Selection of Effective HTRA3 Activators Using Combinatorial

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Selection of effective HTRA3 activators using combinatorial chemistry Magdalena Wysocka, Kamila Sychowska, Natalia Gruba, #ukasz Winiarski, Marcin Skore#ski, Mateusz Psurski, Joanna Makowska, Artur Gieldon, Tomasz Wenta, Miros#aw Jarz#b, Przemyslaw Glaza, Joanna Zdacewicz, Marcin Sie#czyk, Barbara Lipinska, and Adam Lesner ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00051 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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ACS Combinatorial Science

Selection of effective HTRA3 activators using combinatorial chemistry Magdalena Wysocka1, Kamila Sychowska1, Natalia Gruba1, Łukasz Winiarski2, Marcin Skoreński2, Mateusz Psurski2, Joanna Makowska1, Artur Giełdoń1, Tomasz Wenta3, Mirosław Jarząb3, Przemysław Glaza3, Joanna Zdancewicz1, Marcin Sieńczyk2, Barbara Lipińska3, Adam Lesner1 1

Faculty of Chemistry, University of Gdansk, Gdansk, Poland

2

Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland

3

Faculty of Biology, University of Gdansk, Gdansk, Poland

Abstract Herein we report selection, synthesis and enzymatic evaluation of a peptidomimetic library able to increase proteolytic activity of HtrA3 (high temperature requirement A) protease. Iterative deconvolution in solution of synthesized modified pentapeptides yielded two potent HtrA3 activators acting in the micromolar range (HCOO-CH2O-C6H4-OCH2-CO-Tyr-AsnPhe-His-Asn-OH

and

HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Glu-OH).

Both

compounds increased proteolysis of an artificial HtrA3 substrate over 40-fold in a selective manner. Based on molecular modeling, the selected compounds bind strongly to the PDZ domain.

Introduction High-temperature requirement factor A3 (HtrA3) belongs to the HtrA (high-temperature requirement A) family of ATP-independent serine proteases, highly conserved in evolution [1]. All members are oligomeric proteins characterized by the presence of a trypsin-like protease domain with the catalytic triad His-Asp-Ser and a 95 kDa PDZ (postsynaptic density and zonula occludens 1) domain at the C-terminal end. The latter is involved in substrate or regulatory peptide binding and may regulate protease activity via allosteric interactions with the protease domain. HtrA proteins are part of the defense mechanism against cellular stresses (such as heat shock and oxidative stress) [2]. They may also be involved in the degradation of aberrant or regulatory proteins and, in some cases, function as chaperones, protecting the protein structure. To implement their proteolytic function, they need to be activated. In humans, four HtrA proteins (HtrA1-4) have been identified and found to be involved in vital

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physiological processes including the maintenance of mitochondrial homeostasis, cell death (by apoptosis, necrosis, or anoikis) and cell signaling [3]. Disturbances in their functions may contribute to the development of several diseases such as cancer, arthritis, and neurodegenerative disorders [4]. In comparison to HtrA1 and HtrA2, there is limited data regarding the substrate specificity and function of HtrA3. Recently, the structure and limited primary specificity of HtrA3 has been revealed [5]. It shares the common HtrA family fold with regulatory and proteolytic domains forming a homotrimer (Fig. 1). HtrA3 proteolytic activity is known to be essential for the induction of apoptosis and promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [6,7]. HtrA3 recognizes and cleaves peptide bonds after aliphatic residues such as Val, Leu, Ile, and Met. Several reports describe negative correlations between cancer development (breast and lung tumors) and HtrA3 expression [8,9], which may indicate that HtrA3 is involved in tumor suppression or, more likely, in apoptosis induction. Thus, searching for compounds that increase the proteolytic activity of HtrA3 seems to be important. Recently, the pentapeptides Phe-Gly-Arg-Trp-Val and Arg-Ser-Trp-Trp-Val were selected using a phage display but were found to have only a moderate affinity to the studied enzyme [10]. Liu et al., designed and synthesized the peptide Ac-Phe-Gly-Arg-TrpVal with a brominated indole ring (at position 4 or 6) in the Trp residue. The 4-bromo analog was found to bind with a 10-fold greater affinity than the non-brominated parent compound to the PDZ domain of HtrA3. Substitution in position 6 in the same group did not increase the activity of the new compound [11]. With the goal of finding more potent peptide activators, we decided to take another combinatorial chemistry approach. Materials and methods Synthesis The procedure for hydroquinone moiety synthesis has been described previously [12,13]. Briefly, monoalkylation of hydroquinone with tert-butyl bromoacetate led to tert-butyl (4hydroxyphenoxy)acetate, which was further converted into benzyl 2-(4-(2-tert-butoxy-2oxoethoxy)phenoxy)acetate with benzyl bromoacetate (Scheme 1). The final hydrogenolysis


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step allowed us to obtain the target 2-(4-(2-(tert-butoxy)-2-oxoethoxy)phenoxy)acetic acid.

Scheme 1. (a) tert-butyl bromoacetate, NaOH, dioxane/H2O, N2, 22°C, 1 h (yield: 35%); (b) benzyl bromoacetate, Na2CO3, DMF, 22°C, 24 h (yield 73%); (c) Pd/C, H2, AcOEt, 22°C (yield 92%) Peptide synthesis and library preparation The peptide library was synthesized via the Fmoc/tBu approach using the Tenta Gel S AC resin (Rapp Polymeres, Tubingen, Germany) as the solid support. The Fmoc-protected amino acids (Fmoc-AA, 2meq) were used for peptide chain elongation by using DIPCI (N,N′diisopropylcarbodiimide)/HOBt (N-hydroxybenzotriazole) as the coupling agent. The Fmoc deprotection

after

each

coupling

step

was

achieved

with

20%

piperidine

in

dimethylformamide (DMF). After completion of the synthesis, the final libraries or individual peptides were cleaved off the resin by using a TFA/H2O/phenol/triisopropylsilane (88:5:5:2, v/v/v/v) mixture.

Library synthesis The libraries were synthesized by applying the mix and split method [14]. The resin (initial amount used = 8.5 g) after coupling the first amino acid residue was divided into 19 equal portions (hereinafter referred to as systems). Each system was treated with a 20% piperidine/DMF solution to remove the Fmoc protecting group. Next, the second distinct amino acid residue was attached to each of the 19 systems. Simultaneously, 15% of each resin was removed for further library deconvolution steps. The remaining portions of each resin were mixed and again divided into 19 separate systems. The procedure was repeated until the fifth amino acid residue was coupled. At this stage, the Boc-protected hydroquinone function was conjugated to each of the 19 systems followed by cleavage peptides off the resin under the conditions described above. The products were precipitated in diethyl ether, re-dissolved in distilled water, and freeze dried.



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Physicochemical characteristics A MALDI-TOF mass spectrometry analysis was performed to identify all synthesized libraries and compounds (Biflex III MALDI–TOF mass spectrometer, Bruker, Germany) by using the α-cyano-4-hydroxy-cinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB) matrix. The purity of the final products was analyzed by RP-HPLC on a Pro Star system (Varian, Australia) equipped with a Gemini 5-µm C18, 110Å (150 mm × 4.6 mm) column (Phenomenex, USA) and a UV-VIS detector.

Enzymatic studies Initial screening All enzymatic experiments were performed in 96 well black plates (Brand, Merck, Germany). Then, 10 µL of a stock library solution (5 mg of compounds 1-8 each was dissolved in 1 mL of 50% DMSO/HEPES pH buffer) was added to 170 µL of a 10-nM HtrA3 buffered solution (25-mM HEPES, 250-mM NaCl) followed by 15 min of incubation at 37°C. Next, 20 µL of the substrate (Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-ANB-NH2, 3 µM) was added. As the control, the system lacking the activator peptide was used. The change in the fluorescence intensity (Ex. 360 nm, Em. 450 nm) was measured for 30 min. The linear part of the plot was used for the determination of the kinetic parameters. Library screening A volume of 10 µL of the stock library solution (3 mg of each sublibrary with fixed X5 amino acid position was dissolved in 1 mL of a 50% DMSO/HEPES pH buffer) was added to 170 µL of a 10-nM HtrA3 buffered solution (25-mM HEPES, 250-mM NaCl) followed by 15 min of incubation at different temperatures (22, 30, 37, and 45°C). Next, 20 µL of the substrate (Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-ANB-NH2, 3 µM) was added. As the control, the system lacking an activator peptide was used. The change in the fluorescence intensity (Ex. 360 nm, Em. 450 nm) was measured for 30 min. The linear part of the plot was used for the determination of the kinetic parameters. The above procedure was repeated five times for all sublibraries (X5-X1 positions) until the final sequence of the HtrA3 activator was identified. Optimal HtrA3 activation conditions


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A volume of 10 µL of the most potent activator 1 selected from library screening (88.7 µM) or activator 2 (87.3 µM) was added to 170 µL of the HtrA3 solution (25-mM HEPES, 250mM NaCl) at different concentrations ranging from 1.071 nM to 217 nM. The mixture was incubated for 15 min at 37°C before 20 µL of the fluorogenic substrate (Ala(2-MCA)-Ile-ArgArg-Val-Ser-Tyr-Ser-Phe-ANB-NH2, 3 µM) was added. The change in the fluorescence intensity (Em. 360 nm, Em. 450 nm) was measured for 30 min. The linear part of the plot was used for the determination of the kinetic parameters. Activator working range HtrA3 (10 nM) dissolved in 170 µL of the assay buffer (25-mM HEPES, 250-mM NaCl) and incubated for 15 min with activators 1 and 2 at different concentrations ranging from 8.87 µM to 887 µM before the addition of the fluorogenic substrate solution (Ala(2-MCA)-Ile-ArgArg-Val-Ser-Tyr-Ser-Phe-ANB-NH2, 20 µL, 3 µM). The change in the fluorescence intensity resulting from the release of MCA was measured for 30 min (Em. 360 nm, Em. 450 nm). The experiment was performed at 22, 30, 37, and 45°C. EC50 values were calculated based on above data using the non-linear least squares subroutine in the program GraphPad Prism v6.0 in four variable model. Kinetic parameters In the kinetic measurements, the fluorescent substrate was used at concentrations ranging from 0.025 to 15 µM, and the enzyme concentration was 10 nM; the fluorescence was measured at 37°C as a function of time, using a Fluorostar Omega microplate reader (BMG, Germany). At least three measurements were performed, and the standard deviation did not exceed 10%. Steady-state kinetic parameters were obtained by fitting data to Michealis Menten equation. The non-linear least squares subroutine in the program GraphPad Prism v6.0 was used for the fitting. The experiment was performed in presence of activator 1 or activator 2 or its scrambled counterparts at concentration equal 88.7 µM. Selectivity study To examine the influence of activators 1 and 2 on the activity of different members of the HtrA family, we decided to independently incubate both activators (1 and 2) for 15 min with 10-nM solutions of HtrA3, HtrA3 (-) PDZ, HtrA2, HtrA2(-) PDZ, and HtrA1 in the assay buffer. Next, a fluorogenic substrate solution (Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-



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Phe-ANB-NH2, 20 µL, 3 µM) was added. The change in the fluorescence intensity was measured as described above. Proteolytical stability of activators HtrA3 (10 nM) dissolved in 170 µL of the assay buffer (25-mM HEPES, 250-mM NaCl) and incubated for 24 h with activators 1 and 2 each having a concentration of 88.7 µM before the addition of the fluorogenic substrate solution (Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-SerPhe-ANB-NH2, 20 µL, 3 µM). The change in the fluorescence intensity was measured as described above. Since then, the RP HPLC analysis of each system was performed under the conditions described earlier. Molecular modeling The 2,2′-(1,4-phenylenebis(oxy))diacetic acid was parameterized using the antechamber program from the AMBER v.12 package [13,14]. The position of the ligand was approximated from the two crystal structures: mutationally inactivated HtrA3 (4RI0.pdb) and HtrA3 PDZ domain bound to the phage-derived ligand (2P3W.pdb). The newly obtained model was optimized using the steepest descent and continuous gradient minimization methods in repetitive cycles to obtain a low energy structure. To maintain the protein structure as much as possible, positional constraints on all Cα atoms were added. The ligand and the protein side-chains were free to move. The model was analyzed using the RasMol AB [15] program.

Figure 1. Structure of HtrA3 (4RI0.pdb). PDZ domain I marked in blue.


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Aim of the study The aim of this study was to design and synthesize a library of peptidomimetics having a general formula shown in Figure 2. The deconvolution of this library revealed the sequence of the compound able to enhance the proteolytic activity of HtrA3.

Figure 2. General formula of the pentapeptide activators library; positions X1-X5 occupied by 19 proteinogenic amino acid residues except for Cys. Results The incubation of HtrA3 with the recently developed HNE (human neutrophil elastase) inhibitors [17] showed that although some of the tested molecules were able to inhibit HtrA3 (compounds 1, 2, 4, and 5), to our surprise, one of the compounds (3) enhanced the ability of HtrA3 to hydrolyze a fluorogenic substrate (Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-PheANB-NH2, [7]) almost three times (Figure 3). The only difference between phosphonic peptides 1-5 was the structure of an N-terminal capping group. This observation initiated the subsequent analyzes performed to establish the basic requirements of an effective HtrA3 activating motif. We have synthesized a few simplified structural analogs of 3 that were devoid of the phosphonate function (6-8). The obtained results (Figure 4) demonstrated that two general structural factors seem to play a role in the enhancement of the HtrA3 activity: first, the N-terminal aromatic carboxyl moiety (HCOO-CH2O-C6H4-OCH2-COOH = 2,2′-(1,4phenylenebis(oxy))diacetic acid) and second, the length of the potential activator that equals at least five amino acid residues since shorter peptidomimetics did not influence the HtrA3mediated substrate hydrolysis rate. Considering the above structural factors, we have further focused on the activator optimal sequence mapping.



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A O O

S

O

O

O

N H

N

N H

O P O O

S

O N

O HO

S

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O N H

N

O

O P O O

HO

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S

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N H

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N

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N

O P O O

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O

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4

2

Figure 3. A. Structure of HNE inhibitors screened toward HtrA3. B. The influence of peptidyl phosphonate esters (1-5) on the rate of the fluorogenic substrate hydrolysis by HtrA3.

A

O N

N H

S

O

O N H

O N

N H

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OH O

O N H

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OH

O

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O

O P O O

3

O

HO

O

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B

S

O N H

O

O

HO

6

O

8

Figure 4. Structure of the first potential HtrA3 activators—analogs of 3. B. The influence of 3 analogs (6-8) on the HtrA3 proteolytic activity. The obtained preliminary data encouraged us to synthesize a library of peptidomimetics containing the N-terminal HCOO-CH2O-C6H4-OCH2-COOH moiety (Figure 2). At each randomized position, all proteinogenic amino acids, except the Cys residue, were present. Further deconvolution of this library revealed a compound sequence that could enhance the proteolytic activity of HtrA3. Library screening All HtrA3 enzymatic assays were performed using a recently developed fluorogenic substrate Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-ANB-NH2 [martins]. After the addition of each tested sublibrary with a fixed amino acid position, the increase in the fluorescence intensity was monitored in time with respect to the enzyme solution devoid of the tested activator sublibrary (control). The data analysis results obtained for the X5 position screening indicated a high preference toward sublibraries with aromatic X5 residues such as Tyr, while


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Trp, Phe, Leu, and Pro were less accepted (Figure 5A)—a ten-fold increase in the substrate hydrolysis velocity was observed for a sublibrary with Tyr and eight-fold for Trp when compared with the control sample devoid of an activator. For other residues, a less significant effect on the activity of HtrA3 was observed; thus, Tyr as the X5 residue was introduced into the next step of library deconvolution. A subsequent analysis of the cleavage rates of the sublibraries of the general formula HCOO-CH2O-C6H4-OCH2-CO-Tyr-X4-X3-X2-X1-OH showed that X4 residues containing a small aliphatic side chain (Asn, Ala, Gln, Asp, and Thr) displayed an HtrA3 activating effect (Figure 5B). As the highest, approximately three-fold as compared to the control, an increase in the hydrolysis rate was observed for Asn; this residue was fixed at the X4 position, and the library HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-X3-X2X1-OH was subjected to the next step of deconvolution. Based on the obtained results (Figure 5C), the highest increase in the HtrA3-mediated substrate cleavage was observed for libraries containing Leu and Phe as the X3 residues, while for the other residues examined, no significant activation was observed when compared with the activation observed for libraries from the previous deconvolution steps (Tyr-X4-X3-X2-X1 and Tyr-Asn-X3-X2-X1). Fixing the Phe residue at the X3 position led to further deconvolution steps and the analysis of the sublibraries of the known X2 position. Although for most of the amino acid residues examined at the X2 position, an increase in the HtrA3 activity was observed, the highest, approximately 15 times increase, was observed for His (Figure 5D), which was selected for further studies. Final steps of the library deconvolution allowed for the selection of an optimal X1 residue. Two X1 residues, Glu and Asn, displayed a similar (approximately 25×) increase in the HtrA3 activity when compared with the control (Figure 5D). As a result of the performed library deconvolution, two peptides HCOO-CH2O-C6H4OCH2-CO-Tyr-Asn-Phe-His-Asn-OH (Act1) and HCOO-CH2O-C6H4-OCH2-CO-Tyr-AsnPhe-His-Glu-OH (Act2) were selected as the optimal HtrA3 activators and subjected to further analyzes including enzyme titration at different temperatures and establishing a minimal enzyme concentration that responds to activators Act1 or Act2 (equal respectively). As shown in Figure 6, HtrA3 at a concentration higher than 11 nM responded to the presence of both activators (used at concentrations of 8.73 µM and 8.87 µM, respectively); here, the higher activating effect was observed for Act2, which was almost two times more active than Act1 when HtrA3 was used at 10.7 nM. In order to verify if this activation event is related to the peptide sequence not to the peptide length or the N-terminal moiety we decided to synthesized the two scrambled peptides Sc1 (HCOO-CH2O-C6H4-OCH2-CO-Asn-His-Tyr-



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Asn-Phe-OH) and Sc2 (HCOO-CH2O-C6H4-OCH2-CO-Glu-His-Tyr-Asn-Phe-OH). As visible on Figure 7 no significant increase of hydrolysis of the substrate as compare to control system was recorded. This observation seems to prove that activation of HtrA3 is strictly linked with presence of specific sequences of selected peptidomimetics (Act1 or Act2).

Figure 5. Analysis of the HtrA3-mediated hydrolysis rate of the fluorogenic substrate (Ala(2MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-ANB-NH2) in the presence of activator libraries with fixed A) X5 position, B) X4 position, C) X3 position, D) X2 position, and E) X1 position, and F) structures of developed activators.


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Figure 6. Fluorescent substrate hydrolysis rate at increasing HtrA3 concentration influenced by the presence activators Act1 and Act2 (88.7 µM). The assay was performed at 37°C.

Further analysis showed that HtrA3 activation is not temperature dependent, and both activators (Act1 and Act2) showed an increase in the HtrA3-mediated substrate hydrolysis between 23°C and 45°C (Figure 7). Similar to previous experiments, the HtrA3 activating effect was more significant for compound Act2 for which higher hydrolysis rates were observed for all temperatures tested at each concentration value. In the HtrA protease family, of whose HtrA3 is a good example, activation and proteolysis are physiologically linked with a large protein substrate. Binding of such molecules to the enzyme results in subsequent or simultaneous activation of HtrA3 and occurrence of a proteolytical event. To study if the same could be observed for artificial low-molecular weight activators selected in this study, we analyzed a mixture of HtrA3 incubated with Act1 or Act2 along with fluorogenic substrates using chromatographic methods (RP-HPLC). Figure 8 illustrates the results of these experiments. As clearly visible Act1 or Act2 peaks (Rt 16.09 or Rt 16.38, respectively) remain intact during the 24 h of the experiment since the fluorogenic substrate is consumed by the enzyme within 1 h. This finding indicates that its mode of action is not related to the active center of the studied enzyme.



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Figure 7. Substrate Ala(2-MCA)-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-ANB-NH2 hydrolysis rate in the presence of increasing concentration of HtrA3 activator 1 (A) and activator 2 (B) and scramble peptides 1 (C) and 2 (D)

Figure 8. Chromatography analysis of activators Act1 and Act2 after incubation with HtrA3.


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Selectivity study As mentioned above, HtrA3 belongs to a closely related HtrA family of enzymes with overlapping specificities. To study whether the activators can discriminate among individual HtrA members, we decided to perform a selectivity study. Compounds Act1 and Act2 were incubated with HtrA1, HtrA2, and HtrA3 as well as with HtrA2- and HtrA3-PDZ-deficient mutants (HtrA2(-)PDZ and HtrA3(-)PDZ). As shown in Figure 9, increased proteolysis efficiency was observed for HtrA3 but not for HtrA3(-)PDZ; this indicates the involvement of the PDZ domain in the activation event. Surprisingly, a similar observation was not recorded for HtrA2 and its PDZ truncated analog for which more than 20-times increased proteolysis was observed when compared with the activity of full-length HtrA2 after an addition of either of the developed activators Act1 or Act2. Both compounds were able to increase the proteolytic activity of all HtrAs tested in the presented study.

Figure 9. Influence of activators Act1 and Act2 on the proteolytic activity of HtrA members.

To inspect how Act1 or Act2 influences substrate–enzyme interactions, we determined the kinetic parameters (kcat, KM, and specificity parameter kcat/KM) of the substrate in the presence of the mentioned compounds. As presented in table 1 it is clearly visible that presence of activators the fluorescent increase substrate cleavage rate. Insignificant activation is visible for Sc1 and Sc2 peptides. Kinetic parameters obtained for fluorescent substrates for HrA3 incubated with Act1 or Act2 and with its scrambled analogs indicates that substantial change in kinetic parameters is observed for



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two first systems. In case of activators there is a dramatic increase as compare to untreated substrate in maximal velocity of reaction almost 55× 10-8 mol×s-1 (Act1), 63× 10-8 mol×s-1 (Act2) since for substrate alone the maximal velocity is close to 1× 10-8 mol×s-1. Also scrambled controls did not influence this parameter. KM values calculated for activated HtrA3 are greater as compare to control but in much lower extend reaching KM 48 µM and 64 µM. Table 1. Kinetic parameters of Act1 and Act2 and its scrambled analogs. compound

Vmax M/s * × 10

kcat [s-1]

-8

KM [M] × 10

-6

kcat/KM [M/s] × 106

Act1

54.8±4.3

54.8±4.8

48.8±5.9

1.124±0.138

Act2

63.1±7.2

63.1±7.2

63.8±4.4

1.067±0.189

Scrambled

1.67±0.01

1.67±0.01

31.0±4.2

0.054±0.005

2.12±0.03

2.12±0.03

27.9±3.9

0.076±0.016

1.43±0.02

1.43±0.02

32.5±3.2

0.043±0.003

peptide1 Scrambled peptide2 Substrate

*Data were fitted to the Michealis–Menten equation

In order to compare the activating potency of synthesized and tested compounds the EC50 values were determinated. As visible on figure 10 and table 2 the EC50 values for Act1 and Act2 reaching since its scrambled analogs Sc1 and Sc2 display significant lower effect on HtrA3 activity. Table 2. EC50 values for activators and its scrambled analogs compound

EC50

Hill slope

[µM]

Act1

40.7± 8.2

7.2±0.5

Act2

89.7±9.1

18.8±2.1

Scrambled peptide1

1112.5±138.4

1.6±0.2

Scrambled peptide2

892.7±92.2

1.3±0.1


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Figure 10. EC50 values determinated for activators and their scrabbled system.

Results of the antiproliferative effect of both compounds are presented in Table 2S. Both the tested compounds displayed relatively low cytotoxicity against a panel of cancer cell lines,. However the ovarian cancer cell line seems to be most sensitive for activator treatment. We observed a noticeable (24.6% for Act2 and 32.2% for Act1) reduction of the cell number of the A2780 concentration of the tested compound equal to 100 µM. Overall, non-significant cytotoxicity could be an effect of the low cell permeability of negatively charged molecules [21]. One of the explanation of the increased susceptibility of the ovarian cancer derivate cell line (A2780) might be the observation made by Zhao et al. of the suppression of the Htra3 level in this malignancy despite its stage [6]. Molecular modelling The molecular model of HtrA3 with compound (1) or (2) indicates that both peptidomimetics are a nicely fit to the PDZ domain that is responsible for increasing the HtrA3 activity in vivo. The detailed analysis of the HtrA3, (HCOO-CH2O-C6H4-OCH2-CO-YNFHE(N)) model shows the following protein: ligand interactions (Fig. 11). The residue in position 1 (E/N) is in the vicinity of Lys224, and in the case of glutamic acid, it is possible to create a strong slat bridge. The side-chain of the glutamic acid (Glu1) is also long enough to create a hydrogen bond with an asparagine residue (Asn196). For asparagine, which could be present in position 1 instead of Glu1, the only possible interaction with the protein is a hydrogen bond with Lys224. The experimental results show that there is no effect when glutamine (Gln) or aspartic acid (Asp) residue is in position 1. We may suspect that the side-chain of the Asp is not long enough to create a stable hydrogen bond with Lys224. In the case of the Asn residue, the obtained results may be explained by the comparison of the interaction energy with the



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side-chain of the Lys224. When Gln is in position 1 instead of Asn, the interaction energy between the side-chains is about 7 times higher. The His2 amino-acid residue is near Asp223 and Lys225; however, we did not see any significant interaction of this residue with the protein, since it is directed toward the solvent. This result is partially confirmed by the experiment. In this instance, there is no leading residue in position 2. The Phe3 residue of the ligand seems to play two roles. The first is a π-type interaction with Phe 344, and the second is the protection of the salt bridge between Asp221 and Lys347. Since Lys 347 is located at the N-terminal part of the loop connecting the PD and the PDZ domain, we may suspect that this interaction is crucial to maintain the appropriate position of PDZ with respect to the PD domain. This result is partially supported by the experiment, since the second residue working in position 3 is leucine (Leu). Leucine may also be involved in the protection of the abovedescribed salt bridge; however, it cannot create a π-type interaction with Phe344. We observed that Asn4 is directed toward the solvent and does not interact with the protein. Subsequently, we found a hydrogen bond between the backbone of Tyr5 and Ile222. The sidechain of Tyr5 is directed toward the empty space between PD and PDZ. In our model, we observed a hydrogen bond between Tyr5 and Lys350 and/or Asp351. In the case of Lys350, a different rotamer may be present. This may explain the good result in the case of glutamic acid and glutamine, which have side-chains long enough to create a strong interaction. The carboxyl group of the 2,2′-(1,4-phenlenbio(oxy) diacetic acid residue is making the salt bridge with Lys354. Additionally, the phenyl group is making a π-type interaction with the tryptophan residue, Trp352. Results of the theoretical study showed that the affinity of binding the designed hexapeptide to HtrA3 is much higher than of the other known ligands. In our putative model, this effect is observed due to the formation of two strong interactions (two salt bridges).


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Figure 11. Model of the human HtrA3 complexed with A) HOOC-CH2O-C6H4-OCH2-COTyr-Asn-Phe-His-Asn-OH (1) and B) HOOC-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-GluOH (2). In conclusion, the presented study describes the first attempt to apply the combinatorial chemistry methods for the development of peptidomimetics acting as activators of HtrA3 protease. Surprisingly, the screening for potential HtrA3 inhibitors gave the first insight into the structural requirements toward an effective activator molecule. Considering the structure of the N-terminal capping group and five amino acid-long chains of potential activators, we have designed a library of a general formula: HOOC-CH2O-C6H4-OCH2-CO-X5-X4-X3-X2X1. The following deconvolution of the designed library allowed for the selection of highly potent compounds which at the micromolar concentration increased the rate of the HtrA3mediated hydrolysis of the synthetic fluorescent substrate up to 40 times. The molecular model of the complex between HtrA3 and Act1 or Act 2 indicated their strong binding in the PDZ domain of the enzyme. Positive cooperativity of both compounds is clearly visible in terms of their kinetic parameters with the Hill coefficient above a 10-fold increase as compared to the control system. Both compounds reveal low cytotoxicity against the HtrA3-



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deficient malignant ovarian cell line that makes them an excellent starting structure for a further development of anticancer drugs.

Supporting Information Available This file contains physicochemical characteristic of finals compounds and their scrambled analogs (RP-HPLC and mass spectrometry analysis). Additionally results proliferation assay of the panel of cancer cell lines (UM-UC-3, PC-3, HT-29, MV-4-11, Balb/3T3, A-549 MCF7 and A2780) in presence of selected compounds are presented./

Acknowledgments This work was supported by the National Science Center (Poland) (https://www.ncn.gov.pl) project grant no. UMO-2013/09/B/NZ1/01068 to BL. REFERENCES 1. Karagiannis, G. S.; Berk, A.; Dimitromanolakis, A.; Diamandis, E. P., Enrichment map profiling of the cancer invasion front suggests regulation of colorectal cancer progression by the bone morphogenetic protein antagonist, gremlin-1. Mol Oncol. 2013, 7, (4), 826-39. 2. Skorko-Glonek, J.; Zurawa-Janicka, D.; Koper, T.; Jarzab, M.; Figaj, D.; Glaza, P.; Lipinska, B., HtrA protease family as therapeutic targets. Curr. Pharm. Des. 2013, 19, (6), 977-1009. 3. Patterson, V. L.; Thompson, B. S.; Cherry, C.; Wang, S. B.; Chen, B.; Hoh, J., A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging. J. Vis. Exp. 2016, (113). doi: 10.3791/54136. 4. Moriya, Y.; Uzawa, N.; Morita, T.; Mogushi, K.; Miyaguchi, K.; Takahashi, K.; Michikawa, C.; Sumino, J.; Tanaka, H.; Harada, K., The high-temperature requirement factor A3 (HtrA3) is associated with acquisition of the invasive phenotype in oral squamous cell carcinoma cells. Oral. Oncol. 2015, 51, (1), 84-9. 5. Beleford, D.; Rattan, R.; Chien, J.; Shridhar, V., High temperature requirement A3 (HtrA3) promotes etoposide- and cisplatin-induced cytotoxicity in lung cancer cell lines. J. Biol. Chem. 2010, 285, (16), 12011-27. 6. Zhao, J.; Zhang, J.; Zhang, X.; Feng, M.; Qu, J., High temperature requirement A3 (HTRA3) expression predicts postoperative recurrence and survival in patients with nonsmall-cell lung cancer. Oncotarget 2016, 7, (26), 40725-40734. 7. Yin, Y.; Wu, M.;, Nie, G.; Wang, K.; Wei, J.; Zhao, M.; Chen, Q., HtrA3 is negatively correlated with lymph node metastasis in invasive ductal breast cancer. Tumor Biology 2013, 34, (6), 3611-7. 8. Singh, H.; Li, Y.; Fuller, P. J.; Harrison, C.; Rao, J.; Stephens, A. N.; Nie, G., HtrA3 Is Downregulated in Cancer Cell Lines and Significantly Reduced in Primary Serous and Granulosa Cell Ovarian Tumors. J. Cancer. 2013, 4, (2), 152-64.


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9. Runyon, S. T.; Zhang, Y.; Appleton, B. A.; Sazinsky, S. L.; Wu, P.; Pan, B.; Wiesmann, C.; Skeleton, N. J.; Sidhu, S. S., Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3. Protein Science 2007, 16, (11), 2454-2471. 10. Liu, H.; Dou, S. F.; Zhang, X.; Wang, Y.; Wen, Q. L.; Mu, Y.N., Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface. Int. J. Pept. Res. Ther. 2016, 22, (3), 371-376. 11. Inoue, Y.; Omodani, T.; Shiratake, R.; Okazaki, H.; Kuromiya, A.; Kubo, T.; Sato, F., Development of a highly water-soluble peptidebased human neutrophil elastase inhibitor; AE3763 for treatment of acute organ injury. Bioorg. Med. Chem. 2009, 17, (21), 7477−7486. 12. Newman, M. S.; Cella, J. A., Studies on the monoalkylation of hydroquinone. J. Org. Chem. 1974, 39, (2), 214−215. 13. Sole, B.; Barany, G., Optimization of Solid-Phase Synthesis of. [Ala8]-dynorphin A. J. Org. Chem. 1992, 57, (20), 5399–5403. 14. Wysocka, M.; Gruba, N.; Miecznikowska, A.; Popow-Stellmaszyk, J.; Gütschow, M.; Stirnberg, M.; Furtmann, N.; Bajorath, J.; Lesner, A.; Rolka, K., Substrate specificity of human matriptase-2. Biochimie 2014, 97, 121-7. 15. Glaza, P.; Osipiuk, J.; Wenta, T.; Zurawa-Janicka, D.; Jarzab, M.; Lesner, A.; Banecki, B.; Skorko-Glonek, J.; Joachimiak, A.; Lipinska, B., Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains. PLoS One. 2015, 10, (6), 1-24. 16. Jarzab, M.; Wenta, T.; Zurawa-Janicka, D.; Polit, A.; Gieldon, A. J.; Wysocka, M.; Glaza, P.; Skorko-Glonek, J.; Ciarkowski, J.; Lesner, A.; Lipinska, B., Intra- and intersubunit changes accompanying thermal activation of the HtrA2(Omi) protease homotrimer. Biochim. Biophys. Acta. 2016, 1864, (3), 283–296. 17. Pearlman, D.A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.; Cheatham, T. E.; DeBolt, S.; Ferguson, D.; Seibel, G.; Kollman, P., AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comp. Phys. Commun. 1995, 91, 1-41. 18. Case, D.A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K.M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R., The Amber biomolecular simulation programs. J. Comput. Chem. 2005, 26, 1668-1688. 19. Pikora, M.; Gieldon, A., RASMOL AB - New functionalities in the program for structure analysis. Acta Biochimica Polonica, 2015, 62, (3), 629-631. 20. Winiarski, L., Oleksyszyn, J., Sienczyk, M., Human neutrophil elastase phosphonic inhibitors with improved potency of action. J. Med. Chem. 2012, 55, (14), 6541-53. 21. Zou L.-L., Ma J.-L., Wang T., Yang T.-B., Liu C.-B., Cell-Penetrating PeptideMediated Therapeutic Molecule Delivery into the Central Nervous System Curr Neuropharmacol. 2013, 11, 197–208.



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338x190mm (96 x 96 DPI)


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Selection of Effective HTRA3 Activators Using Combinatorial

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