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Structure-Based Design of High-Affinity Macrocyclic Peptidomimetics to Block the Menin-Mixed Lineage Leukemia 1 (MLL1) Protein−Protein Interaction Haibin Zhou,† Liu Liu,† Jing Huang,‡,§ Denzil Bernard,† Hacer Karatas,† Alexandro Navarro,† Ming Lei,‡,§ and Shaomeng Wang*,† †

Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, Medicinal Chemistry, ‡Howard Hughes Medical Institute, §Biological Chemistry, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48109-0934, United States S Supporting Information *

ABSTRACT: Menin is an essential oncogenic cofactor for mixed lineage leukemia 1 (MLL1)-mediated leukemogenesis through its direct interaction with MLL1. Targeting the menin−MLL1 protein−protein interaction represents a promising strategy to block MLL1-mediated leukemogenesis. Employing a structure-based approach and starting from a linear MLL1 octapeptide, we have designed a class of potent macrocyclic peptidomimetic inhibitors of the menin−MLL1 interaction. The most potent macrocyclic peptidomimetic (MCP-1), 34, binds to menin with a Ki value of 4.7 nM and is >600 times more potent than the corresponding acyclic peptide. Compound 34 is also less peptide-like and has a lower molecular weight than the initial MLL1 peptide. Therefore, compound 34 serves as a promising lead structure for the design of potent and cell-permeable inhibitors of the menin−MLL1 interaction.

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A recent study8 has shown that menin interacts with MLL1 via a short bipartite motif, called MLL1MBM (menin-binding motif of MLL1), spanning the region from residue 6 to residue 25 (RWRFPARPGTTGGGGGGGRR). MLL1MBM consists of a highly conserved octameric peptide MLL1 6 − 1 3 (RWRFPARP), a nonstructural polyglycine linker and a double-arginine motif (R24R25) (Figure 1).8 The cocrystal structure of menin complexed with MLL1MBM shows that their interaction is mostly mediated through the conserved octameric MLL1 peptide, which adopts a cyclized conformation stabilized by an intramolecular hydrogen bond between the side chain of Arg8 and the backbone carbonyl of Pro13 (Figure 1). Mutation data7,8 has also suggested that the binding affinity between menin and MLL1MBM is dominated by hydrophobic contacts through residues Phe9, Pro10, Ala11, and Pro13 of MLL1MBM and surface hydrophobic pockets in menin (Figure 1). A class of thienopyrimidine compounds was recently discovered by a high-throughput screening approach as smallmolecule inhibitors of the menin−MLL1 protein−protein interaction.9 It was also shown that these inhibitors of the menin−MLL1 interaction reverse the oncogenic activity of MLL1 fusion proteins in leukemia cells.9 The crystallographic structure of a compound from this class bound to menin shows

INTRODUCTION Chromosomal translocations in the mixed lineage leukemia gene 1 (MLL1) are observed in >70% of infant leukemia, 5− 10% of acute myeloid leukemia in adults, and in therapy-related leukemia in patients previously treated with topoisomerase II inhibitors.1 Rearrangements of the MLL1 gene lead to the fusion of the N-terminus of MLL1 protein to more than 60 functionally diverse partner proteins.2 The resultant MLL1fusion proteins provoke abnormal expression of HOX genes, which is pivotal in leukemia pathogenesis.3 Menin, a product of the MEN1 gene mutated in the human cancer syndrome known as multiple endocrine neoplasia type 1 (MEN1), associates with the extreme N-termini of all the MLL1 fusion proteins and is essential for the recruitment of MLL1 oncoproteins to target genes during leukemogenesis.4−7 A potential therapeutic strategy for MLL1-rearranged leukemia is to block the interaction between menin and MLL1, thus inhibiting recruitment of MLL1 fusion proteins to target loci. It has been shown that expression of short MLL1 peptides containing the menin-binding motif of MLL1 sequence disrupts the in vivo menin−MLL1 interaction and inhibits the growth of MLL1-AF9 transformed cells.5 Thus, design of small-molecule inhibitors of the menin−MLL1 interaction represents a plausible approach for the development of new therapies for the treatment of MLL1 rearranged leukemia. © XXXX American Chemical Society

Received: October 19, 2012

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dx.doi.org/10.1021/jm3015298 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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value of 22 nM, inhibits cell proliferation, suppresses Hoxa9 expression and induce differentiation in MLL1-rearranged leukemia cells.10 To probe the MLL1 binding site in menin and facilitate the design of high-affinity small-molecule menin−MLL1 inhibitors, we report herein our structure-based design of potent macrocyclic peptidomimetic inhibitors of the menin−MLL1 interaction. Our efforts have led to the discovery of compound 34, which binds to menin with a Ki value of 4.7 nM and has a much reduced molecular weight and peptidic characteristics as compared to the initial MLL1 peptide. Chemistry. All the linear peptides were synthesized on an ABI 433 peptide synthesizer using Fmoc chemistry. Rink amide resin was used as the solid support and yielded amide capping at the C-terminus, and all the linear peptides were capped with an acetyl group at the N-terminus. For the synthesis of the macrocyclic compounds, ring-closing metathesis11 (Scheme 1) or lactamization reaction (Scheme 2) was employed to form the ring. In Scheme 1, the general method for the synthesis of final compounds IV (compounds 7, 9, 12−15, and 17−35 in Tables 3−6) are outlined. Briefly, intermediates I with a carboxylic acid at the C-terminus were prepared on an ABI 433 peptide synthesizer using proline preloaded 2-chlorotrityl chloride resin as the solid support, followed by the cleavage of the peptides from resin by treatment with 0.5% TFA in CH2Cl2. Acids I were coupled to corresponding amines (CH 2 CH(CH2)nNH2) to generate intermediates II. Ring closures of intermediates II were achieved by ruthenium-catalyzed olefin metathesis reaction11 to give III, whose CC double bond was

Figure 1. Crystal structure of menin (surface) complexed with MLL1MBM (cyan) (PDB ID: 3U85). Intramolecular hydrogen bond in MLL1MBM is shown with dashed lines.

that these compounds bind to the same site as the MLL1 peptide but have a different binding mode.10 The most potent compound reported from this class binds to menin with a Ki

Scheme 1. General Method for the Synthesis of Macrocyclic Peptidomimetics Using RCM Reactiona

Reagents and conditions: (a) (i) ABI 433 peptide synthesizer, (ii) 0.5% TFA in CH2Cl2; (b) CH2CH(CH2)nNH2, HATU, HOAt, DIEA; (c) Grubbs Catalyst (first generation), CH2Cl2; (d) (i) H2, Pd/C, (ii) TFA:TES:H2O (18 mL:0.5 mL:1 mL) or diethylamine then TFA:TES:H2O (18 mL:0.5 mL:1 mL). The structures for all final compounds are depicted in Tables 3−6. a

B



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Scheme 2. General Method for the Synthesis of Macrocyclic Peptidomimetics Using Lactamization Reactiona

a

Reagents and conditions: (a) (i) ABI 433 peptide synthesizer, (ii) 0.5% TFA in CH2Cl2; (b) (i) HATU, HOAt, DIEA, (ii) TFA:TES:H2O (18 mL:0.5 mL:1 mL). Structures of compounds 10, 11, and 16 are outlined in Tables 3 and 4.

Scheme 3. Synthesis of 37a

a

Reagents and conditions: (a) (i) ABI 433 peptide synthesizer, (ii) 0.5% TFA in CH2Cl2; (b) (i) VII, HATU, HOAt, DIEA, (ii) diethyl amine; (c) (i) 5-FAM.SE, DIEA, (ii) TFA:TES:H2O (18 mL:0.5 mL:1 mL).

carboxy fluorescein succinimide ester (5-FAM, SE) to the side chain of the lysine of the immobilized linear peptide which was synthesized on ABI 433 peptide synthesizer. The fluorescent labeled tracer 37 was prepared as shown in Scheme 3. Intermediate VII was made by the procedure used for intermediates I. Acid VII was coupled to amine VIII, followed by removal of the Fmoc group to give IX. Treatment of IX with 5-FAM, SE fluorophore in the presence of DIEA, followed by removal of the protecting groups, yielded 37.

reduced by catalytic hydrogenation, followed by removal of the protecting groups to yield final compounds IV. The acyclic compound 8 was synthesized using a similar strategy as that for intermediate II (Supporting Information Scheme S1). Final compounds VI (compounds 10, 11, and 16 in Tables 3 and 4) were synthesized through lactamization as shown in Scheme 2. Proline preloaded 2-chlorotrityl chloride resin was used as the solid support on ABI 433 peptide synthesizer. TFA in CH2Cl2 was used to cleave the peptides from the resin, which also led to removal of the protecting groups at the terminal amines to give intermediates V. Cyclization of intermediates V were achieved using HATU and HOAt by coupling the acid at the C-terminus and amine at the Nterminus. Removal of the protecting groups yielded final compounds VI. As shown in Scheme S2 (Supporting Information), fluorescent labeled tracer 36 was synthesized by tethering 5-

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RESULTS AND DISCUSSION Determination of the Minimal Motif in MLL1 for HighAffinity Binding to Menin. The crystal structure of MLL1MBM complexed with menin showed that the octameric peptide MLL16−13 motif at the N-terminus of MLL1MBM sits in a well-defined surface pocket in menin.8 The crystal structure also showed that the seven glycines in the peptide have minimal C



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contacts with menin.8 Indeed, removal of these seven glycines in this peptide had minimal effect on the binding affinity to menin.8 To evaluate the binding contribution of the C terminal double arginines to menin, we synthesized peptide 1 (AcRWRFPARPGTGRR-NH2) and a shorter peptide 2 (AcRWRFPARP-NH2). In our optimized fluorescence-polarization (FP) based competitive binding assay, 1 binds to menin with a Ki value of 2.9 nM, whereas 2 has a Ki value of 71.1 nM, being 25-fold less potent than 1 (Table 1). Consistent with the previous results,8 our data showed that the double arginine motif makes a significant contribution to binding.

Linear peptide 2 has a relatively small size (eight residues) and high affinity (Ki = 71.1 nM) to menin and was thus employed as the starting point for our structure-based design of macrocyclic peptidomimetics. In the cocrystal structure of the menin−MLL1MBM complex, the octapeptide MLL16−13 adopts a cyclized conformation, stabilized by an intramolecular hydrogen bond formed between the side chain of Arg8 and the backbone carbonyl of Pro13 (Figure 1). We designed and synthesized a macrocyclic compound 7 by replacing the side chain of Arg8 with a 7-carbon aliphatic amine in 2 and coupling the amino group to the carboxylic acid group in Pro13 to form an amide (Figure 2). A 7-carbon linker was selected, initially, because there are seven atoms between the backbones of Arg8 and Pro13 in 2, including the hydrogen in the hydrogen bond (Figure 2). Peptide 8, an acyclic counterpart of 7, was synthesized to distinguish the effect of cyclization from any additional interaction introduced by the linker (Figure 2). Compound 7 binds to menin with a Ki value of 23.8 nM and is 3 times more potent than the acyclic, wild-type peptide 2 (Figure 2). In contrast, the acyclic control compound, 8, has a Ki value of 2170 nM, 91 times less potent than compound 7 (Figure 2). This indicates that the intramolecular hydrogen bond has a significant contribution to the high binding affinity of compound 2, and cyclization in compound 7 can successfully replace this intramolecular hydrogen bond to effectively constrain the conformation to mimic the bound conformation of 2 to menin. To understand the structural basis for its high binding affinity to menin, we determined the cocrystal structure of 7 complexed with menin (Figure 3a). The coordinates for the cocrystal structure for compound 7 in complex with menin have been deposited into the Protein Data Bank (PDB ID: 4I80). Detailed data collection and refinement statistics are provided in Table 2. In this cocrystal structure, the alkyl linker of compound 7 has no direct interaction with the protein; however, cyclization preorganizes the key residues to interact with the menin protein. Compared with the menin−MLL1MBM costructure, the backbone of 7 occupies the same position as that of MLL16−13 in MLL1MBM and thus captures all the critical interactions observed in the menin−MLL1MBM complex (Figure 3b). The unusual backbone dihedral geometry of Pro10 in MLL1 causes its intrusion, together with the phenyl ring of Phe9 in MLL1, into the deep hydrophobic well formed by the side chain of residues Leu175, Leu177, Ala182, Phe238, Tyr276, and Met278 of menin (Figure 3a). The side chain of Ala11 interacts with a shallow hydrophobic pocket in menin, while the backbone carbonyl forms a hydrogen bond with Tyr323. The pyrrolidine

Table 1. Binding Affinities of Truncated MLL1 Peptides to Menin compd 1 2 3 4 5 6

sequence Ac-RWRFPARPGTGRRNH2 Ac-RWRFPARP-NH2 Ac-RWRFPAR-NH2 Ac-WRFPARP-NH2 Ac-RFPARP-NH2 Ac-FPARP-NH2

IC50 ± SD (nM)

Ki ± SD (nM)

11.4 ± 3.3

2.9 ± 1.3

232 85300 1060 9570 140000

± ± ± ± ±

54 5830 142 2830 4448

71.1 25500 311 2850 41800

± ± ± ± ±

19.5 1740 55 821 1330

We next determined the minimal sequence of 2 for highaffinity binding to menin. Removal of the C-terminal residue, Pro13, in 2 afforded 3, which binds to menin with a Ki value of 25500 nM and is >300-fold less potent than 2. This dramatic loss in binding affinity upon removal of Pro13 is consistent with its extensive hydrophobic contacts with menin and the formation of an intramolecular hydrogen bond between the backbone carbonyl of Pro13 and the side chain of Arg8. Sequential deletions of N-terminal residues Arg6, Trp7, and Arg8 in 2 resulted in 4, 5, and 6, which have Ki values of 311, 2850, and 41800 nM, respectively (Table 1). Compounds 4, 5, and 6 are thus 4-, 40-, and 588-fold less potent than 2, respectively. The binding data thus show that, consistent with previously published mutagenesis data and cocrystal structure,7,8 each of these four residues (Arg6, Trp7, Arg8, Pro13) makes a significant contribution to the binding of 2 to menin. Structure-Based Design of Macrocyclic Peptidomimetics. As compared to their linear counterparts, macrocyclic molecules can achieve higher affinities and specificities to their target proteins through a conformationally constrained, preorganized ring structure.12,13 We have therefore designed and synthesized macrocyclic peptidomimetics starting from linear MLL1 peptides.

Figure 2. Design of macrocycle 7 and acyclic control compound 8. D



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Figure 3. Crystal structure of menin (gray) complexed with compound 7 (yellow). (a) Overall binding mode for 7 with residues forming the binding pocket shown as sticks. (b) Superposition of the crystal structure of 7 (yellow) onto the crystal structure of MLL1MBM (cyan). Hydrogen bonds are indicated with dashed black lines. Figures were prepared with Pymol.

Table 2. Crystallographic Data Collection and Refinement Statistics

Table 3. Structures and Binding Affinities of Macrocyclic Peptidomimetics with Varying Length of Linker

menin−7 data collection space group cell dimensions a, b, c (Å) α, β, γ (deg) resolution (Å) (high-resolution shell) Rmerge (%) (high-resolution shell) I/σ (high-resolution shell) completeness (%) (high-resolution shell) redundancy (high-resolution shell) Refinement resolution (Å) no. of reflections Rwork/Rfree (%) no. of atoms protein B-factors protein rms deviations bond lengths (Å) bond angles (deg)

P41212 141.379, 141.379, 92.879 90, 90, 90 100−3.10 (3.21−3.10) 9.8 (29.1) 41.7 (8.5) 92.4 (73.1) 25.5 (20.1) 44.7−3.1 16307 21.49/24.54 3895 107.89 0.009 1.246

compd

linker length (n + 3)

2 7 8 9 10 11 12 13

N/A 7 N/A 6 5 4 8 9

IC50 (nM) 232 78.1 7390 63.3 514 11000 25.0 144

± ± ± ± ± ± ± ±

54 11.7 1160 12.1 28 1050 3.4 20

Ki (nM) 71.1 23.8 2170 17.5 136 3320 6.9 42.5

± ± ± ± ± ± ± ±

19.5 7.2 372 2.7 6 359 1.0 10.2

and compound 11 with a 4-methylene linker are 6 and 139 times less potent than compound 7, respectively. These results indicate that linkers shorter than the 6-methylene linker may significantly distort the conformation of these macrocycles from that desired for optimal binding to menin. Compound 12, with an 8-carbon linker, binds to menin with a Ki value of 6.9 nM and is 3 times more potent than 7. However, 13 with a 9-carbon linker is 2 times less potent than 7 (Table 3). Thus, macrocyclic compound 12 is 10 times more potent than the acyclic, wild-type peptide 2 and 300 times more potent than the acyclic control compound 8. These data show that compound 12 in this series of macrocycles has the optimal ring size for effective interaction with menin. Optimization of Compound 12. Although compound 12 has high affinity to menin, it consists of eight amino residues.

ring of Pro13 is sandwiched between the side chains of Tyr319 and Tyr323 in menin (Figure 3a). The positively charged side chain of Arg12 is involved in charge−charge and hydrogen bond interactions with the negatively charged Glu359 and Glu363 residues in menin (Figure 3a). Another hydrogen bond is seen with the backbone carbonyl of peptide Arg6 and Asn244. Superposition of the bound conformations of 7 and MLL1MBM (Figure 3b) clearly shows that our cyclization strategy does not affect the bound conformation but reinforces it. We further investigated the effect of the macrocyclic ring size in compound 7 for binding to menin by varying the length of the alkyl linker, and the results are shown in Table 3. While compound 9 with a 6-methylene linker binds to menin with an affinity similar to 7, compound 10 with a 5-methylene linker E



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six residues, it is an attractive lead structure for further optimization. Further Optimization of Compound 14. We next modified 14 in an effort to enhance its binding affinity to menin. To identify the regions of the binding pocket most likely to contribute to improved binding affinity, we determined the hydrophobic hotspots on the menin surface by molecular dynamics simulations (MD) using isopropyl alcohol and phenol independently as cosolvents (Figure 4). Previous studies have shown the ability of cosolvent simulations to effectively map the hotspot regions as well as identifying protein conformations relevant to ligand binding.14−16 Using two different cosolvents for MD simulations of menin, we mapped convergent regions of favorable hydrophobic interactions that encompassed the Phe9, Pro10, and Ala11 in MLL1MBM for binding to menin. Because the Pro10 residue is critical to maintain the conformation of this macrocycle, we focused our modifications on Phe9 and Ala11 to enhance the hydrophobic interactions between these two residues in 14 and menin. The cocrystal structure of 7 complexed with menin (Figure 3) showed that the methyl side chain group of Ala11 interacts with a small hydrophobic pocket in menin, and modeling analysis (Figure 4) showed that there is room to accommodate a slightly larger group at this site. We therefore designed and synthesized a series of analogues of 14 in which we employed different hydrophobic groups to probe the small binding pocket occupied by the methyl group in Ala11. As shown in Table 5, compound 17 with an additional methyl group on the α-carbon of Ala11, is 3 times more potent than 14, while compound 18, which contains an ethyl group, is 16 times less potent than 14. On the other hand, compound 19, which contains a threemembered ring, is slightly more potent than 17. Encouraged by the data for 19, we synthesized 20 and 21, with a cyclobutyl or cyclopentyl group. Compound 20 binds to menin with a Ki value of 26.7 nM, which is twice as potent as 19 and 7 times more potent than 14. However, compound 21 is 8 times less potent than 14 and 62 times less potent than 20. Thus the cyclobutyl group at this position was determined to be optimal for binding to menin.

We next sought to reduce its size and peptidic characteristics. Because residues Arg6 and Trp7 in 12 are not part of the macro-ring system and are not critical for maintaining the ring conformation for binding to menin, we replaced these two residues with NH2, NHCOCH3, or H, which resulted in 14, 15, and 16, respectively (Table 4). Compounds 14, 15, and 16 Table 4. Structures and Binding Affinities of Compounds 14, 15, and 16

bind to menin with Ki values of 197, 586, and 1840 nM and are 29, 85, and 267 times less potent than 12, respectively (Table 4). These data show that Arg6 and Trp7, although not critical, contribute to the binding of 12 to menin. Interestingly, 14, which has a positively charged amino group, is 3 times more potent than 15 and 9 times more potent than 16, indicating that a positively charged group at this site can enhance binding to menin, presumably due to the highly acidic nature of the binding cavity of menin. Because compound 14 consists of only

Figure 4. Hydrophobic hot spots (mesh) in the MLL1 peptide (cartoon) binding groove detected from cosolvent isopropyl alcohol (a) and phenol (b) mapping method based on 16 ns MD simulations with the menin (1−386) crystal structure (surface). MLL1 residues in the hotspot regions are shown in stick. F



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pocket for the phenyl group can accommodate larger hydrophobic moieties at the ortho and meta positions, the electronic properties of the substituent may also be important. The protein wall along one side of the phenyl binding site is lined by several acidic residues, and the strong electronegativity of fluorine may enhance polarization of the ring, leading to better binding affinity. To further investigate the effect of Fsubstitution at different positions, we synthesized 32 with Fsubstitution at the ortho-position and 33 with F-substitution at the para-position. 32 binds to menin with a Ki value of 27.5 nM, similar to that of 20, while 33 binds to menin with a Ki value of 123 nM and is 5 times less potent than 20. Because Fsubstitution at the meta-position improves the binding affinity to menin, we synthesized and tested compound 34 with two Fsubstitutions at the two meta-positions on the phenyl ring. Compound 34 binds to menin with a Ki value of 4.7 nM and is thus 6 times more potent than 20 (Figure 5). To test the stereospecificity, we changed the 3,5-difluoro-L-Phe9 in 34 to 3,5-difluoro-D-Phe9, which resulted in 35, an epimer of 34. Compound 35 binds to menin with a Ki value of >100 μM and is >10000 times less potent than 34 (Figure 5). Modeling studies suggested that changing the stereochemistry projects the phenyl residue outside the pocket and also disrupts other interactions with the protein, leading to very poor binding to menin compared to 34 (Supporting Information). Development and Optimization of Competitive Binding Assays. To determine quantitatively and accurately the binding affinities of our designed compounds to menin protein, we developed and optimized sensitive fluorescence polarization (FP)-based competitive binding assays. All IC50 and Ki values reported here are averages and standard deviations (SDs) of at least three independent experiments. First, on the basis of peptide 1, we designed and synthesized a fluorescent labeled tracer, 36, containing a 14-residue MLL1 segment, a spacer (Ahx-Ahx), a lysine residue, and a fluorophore tethered to the amino group of the side chain in the lysine residue (Figure 6a). The Kd value of 36 to menin was determined to be 15.9 nM (Figure 6b). Using 36 and menin protein, we established a competitive binding assay and used this assay to evaluate the binding affinities of our initial compounds. During the course of our work, some of our designed macrocyclic peptidomimetics were found to have very high binding affinities to menin, exceeding the lower limits of the initial competitive binding assay. To accurately determine the binding affinities of our designed compounds, we designed and synthesized a new tracer, 37, based on the macrocyclic peptidomimetic 34 (Figure 6a). The Kd value of 37 to menin was determined to be 1.4 nM (Figure 6c). Using 37 and menin protein, we reoptimized our FP-based competitive binding assay. All the binding data reported in the present study were obtained using the optimized assay.

Table 5. Structures and Binding Affinities of Compounds with the Modified Ala11

The cocrystal structure of menin complexed with compound 7 showed that Phe9 is nested in a hydrophobic pocket of menin (Figure 3), and our hotspot analysis also indicated that this region is favorable for hydrophobic interactions (Figure 4). We therefore made extensive modifications of the Phe9 residue in 20 to explore this hydrophobic pocket (Table 6). Replacement of the phenyl group with a cyclohexyl group resulted in 22, which is 32 times less potent than 20. Introduction of a chlorine substituent at the ortho- or meta- or para-position of the phenyl ring in 20 yielded 23, 24, and 25, which bind to menin with Ki values of 30, 44.6, and 549 nM, respectively. These data clearly show that a p-Cl substituent on the phenyl group is detrimental while either an o- or m-Cl substituent has minimal effect. We then focused on modifications at the m-position of the phenyl ring in Phe9 and synthesized compounds containing substituents with varying size and electronic property. The resulting compounds with a methyl, trifluoromethyl, ethyl, isobutyl, or phenyl substituent are 12, 27, 50, 434, and 524 times less potent than compound 20, respectively (Table 6). However, compound 31, with a m-F substitution, binds to menin with a Ki value of 6.8 nM and is 4 times more potent than 20 (Table 6). The surprisingly similar affinities of 20, 23, and 24, and the improved binding of 31, suggested that while the binding

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SUMMARY In this study, we first determined the minimal MLL1 segment needed for high-affinity binding to menin. Our systematic truncation studies showed that peptide 2, which contains an 8residue segment of MLL1, binds to menin with a high affinity (Ki value of 71.1 nM) and further shortening from either the Nor the C-terminus leads to a major decrease in the binding affinity. Utilizing the cocrystal structure of MLL1MBM complexed with menin, we designed and synthesized a series of macrocyclic G



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Table 6. Structures and Binding Affinities of Compounds with the Modified Phe9

peptidomimetics based upon peptide 2 through cyclization of the side chain of Arg8 and backbone carbonyl group of Pro13. Compound 12 with an 8-carbon cyclization linker has a Ki value of 6.9 nM to menin, 10 times more potent than its acyclic, wildtype counterpart. Determination of a cocrystal structure of 7, a potent macrocyclic analogue of 12, in complex with menin, established the structural basis for its high affinity binding to menin and assisted our further structure-based design and optimization. Our extensive optimization of compound 12 resulted in a highly potent macrocyclic peptidomimetic 34. Compound 34 binds to menin with a Ki value of 4.7 nM and is >600 times more potent than the corresponding acyclic peptide and 15 times more potent than the initial starting linear peptide 2. Importantly, compound 34 has much reduced molecular weight and peptidic characteristics as compared to 2. Further optimization of 34 may ultimately yield a class of potent and cell-permeable small-molecule inhibitors of the menin−MLL1 interaction as potential new therapeutics for the treatment of acute leukemia with MLL1 rearrangements.

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Figure 5. (a) Competitive binding curves and Ki values of compounds 2, 5, 34, and 35 to menin as determined using an FP-based binding assay. (b) Chemical structures of 34 and 35.

EXPERIMENTAL SECTION

General Chemistry Information. Unless otherwise stated, all reagents and solvents were used as supplied without further purification. The final products were purified by a C18 reverse phase semipreparative HPLC column with solvent A (0.1% of TFA in water) and solvent B (0.1% of TFA in CH3CN) as eluents. The H



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Figure 6. (a) Chemical structures of fluorescent labeled tracers 36 and 37. (b) Binding isotherm of 36 to menin. (c) Binding isotherm of 37 to menin. mg, 0.15 mmol, 3 equiv), and DIEA (35 μL, 0.20 mmol, 4 equiv) in THF (10 mL) was stirred at room temperature for 2 h then concentrated. Purification of this residue by HPLC afforded compound II. A solution of bis(tricyclohexylphosphine)ben-zylidine ruthenium(IV) dichloride (Grubbs First Generation Catalyst) (5.6 mg, 0.0068 mmol, 0.2 equiv) in DCM (30 mL) was degassed then added to a solution of peptide II (0.034 mmol, 1 equiv) in DCM (30 mL) under N2. The resulting solution was stirred at room temperature for 3 h before filtering through Celite and concentrated. Purification of the residue afforded III. To a solution of peptide III (0.021 mmol, 1 equiv) in MeOH (10 mL) was added 10% Pd−C (20 mg). The solution was stirred under 1 atm of H2 at room temperature for 0.5 h before being filtered through Celite and concentrated. The residue was treated with a mixture of TFA:TES:H2O (18 mL:0.5 mL:1 mL) for 10 min at room temperature and evaporated. Purification of the crude product by HPLC afforded IV. General Procedures for Synthesis of VI (Scheme 2). Compound V was prepared using a similar procedure as that used for compound I in Scheme 1. A solution of V (0.05 mmol, 1 equiv) in THF (20 mL) was added dropwise to a stirred solution of HATU (38 mg, 0.1 mmol, 2 equiv), HOAt (14 mg, 0.1 mmol, 2 equiv), and DIEA (35 μL, 0.2 mmol, 4 equiv) in THF (10 mL) under N2. The resulting solution was stirred at room temperature for 3 h before being concentrated. The residue was purified and then was treated with a

immobilized amino acid residues on the resin were protected as follows: Arg(Pbf), Trp(Boc), Lys(Mtt), Homolys(Boc). The purity was determined by Waters ACQUITY UPLC, and all the final compounds were >95% pure. The characterization of the compounds was accomplished by HRMS (ESI+) (Agilent Q-TOF Electrospray). 1 H NMR and 13C NMR spectra of the representative compounds were acquired at a proton frequency of 300 MHz, and chemical shifts are reported in parts per million (ppm) relative to an internal standard. These data are provided in the Supporting Information. Synthesis of Linear Peptides in Table 1. The linear peptides were synthesized with an ABI 433 peptide synthesizer using Fmoc chemistry. Rink amide resin was used as the solid support yielding amide capping at the C-terminus. All the peptides were capped with acetyl at the N-terminus. TFA:TES:H2O (18 mL:0.5 mL:1 mL) cleavage cocktail was used to cleave the peptides from resin, which also led to removal of the protecting groups. The cleavage solution was evaporated, and the crude product was precipitated with diethyl ether followed by HPLC purification. General Procedures for Synthesis of Final Compounds IV (Scheme 1). Peptide I was synthesized on an ABI 433 peptide synthesizer using proline preloaded 2-chlorotrityl chloride resin as the solid support. The peptide was cleaved from the resin with 0.5% TFA in CH2Cl2, yielding a carboxylic acid at the C-terminus. The cleavage solution was evaporated, and the crude product was purified by HPLC. A solution of acid I (0.05 mmol, 1 equiv), the corresponding amine (0.15 mmol, 3 equiv), HATU (57 mg, 0.15 mmol, 3 equiv), HOAt (20 I



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Crystallographic Studies. The expression and purification of human menin protein and crystallization methods are the same as described previously.8 Briefly, crystallization of menin with 7 was achieved by sitting-drop diffusion with a well solution containing 100 mM Tris-HCl (pH 7.0), 200 mM MgCl2, and 2.3 M NaCl. The menin−7 complex structure was determined by multiwavelength anomalous dispersion to a resolution of 3.1 Å. Data collection and refinement statistics are given in Table 2. Computational Methods. The menin−MLL1MBM complex (PDBID: 3U85)8 was used to extract the MLL1-binding domain (residues 1−386) and used for hotspot detection using cosolvent simulations in Amber18 with the ff99SB force field.19 Then 16 ns simulations were performed in each 20% v/v isopropyl alcohol/water, and 10%v/v phenol/water cosolvent media following the protocol described previously.14−16 Initial alignment of saved conformations was performed using the ptraj utility from the Amber suite. Helices comprising α11 (residues 277−289), α12 (residues 298−312), and α13 (residues 318−331) were used for alignment. Subsequently, hotspots were determined based on grid occupancy of cosolvent atoms and used for design strategy.

mixture of TFA:TES:H2O (18 mL:0.5 mL:1 mL) for 10 min at room temperature and evaporated. Purification of the residue by HPLC afforded VI. Synthesis of 36 (Supporting Information Scheme S2). The immobilized peptide, Ac-SRWRFPARPGTGRR-Ahx-Ahx-K(Mtt) (0.1 mmol), on Rink amide resin was achieved on an ABI 433 peptide synthesizer. The resin was then treated with 1% TFA in CH2Cl2 (10 mL) to remove the 4-methyltrityl (Mtt) protecting group at the lysine side chain, and this was followed by overnight treatment with 5carboxy fluorescein succinimide ester (5-FAM, SE) (71 mg, 0.15 mmol, 1.5 equiv) and DIEA (52 μL, 0.3 mmol, 3 equiv) in DMF. The peptide was cleaved from the resin and followed by HPLC purification. Synthesis of 37 (Scheme 3). Compound VII was prepared using a similar procedure as that used for I. A solution of carboxylic acid VII (45 mg, 0.04 mmol, 1 equiv), VIII (43 mg, 0.04 mmol, 1 equiv), HATU (30 mg, 0.08 mmol, 2 equiv), HOAt (11 mg, 0.08 mmol, 2 equiv), and DIEA (35 μL, 0.20 mmol, 5 equiv) in THF (10 mL) was stirred at room temperature overnight and then concentrated. Then the residue was dissolved in CH3CN (10 mL) and treated with diethylamine (82 μL, 20 equiv) for 10 min. The reaction mixture was concentrated and purified by HPLC to give IX. To a solution of IX (20 mg, 0.01 mmol, 1 equiv) in CH2Cl2 (3 mL), 5-FAM, SE (9.6 mg, 0.02 mmol, 2 equiv), and DIEA (7 μL, 0.04 mmol, 4 equiv) were added, and the resulting solution was stirred for 3 h before being concentrated. The residue was treated with a TFA:TES:H2O (18 mL:0.5 mL:1 mL) cocktail for 10 min and concentrated and purified to yield 37 (8 mg, 12% in four steps). FP-Based Binding Assays. Both fluorescence and fluorescence polarization were measured using the Tecan Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, NC) in Microfluor 1, 96well, black, round-bottom plates (Thermo Scientific). The Kd values of the fluorescent probes (36 and 37) to menin protein were determined by monitoring the total FP of mixtures composed of each fluorescent probe at a fixed concentration and the menin protein with increasing concentrations up to full saturation. Then 3 nM of 36 or 2 nM of 37 and menin protein were added to each well to a final volume of 100 μL in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide [Invitrogen], 2% DMSO, and 0.005% of Triton X-100 for 36 saturation assay or 0.02% of Tween-20 for 37 saturation assay, respectively). Plates were mixed and incubated at room temperature for 60 min with gentle shaking to ensure equilibrium. FP values were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, NC) in Microfluor 1, 96-well, black, round-bottom plates (Thermo Scientific). Kd values were then calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 5.0 software (Graphpad Software, San Diego, CA). Ki values of tested compounds were determined in dose-dependent competitive binding experiments. Mixtures of 2 μL of the tested compound with different concentrations in DMSO and 98 μL of preincubated menin/36 or menin/37 complex with fixed concentrations in the assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02% sodium azide, with 0.005% Triton X-100 for assay using 36 and 0.02% Tween-20 for assay using 37) were added into assay plates and incubated at room temperature with gentle shaking for 60 min. Final concentrations of menin protein and 36 were 30 and 3 nM, and menin protein and 37 were 3 and 2 nM, respectively, and final DMSO concentration was 2%. Negative controls containing menin/36 or menin/37 complex only (equivalent to 0% inhibition), and positive controls containing free 36 or 37 only (equivalent to 100% inhibition) were included in each assay plate. FP values were measured as described above. IC50 values were determined by nonlinear regression fitting of the sigmoidal dose-dependent FP decreases as a function of total compound concentrations using Graphpad Prism 5.0 software (Graphpad Software, San Diego, CA). Ki values of tested compounds to the menin protein were calculated using the measured IC50 values, the Kd values of probes to menin, and the concentrations of menin and probes in the competitive assays.17

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis of 8 and 36, HRMS and NMR spectral data and UPLC purity analysis for designed compounds, and computational modeling of menin complexes with compounds 34 and 35. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 734-615-0362 Fax: 734-647-9647 E-mail: shaomeng@ umich.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.

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ABBREVIATIONS USED MLL, mixed lineage leukemia; MLL1MBM, menin-binding motif of MLL1; MEN1, multiple endocrine neoplasia type 1; FP, fluorescence-polarization; Fmoc, fluorenylmethoxycarbonyl; DIEA, N,N-diisopropylethylamine; 5-FAM, SE, 5-carboxyfluorescein succinimidyl ester; Ahx, 6-aminohexanoic acid; Mtt, 4methyltrityl; TFA, trifluoroacetic acid

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REFERENCES

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