Synthesis, biological evaluation and molecular modeling of pseudo-peptides based statine as inhibitors for human tissue kallikrein 5

Accepted Manuscript Synthesis, Biological Evaluation and Molecular Modeling of Pseudo-Peptides based Statine as Inhibitors for Human Tissue Kallikrein 5 Lucas V.B. Hoelz, Bruna C. Zorzanelli, Pedro Henrique R. de A. Azevedo, Silvia G. Passos, Lucas R. de Souza, Marcelo Zani, Sergio Pinheiro, Luciano Puzer, Luiza R.S. Dias, Estela M.F. Muri PII:

S0223-5234(16)30068-X

DOI:

10.1016/j.ejmech.2016.01.060

Reference:

EJMECH 8350

To appear in:

European Journal of Medicinal Chemistry

Received Date: 14 October 2015 Revised Date:

11 January 2016

Accepted Date: 30 January 2016

Please cite this article as: L.V.B. Hoelz, B.C. Zorzanelli, P.H.R. de A. Azevedo, S.G. Passos, L.R. de Souza, M. Zani, S. Pinheiro, L. Puzer, L.R.S. Dias, E.M.F. Muri, Synthesis, Biological Evaluation and Molecular Modeling of Pseudo-Peptides based Statine as Inhibitors for Human Tissue Kallikrein 5, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Synthesis, Biological Evaluation and Molecular Modeling of Pseudo-Peptides based Statine as Inhibitors for Human Tissue Kallikrein 5

Lucas V. B. Hoelz a, Bruna C. Zorzanelli b, Pedro Henrique R. de A. Azevedo a, Silvia

Luiza R. S. Dias a, Estela M. F. Muri a, 1

Laboratório de Química Medicinal, Departamento de Tecnologia Farmacêutica,

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a

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G. Passos c, Lucas R. de Souza c, Marcelo Zani c, Sergio Pinheiro b, Luciano Puzer c,

Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mario Viana 523,

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Santa Rosa, 24241-000, Niterói, RJ, Brazil

Departamento de Química Orgânica, Instituto de Química, Universidade Federal

Fluminense, Outeiro de S. João Batista s/n Centro 24020-141, Niterói, RJ, Brazil. c

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua

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Arcturus3, São Bernardo do Campo, SP, Brazil.

ABSTRACT: Human kallikrein 5 (KLK5) is a potential target for the treatment of skin

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inflammation and cancer. A new series of statine based peptidomimetic compounds were designed and synthesized through simple and efficient reactions. Some KLK5

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inhibitors (2a-c compounds) were identified with nanomolar affinity showing Ki values of 35−38nM. Our molecular modeling studies suggest that the inhibitors binding at the KLK5 through H-bond interactions with key residues (mainly His108, Gln242, Gly243, Ser245, and Ser260), disrupting the correlated motions mainly among the Ile67-Tyr127, Glu128-Val187, and Gly237-Ser293 sub-domains, which seems to be crucial for KLK5 activity. Therefore, we believe that these findings will significantly facilitate our

1

Corresponding author. E-mail: [email protected]

ACCEPTED MANUSCRIPT understanding of the conformational dynamics in the course of KLK5 inhibition and, consequently, the development of more potent molecules as alternative for cancer treatment.

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Keywords: Peptidomimetics; statines; kallikreins; inhibitors; molecular dynamics.

ACCEPTED MANUSCRIPT Introduction Some enzymes serine proteases are involved in diseases for which no effective therapy is available. In this context merits attention the kallikreins, which are involved in epithelial disorders, bacterial infections and in certain cancer metastatic processes.

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The human tissue kallikreins comprise a family of 15 kallikrein-like serine peptidases (KLK1-KLK15) detected in almost every tissue of the human body and that actively participate in many physiological and pathological events [1-8].

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The KLK5 is a member of the kallikrein family that exhibits trypsin-like activity. Together with KLK7, it seems to be the more abundantly expressed in human

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skin and they have an important role in skin physiology and in the development of diseases related to epithelial disorders [4, 6, 9-11]. In addition, the over expression of KLK5 seems to be involved in endocrine-related malignancies, including ovarian, breast, and testicular cancer [12-13]. Indeed, while KLK7 is moderately expressed in

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the normal ovary, it is over expressed in ovarian carcinoma tissues at the mRNA and/or protein levels. The results of an in vivo model strongly suggest that the over expression of KLK4, KLK5, KLK6, and KLK7 contributes to ovarian cancer progression [14].

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Furthermore, more recently we have described the ability of KLK5 to hydrolyze plasminogen releasing active plasmin [15], which can represent a new way to

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understand its involvement in cancer process, in which KLK5 appears to be upregulated. These observations have made KLK5 and KLK7 as new targets for the discovery of active substances for the treatment of cancer [1, 8, 11]. However, so far there are few inhibitors described for both enzymes [16], including the two natural isocoumarins, Vioxanthin and 8,8’-paepalantine [17]. From the knowledge that peptidomimetics are recognized inhibitors for serine proteases [18-22], more recently we introduced the first isommanide-based peptidomimetics as a novel class of inhibitors

ACCEPTED MANUSCRIPT of the KLK5 and KLK7 in the low micromolar range [23-24]. Based on these previous works, herein we report a new series of isommannide-based statine peptidomimetics conjugates as potent and specific inhibitors for KLK5.

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Results and Discussion Chemistry

The highly stereoselective synthesis of the β-hydroxy-γ-amino acid 1 possessing

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the statine backbone (Scheme 1) was previously described in four steps and good overall yield from L-phenylalanine [25-26]. The condensation reactions of acid 1 and

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the methyl ester hydrochlorides of the natural amino acids L-phenylalanine (L-PheOMe.HCl), L-Valine (L-Val-OMe.HCl) and L-Proline (L-Pro-OMe.HCl) by employing the classical EDC/HOBt/NMM protocol [21] afforded the corresponding pseudopeptides 2a-c in high yields. The subsequent reactions of 2a-c with lithium hydroxide

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under the well known non-epimerizing conditions led to the respective acids 3a-c in good yields. The last step consisted of peptide bond formation by the condensation reactions of acids 3a-c with the known amine 4 (Scheme 1) derived from isommanide

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[19], which furnished the corresponding isomannide-based statine peptidomimetics

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conjugates 5a-c in high yields.

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Scheme 1. Synthetic route of statin based compounds and structure of amine 4. i) LPhe-OMe.HCl or L-Val-OMe.HCl or L-Pro-OMe.HCl, EDC.HCl, HOBt, NMM, CH2Cl2, 0ºC, 24h; 80% for 2a, 72% for 2b and 70% for 2c. ii) LiOH, THF, MeOH, H2O, 0ºC, 17h; 72% for 3a, 85% for 3b and 94% for 3c. iii) 4, EDC.HCl, HOBt, NMM, CH2Cl2, 0ºC, 24h; 90% for 5a, 82% for 5b and 86% for 5c.

Inhibition Assays

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Before to start with the inhibition assays it was carried out tests of solubility with all synthesized compounds, where the compounds 5a-c were insoluble in the aqueous buffer used in the enzymatic assays, in concentrations higher than 1.0 µM. At

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that concentration, it was not detected any inhibitory activity of the compounds 5a-c against KLKs. The inhibitory profile of all other compounds (2a-c and 3a-c), which

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were soluble in the test conditions, against KLK1, KLK5, KLK7 and KLK6 was initially examined in terms of residual activity of the enzyme, using 10 µM of inhibitor concentration and incubation for 5 min with the enzyme, before adding the substrate. These initial tests showed only the compounds 2a-c presented inhibitory activity, and only against KLK5 (Table 1). The compounds 3a-c did not affect the activity of KLK1, KLK5, KLK6 and KLK7, even when the concentration of the inhibitors was increased to 100 µM. We believe this selective inhibition for KLK5 presented by the derivatives 2a-c may be explained by the difficult of the enzyme to interact with the extremities of

ACCEPTED MANUSCRIPT the peptide compounds, once all tissue kallikreins are abroad characterized as endopeptidases. There are examples showing the capacity of endopeptidases to hydrolyze the protected C- or N-terminus of proteins or peptides, where these enzymes are less effective against the same substrate with the unprotected extremity [27].

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The Table 1S presents a comparison among the structures of the compounds 2a, 3a and 5a, where it is possible to observe the minimal differences among them. The compounds 2a and 5a present protected extremities (-CO-OCH3 and isomannide group,

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respectively), while 3a presents a free extremity (-CO-OH). The Table 1S also presents the peptide bond and the residues (P2, P1 and P1’, according to Schechter and Berger

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1967) [28] of interaction with the enzyme.

To a better understanding of the interaction between these kallikreins and the three compounds assayed, we have performed a detailed kinetic study to determine the mechanism of inhibition. Lineweaver–Burk double-reciprocal plots show the intercepts

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of all lines, both in the absence of and at four different inhibitor concentrations, converging at the y-axis (1/Vmax), whereas the slope (Km/Vmax) and x-axis intercepts (1/Km) vary with inhibitor concentration. Therefore, the Vmax values remain constant,

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where as the apparent Km values increase with increasing inhibitor concentrations. This behavior is consistent with a mutually exclusive binding mode between the inhibitor

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and substrate (Figure S1, see supplementary material). Since IC50 values can vary with substrate concentration for competitive inhibitors, the enzyme dissociation constant (Ki) represents the better way to compare different molecules as potential inhibitor scaffolds. Using the data depicted in Figure S1, it was possible to determine the Ki values of each competitive inhibitor by plotting the reciprocal of the initial velocity (1/V0) versus a series of inhibitor concentrations at constant substrate concentrations. On the plots the

ACCEPTED MANUSCRIPT x-intercept indicates the Ki showed in Table 1. It was possible to observe that all three compounds presented similar values of Ki in a range of 0.12-0.13 µM. The KLK5 is well know as a trypsin-like enzyme, presenting specificity for substrate with basic residues at P1 position, which means that the S1 subsite of KLK5

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prefers to interact with residues of Arginine or Lysine. A very good review written by Goettig and co-workers (2010) [29], based on the statistical approach of the MEROPS specificity matrices (http://merops.sanger.ac.uk/), reports that KLK5 also can interact

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with residues of Glycine at P1 position. In fact, of all KLKs, KLK5 is the only one with the capacity to accommodate a residue of Glycine at its S1 subsite. In present work, the

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use of statin based pseudo-peptides has the advantage to allow the placement of a Glycine-like residue in the P1 position. Examining the structure of compounds 2a-c (Table 1), and taking into account the location of the peptide bond, it is possible to infer that all the three compounds, presenting a Glycine-like residue in their probably P1

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position, act as selective inhibitors for KLK5.

Taken a look into the structure of the three compounds, it is possible to observe that the differences between them is most regarding to the substitutes R1, and R1 = R2

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for 2c (scheme 1, and Table 1), where 2a, 2b and 2c present a benzyl, an isopropyl, and methylene groups of pyrrolidine ring, respectively. These substitutes represent the

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lateral chain of the amino acids Phenylalanine, Valine and Proline, respectively, and probably they are interacting with the S1’ subsite of KLK5, which presents a broad specificity for different kinds of residues of amino acids [29].

ACCEPTED MANUSCRIPT Table 1. IC50 and Ki values of Statin based peptidomimetics with three different residues at P1’ position. KLK5 Compound

OH

O OCH3 N H

NHBoc

O

112 ± 21

0.12 ± 0.01

115 ± 11

0.13 ± 0.01

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2b

µM) Ki (µ

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2a

IC50 (nM)

122 ± 17

2c

Molecular Docking

0.13 ± 0.01

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To compare the binding mode of all inhibitors (2a-c) to the KLK5 enzyme, we carried out ten docking simulations for each ligand (totalizing 50 poses per inhibitor)

analysis.

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and, consequently, the poses with the lowest energy for each ligand was selected for

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The molecular docking study showed that all inhibitors have similar modes of interaction with KLK5 (Figure 1, Table 2S; see Supplementary Material). The interaction energy of the complexes between the inhibitors and KLK5 was also analyzed (Table 2S). The hydrogen bonds (H-bonds) and repulsive steric interactions were mapped using a ligand-map algorithm, generated by the MVD program [30]. In summary, all inhibitors interact via H-bonds with the same residues (i.e, Cys241, Gln242, Asp244, Ser245, Ser260) in a similar way to other peptidomimetic inhibitors of KLK5 [23], where 2a and 2c inhibitors make additional interactions with

ACCEPTED MANUSCRIPT Cys73 and Gly243 residues. The repulsive steric interactions are also very similar (Table 2S). In addition, the three inhibitors present close values of H-bond, steric

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interaction, and Moldock Score energies.

Figure 1. (A) Binding modes of the 2a (purple), 2b (green) and 2c (cyan) inhibitors to KLK5 active site. Representation of hydrogen bond interactions in each complex: (B) 2a-KLK5; (C) 2b-KLK5; and (D) 2c-KLK5. The hydrogen bonds are represented by black interrupted lines. The inhibitors and KLK5 residue structures are in stick model and colored by atom: the nitrogen atoms are shown in blue, the oxygen in red, and the carbon chain are filled with the specific color for each inhibitor: 2a-KLK5, purple; 2bKLK5, green; and 2c-KLK5, cyan.

ACCEPTED MANUSCRIPT Structure and Dynamics of KLK5 in the Free and Bound Systems In order to study the dynamic behavior of the enzyme before and after binding to the inhibitor (i.e., free and inhibitor-bound enzyme), we have performed the first study using 150 ns of molecular dynamics (MD) simulations of two KLK5 aqueous systems:

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KLK5APO (free KLK5) and KLK52B (2b-KLK5complex). Taking into account the structural similarity among 2a-c derivatives, we selected 2b structure to carry out our molecular modeling studies. In our MD simulations, the X-ray crystal structure of the

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KLK5 in apo form (PDB ID: 2PSX) [31] was used as the starting structure. The structure of KLK5 catalytic domain contains 227 amino acids, including the catalytic

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triad residues (His108, Asp153 and Ser245; numbering scheme of UniProtKB). The analysis of KLK5 secondary structure elements for both systems (KLK5APO and KLK52B) shows there is no significant variation in the stability during the entire simulation time (150 ns), where the inhibitor binding seems do not change the protein

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fold (see Supplementary Material, Figures S2 and S3).

By comparing the root-mean-square deviation values of all KLK5 Cα-atoms (Cα-RMSD) relative to the starting structures (Figure 2A), we have observed that both

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systems, KLK5APO (black line) and KLK52B (red line), achieve the stability at about 50

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ns, presenting average RMSD values of 1.0 and 2.0 Å, respectively, in the last 100 ns. In the initial 50 ns of the simulation time, the RMSD values for both systems are practically identical, probably because the two systems (KLK5APO and KLK52B) were assembled from the same initial 3D structure (PDB ID: 2PSX) [31]. Then, to analyze the structure stability in each segment of the protein, we have calculated the root-mean-square fluctuations of all KLK5 Cα-atoms (Cα-RMSF) for both systems, KLK5APO (black line) and KLK52B (red line), over the last 100 ns of simulation (Figure 2B). In general, the Cα-RMSF plot shows that there is no major

ACCEPTED MANUSCRIPT variation in the structure fluctuation with the exception of the loop 3 (L3; region between Gly119 and Tyr127) and the loop (L4; region between His142 and Leu154). Interestingly, the presence of the inhibitor decreases the L3 fluctuation (2 Å), while at L4 region the atomic fluctuation is increased (1 Å).

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In addition, the analysis of the radius of gyration (Rg) (Figure 2C) shows only a slight increase of 0.50 Å for KLK5APO system (black line) in comparison with KLK2B system (red line), during the last 50 ns of simulation. Again, as in the RMSD analysis,

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Rg values for both systems are practically identical in the first half of the simulation

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time, probably due to the same reason previously discussed.

Figure 2. (A) The root-mean-square deviation (Cα-RMSD) and (B) root-mean-square fluctuation (Cα-RMSF) analysis of all Cα-atoms of kallikrein 5 (KLK5) in the KLK5APO (black line) and KLK52B (red line) systems. (C) The radius of gyration (Rg) analysis of KLK5 in the KLK5APO (black line) and KLK52B (red line) systems. The CαRMSD and Rg analysis were carried out during all the simulation time, while the CαRMSF analysis shows the residues fluctuation of the residues during the last 100 ns of simulation.

ACCEPTED MANUSCRIPT Analysis of Hydrogen Bonding Interactions in KLK5-2b Complex The 2b inhibitor moved away from its initial position, approximately 0.3 Å (Supplementary Material Figure S4), through 150 ns of MD simulation. During the last 100 ns of the KLK52B simulation, the binding mode of 2b was restricted by hydrogen

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bonds (H-bonds) mainly with His108, Gln242, Gly243, Ser245, and Ser260 residues (Figure 3), similarly to other peptidomimetic inhibitors of KLK5 [23] as discussed before.

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In the KLK52B system (Figure 3), the N2 atom of 2b establishes an H-bond with the backbone carbonyl group of Ser260 (S2 subsite; lifetime = 147.4 ns), which is the

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most persistent H-bond in this system. The inhibitor O1, O3 and O6 atoms make three H-bonds with the side chain amino group of Gln242 (between S1 and S2’ subsites; lifetimes = 11.72 ns, 15.18 ns and 21.34 ns, respectively), where O1 of 2b also interacts with the backbone amino group of Gly243 (S2’ subsite; lifetime = 16.47 ns) (Figure 3).

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In addition, O4 interacts with imidazole group of His108 and with backbone carbonyl group of Ser245 (S2 subsite; lifetimes = 66.12 ns and 90.81 ns, respectively). Other H-bonds are also observed in the 2b-KLK5 complex, but during only a

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few picoseconds of the simulation time (Supplementary Material, Figure S5). These interactions include all heteroatoms of the inhibitor, with the exception of O2 atom.

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Thus, O1 forms H-bond with the side chain hydroxyl group of Ser260 (lifetime = 1.32 ns), O2 interacts with the side chain amino group of Gln242 (lifetime = 0.002 ns), O4 establishes H-bond with Gln242 side chain amino group (lifetime = 0.010 ns), O5 makes H-bond with side chain OH group of Ser245 (lifetime = 0.081 ns) and O6 makes H-bonds with side chain OH group of Ser245 and with backbone amino group of Gln242 (lifetimes = 0.051 ns and 0.006 ns, respectively). Moreover, N1 interacts with the side chain amino and carbonyl groups (amide group) of Gln242 (lifetime = 0.087 ns

ACCEPTED MANUSCRIPT and 0.078 ns, respectively), while N2 makes two H-Bonds with the side chain hydroxyl group of Ser245 (lifetime = 0.273 ns) and with backbone amino group of Trp261 (S4

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subsite; lifetime = 0.004 ns) (Supplementary Material; Figure S5).

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Figure 3. Close view of the main hydrogen bonds (H-bonds, black dotted lines) among 2b inhibitor and kallikrein 5 (KLK5) residues in the KLK5-2b complex, during the last 100 ns of molecular dynamics simulation in aqueous solution. The H-bonds lifetimes are 147.4 ns (Ser260), 90.81 ns (Ser245), 66.12 ns (His108), 16.47 ns (Gly243), 15.18 ns and 11.72 ns (both with Gln242). 2b and KLK5 residues are in stick model and colored by atom (carbon, cyan or white; nitrogen, blue; oxygen, red), where the hydrogen atoms were omitted for clarity, and the S1, S2, S1’ and S2’ subsites are represented by black lines.

In addition we investigate a potential nucleophillic attack of the catalytic serine

on amide carbonyl of the inhibitor. The minimum distance between the oxygen atom of Ser245 and the carbon atom of 2b carbonyl group was monitored over the last 100 ns simulation. The distance between these two atoms has remained at about 9 Å, oscillating between 8.5 and 11 Å. Thus, the hydroxyl group of Ser245 residue does not appear to attack the amide carbonyl group of 2b inhibitor during the simulation time (Supplementary Material; Figure S6).

ACCEPTED MANUSCRIPT Cross-Correlation Map Analysis in the Free and Bound Enzyme Systems To gain further insight into the perturbation of the enzyme structure after binding to the inhibitor, we have investigated the correlation between the motions of the Cα-atom residues by performing cross-correlation maps analysis for both KLK5APO

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(Figure 4A) and KLK52B (Figure 4B) systems, where these coefficients provide information about the correlation between the fluctuations of the positions of the Cαatom residues.

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Overall, in the KLK5APO system (Figure 4A), high values are obtained for correlated motions among the Cα-atoms mainly among the regions Ile67-Tyr127 (β-

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strands B1 to B5), Glu128-Val187 (β-strands B6 to B8), and Gly237-Ser293 (β-strands B11, B12 and helix H2) of KLK5, sub-domains in which the residues of the catalytic triad (His108, Asp153 and Ser245) are found. On the other hand, the correlated motion analysis reveals an expressive reduction of correlations in the KLK52B system (Figure

3, 4, and 5 of KLK5.

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4B). Particularly, the largest reductions of correlations are observed among the regions

Therefore, we suggest that the binding of the 2b inhibitor is capable of

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disrupting the correlated motions of the KLK5 structure, which probably is associated

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with the enzymatic function.

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ACCEPTED MANUSCRIPT

Conclusions

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Figure 4. Maps of the correlated motions among the KLK5 Cα-atoms of the (A) KLK5APO (free enzyme) and (B) KLK52B (inhibitor-bound enzyme) systems, during the last 100 ns of simulation time. The strength of the computed correlation between two respective CRZ Cα-atoms is color-coded (see the color baron the bottom), where highly correlated motions are in red and poorly correlated motions are in blue.

We reported a new class of small statine based peptidomimetic compounds that

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selectively inhibited the activity of KLK5 in a nanomolar order. We have carried out 150 ns of molecular dynamics simulation to study the structure and dynamic behavior of

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KLK5 in a complex with the 2b inhibitor. The inhibitor binding at the KLK5 through the H-bond interactions with key residues (mainly His108, Gln242, Gly243, Ser245, and Ser206), disrupting the correlated motions mainly among the Ile67-Tyr127, Glu128-Val187, and Gly237-Ser293 sub-domains, which seems to be crucial for KLK5 activity. Therefore, we believed that these findings will help to understand the conformational dynamics in the course of KLK5 inhibition, and, probably, could guide the design and synthesis of new more potent inhibitors of KLK5, a therapeutic target for cancer.

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Experimental Chemistry L-Phenylalanine methyl ester hydrochloride (L-Phe-OMe.HCl), L-Valine methyl

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ester hydrochloride (L-Val-OMe.HCl) and L-Proline methyl ester hydrochloride (L-ProOMe.HCl) were purchased from Aldrich Chem. Co. All solvents were purchased as reagent grade, dried using standard conditions and stored over molecular sieves.

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Purification of products was carried out using silica gel flash chromatography (Whatman 60, 230-400 mesh). Melting points were obtained on a Unit-Melt Thomas

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Hoover capillary melting point apparatus and are uncorrected. IR spectra were obtained on a Perkin-Elmer spectrometer model Spectrum One in liquid film or KBr pellets. NMR spectra were recorded on a Varian VNMRS (500MHz) spectrometer.

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General Procedure for Compounds 2a-c

To a 0oC cooled mixture of compound 1 (0.2 g; 0.647 mmol) and the appropriate hydrochloride (L-Phe-OMe.HCl, L-Val-OMe.HCl or L-Pro-OMe.HCl) (1.15 mmol) in

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dry CH2Cl2 (10 mL) were added EDC.HCl (0.186 g; 0.970 mmol), HOBt (0.13 g; 0.970 mmol) and N-methylmorpholine (0.21 mL; 1.94 mmol). The mixture was stirred at

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room temperature for 24 hours and the volatiles were removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (50 mL) and successively washed with 5% H3PO4 (50 mL), 20% Na2CO3 (50 mL), water (40 mL) and brine (50 mL) and dried with Na2SO4 after which it was filtered and evaporated under reduced pressure. The products were purified by flash chromatography on silica gel using EtOAc/hexane as eluents.

ACCEPTED MANUSCRIPT (S)-methyl-2-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’-phenylpentanamido)-3-phenylpropanoate (2a) Yield: 80%; white solid; mp 78-79 oC. IR (KBr, ν cm-1): 3389, 3287, 3062, 2976, 1749, 1682, 1665, 1646, 1542, 1496, 1366,

1

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1292, 1165, 1049, 702.

H NMR (500 MHz, CDCl3): δ=7.30-7.09 (10H, m, Ar-H), 6.50 (1H, sl, NH), 4.90 (1H,

sl,OH), 4.80-4.79 (1H, m, H2), 3.92 (1H, m, H4’), 3.77-3.72 (1H, m, H3’), 3.70 (3H, s,

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OCH3), 3.16-3.12 (1H, m, H3), 3.02 (1H, dd, J= 13.9, 6.6 Hz, H3), 2.88 (2H, d, J= 7.4 Hz, H5’), 2.46-2.40 (1H, m, H2’), 2.19 (1H, d, J= 15 Hz, H2’), 1.40 (9H, s, 3xCH3).

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C NMR (125.6 MHz, CDCl3): δ = 172.2 (C1’), 171.7 (C1), 156.2 (C12’), 138.1 (C6’),

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135.7 (C4), 129.3 (C8’), 129.1 (C6), 128.5 (C7’), 128.4 (C5), 127.1 (C9’), 126.3 (C7), 79.4 (C13’), 68.0 (C3’), 55.3 (C4’), 53.2 (C2), 52.3 (OCH3), 40.0 (C2’), 38.4 (C3), 37.6 (C5’), 28.3 (2xCH3), 27.9 (CH3).

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HRMS-FAB: m/z [M + 1] Calcd for C26H34N2O6: 471.2490 (+H); found: 471.2492. [α]D20 +25,2 (c 1.0, CH2Cl2).

(S)-methyl-2-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’-phenylpentanami-

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do)-3-methylbutanoate (2b)

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Yield: 72%; yellowish solid; mp 47-48 oC. IR (KBr, ν cm-1): 3288, 3062, 2966, 1743, 1685, 1648, 1536, 1453, 1391, 1365, 1250, 1207, 1163, 1046, 1020, 849, 733, 698. 1

H NMR (500 MHz, CDCl3): δ =7.29-7.18 (5H, m, Ar-H), 6.58 (1H, sl, NH), 4.92 (1H,

sl, OH), 4.50-4.47 (1H, m, H2), 3.99-3.96 (1H, d, J = 7.6 Hz, H4’), 3.81- 3.73 (1H, m, H3’), 3.72 (3H, s, OCH3), 2.90 (2H, d, J= 7.4 Hz, H5’), 2.58-2.53 (1H, m, H3), 2.28 (1H, d, J= 13.3 Hz, H2’), 2.14 (1H, dd, J= 12.0, 6.8 Hz, H2’), 1.40 (9H, s, 3xCH3), 0.93 (3H, d, J= 7.0 Hz, H4 or H5), 0.91 (3H, d, J= 7.0 Hz, H5 or H4).

ACCEPTED MANUSCRIPT C NMR (125.6 MHz, CDCl3): δ =172.5 (C1’), 172.1 (C1), 156.2 (C12’), 138.1 (C6’),

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129.3 (C8’), 128.3 (C7’), 126.3 (C9’),79.4 (C13’),68.1 (C3’), 57.1 (C2), 55.4 (C4’), 52.1 (OCH3), 40.0 (C2’), 38.5 (C5’), 30.9 (C3), 28.3 (3xCH3), 18.9 (C4), 17.8 (C5).

[α]D20 -13 (c 1.0, CH2Cl2).

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HRMS-FAB: m/z [M + 1] Calcd for C22H34N2O6: 423.24896 (+H); found: 423.2485.

pyrrolidine-2-carboxylate (2c) Yield: 70%; white solid; mp 119-120 oC.

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(S)-methyl-1-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’-phenylpentanoyl)

1244, 1200, 1170, 1049, 887, 702. 1

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IR (KBr, ν cm-1): 3381, 3263, 2967, 2861, 1747, 1697, 1632, 1511, 1464, 1436, 1365,

H NMR (500 MHz, CDCl3): δ=7.29-7.18 (5H, m, Ar-H), 4.45-4.42 (1H, m, H2), 4.03

(1H, d, J= 9.6 Hz, H4’), 3.75-3.66 (1H, m, H3’), 3.71 (3H, s, OCH3), 3.57-3.52 (1H, m, H5), 3.46-3.42 (1H, m, H5), 2.91 (2H, d, J= 7.7 Hz, H5’), 2.54-2.48 (1H, m, H2’), 2.35

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(1H, d, J= 16.6 Hz, H2’), 2.21-2.13 (1H, m, H3), 2.07-2.02 (1H, m, H3), 2.00-1.96 (2H, m, H4), 1.40 (9H, s, 3xCH3).

C NMR (125.6 MHz, CDCl3): δ=172.3 (C1’), 171.9 (C1), 155.9 (C12’), 138.3 (C6’),

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13

129.4 (C8’), 128.3 (C7’), 126.2 (C9’),79.1 (C13’), 66.8 (C3’), 58.5 (C2), 55.6 (C4’), 52.2

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(OCH3), 47.0 (C5), 38.7 (C2’), 37.8 (C5’), 29.2 (C3), 28.3 (3xCH3), 24.5 (C4). HRMS-FAB: m/z [M + 1] Calcd for C22H32N2O6: 421.23331 (+H); found: 421.2331. [α]D20-62.8 (c 1.0, CH2Cl2).

General Procedure for Compounds 3a-c To 0oC cooled solutions of compounds 2a-c (1.5 mmol) in THF (30 mL) were added methanol (9 mL), water (9 mL) and LiOH (0.387 g; 16.158 mmol). The mixture was stirred for 17h and the volatiles were removed under reduced pressure. The

ACCEPTED MANUSCRIPT resulting aqueous solution was cooled to 0oC, acidified with 10% HCl until pH 4 and extracted with EtOAc (5 x 30 mL). The combined organic layers were washed with brine (10 mL) and dried with Na2SO4. Filtration and evaporation of the solvent furnished the products as solids which were recrystallized in Et2O/ hexane.

phenylpropanoic acid (3a) Yield: 72%; white solid; mp 154-156 oC.

phenylpentanamido)-3-

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(S)-2-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’

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IR (KBr, ν cm-1): 3551, 3437, 3271, 2980, 2594, 1724, 1686, 1619, 1572, 1531, 1506, 1498, 1368, 1248, 1185, 1057, 700.

H NMR (500 MHz, CDCl3): δ=7.30-7.20 (10H, m, Ar-H), 6.00 (1H, sl, NH), 4.93 (1H,

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1

d, J= 9.0 Hz, H2), 4.00 (1H, d, J= 9.5 Hz, H4’), 3.75-3.6571 (1H, m, H3’), 2.90 (2H, d, J= 8.0 Hz, H3), 2.63 (2H, dd, J= 16.9, 10.0 Hz, H5’), 2.45 (2H, d, J= 16.9 Hz, H2’), 1.41 (9H, s, 3xCH3).

C NMR (125.6 MHz, CDCl3): δ=173.3 (C1),173.0 (C1’), 156.6 (C12’), 137.9 (C6’),

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136.0 (C4), 129.4 (C8’), 129.2 (C6), 128.4 (C7’), 128.3 (C5), 126.9 (C9’), 126.3 (C7), 80.3

27.9 (CH3).

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(C13’), 68.3 (C3’), 55.5 (C4’), 53.1 (C2), 39.6 (C2’), 38.2 (C3), 37.7 (C5’), 28.3 (2xCH3),

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HRMS-FAB: m/z [M + 1] Calcd for C25H32N2O6: 457.23331 (+H); found: 457.2334. [α]D20 +23.4 (c 1.0, CH2Cl2). (S)-2-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’-phenylpentanamido)-3methylbutanoic acid (3b) Yield: 85%; white solid; mp 70-73 oC. IR (KBr, ν cm-1): 3315, 2966, 2930, 1685, 1641, 1532, 1392, 1366, 1252, 1164, 1047, 732, 699.

ACCEPTED MANUSCRIPT 1

H NMR (500 MHz, CDCl3): δ=7.28-7.14 (5H, m, Ar-H), 5.09 (1H, d, J= 9.3 Hz, OH),

4.54-4.52 (1H, m, H2), 4.01-3.93 (1H, m, H4’), 3.83-3.74 (1H, m, H3’), 2.91 (1H, dd, J= 13.6, 7.4 Hz, H5’), 2.87-2.83 (1H, m, H5’), 2.66 (1H, d, J= 15.4 Hz, H3), 2.50-2.39 (1H, m, H2’), 2.27-2.15 (1H, m, H2’), 1.37 (9H, s, 3xCH3), 0.93 (6H, d, J= 5.7 Hz, H4and H5). C NMR (125.6 MHz, CDCl3): δ=174.0 (C1), 173.3 (C1’), 156.6 (C12’), 138.0 (C6’),

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129.2 (C8’), 128.3 (C7’), 126.3 (C9’), 80.1 (C13’), 68.4 (C3’), 56.9 (C2), 55.5 (C4’), 39.8 (C2’), 38.2 (C5’), 31.1 (C3), 28.2 (2xCH3), 28.0 (CH3), 18.7 and 17.7 (C4 and C5).

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HRMS-FAB: m/z [M + 1] Calcd for C21H32N2O6: 409.23331 (+H); found: 409.2331. [α]D20 +2 (c 1.0, CH2Cl2).

dine-2- carboxylic acid (3c)

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(S)-1-((3’S,4’S)-4’-(tert-butoxycarbonylamino)-3’-hydroxy-5’-phenylpentanoyl)pyrroli-

Yield: 94%; white solid; mp 74-76 oC.

IR (KBr, ν cm-1): 3408, 2977, 2930, 1711, 1624, 1497, 1454, 1367, 1249, 1169, 1049,

1

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695.

H NMR (500 MHz, CDCl3): δ=7.29-7.15 (5H, m, Ar-H), 5.05 (1H, d, J= 9.6 Hz, H2),

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4.48 (1H, m, H4’), 4.05 (1H, d, J= 9.6 Hz, H3’), 3.71 (1H, dd, J= 16.3, 8.0 Hz, H5), 3.533.40 (2H, m, H5 and H5’), 2.90 (1H, d, J= 7.5 Hz, H5’), 2.57 (1H, dd, J= 16.7, 10.0 Hz,

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H2’), 2.39 (1H, d, J= 14.9 Hz, H2’), 2.28-2.18 (1H, m, H3), 2.10-2.02 (3H, m, H3and H4), 1.39 (9H, s, 3xCH3).

C NMR (125.6 MHz, CDCl3): δ=173.8 (C1), 173.0 (C1’), 156.1 (C12’), 138.2 (C6’),

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129.4 (C8’), 128.3 (C7’), 126.2 (C9’), 79.4 (C13’), 67.1 (C3’), 59.1 (C2), 55.6 (C4’), 47.5 (C5), 38.6 (C2’), 38.2 (C5’), 28.5 (C3), 28.3 (3xCH3), 24.5 (C4). HRMS-FAB: m/z [M + 1] Calcd for C21H30N2O6: 407.21766 (+H); found: 407.2176. [α]D20-80.8 (c 1.0, CH2Cl2).

ACCEPTED MANUSCRIPT General Procedure for Compounds 5a-c To 0oC cooled solutions of compounds 3a-c (0.369 mmol) in dry CH2Cl2 (7.2 mL) were added amine 4 (0.104 g; 0.443 mmol), EDC.HCl (0.089g; 0.566 mmol), HOBt (0.076 g; 0.566 mmol) and N-methyl morpholine (0.12 mL). The mixture was

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stirred at room temperature for 24h and the volatiles were removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (50 mL) and successively washed with 5% H3PO4 (50 mL), 20% Na2CO3 (50 mL), water (40 mL) and brine (50

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mL) and dried with Na2SO4 after which it was filtered and evaporated under reduced

EtOAc/hexane as eluents.

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pressure. The products were purified by flash chromatography on silica gel using

t-butyl (2S,3S)-5-((S)-1-((3S,3aR,6R,6aS)-6-(benzyloxy)hexahydrofuro-[3,2-b]furan-3ylamino)-1-oxo-3-phenylpropan-2-ylamino)-3-hydroxy-5-oxo-1-phenylpentan-2-yl carbamate (5a)

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Yield: 90%; white solid; mp 84-86 oC.

IR (KBr, ν cm-1): 3280, 3062, 2930, 2877, 1680, 1638, 1538, 1496, 1454, 1365, 1249, 1166, 1095, 1020, 739, 698.

H NMR (500 MHz, CDCl3): δ=7.36-7.15 (15H, m, Ar-H), 4.66 (1H, d, J= 11.8 Hz,

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1

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H7’’), 4.60-4.55 (1H, m, H4’), 4.50 (1H, d, J= 11.8 Hz, H7’’), 4.29-4.26 (1H, m, H3’), 4.14-4.10 (1H, m, H5’’), 4.02-4.00 (1H, m, H2’’), 3.95-3.89 (3H, m, H3’’, H4’’and H6’’), 3.77 (1H, t, J= 8.2 Hz, H1’’), 3.71-3.67 (2H, m, H4’’and H2), 3.59 (1H, t, J= 8.2 Hz, H1’’), 3.09-3.05 (1H, m, H3), 2.96-2.92 (1H, m, H3), 2.86 (2H, d, J= 7.0 Hz, H5’), 2.45-2.41 (1H, m, H2’), 2.27-2.25 (1H, m, H2’), 1.38 (9H, s, 3xCH3). C NMR (125.6 MHz, CDCl3): δ=172.1 (C1’), 170.3 (C1), 156.2 (C12’), 138.1 (C6’),

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137.6 (C4), 136.4 (C8’’), 129.3 (C8’), 128.6 (C6), 128.4 (C10’’), 128.3 (C7’), 127.9 (C5), 127.8 (C9’’), 127.0 (C9’), 126.3 (C7 and C11’’), 86.6 (C2’’), 80.0 (C5’’), 79.5 (C13’), 78.9

ACCEPTED MANUSCRIPT (C6’’), 72.9 (C4’’), 72.3 (C7’’), 70.5 (C1’’), 68.6 (C3’’), 56.8 (C4’), 55.3 (C2), 54.5 (C3’), 40.5 (C2’), 38.6 (C3), 38.4 (C5’), 28.3 (3xCH3). HRMS-FAB: m/z [M + 1] Calcd for C38H47N3O8: 674.34359 (+H); found: 674.3435. [α]D20+30.8 (c 1.0, CH2Cl2).

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t-butyl (2S,3S)-5-((S)-1-((3S,3aR,6R,6aS)-6-(benzyloxy)hexahydrofuro-[3,2-b]furan-3ylamino)-3-methyl-1-oxobutan-2-ylamino)-3-hydroxy-5-oxo-1-phenylpentan-2-yl

Yield: 82%; white solid; mp 160-163 oC.

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carbamate (5b)

IR (KBr, ν cm-1): 3571, 3317, 2960, 2925, 1683, 1634, 1528, 1453, 1390, 1367, 1316,

1

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1259, 1165, 1082, 1048, 1018, 887, 854, 800, 745, 697.

H NMR (500 MHz, CDCl3): δ=7.33-7.15 (10H, m, Ar-H), 4.71 (1H, d, J= 11.8 Hz,

H7’’), 4.58-4.54 (1H, m, H5’’), 4.51 (1H, d, J= 11.8 Hz, H7’’), 4.41-4.38 (1H, m, H2’’), 4.38-4.34 (1H, m, H2), 4.26-4.19 (1H, m, H4’), 4.04-3.95 (3H, m, H63’’, H4’’and H6’’),

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3.81 (2H, t, J= 8.2 Hz, H1’’and H4’’), 3.75-3.69 (1H, m, H3’), 3.62 (1H, t, J= 8.2 Hz,; H1’’), 2.87 (2H, d, J= 6.6 Hz, H5’), 2.53-2.46 (1H, m, H2’), 2.37-2.29 (1H, m, H2’), 2.102.03 (1H, m, H3), 1.37 (9H, s, 3xCH3), 0.90 (6H, t, J= 7.0 Hz, 2xCH3).

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C NMR (125.6 MHz, CDCl3): δ=172.4 (C1’), 170.9 (C1), 156.3 (C12’), 138.1 (C6’),

13

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137.6 (C8’’), 129.2 (C8’), 128.4 (C7’), 128.3 (C9’), 127.9 (C10’’), 127.8 (C9’’), 126.3 (C11’’), 86.8 (C2’’), 80.3 (C5’’), 79.6 (C13’), 79.0 (C6’’), 73.2 (C4’’), 72.4 (C7’’), 70.7 (C1’’), 68.7 (C3’’), 58.6 (C4’), 57.1 (C2), 55.4 (C3’), 40.6 (C2’), 38.4 (C5’), 30.8 (C3), 28.3 (3xCH3), 19.2 (C4), 18.1 (C5). HRMS-FAB: m/z [M + 1] Calcd for C34H47N3O8: 626.34359 (+H); found: 626.3434. [α]D20+37 (c 1.0, CH2Cl2). t-butyl (2S,3S)-5-((S)-2-((3S,3aR,6R,6aS)-6-(benzyloxy)hexahydrofuro-[3,2-b]furan-3ylcarbamoyl)pyrrolidin-1-yl)-3-hydroxy-5-oxo-1-phenylpentan-2-yl carbamate (5c)

ACCEPTED MANUSCRIPT Yield: 86%; white solid; mp 68-70 oC. IR (KBr, ν cm-1): 3422, 3324, 3034, 2971, 2881, 1688, 1646, 1531, 1453, 1364, 1261, 1166, 1084, 1050, 1027, 804, 698. 1

H NMR (500 MHz, CDCl3): δ=7.26-7.05 (10H, m, Ar-H), 4.64 (1H, d, J= 11.8 Hz,

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H7’’), 4.44 (2H, d, J= 11.,8 Hz, H5’’and H7’’), 4.40-4.36 (1H, m, H4’), 4.34-4.31 (1H, m, H2’’), 4.25-4.21 (1H, m, H2), 4.05-4.00 (1H, m, H3’’), 3.96-3.87 (2H, m, H4’’and H6’’), 3.75 (1H, t, J= 8.2 Hz, H1’’), 3.67 (2H, d, J= 9.0 Hz, H4’’and H3’), 3.58 (1H, t, J= 8.2 Hz,

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H1’’), 3.40-3.37 (1H, m, H5), 3.32-3.27 (1H, m, H5), 2.84 (2H, d, J= 7.4 Hz, H5’), 2.46-

H3and H4), 1.30 (9H, s, 3xCH3).

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2.31 (2H, m, H2’), 2.22-2.15 (1H, m, H3), 1.97-1.91 (1H, m, H4), 1.84-1.78 (2H, m,

C NMR (125.6 MHz, CDCl3): δ=172.4 (C1’), 170.8 (C1), 156.0 (C12’), 138.2 (C6’),

13

137.7 (C8’’), 129.3 (C8’), 128.4 (C7’), 128.3 (C9’), 127.8 (C10’’), 127.7 (C9’’), 126.3 (C11’’), 87.0 (C2’’), 80.2 (C5’’), 79.3 (C13’), 78.9 (C6’’), 73.5 (C4’’), 72.4 (C7’’), 70.4 (C1’’),

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67.9 (C3’’), 59.9 (C4’), 57.1 (C2), 55.1 (C3’), 47.6 (C5), 38.6 (C5’), 38.4 (C2’), 28.3 (3xCH3), 28.0 (C3), 24.6 (C4).

HRMS-FAB: m/z [M + 1] Calcd for C34H45N3O8: 624.32794 (+H); found: 624.3279.

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[α]D20-4 (c 1.0, CH2Cl2).

Kinetics Assays

The human tissue kallikreins were obtained as previously described [15]. The

Fluorescence Resonance Energy Transfer (FRET) peptides Abz-KLYSSKQ-EDDnp (Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine) and AbzKLRSSKQ-EDDnp were kindly provided by Dr Maria Juliano, from Federal University

São Paulo (Unifesp). All enzymatic reactions were performed in 50

mMTris–HCl, pH 7.5.

ACCEPTED MANUSCRIPT Stock solutions (DMSO:H2O/1:1) of the compounds were diluted to the appropriate concentration prior to the assays. The inhibitors were screened against human kallikreins (KLK5s) at an initial concentration of 10 µM. The cuvet contained 900 µL of buffer, 0.02 µg of KLK5s and the inhibitor was incubated during 5 minutes at

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37°C. The reactions were started by the addition of the FRET substrate AbzKLRSSKQ-EDDnp (for KLK1, KLK5 and KLK6) or Abz- KLYSSKQ-EDDnp (for KLK7), and measurements were conduct using a spectrofluorimeter Hitachi F2500

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(wavelength of excitation 320 nm, and emission 420 nm). Control assays were performed without inhibitor (negative control). The IC50 values were independently

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determined by making rate measurements for at least five inhibitor concentrations in a range of 0.01 µM to 1.0 µM. The values represent means ± SD of three individual experiments. The IC50 results were obtained using GraFit 7 program [32]. The Ki values were determined by Lineweaver–Burk plots.

Molecular Docking

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Molecular Modeling Procedure

The molecular docking procedure was validated by removing the Leupeptin

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inhibitor complexed to KLK5 structure from its binding site and redocking it to the enzyme (PDB ID: 2PSX) [31]. The validation protocol was performed using the

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Molegro Virtual Docker software [30], which uses a heuristic search algorithm that combines differential evolution with a cavity prediction algorithm. The root mean square deviation (RMSD) between the predicted pose and the observed X-ray crystallographic binding mode of the Leupeptin (PDB ID: 2PSX) equal to 1.49 Å suggests the reliability of the docking protocol. Then, the selected parameter set was used to predict the binding mode of 2a-c inhibitors.

ACCEPTED MANUSCRIPT Consequently, the 2a-c structures were prepared using the Spartan’10 software [33] and the docking of all inhibitors to KLK5 binding site was also performed in Molegro Virtual Docker program [30], where the MolDock Optmizer search algorithm was used with a minimum of 100 runs, the whole enzyme was chosen as the search

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space, and the parameter settings were: population size = 300; maximum iteration = 2000; scaling factor = 0.50; offspring scheme = scheme 1; termination scheme = variance based; and crossover rate = 0.90.

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Setup and Molecular Dynamics Simulation

The free (KLK5APO) system was prepared deleting all crystallographic water and

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ion atoms, and, subsequently, adding hydrogen atoms to theKLK5 crystal structure [31] and the bound (KLK52B) system was constructed using the 2b-KLK5 complex predicted by the docking procedure. Thus, all the amino acids were modeled in the protonation state they exhibit as free amino acids in water under pH 7.0.

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The construction of the ligand topology was made using MKTOP program [34] and the partial charges were calculated at Resp Esp charge Derive (R.E.D.) server [35]. The MD simulations were carried out using the GROMACS 4.6.7 package [36].

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The KLK5 enzyme was solvated within a cubic box (box length 80.4 Å) containing 15,979 TIP4P water molecules [37], in which 8 of them were replaced by 11 chloride

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counterions to neutralize the system. Then, the AMBER99sb-ildn parameter set [38-39] was employed for the KLK5, TIP4P water model [37] and counter-ions. All simulations were carried out in the NpT ensemble, considering periodic boundary conditions. The temperature was maintained at 300 K, using the Nosé-Hoover thermostat coupling [40], and isotropic pressure coupling was applied, using the Parrinello-Rahman barostat algorithm [41]. Electrostatic interactions were treated with the Particle-Mesh Ewald (PME) method, using a cut-off of 1.0 nm, and Lennard-Jones interactions were also cut

ACCEPTED MANUSCRIPT off at 1.0 nm [36]. The LINCS [42] and SETTLE [43] algorithms were used to constrain all bonds and water molecules, respectively. The time-step for integration was set to 2.0 fs. Initial water relaxation dynamics were carried out during 2.0 ns for system equilibration, keeping the complex of 2b-KLK5 position restrained. Subsequently,

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production run was performed during 150 ns, without position restraints.

Analyses of the Protein Structure and Dynamics in the Free and Bound Systems

The protein structure and dynamics in the KLK5APO and KLK52b aqueous

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systems were evaluated by the analyses of the secondary structure (SS) elements, root mean square deviations (RMSD) and fluctuations (RMSF), and radius of gyration (Rg)

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using the DO_DSSP, G_RMS, G_RMSF, and G_GYRATE programs, respectively, of GROMACS 4.6.7 package [36]. With the exception of RMSF, calculated for the last 100 ns of simulation time, the other parameters (SS elements, RMSD, and Rg) were calculated for the entire simulation time (150 ns). All the graphs were plotted using

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XMGRACE 5.1.19 program [44].

Intermolecular Hydrogen Bonds Analysis in the Protein Bound System The number of distinct hydrogen bonds (H-bond) formed by the 2b inhibitor to

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the amino acids of the enzyme during the 150 ns of MD simulation of the KLK52B system was calculated using the G_HBOND program of GROMACS 4.6.7 package

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[36].

Analysis of the Correlated Movements in the Free and Bound Protein Systems The residue-by-residue cross-correlation in the KLK5APO and KLK52B systems

were calculated using the generalized cross-correlation approach applied to the atomic coordinates of the Cα-atoms (backbone), during the last 70 ns of MD simulation in aqueous solvent. This approach is based on the mutual information method developed

ACCEPTED MANUSCRIPT by the Grubmüller group using the G_CORRELATION program [45-46] available in the GROMACS 3.3.3 package [47].

Acknowledgments

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We gratefully acknowledge financial support from Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES), National Council for Scientific and Technological

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Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico— CNPq), the Carlos Chagas Filho Foundation for Support of Research of the State of Rio

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de Janeiro (Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro—FAPERJ), and Foundation for Support of Research of the State of São Paulo (Fundação de Amparo a Pesquisa do Estado de São Paulo—FAPESP).

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References

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[1] K. Mavridis, M. Avgeris, A. Scorilas, Targeting kallikrein-related peptidases in prostate cancer, Expert Opin Ther Targets, 18 (2014) 365-383. [2] G. Liang, X. Chen, S. Aldous, S.F. Pu, S. Mehdi, E. Powers, A. Giovanni, S. Kongsamut, T. Xia, Y. Zhang, R. Wang, Z. Gao, G. Merriman, L.R. McLean, I. Morize, Virtual Screening and X-ray Crystallography for Human Kallikrein 6 Inhibitors with an Amidinothiophene P1 Group, ACS Med Chem Lett, 3 (2012) 159-164. [3] N. Emami, E.P. Diamandis, Human tissue kallikreins: a road under construction, Clin Chim Acta, 381 (2007) 78-84. [4] G. Pampalakis, O. Obasuyi, O. Papadodima, A. Chatziioannou, V. Zoumpourlis, G. Sotiropoulou, The KLK5 protease suppresses breast cancer by repressing the mevalonate pathway, Oncotarget, 5 (2014) 2390-2403. [5] G. Liang, X. Chen, S. Aldous, S.F. Pu, S. Mehdi, E. Powers, T. Xia, R. Wang, Human kallikrein 6 inhibitors with a para-amidobenzylanmine P1 group identified through virtual screening, Bioorg Med Chem Lett, 22 (2012) 2450-2455. [6] X. Tan, F. Soualmia, L. Furio, J.F. Renard, I. Kempen, L. Qin, M. Pagano, B. Pirotte, C. El Amri, A. Hovnanian, M. Reboud-Ravaux, Toward the first class of suicide inhibitors of kallikreins involved in skin diseases, J Med Chem, 58 (2015) 598-612. [7] H. Chung, M. Hamza, K. Oikonomopoulou, V. Gratio, M. Saifeddine, G.D. Virca, E.P. Diamandis, M.D. Hollenberg, D. Darmoul, Kallikrein-related peptidase signaling in colon carcinoma cells: targeting proteinase-activated receptors, Biol Chem, 393 (2012) 413-420.

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Highlights A new series of statine based peptidomimetics is exposed. The enzymatic activity against human kallikrein 5 (KLK5) was studied. Some KLK5 inhibitors were identified with nanomolar affinity. Molecular dynamic simulation of KLK5-inhibitor complex was carried out.

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1. 2. 3. 4.