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. 2023 Apr 17;14(5):606–613. doi: 10.1021/acsmedchemlett.3c00029

Rational Design of Highly Potent and Selective Covalent MAP2K7 Inhibitors

Dalton R Kim †, Meghan J Orr †, Ada J Kwong †, Kristine K Deibler †, Hasan H Munshi †, Cory Seth Bridges ‡, Taylor Jie Chen ‡, Xiaoyu Zhang †,§, H Daniel Lacorazza ‡, Karl A Scheidt †,§,∥,*
PMCID: PMC10184151  PMID: 37197477

Abstract

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The mitogen-activated protein kinase signaling cascade is conserved across eukaryotes, where it plays a critical role in the regulation of activities including proliferation, differentiation, and stress responses. This pathway propagates external stimuli through a series of phosphorylation events, which allows external signals to influence metabolic and transcriptional activities. Within the cascade, MEK, or MAP2K, enzymes occupy a molecular crossroads immediately upstream to significant signal divergence and cross-talk. One such kinase, MAP2K7, also known as MEK7 and MKK7, is a protein of great interest in the molecular pathophysiology underlying pediatric T cell acute lymphoblastic leukemia (T-ALL). Herein, we describe the rational design, synthesis, evaluation, and optimization of a novel class of irreversible MAP2K7 inhibitors. With a streamlined one-pot synthesis, favorable in vitro potency and selectivity, and promising cellular activity, this novel class of compounds wields promise as a powerful tool in the study of pediatric T-ALL.

Keywords: mitogen-activated protein kinases, drug discovery, small molecule inhibitors, lead optimization, covalent inhibitors, irreversible inhibitors, T cell acute lymphoblastic leukemia, MAP2K7, MEK7, MKK7

Acute lymphoblastic leukemia (ALL) is a deadly blood cancer and the most common hematological malignancy in pediatric patients under 14 years of age. Roughly 6000 cases of ALL are diagnosed annually in the United States,1 with half of these cases occurring in children and adolescents.2 Despite advancements in treatments and improvement in outcomes,3,4 ALL remains the most frequent cause of death from cancer before the age of 20,5,6 and relapse continues to be the leading cause of cancer-related mortality in children.7 Nearly a quarter of pediatric patients and over half of adult patients with T cell ALL (T-ALL) exhibit resistance and ultimately relapse.4 Further, survivors of childhood ALL are at risk for a multitude of sequelae because of multi-agent chemotherapy,8 which has evolved only incrementally since “total therapy” of T-ALL was described by Pinkel.9 Targeted therapies for children with high-risk T-ALL have been an area of active research.10,11

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor12 known to function as either an oncogene or a tumor suppressor in a context-dependent manner.13,14 Epigenetic silencing of Klf4 by CpG methylation occurs in pediatric ALL, and decreased KLF4 expression has been identified in treatment-resistant cases of ALL.15,16 Physiologically, KLF4 represses transcription of Map2k7, a gene that encodes mitogen-activated protein kinase kinase 7 (MAP2K7), also known as MAPK/Erk kinase 7 (MEK7 or MKK7).11 Loss of Map2k7 repression and consequent amplification of downstream MAPK signaling is thought to contribute to T-ALL pathology.11,17 Inhibition of MEK718,19 or its substrate c-Jun N-terminal kinase (JNK)11 has been shown to reduce leukemic expansion in patient-derived xenograft mouse models.

Since JNK is the only known substrate of MAP2K7,11,20,22 selective inhibitors of JNK are logical chemical probes for the study of MAPK dysregulation in T-ALL. However, JNK is also implicated in genomic stability through its roles in the repair of DNA double-strand breaks23 and photodamage,24 which are not known to be MAP2K7-dependent. Direct inhibition of MAP2K7 would facilitate a more valid investigation of Klf4 inactivation and consequent MAPK signaling amplification in T-ALL.22

Recent efforts toward the development of novel MAP2K7 inhibitors have been the subject of a review.25 The fungal natural product 5(Z)-7-oxozeaenol (5Z7O, 1) was found to covalently engage MAP2K7 at Cys218, as determined by X-ray crystallography (Figure 1B).26 Inhibition of MAP2K7 by 5Z7O induced apoptosis in T-ALL in a dose-dependent manner.18 However, 5Z7O potently inhibits a variety of other kinases, including TAK1, which is a MAP3K.27 Furthermore, 1 boasts only moderate potency against MEK7 (IC50 = 1.3 μM).26 In 2019, a high-throughput in silico screen of over 100 000 acrylamide compounds identified the first potent and selective MAP2K7 inhibitors (2a-d).28 The covalent engagement of these indazole-based arylacylamides was confirmed by X-ray crystallography and mass spectrometry. An additional class of covalent MAP2K7 inhibitors based on the pyrazolopyrimidine scaffold has also been disclosed (3a,b),29 the design of which leveraged similarities between the MAP2K7 and EGFR binding pockets. The alkynyl precursor of 3b was diversified to generate a library of triazoles, thereby tuning potency.30 A series of dual MAP2K4/MAP2K7 inhibitors were reported in 2020.31 Knapp and Chaikuad et al. demonstrated that the N-terminal regulatory helix of MAP2K7 stabilizes the active state of this kinase allosterically. They identified a host of type I (e.g., 4) and type II inhibitors targeting this flexible region of the kinase.32

Figure 1.

Figure 1

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(A) Simplified MAPK signaling cascade. (B) Structures of known MAP2K7 inhibitors. (C) This work constitutes a structure-based approach toward the optimization of AST-487 as a MAP2K7 inhibitor. Enablement of a key cation−π interaction and the employment of an electrophilic moiety endowed the optimized compound with markedly higher potency and selectivity.

We previously reported a chemical probe strategy interrogating selectivity across all seven MEK isoforms.33 Therein, we described a functional in vitro platform utilizing the ADP-Glo kinase assay34 to assess commercially available kinase inhibitors with known off-target binding affinity to MAP2K4. Despite sharing structural homology and a common phosphorylation substrate in JNK, MAP2K4 and MAP2K7 exhibited strikingly dissimilar susceptibility to the selected inhibitors. Notably, only FMS-like tyrosine kinase 3 (FLT3) inhibitor AST-487 (5, Figure 1C)35−37 demonstrated submicromolar MAP2K7 inhibition; similar AST-487 potency was otherwise seen only against the promiscuous MAP2K5 isoform.

Structural analysis of MEK ATP binding pockets identified MAP2K7 to have the shallowest depth and smallest volume among all isoforms with available crystal structures.33 MAP2K7 is also unique among the MEK family for the presence of four cysteine residues in its active site.33 It is the only MEK isoform featuring a hinge region cysteine (Cys218),33 and only 11 human kinases bear a cysteine at the same relative position.38 We envisioned the development of a novel irreversible MAP2K7 inhibitor leveraging these combined structural insights (Figure 1C). Preliminary modeling involved docking AST-487 with early MEK7 crystal structures (PDB: 3WZU).26 Despite this compound’s type II binding mode with other kinases,36,37,39,40 computation and spectroscopic experiments support a type I binding mode with MAP2K7.33

We commenced with the synthesis of truncated AST-487 derivatives retaining motifs thought to participate in key interactions.33 A streamlined synthesis inspired by a process35 for AST-487 was developed to access analogues. Each compound was prepared by a one-pot process that afforded the desired MAP2K7 inhibitor following workup and chromatography (Scheme 1). This expedient and high-yielding procedure enabled the rapid generation of chemical diversity.

Scheme 1. One-Pot Process Towards the Synthesis of Novel MAP2K7 Inhibitors.

Scheme 1

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With preliminary derivatives and controls in hand, we commenced their biological characterization by surveying in vitro activity against MAP2K7. As shown in Table 1, 8 demonstrated impressive inhibitory capacity (IC50 = 60 nM) that surpasses that of AST-487 (5, IC50 = 260 nM). In contrast, compounds 6, 7, and 9 were significantly less active than the lead compound. Notably, 6 shares significant structural homology with type I MAP2K7 inhibitor 4, which represents a conformationally confined analogue of this compound. To probe steric congestion in the proximity of the electrophile, compounds 10 and 11 were prepared and assayed. Surprisingly, neither of these compounds demonstrated measurable activity. This finding is rationalized by computational modeling of 8, which suggested a great deal of steric congestion in proximity to Cys218 (Figure 2). An exchange of the electrophilic α-chloroacetamide for the less reactive α-fluoroacetamide (12) or α-methoxyacetamide (13) resulted in greatly attenuated activity, which implies a covalent mechanism.

Table 1. In Vitro Potency of Preliminary MAP2K7 Inhibitors as Determined by the ADP-Glo Assay.

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Figure 2.

Figure 2

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Compound 8 (60 nM) was modeled to bind with its appendant electrophile in proximity to Cys218. However, steric congestion around the electrophilic center leads to less potent analogues with substitution nearby (e.g., 10 and 11).

Given the greater potency endowed by the α-chloroacetamide electrophilic motif and meta substitution around the arene, we focused our efforts on compounds of this nature. Although all FDA-approved covalent inhibitors to date feature electrophilic acrylamides or epoxyketones,41 the half-life of α-chloroacetamides surpasses that of acrylamides in the presence of glutathione at physiological temperature and pH.42 We hoped to leverage this preliminary structure–activity relationship to explore this reactive and less frequently employed electrophile43 that has enjoyed recent success.44,45

We next probed the chemical space with respect to aminopyrimidine functionality. The chloropyrimidine intermediate (SI-1) was derivatized with a variety of phenethylamines, other primary amines, as well as secondary amines (Table 2). As a whole, compounds with less steric congestion in close proximity to the pyrimidine fared better, with select arylethylamine-derived inhibitors (15 and 19) demonstrating the greatest potency. Even moderately sterically demanding substitution along the appendant arene was associated with lowered potency (e.g., 21–23).

Table 2. In Vitro Potency of MAP2K7 Inhibitors with Varying Pyrimidine Substitution.

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These findings were paralleled by docking studies (PDB: 6YG3):32 substituted phenethylamines suffered from steric bulk at the nonelectrophilic end of the molecule without gaining additional favorable interactions. This observation is in stark contrast with benzylamine-derived inhibitors, which computational studies predicted to engage in an additional cation−π interaction with Lys165 (Figure 3). This interaction additionally appeared to steer the carbon skeleton of the inhibitor away from unfavorable steric interactions.

Figure 3.

Figure 3

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Docking comparison of 3,4-methylenedioxyphenethylamine-derived inhibitor (23) and benzylamine-derived 25. Sterics are thought to underlie poor potency of substituted phenethylamine derivatives and better fit of benzylamine analogues.

Guided by these new insights, we next designed, synthesized, and evaluated a new class of benzylamine-functionalized pyrimidines (Table 3). Gratifyingly, the simplest of these compounds, 25 (DK2403), demonstrated extremely high potency (IC50 = 10 nM; IC50 = 93 nM without preincubation;46 see Supporting Information). The potency of its N-methylated derivative (24) suffered 10-fold attenuation, thereby highlighting the importance of this key hydrogen bond. Arene substitution generally led to a decrease in potency, with some positions and substitutions tolerated better than others.

Table 3. In Vitro Potency of Benzylamine-Derived MAP2K7 Inhibitors.

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Small substituents, such as fluorine atoms, were well tolerated (26–29), with the 3,4-difluorinated derivative (29) demonstrating 42 nM potency. Substituents as large as a methyl group invariably led to decreased potencies, and substitution at the ortho position (30, 32) pushed potencies toward the micromolar range. Larger substituents were better tolerated in the para (34, 36) and meta (35) positions, although disubstitution at the meta positions was poorly tolerated (33).

With a series of highly potent MEK7 inhibitors in hand, we next probed the selectivity of these compounds. Among the MEK isoforms, MEK7 shares the greatest structural homology with MEK4, thereby making it the key kinase to test as we commence selectivity studies.47,48 Potency was determined against MEK4 employing our standard protocol (vide supra). Much to our surprise, compounds 8, 9, and 25 showed virtually no activity against MEK4 (IC50 > 80 μM, Figure 4). When studied against the MEK3, 5, and 6 isoforms, 8, 9, and 25 were again found to be inactive (see Figure SI-1). MEK4 and MEK7 feature high structural33 and functional20 homology, and selective inhibition of one in the presence of the other is often difficult.31,47 We were surprised to not induce MEK4 inhibition at concentrations as high as 80 μM, which prompted us to pursue further selectivity studies.

Figure 4.

Figure 4

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MEK4/MEK7 selectivity profiles of select inhibitors.

To better understand the selectivity profile of this class of inhibitors, 25 was subjected to a 97-kinase selectivity screen (Figure 5, ScanEDGE, DiscoverX/Eurofins; see Figure SI-9 for details). Among the 97 off-target kinases surveyed, our lead compound only interacted significantly with EGFR and the EGFR(L858R) mutant at 1 μM (selectivity scores:49 S(35) = S(10) = 0.011). Additional assays confirmed a lack of binding at MEK3, MEK5, and MEK6 (Figure SI-1). Notably, 25 had no appreciable activity on MEK1/2, p38α/β, or JNK1/2/3, which are relevant kinases in our model for molecular pathophysiology in T-ALL.11,17,18,50 This compound also did not engage FLT3, a potential anticipated pitfall given the structural similarities between 25 and 5, a reversible FLT3 inhibitor. MAP2K7 was not among the 90 wildtype kinases assayed and represented in Figure 5.

Figure 5.

Figure 5

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Off-target activity profile of 25. Among 90 wildtype kinases surveyed, only compound 25 is appreciably bound by one kinase (EGFR, red dot) at 1 μM.

To confirm the suspected covalent mechanism of DK2403 (25), full-length MAP2K7 N-terminal GST fusion protein (74 kDa) was incubated with 25 (2 equiv), and the incubate was analyzed by LC-TOF MS. To our delight, MS analysis demonstrated covalent engagement (see Figures SI-5 and SI-6). The incubates were then digested with either trypsin/chymotrypsin combination or GluC. The former digest revealed covalent engagement of 25 with the intended Cys218 residue (Figures 6 and SI-7). Adducts involving other active site residues were not observed, although labeling of solvent-exposed Cys341 was observed following GluC digestion (Figure SI-8). Selectivity of our most potent compound likely derives from targeting Cys218, as only 11 human kinases bear an analogous cysteine (Figure 6A).38

Figure 6.

Figure 6

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(A) Summary of active site cysteine residues among the MEK isoforms. Cys218, targeted by DK2403 (25), is highlighted in red. (B) Observed tandem mass spectrometry adduct confirming DK2403 covalently engages Cys218.

To study the action of DK2403 in living cells, the dose–response cytotoxicity of the compound was assessed in a variety of T-ALL cell lines at 48 h.51 As shown in Figure 7A, 25 displayed marked cytotoxicity, which surpassed the potency of 8 (Figure SI-3) and JNK-IN-8.11 This effect was resistant to washout, thereby supporting a covalent mechanism (Figure 7B). To our knowledge, this represents the most cytotoxic MAP2K7 inhibitor toward T-ALL lines of interest with the exception of 1(18) and OTSSP167.52 The cytotoxicity of these compounds is likely multifactorial given their promiscuity,53,54 and for 1, low MAP2K7 potency (IC50 = 1.3 μM vs 80 nM for MAP2K1 and 80 nM for TAK1).26 EGFR inhibitor erlotinib55 failed to induce cytotoxicity at similar concentrations, thereby suggesting that the effect on cell viability is likely driven by MAP2K7 inhibition (Figure SI-11).

Figure 7.

Figure 7

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(A) Dose–response cytotoxicity of DK2403 (25) in T-ALL cell lines. (B) Washout cytotoxicity studies of DK2403. (C) Western blot analysis demonstrating dose–response (in μM) decreased phosphorylation of JNK and ATF2 in T-ALL cells following treatment with 25.

Further probing the cellular efficacy of our novel MAP2K7 inhibitor, we examined the dose–response effect of 25 on JNK and ATF2 phosphorylation after 24 h of incubation to ensure no treatment-associated cytotoxicity. Our studies assessed endogenous MAP2K7 inhibition, rather than the inhibition of induced MAP2K7/JNK, as was done previously.32 Treatment with 25 potently attenuated phosphorylation at 5–10 μM (Figure 7C). Combined with our clean selectivity profile, this finding suggests that cytotoxicity is derived from attenuating aberrant JNK signaling described in these cell lines.11,17,18,50

In conclusion, we have developed novel potent and selective MAP2K7 inhibitors that covalently engage the unique Cys218 residue within the active site. Our investigation commenced with a known FLT3 inhibitor with off-target MAP2K7 activity previously identified by our group. Lead optimization activities were guided by an iterative cycle of computational modeling, synthesis, and in vitro evaluation. The rapid generation of chemical diversity was enabled by a streamlined one-pot process, much unlike involved synthetic approaches required by compounds exhibiting similar selectivity.56,57 The preliminary selectivity studies described herein support that DK2403 (25) potently inhibits MAP2K7 without significantly disrupting the greater kinome, thereby rendering it an excellent candidate for the study of MAP2K7 in pediatric T-ALL.

Acknowledgments

We thank the National Cancer Institute of the National Institutes of Health (NCI; R01CA188015 and R01CA207086) and Northwestern University for financial support. D.R.K. was supported by the NIH under award numbers F31CA228431 and T32GM008152. M.J.O. was supported by the NIH under award number F30DA050445. H.H.M. thanks Northwestern University for support through a Summer Undergraduate Research Grant and NU Chemistry of Life Processes Institute for an Academic Year Undergraduate Research Grant. A.J.K. was supported by the NIH under award numbers T32GM105538 and F31CA250353. K.K.D. thanks the National Science Foundation (NSF) for support through a graduate research fellowship (DGE-1324585). T.J.C. was supported by the NIH under T32 GM008231. This work employed the facilities of the High Throughput Analysis Laboratory (HTAL, NU) and the Integrated Molecular Structure Education and Research Center (IMSERC, NU) NMR and MS facilities. The kinome tree image was generated using the TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of DiscoverX Corporation.

Glossary

Abbreviations

ALL

acute lymphoblastic leukemia

KLF4

Krüppel-like factor 4

MAP2K7

mitogen-activated protein kinase kinase 7

MEK7

MAPK/Erk kinase 7

JNK

c-Jun N-terminal kinase

5Z7O

5(Z)-7-oxozeaenol

TAK1

transforming growth factor-β (TGF-β)-activated kinase 1

EGFR

epidermal growth factor receptor

FLT3

FMS-like tyrosine kinase 3

GST

glutathione S-transferase

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00029.

  • Additional in vitro results; cytotoxicity data; selectivity data; PAMPA data; TOF-MS and MS-MS data; experimental details for in vitro, cellular, and MS experiments; modeling protocols; compound synthesis; and characterization (PDF)

Author Contributions

D.R.K., K.K.D., and K.A.S. conceived the project. D.R.K. and K.A.S. directed the project. D.R.K. and H.H.M. conducted all organic synthesis and characterization. M.J.O. conducted all docking studies and computational modeling. D.R.K., M.J.O., and A.J.K completed all in vitro assays. C.S.B. and T.J.C. completed all cellular assays with direction from H.D.L. D.R.K. completed whole-protein LC/MS-TOF experiments. X.Z. completed protein digestion and MS-MS experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): Intellectual property filings.

Supplementary Material

ml3c00029_si_001.pdf (4.8MB, pdf)

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