (6): Arg (Mtr)-kbt HCl (4, 02 mmol) was coupled with 19 of the natural Fmoc-L-amino acids (Fmoc-X) with standard protecting groups (5, 0.02 mmol) using HATU (0.02 mmol) as coupling reagent in presence of iPr2NEt (0.1 mmol) in



DMF (0.5 mL) under a nitrogen atmosphere. After stirring for 4h at RT, the reaction was concentrated in vacuo and deprotection of the crude product was accomplished by stirring in 0.3 mL of a TFA-thioanisole-water mixture (95:2.5:2.5) for 4-5h. After concentrating in vacuo, the crude material was dissolved in DMSO and purified using reverse phase HPLC (0.05% TFA/acetonitrile/water gradient). The pure fractions were pooled, frozen and lyophilized to give the pure dipeptides, Fmoc-XR-kbt (6) as white powders.

Fmoc-RR kbt (6a). Yield (75%). 1H NMR (400 MHz, CD3OD)  ppm 8.20 (d, J=7.83 Hz, 1 H), 8.11 (d, J=7.43 Hz, 1 H), 7.79 (d, J=7.43 Hz, 2 H) 7.53 - 7.69 (m, 6 H), 7.22 - 7.43 (m, 2 H), 5.70 (d, J=8.61 Hz, 1 H), 4.36 (dd, J=13.89, 6.85 Hz, 3 H) 4.10 - 4.27 (m, 3 H), 3.05 - 3.25 (m, 6 H), 2.19 (br. s., 1 H), 2.04 (br. s., 1 H), 1.51 - 1.89 (m, 9 H), , ,LCMS (ESI+), calculated m/z 669.3, found 670.5 (M+H+).

Fmoc-GR kbt (6b). Yield (75%). 1H NMR (400 MHz, CD3OD)  ppm 8.02 - 8.26 (m, 2 H), 7.50 - 7.84 (m, 6 H), 7.19 - 7.46 (m, 4 H), 5.70-5.81 (m, 1 H), 4.17 - 4.39 (m, 3 H), 3.72 - 3.94 (m, 2 H), 3.24 (br. s., 2 H), 2.18 (br. s., 1 H), 1.65 - 1.87 (m, 3 H). LCMS (ESI+), calculated m/z 570.2, found 571.4 (M+H+).

Fmoc-KR kbt (6c). Yield (84%). 1H NMR (400 MHz, CD3OD)  ppm 7.98 - 8.09 (m, 1 H), 7.92 - 7.99 (m, 1 H), 7.63 - 7.67 (m, 3 H), 7.41 - 7.53 (m, 3 H), 7.13 - 7.33 (m, 2 H), 6.93 - 7.09 (m, 2 H), 4.95 (q, J=7.30 Hz, 2 H), 4.83 (m, 1 H), 4.58 – 4,72 (m, 2 H), 3.15 - 3.17 (m, 2 H), 2.49 - 2.52 (m, 2 H), 2.17 (br. s., 2 H), 1.84 - 1.91 (m, 4 H), 1.15 (br. s., 2 H), 1.09 (t, J=7.24 Hz, 2 H),LCMS (ESI+), calculated m/z 641.3, found 642.5 (M+H+).


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Fmoc-AR kbt (6d). Yield (65%). 1H NMR (400 MHz, CD3OD)  ppm 8.19 (d, J=7.83 Hz, 1 H), 8.10 (d, J=7.43 Hz, 1 H), 7.78 (d, J=7.43 Hz, 2 H), 7.61 (dd, J=15.46, 6.46 Hz, 4 H), 7.22 - 7.42 (m, 4 H), 5.68 (d, J=9.00 Hz, 1 H), 5.44 (d, J=12.91 Hz, 1 H), 4.69 (br. s., 2 H), 4.10 - 4.38 (m, 2 H), 3.87 (br. s., 1 H), 3.14 (br. s., 3 H), 1.78 (br. s., 2 H), 1.33 (d, J=7.04 Hz, 2 H). LCMS (ESI+), calculated m/z 584.2, found 585.4 (M+H+).

Fmoc-QR kbt (6e). Yield (65%). 1H NMR (400 MHz, CD3OD)  ppm 8.04 - 8.24 (m, 2 H), 7.78 (d, J=7.04 Hz, 2 H), 7.49 - 7.68 (m, 4 H), 7.21 - 7.44 (m, 4 H), 5.63 - 5.74 (m, 1 H), 4.05 - 4.42 (m, 3 H), 3.34 - 3.48 (m, 1 H), 3.08 - 3.24 (m, 2 H), 2.26 - 2.40 (m, 2 H), 2.00 - 2.23 (m, 2 H), 1.64 - 1.96 (m, 4 H). LCMS (ESI+), calculated m/z 641.2, found 642.5 (M+H+).

Fmoc-ER kbt (6f). Yield (65%). 1H NMR (400 MHz, CD3OD)  ppm 8.03 - 8.24 (m, 2 H), 7.79 (br. s., 2 H), 7.62 (dd, J=14.28, 5.28 Hz, 4 H), 7.20 - 7.45 (m, 4 H), 5.67 (br. s., 1 H), 4.11 - 4.41 (m, 5 H), 3.24 (br. s., 2 H), 2.40 (br. s., 2 H), 2.00 - 2.25 (m, 2 H), 1.65 - 1.96 (m, 4 H). LCMS (ESI+), calculated m/z 642.2, found 643.5 (M+H+).

Fmoc-HR kbt (6g). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.76 (br. s., 2 H), 8.03 8.26 (m, 2 H), 7.50 - 7.85 (m, 6 H), 7.15 - 7.44 (m, 4 H), 5.71 (br. s., 1 H), 4.26 - 4.57 (m, 3 H), 4.18 (br. s., 2 H), 2.96 - 3.25 (m, 4 H), 2.19 (br. s., 2 H), 1.77 (br. s., 2 H). LCMS (ESI+), calculated m/z 650.2, found 651.5 (M+H+).



Fmoc-PR kbt (6h). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.19 (m, 1 H). 8.12 (m, 1 H), 7.82 (m, 2 H), 7.64 (m, 3 H), 7.57 (m, 1 H), 7.41 (m., 1 H), 7.16 - 7.37 (m, 3 H), 5.69 (m, 1 H), 4.18 - 4.48 (m, 4 H), 4.09 (m, 1 H), 3.58 (m, 2 H), 3.13 (m, 1 H), 2.31 (m, 1 H), 2.22 (m, 2 H), 1.92 (m, 2 H), 1.82 (m, 2 H), 1.67 (m, 1 H). LCMS (ESI+) expected m/z 610.2, found 611.5 (M+H+).

Fmoc-MR kbt (6i). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.03 - 8.23 (m, 2 H), 7.78 (d, J=7.04 Hz, 2 H), 7.53 - 7.68 (m, 4 H), 7.22 - 7.44 (m, 4 H), 5.67 (br. s., 1 H), 4.12 - 4.44 (m, 4 H), 3.24 (d, J=7.04 Hz, 2 H), 2.51 (d, J=6.26 Hz, 2 H), 2.18 (br. s., 2 H), 2.02 (s, 3 H), 1.67 - 1.92 (m, 4 H). LCMS (ESI+), calculated m/z 644.2, found 645.5 (M+H+).

Fmoc-SR kbt (6j). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.00 - 8.30 (m, 2 H), 7.81 (br. s., 2 H), 7.65 (d, J=16.04 Hz, 4 H), 7.15 - 7.46 (m, 4 H), 5.70 -5.81 (m, 1 H), 4.05 - 4.53 (m, 4 H), 3.80 (br. s., 2 H), 3.13 (br. s., 2 H), 1.78 (br. s., 2 H). LCMS (ESI+), calculated m/z 600.2, found 601.5 (M+H+).

Fmoc-NR kbt (6k). Yield (80%). 1H NMR (400 MHz, CD3OD)  ppm 8.15 - 8.25 (m, 1 H), 8.03 - 8.16 (m, 1 H), 7.79 (d, J=7.43 Hz, 2 H), 7.52 - 7.69 (m, 4 H), 7.21 - 7.44 (m, 4 H), 5.64 - 5.78 (m, 1 H), 4.53 - 4.67 (m, 1 H), 4.29 (d, J=7.43 Hz, 2 H), 4.12 -4.24 (m, 1 H), 3.17 - 3.30 (m, 2 H), 2.68 - 2.85 (m, 2 H), 2.10 - 2.27 (m, 2 H), 1.63 - 1.96 (m, 4 H). LCMS (ESI+), calculated m/z 627.2, found 628.4 (M+H+).


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Fmoc-LR kbt (6l). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.04 - 8.24 (m, 2 H), 7.78 (d, J=7.43 Hz, 2 H), 7.54 - 7.69 (m, 4 H), 7.22 - 7.45 (m, 4 H), 5.65 (br. s., 1 H), 4.13 - 4.41 (m, 5 H), 3.33 - 3.50 (m, 2 H), 3.23 (br. s., 2 H), 2.17 (br. s., 1 H), 1.44 - 1.90 (m, 4 H), 0.79 - 0.97 (m, 6 H). LCMS (ESI+), calculated m/z 626.3, found 627.5 (M+H+). Fmoc-DR kbt (6m). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.19 (d, J=7.43 Hz, 1 H), 8.03 - 8.12 (m, 1 H), 7.78 (d, J=6.65 Hz, 2 H), 7.50 - 7.69 (m, 4 H), 7.20 - 7.44 (m, 4 H), 5.66 (d, J=4.70 Hz, 1 H), 4.55 (d, J=5.87 Hz, 1 H), 4.12 - 4.42 (m, 3 H), 3.46 (br. s., 1 H), 3.22 (br. s., 2 H), 2.61 - 2.95 (m, 2 H), 2.17 (br. s., 1 H), 1.65 - 1.93 (m, 3 H). LCMS (ESI+), calculated m/z 628.2, found 629.4 (M+H+).

Fmoc-YR kbt (6n). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.06 - 8.16 (m, 1 H), 7.79 (br. s., 4 H), 7.58 (br. s., 4 H), 7.12 - 7.43 (m, 2 H), 6.87 - 7.11 (m, 2 H), 6.54 - 6.66 (m, 2 H), 5.61 - 5.75 (m, 1 H), 4.22 - 4.44 (m, 2 H), .15 (br. s., 1 H), 48.16 - 8.25 (m, 1 H), 2.84 - 3.07 (m, 2 H), 2.66 - 2.84 (m, 2 H), 1.96 - 2.24 (m, 2 H), 1.58 - 1.90 (m, 2 H). LCMS (ESI+), calculated m/z 676.2, found 677.5 (M+H+).

Fmoc-IR kbt (6o). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.04 - 8.23 (m, 2 H), 7.79 (d, J=7.04 Hz, 2 H), 7.51 - 7.68 (m, 4 H), 7.22 - 7.43 (m, 4 H), 5.69 (d, J=5.09 Hz, 1 H), 4.11 4.42 (m, 4 H), 3.98 (d, J=7.83 Hz, 1 H), 3.16 - 3.24 (m, 2 H), 2.16 (m, 1 H), 1.65 - 1.86 (m, 3 H), 1.09 - 1.23 (m, 1 H), 0.77 - 0.97 (m, 8 H). LCMS (ESI+), calculated m/z 626.3, found 627.5 (M+H+).



Fmoc-WR kbt (6p). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.06 - 8.24 (m, 3 H), 7.70 - 7.87 (m, 4 H), 7.59 (d, J=8.61 Hz, 2 H), 7.17 - 7.43 (m, 4H), 7.03 - 7.14 (m, 2 H), 6.81 - 6.95 (m, 2 H), 5.54 - 5.68 (m, 1 H), 5.05 - 5.22 (m, 1 H), 4.39 - 4.60 (m, 2 H), 4.26 - 4.39 (m, 1 H), 4.06 - 4.25 (m,1 H), 3.20 (d, J=4.70 Hz, 2 H), 1.93 - 2.17 (m, 2 H), 1.50 - 1.86 (m, 2 H). LCMS (ESI+), calculated m/z 699.3, found 700.5 (M+H+).

Fmoc-TR kbt (6q). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.17 - 8.27 (m, 1 H), 8.07 - 8.14 (m, 1 H), 7.74 - 7.86 (m, 1 H), 7.54 - 7.70 (m, 2 H), 7.19 - 7.44 (m, 3 H), 5.71 - 5.83 (m, 1 H), 4.05 - 4.54 (m, 4 H), 2.12 - 2.28 (m, 2 H), 1.64 - 1.95 (m, 2 H), 1.49 - 1.56 (m, 2 H), 1.19 (d, 3 H). LCMS (ESI+), calculated m/z 614.2, found 615.5 (M+H+).

Fmoc-FR kbt (6r). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.04 - 8.25 (m, 2 H), 7.50 - 7.83 (m, 7 H), 7.04 - 7.45 (m, 9 H), 5.66 (br. s., 1 H), 4.04 - 4.49 (m, 5 H), 3.46 (br. s., 2 H), 2.80 - 3.24 (m, 4 H), 1.72 (br. s., 2 H). LCMS (ESI+), calculated m/z 660.2, found 661.5 (M+H+).

Fmoc-VR-kbt (6s). Yield (70%). 1H NMR (400 MHz, CD3OD)  ppm 8.16 - 8.27 (m, 1 H), 8.03 - 8.13 (m, 1 H), 7.80 (s, 2 H), 7.65 (br. s., 4 H), 7.25 - 7.46 (m, 4 H), 5.66 - 5.78 (m, 1 H), 4.33 4.44 (m, 1 H), 4.14 - 4.32 (m, 2 H), 3.91 - 4.04 (m, 2 H), 2.12 - 2.27 (m, 1 H), 1.96 - 2.10 (m, 1 H), 1.65 - 1.94 (m, 4 H), 0.84 - 1.07 (m, 6 H). LCMS (ESI+), calculated m/z 612.2, found 613.5 (M+H+).

Fluorescent Kinetic Enzyme Inhibitor Assays of HGFA, matriptase, and hepsin: Inhibitors were serially diluted to 11 concentrations (0-20 µM final concentration in reaction) in DMSO (2% 24 ACS Paragon Plus Environment

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DMSO final concentration) and then mixed with either recombinant catalytic domains of HGFA65, matriptase (#3946-SEB, R&D Systems) or hepsin* (#4776-SE, R&D Systems) in black 384 well plates (Corning # 3575. Corning, NY). The final assay concentration for HGFA, matriptase and hepsin 7.5 nM, 0.2 nM, and 0.3 nM, respectively in TNC buffer (25 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.01% Triton X-100, pH 8). After thirty minutes incubation at room temperature, BocQLR-AMC substrate (Km = 37 µM) was added to the HGFA assays and Boc-QAR-AMC substrate was added to the matriptase (Km = 93 µM) and hepsin (Km = 156 µM) assays. The final substrate concentrations for all assays were at the Km for the respective enzymes. Changes in fluorescence (excitation at 380 nm and emission at 460 nm) were measured at room temperature over time in a Biotek Synergy 2 plate reader (Winnoski, VT). Using GraphPad Prism version 6.04 software program, (GraphPad Software, San Diego, CA, www.graphpad.com), a four-parameter curve fit was used to determine the inhibitor IC50s from a plot of the mean reaction velocity versus the inhibitor concentration. The IC50 values represent the average of three separate experimental determinations. Ki* values were calculated using the Cheng and Prusoff equation75 (Ki= IC50/(1+[S]/Km).

*Hepsin Activation: Recombinant Hepsin (10 μg, 0.44mg/mL) was diluted to 2.4 μM in TNC buffer (25 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.01% Triton X-100, pH 8) and incubated at 37°C. After twenty-four hours, the hepsin was diluted in glycerol to 50%. This stock hepsin (1.2 μM) was stored in a -20°C freezer and diluted in TNC buffer for use in assays.

Cell lines



MCF10A cells were obtained from ATCC (Manassas, VA, USA) and maintained in low passages and regularly tested for mycoplasma contamination. Culture protocols for MCF10A have been described in Partanen et al. 2012. Generation and characterization of doxycycline inducible hepsin in MCF10A cells (MCF10A-Indu20-hepsin) is described in Tervonen et. al.52 Cell based activity assay Fluorogenic peptide substrate for hepsin, Boc-QRR-AMC (#4017093, Bachem) was used to analyze the proteolytic activity of hepsin in a cell-based assay. Fifty thousand MCF10A-indu20hepsin cells were seeded per well in 96 well plates and incubated for 24h at the cell. Hepsin was induced by adding 100 ng/mL doxycycline and incubated for additional 24 h. MCF10A-Indu20hepsin cells were pre-treated with either DMSO or increasing concentration of inhibitors for 30 min, followed by the addition of 30 µM substrate. The amount of peptide cleavage was then detected by using FLUOstar Omega (BMG Labtech) at 350em/450ex nm. The amount of substrate cleaved was expressed in terms of % activity with respect to the DMSO control. Inhibitors IC50s were determined using a four-parameter curve fit in GraphPad Inc. Prism software. (Tervonen et. al.52 and Pant et al79).

Immunofluorescence staining protocols, confocal fluorescence microscopy The immunofluorescence staining and confocal fluorescence microscopy protocols are described in Tervonen et al.52 For immunostaining cells were cultured in 2D monolayer on coverslips. The cells were fixed and permeabilized with ice-cold 100% methanol for 5 min at room temperature (RT). Cells were then washed once with PBS and blocked with in 0.1% 26 ACS Paragon Plus Environment

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bovine serum albumin (BSA) in PBS for 30 minutes in RT. Cell were then incubated in primary antibodies Hepsin (Cayman Chemicals) and Desmoglein 2 (Abcam) followed by three washes before incubation with secondary antibodies (Alexa-488/546-conjugated secondary antibodies, Invitrogen) for 45 min at RT. Nuclei were counterstained with Hoechst 33258 (Sigma). Cover slips were mounted on objective glasses with ImmuMount (Thermo Scientific). For quantitative analysis of immunostained cells, digital images were acquired with confocal microscopes Leica TCS SP8 CARS. Contrast and brightness for images were optimized with uniform settings across single experiment using Adobe Photoshop CS5.

Ancillary Information Supplementary Material. 1H NMR, HPLC purity and MS spectral data for all compounds are provided. Molecular formula strings (CSV) *Corresponding Author Email: [email protected]; Phone: 314-362-0509.

Acknowledgements.



Funding for this work was generously provided by the Alvin J. Siteman Cancer Research Fund (Washington University School of Medicine), Susan G. Komen for the Cure Foundation Grants CCR12222792 and CCR499051, and the Academy of Finland and Finnish Cancer Organization.

Abbreviations. TTSPs, Type-II transmembrane serine proteases; HGFA, hepatocyte growth factor activator; HGF, hepatocyte growth factor; SAR, structure activity relationships; kbt, α-ketobenozothiazole; PSSCL, positional scanning of substrate combinatorial libraries; MSP, macrophage stimulating protein; TRKs, receptor tyrosine kinases; EMT, epithelial to mesenchymal transition; HAI-1, hepatocyte growth factor activator inhibitor type-1; HAI-2, hepatocyte growth factor activator inhibitor type-2.

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