European Journal of Medicinal Chemistry 

Covalent and noncovalent constraints yield a figure eight-like conformation of a peptide inhibiting the menin-MLL interaction

Paulina Fortuna a, Brian M. Linhares b, Trupta Purohit b, Jonathan Pollock b,
Tomasz Cierpicki b, Jolanta Grembecka b, Łukasz Berlicki a, *
a Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzez_ e Wyspian´skiego 27, 50-370, Wrocław,
Poland
b Department of Pathology, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, United States

A R T I C L E I N F O

Article history:
Received 9 July 2020 Received in revised form 4 August 2020
Accepted 8 August 2020
Available online 20 August 2020

Keywords: Leukemia Cyclization
Protein-protein interactions

A B S T R A C T

The interaction between menin and mixed lineage leukemia (MLL) was identified as an interesting target for treating some cancers including acute leukemia. On the basis of the known crystal structure of the MBM1-menin complex (MBM – menin binding motif), several cyclic peptides were designed. Elaboration of the effective cyclization strategy using a metathesis reaction allowed for a successfully large number of derivatives to be obtained. Subsequent optimization of the loop size, as well as N-terminal, central and C- terminal parts of the studied peptides resulted in structures exhibiting low nanomolar activities. A crystal structure of an inhibitor-menin complex revealed a compact conformation of the ligand molecule, which is stabilized not only by the introduction of a covalent linker but also three intramolecular hydrogen bonds. The inhibitor adopts a figure eight-like conformation, which perfectly fits the cleft of menin. We demonstrated that the development of compact, miniprotein-like structures is a highly effective approach for inhibition of protein-protein interactions.

 1. Introduction

Peptide-based therapeutics have received considerable atten- tion because numerous peptidomimetic drugs have been success- fully introduced to the market [1e3]. Their typically considered drawbacks, namely, low proteolytic stability and unpredictable conformation, can be effectively reduced by appropriate modifi- cations to either the side chains or the backbone [4,5]. In particular, protein-protein interactions (PPIs) can be effectively inhibited by peptide-based compounds, due to the possibility of the construc- tion of structurally extended molecules [6e8], although small molecule inhibitors of PPI are still in focus [9e12]. However, the construction of peptidomimetics with the application of appro- priate structural constraints allows for rationally predicted three- dimensional arrangements [13e15]. In addition, these modifica- tions also increases the proteolytic stability of peptide-based in- hibitors. The synthetically feasible building of large molecules allowing numerous interaction points leads to effective inhibition of protein interactions, in particular, those with a large interaction surface [16].

The interaction of mixed lineage leukemia (MLL) fusion proteins with menin was identified as a valuable target for the development of specific anticancer drugs [17].
Initially, it was found that the progress of acute leukemias with translocations of the MLL gene (ca. 5e10% of all leukemia cases) [18], for which patients have low survival prognosis [19], is related to the MLL-menin interaction. This finding was confirmed by ge- netic approaches [20], as well as by the use of the small molecule inhibitors of this PPI [21e25]. Recent studies have also proven that the menin-wild type MLL interaction is important for the growth of some solid tumors such as hepatocellular carcinoma [26,27], pe- diatric gliomas [28], castration resistant prostate cancer [29], and Ewing sarcoma [30].
Two N-terminal fragments of MLL consisting of ~40 amino acid residues interact with menin, namely, menin binding motif 1 (MBM1, MLL4-15) and 2 (MBM2, MLL23-43). MBM1 binds to a large central cavity of menin, and its affinity is much stronger than MBM2 [31]. The MBM1-menin interaction is well-characterized by X-ray crystallography and involves lipophilic interactions with Trp7, Phe9, Pro10 and Pro13 as well as hydrogen bonds to the main chain of MBM1 and Arg12 sidechain (Fig. 1) [31]. MBM1 adopts a U- shaped conformation with a b-turn formed by residues 9e12 and is stabilized by an intramolecular hydrogen bond between Phe9 and Arg12.

The possible therapeutic applications have stimulated the development of several classes of menin-MLL interaction in- hibitors, which mainly include heteroaromatic compounds, e.g., thienopyrimidines [32], benzylpiperidines [33], amino- thienylpyrimidines [34], and aminoglycosides [35], which bind to the central cavity of menin, i.e., the MBM1 binding site. Recent findings show that a structural extension of thienopyrimidine- based inhibitors, which allow compounds to interact with a large surface of menin, may help to enhance activity [36]. The other approach is based on the construction of peptidomimetics mimicking the structure and conformation of the MBM1 peptide [37]. The cyclization by stapling the N- and C-termini of the MBM1
peptide by an eight carbon linker and subsequent optimization of the chosen side chains led to compound with Ki ¼ 4.7 nM.
In this paper, the development of constrained cyclic peptide-
based inhibitors of the menin-MLL interaction is described. The use of a metathesis reaction between two sidechains leads to small macrocycles containing a double bond. The structure covalent constrains are designed for pre-organizing the conformation of peptide for efficient binding. Moreover, the applied synthetic approach allows for the construction of molecules that can be optimized within a macrocycle as well as at the N- and C-termini. Therefore, the development of a structurally extended molecule that forms numerous intermolecular interactions and shows high inhibitory activity is feasible. The interaction of a chosen peptido- mimetic inhibitor with menin was characterized using X-ray crystallography.

2. Results and discussion

2.1. Design

The introduction of covalent linkers as constraints in the peptide allows for the construction of molecules with a well-defined three-

Fig. 1. Crystal structure of the MBM1-menin complex (PDB id 4GQ6) [31]. MBM1 is shown as sticks and menin is presented as a green ribbon. Intramolecular hydrogen bonds are shown as orange dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
dimensional structure. This approach has already been proven to be useful for building biologically active compounds that mimic an appropriate fragment of the native ligand of the target. The active conformation of the fragment of MLL which binds to menin, pre- cisely determined by the crystal structure of the MBM1-menin complex (Fig. 2), can be constrained by covalent linkage of the side chains of two arginine residues [31].
Such construction of the scaffold has some advantages. First, the macrocycle contains a hydrocarbon linker and a proline residue, which induce the appropriate rigidity. Second, both residues within the cycle as well as at the N- and C-termini can be optimized. Third, a double bond-containing linker can feasibly be included in the structure through the application of a ring-closing metathesis re- action and further reduced its saturated form. Fourth, the size of the macrocyclic ring can be modulated by the length of the linker, which is related to applied amino acids with double bond- containing side chains. Importantly, interactions with the target that are lost due to the removal of an arginine residue can be recreated by incorporation of arginine or related residues at the N- or C-terminus of the peptide.

2.2. Chemistry

All peptides were synthesized on solid support (Rink MBH amide resin) using Fmoc chemistry and an automated microwave peptide synthesizer. All residues were coupled twice (5 eq., DIC/
Oxyma) for 15 min at 75 ◦C. Allyl-glycine or (S)-2-aminohexenoic
acid have been incorporated at proposed cyclization positions. Fully protected peptidyl resin was the starting material for the ring- closing metathesis reaction. The reaction was performed at room temperature in the presence of a second generation Grubbs catalyst (Scheme 1). Subsequently, peptides were cleaved from the resin using a TFA/thioanisole/TIS/H2O cocktail to obtain unsaturated derivatives. In the case of derivatives containing a saturated linker, hydrogenation was done before cleavage. The reaction was per- formed with the application of homogenous Wilkinson’s catalyst under a H2 atmosphere (6 bar) for 7 days at room temperature. All final products were purified using preparative HPLC equipped with a C18 column (0.05% TFA in water/0.05% TFA in acetonitrile). The applied synthetic methodology is compatible with any applied peptide sequence.

2.3. Inhibitory activity

Inhibitory activities of all studied peptides were evaluated using a fluorescence polarization assay with the use of fluorescein- labeled MBM1 peptide. The dodecapeptide menin binding frag- ment of MLL (MLL12, IC50 ¼ 84 nM, Table 1) was used as a reference and starting point for all modifications. Initially, two cyclized ana-
logs (5 and 6) and their linear counterparts (1 and 2) were syn- thesized. All of these compounds showed significantly lower activity (two to three orders of magnitude) than the reference MLL12 peptide. This substantial loss of activity was attributed to the absence of the arginine side chain interactions with the target, therefore, an arginine residue was incorporated into the C-terminal part of the peptide in place of a glycine residue. This subset of linear and cyclic undeca- and dodecapeptides (structures 3, 4, 7 and 8) with an additional arginine showed much higher activity, Table 1, confirming the beneficial interactions of this residue with the target molecule. At this point, the highest affinity was achieved for
the cyclic peptide 7 with IC50 ¼ 770 nM, but it was still weaker than
the linear MLL12 peptide. Surprisingly, in all abovementioned cases, dodecapeptides with an N-terminal threonine were less active then the shorter analogs (e.g., 1 vs 2 and 7 vs 8).
Subsequently, the influence of the length and saturation of the

Fig. 2. Design strategy of peptidomimetics (right) based on covalent linkage of the amino acid side chains of the MBM1 peptide (left) that are in spatial proximity in the complex to menin.linker on the activity of peptides was investigated. Cyclic peptides 9e12 have a linker composed of 4e6 carbon atoms and an arginine residue placed at the C-terminus as seen in the most active cyclic compound 7. A saturated analog of peptide 7, namely, compound 9, exhibited increased activity (IC50 ¼ 122 nM versus 770 nM, for 9

and 7, respectively) and importantly, it achieved a similar level of
inhibitory activity to the reference MLL12 peptide. The increase in the length of the linker by one or two carbon atoms (peptides 10 and 11) did not lead to the improvement of inhibitory activity. Again, saturated cyclic peptide 12 (IC50 ¼ 660 nM) was more potent
than its unsaturated counterpart 11 (IC50 ¼ 1100 nM), indicating
better complementarity of the more flexible compound to the target.
The next step of the optimization included modifications at the C- and N-termini (Table 2, peptides 13e18). In this part of the study, we used peptide 7 as a reference. First, a series of peptides with a truncated N-terminus were investigated (structures 7, 13e15). Interestingly, the highest activity in this group was achieved with peptide 13 (IC50 ¼ 151 nM), for which the sequence was shortened
by one residue with respect to parent compound 7. However, the
largest difference in inhibitory activity was found between com- pounds 14 and 15, differing by an arginine residue, which resulted in over two orders of magnitude difference in their inhibitory ac- tivity. This finding indicates the critical importance of the interac- tion of the Arg residue with menin.
In addition, a series of peptides in which the C-terminal arginine residue was exchanged for other positively charged amino acids, including lysine (peptide 16), ornithine (peptide 17) and dia- minopentanoic acid (Dap, peptide 18) were tested (Table 2). The lysine- and Dap-substituted peptides showed similar potency to the parent peptide, while the ornithine-based structure (17) ach-
ieved over 5-fold higher activity (IC50 ¼ 142 nM) over peptide 7. Within this optimization round, the central part of the sequence,
i.e., residues Phe9 and Ala11, has also been investigated, and pep- tides 19e21 have been prepared (Table 3). The substitution pattern was chosen on the basis of previous studies [37] and includes the substitution of phenylalanine with m-fluorophenylalanine (peptide 19), and alanine with 1-aminocyclobutanecarboxylic acid (ACBCA, peptide 20). As expected, each tested modification was beneficial, and peptide 21, which included both of them, showed a significant
enhancement in the biological activity with IC50 ¼ 64 nM.
In the final step of optimization, all preferential changes found for the C-terminal, central and N-terminal parts of the peptide structure were combined (Table 4). Due to the high inhibitory ac- tivity of peptides 21e28 reaching the limit of the fluorescence polarization assay with FLSN_MLL12 [36], we tested these com- pounds using the FP assay utilizing the FLSN_MLL4-43 peptide. At the applied conditions, two most potent peptides from previous

series d 21 and MLL12 showed ICMLL4-43 in high nanomolar or low micromolar range, 657 nM and 3588 nM, respectively. The incor- poration of ornithine to peptide 21 leading to 22 and its analogue 23, did not increase their inhibitory activity. However, subsequent addition of one or three Arg residues on C-terminus resulted in peptides 24 and 25 of exceptionally high inhibitory activity: 58 nM and 2.35 nM, respectively. The reduction of double CeC bond of peptides 23e25 leading to peptides 26e28 did not influence their inhibitory activity substantially. Therefore, the two most active peptides 25 and 28 showed inhibitory activity about three orders of magnitude better than the native sequence in the MLL12 peptide. Activities of obtained peptides have been also compared with previously published peptidomimetics inhibiting menin-MLL interaction [37] by analysis of the reference peptide MLL13 (used in both studies). The most active macrocyclic peptide obtained previously (MCP-1) has been two times less potent then MLL13 [37], while peptide 25 obtained in this study was ca. twenty times more active than MLL13. Therefore, the compounds showed in this study constitute a significant improvement in inhibitory activity in comparison to previously published peptidomimetics. Moreover, these compounds rank among the most active inhibitors of the MLL-menin interaction with inhibitory activity at the same range as
recently published MI-1481 compound with IC50 ¼ 3.6 nM
(measured in the same conditions) [36].

2.4. Structural studies

To understand better the molecular basis of the activity of the cyclic peptides we co-crystallized selected peptides with menin. These efforts resulted in well-diffracting crystals of the peptide 25- menin complex, which allowed to obtain a high resolution crystal structure of the complex (1.50 Å, PDB id 6OPJ). The crystal structure revealed that peptide 25 binds to the central cavity of menin in a manner similar to the native peptide (MML12, PDB id 4GQ6, see Fig. S1 for superimposition). In particular, the central part of pep- tide 25 is forming the same set of interactions, including hydro- phobic contacts between Trp, Pro and Phe residues of the peptide and menin and the hydrogen bonds between the Trp side chain of 25 to Asp153 and the main chain carbonyl (of ACBCA) to Tyr323. The backbone of the macrocycle of this peptide contains 17 heavy atoms and several constraints, including four planar peptide bonds, two cyclic residues (Pro and ACBCA) and one intramolecular hydrogen bonds, Fig. 3. Thus, the macrocyclic part of the peptide can be considered nearly rigid, although it mimics the native, flexible peptide fragment surprisingly well. The interaction pattern of the rest of the molecule is also extended as the conformations of the C- and N-termini are constrained by two additional intra- molecular hydrogen bonds. A guanidine fragment of Arg11 is Scheme 1. aReagents and conditions: (a) second generation Grubbs catalyst, dichloromethane, rt.; (b) TFA/thioanisole/TIS/H2O, (90:5:2.5:2.5, v/v/v/v); (c) H2, 6 bar, Wilkinson’s catalyst, 7 days, rt.involved in a hydrogen bond with the Arg2 and Trp3 carbonyl groups, closing the second macrocycle in a noncovalent way and forming a figure eight-like conformation of the ligand molecule, Fig. 3. Moreover, two guanidine side chains of arginine residues Arg2 and Arg11 of peptide 25 form three charge-assisted hydrogen bonds with Glu363 and Glu359 of menin. Importantly, the Arg11 of peptide 25, which was added during the optimization process, is creating hydrogen bonds with Glu363 and Glu359, which were also observed for Arg12 of the native MLL12 peptide. These hydrogen bonds most likely contribute to strong inhibitory activity of 25 and other cyclic peptides harboring this residue, Table 3. The two C-terminal arginine residues (Arg12 and Arg13) of peptide 25 are not visible in electron density in the crystal structure, but their incor- poration increases significantly the inhibitory activity. It could be reasonably supposed that their side chains could interact with the negatively charged residues of menin placed near peptide 25 C- terminus, namely Glu366 and Asp370 (Fig. 3). Besides, long-range electrostatic interactions between these positively charged resi- dues of 25 and negatively charged binding site on menin could further rationalize the superior inhibitory activity of 25 and its close analogs, Table 3 and Fig. 3. Therefore, the very high inhibitory ac- tivity of the optimized peptides, which are up to three orders of
Table 1
Inhibitory activity of the MLL12 peptide and its linear and cyclic analogs with var- iations in the C-termini, linker lengths and linker saturations against the MLL-menin interaction.

Table 2
Inhibitory activities of peptides containing various N- and C-termini.No Structure IC50 [nM]

7 Ac-Ser-Ala-Arg- -Arg-NH2 770 ± 80
13 Ac-Ala-Arg- -Arg-NH2 151 ± 30
14 Ac-Arg- -Arg-NH2 372 ± 20
15 Ac- -Arg-NH2 77730 ± 90
16 Ac-Ser-Ala-Arg- -Lys-NH2 1390 ± 50
17 Ac-Ser-Ala-Arg- -Orn-NH2 142 ± 6
18 Ac-Ser-Ala-Arg- -Dap-NH2 649 ± 40
Table 3
Inhibitory activity of peptides with various residues within the loop.

magnitude stronger than the native linear MLL12 peptide, is a result of constraining the peptide structure by covalent and noncovalent interactions in a conformation that perfectly fits the central cavity on menin (Fig. S2). Furthermore, incorporation of the C-terminal Arg residues that interact with negatively charged protein surface (Glu359, Glu363, Glu366 and Asp 370) increases further the inhibitory activity of these peptides.
Moreover, to gain an insight in preorganization of inhibitor structure in its unbound state, molecular dynamics calculations were done for two most active inhibitors 25 and 28. In case of unsaturated compounds 25, the conformation of macrocyclic ring is fully stable during molecular dynamics and highly similar to its bound state observed in solved crystal structure (Fig. S4, S6, and S7). For peptide 28 without double bond in the macrocyclic ring, the conformational rigidity is smaller, but both observed confor- mations are similar to that observed in bound state (Fig. S5, S6 and S7). Therefore, the introduced covalent constrains to form a macrocyclic ring within the peptide structure are properly pre- organize the inhibitor for binding to its molecular target.
3. Conclusions

Starting from the structure of the native MBM1 peptide, highly active inhibitors of menin-MLL interactions were discovered after three rounds of optimization. The introduction of a covalent linker incorporating a hydrocarbon chain and subsequent optimization of N-terminal, central and C-terminal parts gave inhibitors with low nanomolar activity (IC50 ~2 nM for peptide 25). The key step in the design process was based on the introduction of C-terminal posi- tively charged amino acid residues (arginine/ornithine), which are able to create charge-assisted hydrogen bonds as well as long range electrostatic interactions with several negatively charged residues of menin.

The crystal structure of the inhibitor-menin complex revealed that the bound peptide folds into a compact conformation, which is

Table 4
Inhibitory activities of peptides containing all optimized fragments.No Structure/sequence ICMLL4-43 [nM]
X Y Z
21 Ac-Ser- -Arg-NH2 CH=CH 657 ± 97
22 Ac-Ser- -Orn-NH2 CH=CH 777 ± 120
23 Ac- -Orn-NH2 CH=CH 1230 ± 520
24 Ac- -Orn-Arg-NH2 CH=CH 58 ± 3
25 Ac- -Orn-Arg-Arg-Arg-NH2 CH=CH 2.35 ± 0.35
26 Ac-Ser- -Orn-NH2 CH2eCH2 569 ± 45
27 Ac- -Orn-Arg-NH2 CH2eCH2 14.0 ± 4.2
28 Ac- -Orn-Arg-Arg-Arg-NH2 CH2eCH2 4.60 ± 0.42
MLL12 Ac-SARWRFPARPGT-NH2 3588 ± 104
MLL13 Ac-RWRFPARPGTGRR-NH2 49 ± 8

stabilized by both a covalently linked, strongly constrained mac- rocycle and numerous intramolecular hydrogen bonds. The non- covalent interactions impose the formation of a second macrocycle (closed by hydrogen bonds), that in total creates a figure eight-like conformation. This tightly bound three-dimensional arrangement can even be considered as a protein-like structure due to the numerous intramolecular interactions and its compactness. It can be concluded that the high inhibitory potential of this structure is a result of the formation of a ‘mini-protein’ that is perfectly compatible with the target cavity. Therefore, we consider these results to be a substantial step forward in the development of an effective methodology for protein-protein interaction inhibitor design.

4. Experimental section

4.1. General

All solvents and reagents were from commercial suppliers (Avantor, Sigma-Aldrich, Merck, Lipopharm, Bachem, and Iris Biotech) and were of analytical grade unless otherwise stated. Preparative HPLC was done using a Dionex UltiMate 3000 LC Sys- tem or Knauer Azura ASM 2.1L equipped with a C18 column (Thermo Scientific, Hypersil Gold 12 m, 250 mm × 20 mm) in water/
acetonitrile (0.05% TFA). Analytical HPLC was performed using a
C18 column (Kinetex 5m, EVO C18 100A, 150 × 4.6 mm).
HR-MS spectra were obtained on a Waters LCT Premier XE spectrometer with a time of flight (TOF) detector. The electrospray ionization method was applied.
All the obtained compounds showed purity over 95% as esti- mated by analytical HPLC.

4.2. Peptide synthesis

Peptide synthesis was performed using an automated peptide synthesizer with microwave irradiation Initiator + Alstra (Biotage). Syntheses were done on 0.06 mmol scale using Rink amide AM resin (100e200 mesh; 0.4e0.74 mmol/g). Initially, resin was
swelled in DMF (20 min, 70 ◦C). Coupling of 5 equivalents of Fmoc- amino acids were done with a DIC:Oxyma mixture (0.5 M: 0.5 M);
in the case where derivatives of canonical a-amino acids were used, the reaction was performed twice (15 min at 70 ◦C). Deprotection
was done using a solution of 20% piperidine in DMF (3 min + 10 min at room temperature).

4.2.1. Cyclization method
The cyclization reaction was performed using an Initiator + SP Wave (Biotage) microwave synthesizer. Grubbs second generation catalyst (8 mg) was dissolved in dichloromethane (1.5 mL) and
placed in a reaction vial. Then, peptidyl resin (0.0472 mmol) and
LiCl solution (25 mg in 0.15 mL DMF) was added. The reactor was closed and heated at 100 ◦C for 2 h. Subsequently, the peptidyl resin
was washed with DMF (3 times 3 mL), methanol (3 times 3 mL) and dichloromethane (3 times 3 mL).

4.2.2. Hydrogenation method

Peptidyl resin, Wilkinson’s catalyst (1 mol%), dichloromethane (9.0 mL) and methanol (1.0 mL) were placed in a high pressure reactor. Subsequently, the reactor was filled with hydrogen up to pressure of 6.0 bar. The reaction mixture was stirred for 7 days at room temperature. Then, the peptidyl resin was filtered off and washed with dichloromethane (3 times 7.0 mL), DMF (3 times 7.0 mL) and methanol (3 times 7.0 mL).

4.2.3. Peptide cleavage
Peptidyl resin was placed in a PE vial with a frit and the deprotection cocktail (TFA/thioanisole/TIS/H2O, 90:5:2.5:2.5, v/v/v/ v) was added. The vial was shaken for 3 h and then the filtrate was collected. The peptide was precipitated using ice-cold diethyl ether. The mixture was centrifuged and precipitate was washed with second portion of ice-cold diethyl ether. Finally, the peptide was purified using preparative HPLC (detailed data are summarized in Table S1).

Fig. 3. Top (A) and side (B) views of the crystal structure of the peptide 25-menin complex (PDB id 6OPJ). Peptide inhibitor is shown as sticks with grey carbon atoms, while selected residues of menin are presented as sticks with green carbon atoms. Hydrogen bonds are shown as orange dashed lines. Residues of menin are labeled in green and those of peptide in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4.3. Inhibition studies

The expression and purification of menin has been described previously [21]. Inhibition of the menin—MLL interaction by stud- ied peptides was measured by a fluorescence polarization (FP) assay using the method described previously [36]. Briefly, IC50 determination was carried out using the fluorescein-labeled (FLSN)
MLL (MBM1) peptide at a concentration of 10 nM, menin at 100 nM or FLSN-MLL4-43 (Kd = 1.0 nM) at 4 nM and menin at 4 nM which were incubated for 1 h in the FP buffer (50 mM Tris, pH 7.5, 50 mM

NaCl, 1 mM DTT). Varying concentrations of peptides were added to the menin—MLL peptide complex and incubated for 1 h before changes in fluorescence polarization were measured using the PHERAstar microplate reader (BMG). The IC50 values were calcu- lated in Origin 7.0 (OriginLab) by plotting the mP values measured
for each compound as a function of compound concentration.

4.4. Crystallization of menin complexes with cyclic peptide inhibitor
25

For co-crystallization experiments 2.8 mg/mL menin was incu- bated with peptide 25 at 1:8 M ratio. Crystals were obtained using
sitting-drop technique at 4 ◦C in 0.2 M lithium sulfate, 0.1 M HEPES,
pH 7.5, and 25% wt/vol PEG-3350. Prior to data collection, crystals were transferred into a cryo-solution containing 20% PEG-550 MME and flash-frozen in liquid nitrogen.

4.5. Crystallographic data collection and structure determination

Diffraction data for menin complex were collected at the 21-ID- G beamline at the Life Sciences Collaborative Access Team at the Advanced Photon Source. Data were processed with HKL-2000 [38]. Structure of the menin-25 complex was determined by molecular replacement using MOLREP with the apo-structure of human menin (PDB code 4GPQ) as a search model in molecular replace- ment. The model was refined using REFMAC [39], COOT [40], CCP4 package [41] and PHENIX [42]. Validation of the structures was performed using MOLPROBITY [43] and ADIT [44]. Details of data processing and refinement are summarized in Supplementary Table S2. Coordinates and structure factors for menine25 com- plex have been deposited in the Protein Data Bank under the following code 6OPJ.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following competing financial interest(s): Drs. Grembecka and Cierpicki received research support from Kura Oncology, Inc. They had also served as consultants for Kura Oncology and have equity ownership in the company. Other coauthors declare no potential conflict of interest.

Acknowledgments

The Discovery Studio package was used under a Polish coun- trywide license. The software resources (BIOVIA Discovery Studio program package) from the Wrocław Centre for Networking and Supercomputing were used in the present study.

Appendix A. Supplementary data

Abbreviations

ACBCA 1-aminocyclobutanecarboxylic acid Boc tert-bytyloxycarbonyl
Dap diaminopentanoic acid
DIC diisopropylcarbodiimide DMF N,N-dimethylformamide DTT dithiotreitol
Fmoc fluorenylmethyloxycarbonyl MBM menin binding motif

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