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. 2019 Nov 11;10(12):1635–1640. doi: 10.1021/acsmedchemlett.9b00394

Tetrazole as a Replacement of the Electrophilic Group in Characteristic Prolyl Oligopeptidase Inhibitors

Tommi P Kilpeläinen †, Jonna K Tyni ‡, Maija K Lahtela-Kakkonen ‡, Tony S Eteläinen †, Timo T Myöhänen †, Erik A A Wallén ⊥,*
PMCID: PMC6912865  PMID: 31857839

Abstract

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4-Phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines were studied as prolyl oligopeptidase inhibitors. The compounds were more potent than expected from the assumption that the tetrazole would also here be a bioisostere of the carboxylic acid group and the corresponding carboxylic acids are at their best only weak inhibitors. The aminoacyl groups l-prolyl and l-alanyl gave potent inhibitors with IC50 values of 12 and 129 nM, respectively. This was in line with typical prolyl oligopeptidase inhibitors; however, we did observe a difference with N-methyl-l-alanyl, which gave potent inhibitors in typical prolyl oligopeptidase inhibitors but not in our novel compound series. Furthermore, all studied 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines decreased α-synuclein dimerization at the concentration of 10 μM, also when they were only weak inhibitors of the proteolytic activity of the enzyme with an IC50 value of 205 μM. Molecular docking studies revealed that the compounds are likely to bind differently to the enzyme compared to typical prolyl oligopeptidase inhibitors represented in this study by 4-phenylbutanoyl-aminoacyl-2(S)-cyanopyrrolidines.

Keywords: Prolyl oligopeptidase, inhibitor, synthesis, tetrazole, alpha-synuclein aggregation, molecular docking

Prolyl oligopeptidase (PREP) is a serine protease with endopeptidase activity on short proline containing peptides, cleaving the peptide on the carboxyl side of the proline residue, and many proline containing peptides, such as oxytocin and several neuropeptides, have been identified as its substrates in vitro (see for review Garcia-Horsman et al.1). PREP is mainly found in the brain, more specifically in caudate nucleus and putamen, hippocampus, and cortex.2 However, significant PREP activities and protein levels have been measured also in peripheral tissues such as skeletal muscle,3 renal cortex, liver, ovaries, and testis, and in ovarian and colorectal tumors.4−6 Alterations in the enzyme activity of PREP have been observed in several diseases, including Parkinson’s disease, Alzheimer’s disease, and Lewy body dementia,7 and in several types of tumors.8,9 Apart from its proteolytic activity, PREP has also other biologically important functions mediated by protein–protein interactions.10,11 PREP is able to enhance dimerization of α-synuclein (αSyn), the key player in cellular toxicity in Parkinson’s disease, via a direct protein–protein interaction.12 PREP has highly flexible regions that are in equilibrium among many conformations, and inhibitor binding stabilizes these flexible regions into one conformation.13 Inhibited PREP has been shown to increase autophagy, which is known to enhance clearance of aggregated forms of proteins and decrease dimerization of αSyn in vitro and in vivo.14,15

Almost all known highly potent PREP inhibitors are structurally related to or recognizable mimetics of the peptide substrates with distinct P1, P2, and P3 sites.16 The P1 moiety mimics the important l-proline (Pro) residue in the substrate, and it is typically a pyrrolidine with or without an electrophilic group in its 2-position. However, also five-membered heteroaryls such as 2-thienyl and 2-furanyl in the P1 position have resulted in potent inhibitors.17 The P2 moiety can be a wide variety of aminoacyl groups or aminoacyl mimetics with Pro being the most preferred natural aminoacyl group, and the P3 moiety is typically a lipophilic acyl group. There are many examples of variations among the published inhibitors;16 however, the two carbonyl groups of the connecting amide bonds have been difficult to replace and only a few inhibitors lack one or both of them in their structures.18−21

The design for the new compounds came from one compound synthesized by the last author of this paper in his earlier research in the search for different replacements for the electrophilic group in PREP inhibitors, where the tetrazolyl group gave a surprisingly potent compound (unpublished data). In the present study, our primary aim was to investigate if 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines as PREP inhibitors had the same structure–activity relationship as typical PREP inhibitor compound series such as 4-phenylbutanoyl-aminoacyl-pyrrolidines and 4-phenylbutanoyl-aminoacyl-2(S)-cyanopyrrolidines. A further aim was to study what effect the novel compound series had on protein–protein-mediated functions of PREP, such as catalysis of αSyn dimerization. We were intrigued by the fact that the tetrazole ring is quite different from typical 2-substituents on the P1 pyrrolidine ring, especially its polar character is exceptional.

There are PREP inhibitors with five-membered heteroaromatics, which look slightly similar to our tetrazoles.22,23 However, all of these have an electrophilic carbonyl group in exactly the same position as the typical electrophile. In addition, the tetrazole ring is negatively charged at physiological pH unlike the five-membered heteroaromatics in the published PREP inhibitors.

The synthetic routes for accessing the compounds in this study are presented in Scheme 1. Compounds 7a (SUAM-1221), 7b, and 14a (KYP-2047) were obtained from our old compound library at the University of Eastern Finland (14a was also synthesized here only as a synthetic intermediate on the route to 15a but not used in the testing). The connecting amide bonds were synthesized using activation of carboxylic acid groups to corresponding acid chlorides or mixed anhydrides. These activation methods could not be used for non-N-alkylated chiral amino acids due to their susceptibility for racemization, and a milder activation to hydroxysuccinimide esters was used for them. Furthermore, in the case of 2-aminoisobutyric acid (Aib) the low solubility of the amino acid in the solvent of the first step of the synthetic route had to be circumvented, and in the case of N-methyl-l-alanine (MeAla), the slightly modified synthetic route gave improved yields and more easily separable intermediates. The 4-phenylbutanoyl-aminoacyl-l-prolinamides were converted to the corresponding nitriles by dehydration with trifluoroacetic anhydride and then further to the corresponding tetrazoles with sodium azide.

Scheme 1. Synthesis of the Compounds, Where a Specific Aminoacyl Group Is Always with the Same Lower Case Letter in Compound Numbering.

Scheme 1

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Reagents and conditions: (a) (1) 10% aqueous Na2CO3, 4-phenylbutanoyl chloride/Et2O; (b) SOCl2/MeOH, reflux; (c) 4-phenylbutanoyl chloride, DIPEA/DCM; (d) LiOH/water, MeOH; (e) (1) pivaloyl chloride (or in its place ethyl chloroformate for 4g), Et3N/DCM, 0 °C, (2) pyrrolidine, Et3N/DCM; (f) pyrrolidine/DCM; (g) (1) TFA/DCM, 0 °C, (2) 4-phenylbutanoyl chloride, Et3N/DCM (or alternatively for 6f, (2) 1 M NaOH, 4-phenylbutanoyl chloride,/Et2O); (h) l-proline methyl ester, DIPEA/DCM; (i) (1) ethyl chloroformate, Et3N/DCM, 0 °C, (2) l-proline methyl ester, Et3N/DCM; (j) (1) pivaloyl chloride, Et3N/DCM, 0 °C, (2) l-prolinamide, Et3N,/DCM; (k) (1) ethyl chloroformate, Et3N/THF, −10 °C, (2) NH3 (7 M in MeOH); (l) (1) ethyl chloroformate, Et3N/DCM, 0 °C, (2) l-prolinamide, Et3N/DCM; (m) TFAA, Et3N/THF; (n) NaN3, NH4Cl/DMF, 100 °C.

The inhibition of the proteolytic activity of PREP (IC50) for the compounds is presented in Table 1. The IC50 values were in the beginning of the project determined using mouse brain homogenate, but we changed later to purified recombinant porcine PREP. Most unsubstituted pyrrolidines and prolinamides were tested with brain homogenate, and all nitriles and tetrazoles were tested with purified porcine PREP. The IC50 values for a few compounds were determined with both homogenate and pure enzyme to verify that the values were comparable.

Table 1. Inhibitory Activities of the Synthesized Compounds.

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compd Xaa R IC50 (nM) enzyme IC50 95% CI IC50 (nM) homogenate IC50 95% CI
7a (SUAM-1221) Pro H 12 10–15    
7b Ala H 147 115–191    
7c MeAla H     298 142–571
7e Sar H     5141 3950–6604
7f d-Ala H     35 180 12 600–10 5300
7g Aib H     13 510 8752–22570
7h βAla H     12 080 7912–17 790
13a Pro CONH2 4371 3841–4965    
13b Ala CONH2     6213 3325–11 340
13c MeAla CONH2     28 800 9792–97 350
13d Gly CONH2     153 800 95 170–249 800
13e Sar CONH2     457 000 329 600–637 700
14a (KYP-2047) Pro CN 0.86 a 0.3 a
14b Ala CN 4.06 2.0–7.7 3.28 2.06–5.22
14c MeAla CN 5.4 4.5–6.4    
14d Gly CN 220 117–454 264 138–493
14e Sar CN 269 b    
15a Pro tetrazolyl 12 9.9–14.7    
15b Ala tetrazolyl 129 71–225 91 55–146
15c MeAla tetrazolyl 27 180 22 150–33 410    
15d Gly tetrazolyl 205 400 c    
15e Sar tetrazolyl 10 640 8110–14 390    
10a Pro CO2H 3626 2821–4965    
10b Ala CO2H 17 540 14 100–22 100    
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a

Confidence interval could not be determined since IC50 value is lower than lowest used concentration.

b

Confidence interval could not be determined since Hill slope is greater than −2.

c

Confidence interval could not be determined since IC50 value is higher than highest used concentration.

In the beginning of the project earlier uncharted aminoacyl groups, Aib, sarcosine (Sar), MeAla, and β-alanine (βAla), were evaluated at the P2 site in 4-phenylbutanoyl-aminoacyl-pyrrolidines, which is the most typical PREP inhibitor structure, and compared to the two natural aminoacyl groups Pro and l-alanine (Ala), which have been reported to give the most potent compounds.24 In this study, we did not carry out comparisons to l-phenylalanine, l-methionine, l-leucine, l-valine, and glycine (Gly) as they have been previously shown to strongly decrease the inhibitory activity.24

All explored aminoacyl groups were in fact one methyl or methylene group changes from Ala, and of these only MeAla gave a potent compound, 7c. The other aminoacyl groups, Sar, d-Ala, Aib, and βAla, gave compounds, 7e, 7f, 7g, and 7h, respectively, with significantly reduced inhibitory activities.

Pro, Ala, MeAla, Gly, and Sar were selected as the aminoacyl groups for our novel tetrazoles. They were synthesized via the corresponding prolinamide and nitrile intermediates, which were also tested for their inhibitory activities. As expected, the prolinamide intermediates 13a–13e had only weak inhibitory activities and the nitrile intermediates 14a–14e were highly potent inhibitors. The electrophilic nitrile group is known to have favorable interaction with the catalytic Ser554 residue at the active site of the enzyme, and for 14a (which is the well-known reference compound KYP-2047),25 this has been described as a reversible covalent interaction.26,27 For the nitriles, also Pro, Ala, and MeAla gave the most potent inhibitors, 14a, 14b, and 14c, with IC50 values of 0.86, 4.06, and 5.4 nM, respectively. These IC50 values should be used with caution as at least 14a is a covalently binding compound with slow, tight binding enzyme kinetics.

Among our target tetrazoles 15a–15e, Pro and Ala gave the most potent inhibitors 15a and 15b, with IC50 values of 12 and 129 nM, respectively. Surprisingly, MeAla gave only a weak inhibitor, 15c, which was over 200-fold lower in inhibitory activity than 15b. It is obvious that the N-methyl group on Ala is not favorable in combination with the tetrazole group. Gly and Sar gave the tetrazoles 15d and 15e, which had weak inhibitory activities of 205 and 10.6 μM, respectively. The carboxylic acid analogues 10a and 10b of the most potent tetrazoles 15a and 15b, respectively, had only weak inhibitory activities in the micromolar range, indicating no bioisosterism between the tetrazole and the carboxylic acid group in this series of compounds.

For evaluating the ability of the tetrazoles 15a–15e to reduce αSyn dimerization, we used a slightly modified PCA (protein fragment complementation assay) method described in Savolainen et al.12 We used mouse N2A cells in the assay, which have high endogenous PREP activity (Suppl. Figure 1). Results of the assay are shown in Figure 1. Interestingly, all tetrazoles 15a–e decreased αSyn dimerization. The αSyn dimerization assay was performed at a concentration of 10 μM and even tetrazole 15d with an IC50 value of 205 μM reduced αSyn dimerization with the same magnitude as tetrazoles 15a and 15b with IC50 values 12 and 129 nM, respectively.

Figure 1.

Figure 1

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Results of the αSyn−αSyn2 dimerization in PCA (protein fragment complementation assay) with 4 h treatment and 10 μM concentration. Tetrazoles 15a–e reduced the amount of αSyn dimers 18–25% compared to control. Compounds 14a (KYP-2047), 14d, and 14e were the only compounds from the nitrile group that were able to reduce αSyn dimerization. Carboxyl acids 10a and 10b were not able to reduce formation of αSyn dimers. Lactacystin serves as positive control for αSyn dimerization. Data are presented as means + SEM (n ≥ 3, ***p < 0.001, **p < 0.01, *p < 0.05).

We decided also to examine the nitrile intermediates as a comparison. The nitrile 14a (KYP-2047) had been verified to reduced αSyn dimerization in several earlier studies, but the other nitriles had not been studied.12,14 To our surprise, the nitriles 14b and 14c with aminoacyl groups Ala and MeAla did not have an effect on αSyn dimerization although they are 4–5 nM inhibitors of PREP, and on the other hand, the nitriles 14d and 14e with aminoacyl groups Gly and Sar had an effect although they are only 220–260 nM inhibitors of PREP. It is important to highlight here that only compounds 14a (89%, p < 0.05), 14d (81%, p < 0.05), 15b (75%, p < 0.01), 15c (75%, p < 0.05), and 15d (77%, p < 0.05) had statistically significant decrease in αSyn dimerization (Student’s t test compared to DMSO control, n ≥ 3).

The αSyn dimerization assay results for both nitriles and tetrazoles clearly indicate that the structure–activity relationship for affecting this function of PREP is slightly different from inhibiting the proteolytic activity. To study the binding of the nitriles 14a–e and tetrazoles 15a–e to PREP, molecular docking studies were performed (Figure 2A–F). The binding pocket included the commonly known S1, S2, and S3 subsites (Figure 2C). Among the nitriles, 14a is known to bind covalently to the catalytically active serine residue (Ser554) at S1.28 Other nitriles could be assumed to orient similarly to 14a directing the nitrile group toward S1 and Ser554 (Figure 2D). Indeed, all nitriles could place the nitrile group at S1 and the phenyl group at S3. The most potent nitriles 14a, 14b, and 14c directed the nitrile group toward Ser554. However, in docking studies 14d and 14e could not orient the nitrile group toward Ser554. This could maybe explain why they are less potent inhibitors than the other nitriles. In the docking protocol the covalent interaction between the nitrile group and Ser554 was not assessed. Thus, the possible covalent bond formation could actually force the nitrile groups to S1.

Figure 2.

Figure 2

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Putative binding site of the tetrazoles with PREP. (A) Crystal structure of PREP. The catalytically active serine residue (Ser554) is marked with black, and the inhibitor-binding site is marked with green mesh. (B) Crystal structure of PREP from site of panels C–F. (C) Ligand-binding pocket with the S1, S2, and S3 subsites. Green indicates lipophilic, yellow aromatic, red electronegative, and blue electropositive areas. (D) Compound 14a at the inhibitor-binding site. The nitrile points toward Ser554 and forms a hydrogen bond to it (not shown in the figure). (E) Compound 15a at the inhibitor-binding site in the commonly known binding mode. (F) Suggested hypothetical binding mode for the tetrazoles with compound 15a as a representative compound.

Interestingly, none of the tetrazoles formed an interaction to Ser554 even though they could place the tetrazole ring at S1. The poses with the tetrazole ring at S1 were compared to the poses of the corresponding nitriles with the nitrile group at S1 (Suppl. Figures 2–6). The comparison revealed that the tetrazole ring might be positioned in the binding pocket slightly differently than the nitrile group. This can be seen most clearly between compounds 14a and 15a and compounds 14d and 15d. The two most potent tetrazoles 15a and 15b were inclined to form an interaction between their negatively charged tetrazole group and the positively charged Arg643 instead of Ser554 (Suppl. Figures 7 and 8). For the other tetrazoles, the tetrazole ring at S1 was not forming any interactions with amino acid residues in the binding pocket (Suppl. Figures 9–11). Moreover, the pose of 15e was tilted when compared to other tetrazoles or nitriles, and its phenyl group positioned outside the pocket (Suppl. Figure 11).

The docking results proposed a putative binding pose in which all the tetrazoles could fit to the binding pocket. In this hypothetical pose, the phenyl group was at S1 instead of S3 (Suppl. Figures 12–16). Tetrazoles might prefer to position the lipophilic benzene ring rather than the hydrophilic tetrazole ring into the hydrophobic S1 pocket. The tetrazole ring does not have a similar ability as the nitrile group to form a covalent bond to Ser554, which is an important interaction for at least some nitriles in anchoring them at S1. As a less hydrophilic moiety, the nitrile group might also be more easily placed into the S1 pocket than the tetrazole ring. Overall, these results suggest that the tetrazoles might have two putative binding poses: the phenyl group at S3 (Figure 2E) or at S1 (Figure 2F).

In conclusion, although the tetrazole group is a known common bioisostere for the carboxylic acid group, the tetrazole is clearly not a bioisostere of a carboxylic acid group in PREP inhibitors. The tetrazoles were more potent inhibitors of the proteolytic activity and αSyn dimerization catalyzing effect of PREP than the corresponding carboxylic acids. The present study further hypothesized that the tetrazoles might have another binding mode in addition to the known binding mode of PREP inhibitors. In this putative binding mode, the tetrazole ring is placed at S3 and the phenyl group at S1. Remarkably, all tetrazoles reduced αSyn dimerization despite the fact that some of them only were weak inhibitors of the proteolytic activity. These findings taken together possibly also indicate that there could be another alternative binding site in enzyme, which affects more the αSyn dimerization catalyzing effect than the proteolytic activity of the enzyme.

Experimental Section

Synthesis and Characterization of Novel Compounds

Synthesis and characterization of all compounds are reported in detail in Supporting Information.

Expression and Purification of Recombinant Porcine PREP

Porcine PREP enzyme was expressed and purified according protocol described by Venäläinen et al.29

Determination of IC50 Value

Preparation of mice brain homogenates and detailed procedure for in vitro assay for inhibitory activity are found in Supporting Information.

PCA for αSyn Dimerization

Detailed cell culture, procedure and used DNA constructs are found in Supporting Information.

Molecular Docking Studies

Protocol is described in Supporting Information.

Acknowledgments

Reinis Svarcbahs is acknowledged for assistance with PREP-purification and Nina Sipari for mass spectrometry. Reference compounds 7a (SUAM-1221), 7b, and 14a (KYP-2047) were obtained from our old compound library at the University of Eastern Finland (earlier University of Kuopio). We thank CSC-IT Center for Science, Finland, for computational capacity and licenses support, and the DDCB core facility for providing a Varioskan plate reader.

Glossary

Abbreviations

PREP

prolyl oligopeptidase

αSyn

α-synuclein

Aib

2-aminoisobutyric acid

Sar

sarcosine

MeAla

N-methyl-l-alanine

βAla

β-alanine

PCA

protein fragment complementation assay

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00394.

  • Experimental methods, PREP activity in N2A cells and N2A cells transfected with hPREP, representative binding poses of 14a–e and 15a–e, interactions of 15a–e with tetrazole ring towards S1, and pose of 15a–e with benzene ring at S1 (PDF)

Author Contributions

The manuscript was written through contributions of all authors. Chemistry was contributed by T.P.K. and E.A.A.W., pharmacology by T.P.K., T.S.E., and T.T.M., and molecular docking studies by J.K.T. and M.K.L.K. All authors have given approval to the final version of the manuscript.

This study was supported by grants from Academy of Finland (318327, 305710), HiLIFE (proof-of-concept), Jane and Aatos Erkko Foundation, Business Finland TUTLI funding, and Sigrid Juselius Foundation to T.T.M., and by salary support from Drug Research Doctoral Program at University of Helsinki to T.P.K. and Drug Research Doctoral Program at University of Eastern Finland to J.K.T.

The authors declare no competing financial interest.

Supplementary Material

ml9b00394_si_001.pdf (1,022.8KB, pdf)

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