A Structure–Activity Relationship Study of SNAIL1 Peptides as Inhibitors of Lysine-Specific Demethylase 1

Abstract

Peptides have recently garnered attention as middle-molecular-weight drugs with the characteristics of small molecules and macromolecules. Lysine-specific demethylase 1 (LSD1) is a potential therapeutic target for lung cancer, neuroblastoma, and leukemia, and some peptide-based LSD1 inhibitors designed based on the N-terminus of SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors, have been reported. The N-terminus of SNAIL1 peptide acts as a cap of the catalytic site of LSD1, inhibiting interactions with LSD1. However, the structure–activity relationship (SAR) of these inhibitors is not yet fully understood. Therefore, in the present study, we aimed to uncover the SAR and to identify novel SNAIL1 peptide-based LSD1 inhibitors. We synthesized peptide inhibitor candidates based on truncating the N-terminus of SNAIL1 or substituting its amino acid residues. In the truncation study, we found that SNAIL1 1–16 (2), which was composed of 16 residues, strongly inhibited LSD1. Furthermore, we investigated the SAR at residues-3 and -5 from the N-terminus and found that peptides 2j and 2k, in which leucine 5 of the parent peptide is substituted with unnatural amino acids, cyclohexylalanine and norleucine, respectively, strongly inhibited LSD1. This result suggests that the hydrophobic interaction between the inhibitor peptides and LSD1 affects the LSD1-inhibitory activity. We believe that this SAR information provides a basis for the development of more potent LSD1 inhibitors.

Introduction

In recent years, middle-molecular-weight drugs have garnered attention as a new drug modality.^1,2) Peptide-based drugs are categorized into middle-molecular-weight drugs and have the characteristics of small molecules such as low immunogenicity and macromolecules such as high target specificity and fewer side effects. In addition, they are provided at a low production cost because they can be obtained by chemical synthesis. Thus, peptide-based drugs are expected to complement small-molecule and macromolecule drugs. In this study, we investigated peptide-based inhibitors for lysine-specific demethylase 1 (LSD1), which is an important drug target.

Among the various post-translational histone modifications, lysine methylation has been extensively studied.^3–7) The methylation of histone lysine residues is a reversible process managed by enzymes known as histone lysine methyltransferases and demethylases.^3–11) Among the lysine demethylases, LSD1 removes methyl groups from mono- and di-methylated lysine 4 of histone H3 (H3K4me1/2) through enzymatic oxidation reliant on flavin adenine dinucleotide (FAD).^8,12–15) LSD1 interacts with multiple proteins, such as CoREST, HDAC1/2, NuRD, SIN3A, and BRMS1, to regulate the expression of numerous genes.^16–21) LSD1 is also involved in various diseases; it promotes the proliferation of lung cancer, neuroblastoma, and leukemia cells^22–25) and is implicated in α-herpesvirus infections and globin disorder.^26,27) Therefore, LSD1 inhibitors are of interest both as tools for studying LSD1 functions and as potential therapeutic agents.^28–34)

Thus far, many LSD1 inhibitors have been reported.^28–34) However, most of these inhibitors are small molecules, and peptide-based inhibitors are relatively scarce.^12,35–46) A series of the reported peptide-based LSD1 inhibitors were designed based on SNAIL1,^17,43–47) a member of the SNAIL/SCRATCH family of transcription factors.^48–50) The N-terminus of SNAIL1 interacts with LSD1 by binding to its catalytic site. Based on this behavior of SNAIL1, we previously reported hydrazine-containing SNAIL1-based peptides designed as irreversible LSD1 inhibitors.⁴⁴⁾ In addition, Tortorici et al. reported SNAIL1-derived peptides that act as reversible LSD1/CoREST complex inhibitors.⁴³⁾ However, the structure–activity relationship (SAR) of the reported peptide-based LSD1 inhibitors is not yet fully understood, and especially SAR of each amino acid residue remains unclear.

In the present study, we aimed to identify more potent SNAIL1-based peptide inhibitors for LSD1 and investigate their SAR. We identified potent peptide-based LSD1 inhibitors and described their design, synthesis, LSD1 inhibitory activities, and SAR.

Results and Discussion

Initially, we performed the truncation study on a peptide consisting of 20 amino acids from the N-terminus of SNAIL1 (SNAIL1 1–20 (1), Fig. 1), which has been reported to strongly inhibit LSD1.⁴³⁾ In addition to 1, two peptides SNAIL1 1–16 (2, Fig. 1) and SNAIL1 1–12 (3, Fig. 1), which consist of 16 and 12 residues, respectively, were synthesized using Fmoc-based solid-phase synthesis (Supplementary Fig. S1). The desired peptides were purified using HPLC (purity >95%).

Fig. 1. Structures of SNAIL1 1–20 (1), SNAIL1 1–16 (2), and SNAIL1 1–12 (3), and Their IC₅₀ Values for LSD1 Inhibition

The IC₅₀ values were determined using the AlphaScreen assay.

Subsequently, LSD1-inhibitory activities of peptides 1, 2, and 3 were evaluated using the AlphaScreen system. In this assay system, we used commercially available recombinant LSD1 and a biotin-labeled H3K4me2 peptide as an LSD1 substrate. We also used acceptor beads immobilized by an H3K4me1 antibody and streptavidin donor beads. When the acceptor and donor beads approach each other via the biotin-labeled H3K4me1 peptide, strong luminescence is observed upon light excitation. In these experiments, we incubated LSD1 and the H3K4me2 peptide substrate in the absence or the presence of test peptides. In the absence of the peptides or the presence of inactive peptides, LSD1 induces demethylation of the H3K4me2 peptide to generate the corresponding H3K4me1 peptide. Therefore, after the addition of the acceptor and donor beads to the mixtures of the enzymatic reaction, strong luminescence is observed due to the formation of a tripartite complex involving the two beads mediated by the H3K4me1 peptide. On the other hand, when a test peptide inhibits the LSD1 catalytic activity and the generation of the H3K4me1 peptide, the luminescence intensity is lower than that in the absence of the test peptide after the addition of the acceptor and donor beads. Thus, the LSD1-inhibitory activity of active peptides was quantitatively estimated based on the luminescence intensity. Analysis of the LSD1-inhibitory activities of peptides 1, 2, and 3 in this system revealed that peptide 2 exhibited slightly stronger LSD1-inhibitory activity than peptide 1, whereas peptide 3 did not strongly inhibit LSD1 (Fig. 1, and Supplementary Fig. S2). These results reveal that 16 residues are required to maintain the LSD1-inhibitory activity.

In addition, we assessed the SAR of peptide 2. The X-ray crystal structure analysis of the interaction of LSD1 with the SNAIL1 N-terminal peptide indicates that residues-3, -4, and -5 are located at or near the catalytic site of LSD1.⁴³⁾ In particular, phenylalanine 4 (residue-4) interacts with coenzyme FAD through π–π interaction (Fig. 2a), which is likely essential to LSD1 inhibition. On the other hand, serine 3 (residue-3) and leucine 5 (residue-5) are situated in positions where they can interact with several amino acid residues of LSD1 (Figs. 2b, c). However, it is unclear which interaction contributes to the LSD1-inhibitory activity. Therefore, we focused on residues-3 and -5 and synthesized various peptides in which these residues were substituted to other amino acids (Supplementary Fig. S3) to uncover the SAR.

Fig. 2. Three-Dimensional Views of the N-Terminal Nonapeptide of SNAIL1 Binding to the Active Site of LSD1 (PDB ID: 2Y48)

Each angle focuses on (a) phenylalanine 4 (residue-4) and FAD, (b) serine 3 (residue-3) and Its neighboring amino acid residues, or (c) leucine 5 (residue-5) and its neighboring amino acid residues.

The replacement of serine 3 (residue-3) in SNAIL1 peptides consisting of 6 residues with alanine reportedly maintains the LSD1/CoREST-inhibitory activity,⁴³⁾ although serine 3 is surrounded by hydrophilic amino acids Asp 555, Asp 556, Thr 335, His 564, and Glu 559 of LSD1 (Fig. 2b). To confirm the influence of serine 3 of peptide 2 on the LSD1-inhibitory activity, we substituted it with phenylalanine or leucine. Phenylalanine-substituted peptide 2a at 10 µM of concentration did not strongly inhibit LSD1 (Fig. 3, and Supplementary Fig. S4). Moreover, the LSD1-inhibitory activity of leucine-substituted peptide 2b was 6.6 times lower than that of peptide 2 (Fig. 3, and Supplementary Fig. S4). This result suggests that the hydroxy group in the SNAIL1 peptides consisting of 16 amino acids is essential for the LSD1-inhibitory activity (Fig. 2b). Therefore, we tested tyrosine-, homoserine-, and threonine-substituted peptides 2c–2e. Homoserine- and threonine-substituted peptides 2d and 2e maintained moderate LSD1-inhibitory activity, although they were slightly inferior to peptide 2. In contrast, tyrosine-substituted 2c exhibited an LSD1-inhibitory activity that was lower than that of peptides 2d and 2e but stronger than that of phenylalanine-substituted peptide 2a. These results suggest that amino acids containing a hydroxy group are preferred to hydrophobic amino acids (Fig. 3, and Supplementary Figs. S4 and S5). However, tyrosine is unsuitable owing to the incompatibility of its benzene ring as a spacer between the main chain and hydroxy group.

Fig. 3. Structures of SNAIL1 1–16 (2) and Peptides 2a–2k and Their IC₅₀ Values for LSD1 Inhibition

We then investigated residue-5. Leucine 5 (residue-5) is located in the hydrophobic space surrounded by Leu 536, Phe 382, Phe 538, and Tyr 761 (Fig. 2c). However, it did not appear to completely fit the hydrophobic space. Therefore, we substituted it with another group to increase the LSD1 inhibitory activity. We prepared and tested peptides 2f and 2g, in which leucine 5 was substituted with aromatic amino acid residues phenylalanine and tyrosine, respectively, in anticipation of the formation of π–π interactions with Phe538 and Tyr761. However, peptides 2f and 2g did not show strong LSD1-inhibitory activities, suggesting that the introduction of aromatic and hydroxy groups is unfavorable for LSD1 inhibition (Fig. 3, and Supplementary Fig. S5). Next, we substituted leucine 5 with other hydrophobic amino acids containing an alkyl group, such as valine, isoleucine, or unnatural amino acids cyclohexylalanine and norleucine, and tested peptides 2h–2k. As shown in Fig. 3, and Supplementary Figs. S5 and S6, the LSD1-inhibitory activities of valine-substituted peptide 2h and isoleucine-substituted peptide 2i decreased compared to that of original peptide SNAIL1 1–16 (2), whereas cyclohexylalanine- and norleucine-substituted peptides 2j and 2k, respectively, exhibited potent LSD1-inhibitory activity. Interestingly, norleucine-substituted peptide 2k showed stronger LSD1-inhibitory activity than the original peptide SNAIL1 1–16 (2). These results suggest that an aliphatic group is more favorable than an aromatic group at residue-5. In addition, a linear alkyl group is more favorable than branched ones; a methyl group of the valine or isoleucine residue is believed to prevent the peptides from forming the active conformation, and three or more methylene groups from the α-position are required for interactions with Phe 382 and Leu 536. However, we do not make sure whether norleucine is the best. Further, the LSD1 inhibitory activities of peptides incorporating amino acids that have more long-chain alkyl groups are also interesting. This is an issue that needs to be investigated in the future.

Finally, we examined the inhibitory activity of peptide 2k toward monoamine oxidase (MAO) A/B, which are other FAD-dependent oxidases.^51,52) Peptide 2k did not inhibit MAOs at 30 µM and exhibited LSD1-selectivity over MAO A/B (Supplementary Table S1). In addition, the affinity of SNAIL1-based peptides to LSD2 is known to be lower than that to LSD1.^17,44) Therefore, peptide 2k is also anticipated to be LSD1-selective over LSD2.

Conclusion

In this study, we identified novel SNAIL1-based LSD1 inhibitors and revealed their SAR. We examined the truncation and replacement of residues-3 and -5 in the SNAIL1 N-terminus. We prepared and tested peptides with residue lengths of 20, 16, and 12 from the N-terminus of SNAIL1 and evaluated their LSD1-inhibitory activity using AlphaScreen systems. SNAIL1 1–16 (2) showed more potent LSD1-inhibitory activity than SNAIL1 1–20 (1) and SNAIL1 1–12 (3). We also investigated the SAR study of SNAIL1 1–16 (2) and found that moderate LSD1-inhibitory activity was maintained even if serine 3 was replaced with homoserine (2d) or threonine (2e), which have a hydroxy group. The substitution of leucine 5 to a hydrophobic amino acid valine or isoleucine reduced the LSD1-inhibitory activity. However, the activity of the peptides whose leucine 5 was substituted to cyclohexylalanine (2j) or norleucine (2k) was similar or superior to that of SNAIL1 1–16 (2). Thus, we successfully discovered novel LSD1-inhibitory peptides incorporating unnatural amino acids and provided the SAR of residues-3 and -5 of the SNAIL1-based LSD1 inhibitors. We believe that this SAR information, targeting LSD1, provides a basis for the development of potent selective inhibitors and biological probes for LSD1.

Experimental

LSD1 Inhibitory Assay Using AlphaScreen^® Assay

Activity assays of LSD1 were performed with the AlphaScreen^® assay system. The purified peptides 1–3 and 2a–2k were diluted with dimethyl sulfoxide (DMSO) to various concentrations. Aliquots of 2.5 µL of peptide solutions [4.0% DMSO in buffer solution (buffer1: 40 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol (DTT) 0.02% (v/v) Tween 20, 10% (v/v) glycerol)] or assay buffer1 containing 4% DMSO as control or blank were added to the wells of a white opaque OptiPlateTM-384. Then, 5.0 µL of an LSD1 (BPS Bioscience, 50097) solution (60 nM, diluted with buffer1) was added to each well (for the blank, 5.0 µL of buffer1 was added instead). The plate was incubated for 30 min at 25 °C with gentle shaking (250 rpm). Aliquots of 2.5 µL of substrate peptide [Lys(Me2)4]-Histon H3 (1-21)-GGK(Biotin) (ANASPEC, AS-64356) solution (10 nM, diluted with buffer1) was added to each well. The plate was incubated for 60 min at 25 °C with gentle shaking (250 rpm). Then, 10.0 µL of acceptor beads (Alpha LISA Protein A Acceptor beads AL101C) with antibody [Anti-Histone H3 (mono methyl K4) antibody [ERP16597]-ChIP Grade, ab176877] in buffer [buffer2: 40 mM HEPES (pH 7.5), 40 mM NaCl, 5% (v/v) glycerol, 0.125% bovine serum albumin (BSA)] (acceptor beads: 120 µg/mL, antibody: 500 nM) was added to each well, and the plate was incubated for 30 min at 25 °C with gentle shaking (250 rpm). Next, 10 µL of donor beads (Alpha Screen Streptavidin Donor beads 6760002S) in buffer 2 (60 µg/mL) was added to each well, and incubated for 30 min at 25 °C in the dark. The Alpha signal of the wells was measured using the EnsightTM multilabel reader (PerkinElmer, Inc., CT, U.S.A.) with excitation at 615 nm and emission at 655 nm. The percent inhibition was calculated from the Alpha signal readings of inhibited wells relative to those of control wells after subtracting blank wells. The LSD1 inhibitory activities were evaluated as IC₅₀ (µM) by means of three replicates. Sigmoidal curve fittings were performed on GraFit Data Analysis Software (Version 7.0.3) (Supplementary Figs. S2 and S4–S6).

Acknowledgments

The authors would like to thank the members of the Comprehensive Analysis Center, SANKEN, Osaka University for the HRMS analyses and Ms. Miho Sawada for technical support. This research was financially supported by Grants-in-Aid for Research Activity start-up (JSPS, JP21K20719) and Early Career Scientists (JSPS, JP22K15251) to Y.T., the Astellas Foundation for Research on Metabolic Disorders to Y.T., and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research; BINDS) from the Japan Agency for Medical Research and Development (AMED) (JP23ama121054). “The diversity female researcher support” and “AIRC-SANKEN Grant” from SANKEN, Osaka University to Y.T. is gratefully acknowledged.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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