Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity.

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Low entropic cost of binding confers high selectivity on an allosteric ERK2 inhibitor.

Extracellular signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK), plays an essential role in physiological cellular processes and is a drug target for treating cancers and type 2 diabetes. A previous in silico screening study focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). In this report, compound 1 was found to show high selectivity toward ERK2 compared with the nearest off-target p38α MAPK, and the crystal structure revealed that compound 1 binds to the allosteric site of ERK2. Fragment molecular orbital calculations based upon this crystal structure provided the structural basis to improve potency of compound 1 derivatives. Further computational studies uncovered that the low entropic cost of binding conferred the high selectivity of compound 1 toward ERK2 over p38α MAPK. These findings demonstrate the feasibility of developing potent and selective ERK2 inhibitors.

Design, Synthesis and Structure-Activity Relationship Studies of Protein Kinase CK2 Inhibitors Containing a Purine Scaffold.

Protein kinase CK2 (CK2) is involved in the suppression of gene expression, protein synthesis, cell proliferation, and apoptosis, thus making it a target protein for the development of therapeutics toward cancer, nephritis, and coronavirus disease 2019. Using the solvent dipole ordering-based method for virtual screening, we identified and designed new candidate CK2α inhibitors containing purine scaffolds. Virtual docking experiments supported by experimental structure-activity relationship studies identified the importance of the 4-carboxyphenyl group at the 2-position, a carboxamide group at the 6-position, and an electron-rich phenyl group at the 9-position of the purine scaffold. Docking studies based on the crystal structures of CK2α and inhibitor (PDBID: 5B0X) successfully predicted the binding mode of 4-(6-carbamoyl-8-oxo-9-phenyl-8,9-dihydro-7H-purin-2-yl) benzoic acid (11), and the results were used to design stronger small molecule targets for CK2α inhibition. Interaction energy analysis suggested that 11 bound around the hinge region without the water molecule (W1) near Trp176 and Glu81 that is frequently reported in crystal structures of CK2α inhibitor complexes. X-ray crystallographic data for 11 bound to CK2α was in very good agreement with the docking experiments, and consistent with activity. From the structure-activity relationship (SAR) studies presented here, 4-(6-Carbamoyl-9-(4-(dimethylamino)phenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl) benzoic acid (12) was identified as an improved active purine-based CK2α inhibitor with an IC50 of 4.3 µM. These active compounds with an unusual binding mode are expected to inspire new CK2α inhibitors and the development of therapeutics targeting CK2 inhibition.

Identification of a novel target site for ATP-independent ERK2 inhibitors.

Extracellular signal-regulated kinase 2 (ERK2) controls vital physiological processes involving proliferation and differentiation and is a drug target molecule for many diseases such as cancers. In silico screening focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). However, a competitive binding assay indicated that compound 1 did not bind to the allosteric site. Here, the crystal structure of ERK2 in complex with compound 1 revealed a novel binding site. This finding demonstrates the feasibility of developing new types of ERK2 inhibitors.

Bivalent binding mode of an amino-pyrazole inhibitor indicates the potentials for CK2α1-selective inhibitors.

Casein kinase 2 (CK2) is a vital protein kinase that consists of two catalytic subunits (CK2α1 and/or CK2α2) and two regulatory subunits (CK2ß). CK2α1 is a drug target for nephritis and cancers, while CK2α2 is a serious off-target because its inhibition causes testicular toxicity. High similarity between the isozymes CK2α1 and CK2α2 make it difficult to design CK2α1-specific inhibitors. Herein, the crystal structures of CK2α1 and CK2α2 complexed with a 3-amino-pyrazole inhibitor revealed the remarkable differences in the protein-inhibitor interaction modes. This inhibitor bound to the ATP binding sites of both isozymes in apparently distinct orientations. In addition, another molecule of this inhibitor bound to CK2α1, but not to CK2α2, at the CK2ß protein-protein interface. Binding energy calculations and biochemical experiments suggested that this inhibitor possesses the conventional ATP-competitive characteristics with moderate allosteric function in a molecular glue mechanism. These results will assist the potential design of potent and selective CK2α1 inhibitors.

Identification of an allosteric and Smad3-selective inhibitor of p38αMAPK using a substrate-based approach.

p38α mitogen activated protein kinase (MAPK) plays important roles in multiple cellular functions by phosphorylating a wide variety of substrates, and therefore, p38α MAPK has been considered as an important drug target. In this study, we designed peptide-based inhibitors for p38α MAPK, which can only inhibit the Smad3 phosphorylation specifically, by targeting the KIM binding site of p38α MAPK. Peptide 6 showed a significant inhibitory potency for the Smad3 phosphorylation by p38α MAPK. Peptide 6 showed no ATP dependency, and did not inhibit the phosphorylation of other substrates by p38α MAPK. The discovery of peptide 6 by targeting the KIM binding site likely provide an opportunity for the discovery of a novel class of allosteric and substrate-specific p38α MAPK inhibitors.

Ensemble structural analyses depict the regulatory mechanism of non-phosphorylated human MAP2K4.

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a critical role in regulating the stress-activated protein kinase signaling cascade. A small angle X-ray scattering experiment, a powerful technique for analyzing a solution structure cleared from the structural artifacts due to crystal packing, provided the ensemble structures of human non-phosphorylated MAP2K4 in three states involving the apo form, the binary complex with an ATP analogue, and the ternary complex with the ATP analogue and substrate peptide. These ensemble structures provided more detailed mechanisms for regulating MAP2K4 in addition to those delineated only by the crystal structures in three states.

Structural basis for producing selective MAP2K7 inhibitors.

Mitogen-activated protein kinase kinase 7 (MAP2K7) in the c-Jun N-terminal kinase signal cascade is an attractive drug target for a variety of diseases. The selectivity of MAP2K7 inhibitors against off-target kinases is a major barrier in drug development. We report a crystal structure of MAP2K7 complexed with a potent covalent inhibitor bearing an acrylamide moiety as an electrophile, which discloses a structural basis for producing selective and potent MAP2K7 inhibitors.

Structural insights for producing CK2α1-specific inhibitors.

Casein kinase 2 catalytic subunit (CK2α) is classified into two subtypes CK2α1 and CK2α2. CK2α1 is a drug discovery target, whereas CK2α2 is an off-target of CK2α1 inhibitors. High amino acid sequence homology between these subtypes hampers efforts to produce ATP competitive inhibitors that are highly selective to CK2α1. Hematein was identified previously as a non-ATP-competitive inhibitor for CK2α1, whereas this compound acts as an ATP competitive CK2α2 inhibitor. Crystal structures of CK2α1 and CK2α2 in complex with hematein revealed distinct binding features that provide structural insights for producing CK2α1-selective inhibitors.

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue.

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.

Protein Allostery in Rational Drug Design.

This chapter focuses on protein kinases that transfer the phosphate group of ATP to the hydroxyl group of a substrate protein. Five hundred eighteen human protein kinases are classified into serine/threonine kinases and tyrosine kinases and individually or synergistically transduce physiologic stimuli into cell to promote cell proliferation or apoptosis, etc. Protein kinases are identified as drug targets because dysfunction of kinases leads to severe diseases such as cancers and autoimmune diseases. A large number of the crystal structures of the protein kinase inhibitor complex are available in Protein Data Bank and facilitated the drug discovery targeting protein kinases. The protein kinase inhibitors are classified into categories, Type-I, Type-II, Type-III, Type-IV, and Type-V, and as a separate class, covalent-type inhibitors. In any type, a protein kinase inhibitor bound to the allosteric region is advantageous in terms of selectivity compared to the traditional ATP-competitive one. In the following sections, the successful and promising examples of the partially or fully allosteric protein kinase inhibitors are illustrated in the following pages.

High-resolution structure discloses the potential for allosteric regulation of mitogen-activated protein kinase kinase 7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) regulates stress and inflammatory responses, and is an attractive drug discovery target for several diseases including arthritis and cardiac hypertrophy. Intracellular proteins such as MAP2K7 are prone to aggregation due to cysteine-driven oxidation in in vitro experiments. MAP2K7 instability due to the four free cysteine residues on the molecular surface abrogated the crystal growth and led to a low-resolution structure with large residual errors. To acquire a higher resolution structure for promoting rational drug discovery, we explored stable mutants of MAP2K7 by replacing the surface cysteine residues, Cys147, Cys218, Cys276 and Cys296. Single-site mutations, except for Cys147, maintained the specific activity and increased the protein yield, while all the multi-site mutations massively reduced the activity. The C218S mutation drastically augmented the protein production and crystallographic resolution. Furthermore, the C218S crystals grown under microgravity in a space environment yielded a 1.3 Å resolution structure, providing novel insights for drug discovery: the precisely assigned water molecules in the active site, the double conformations in the flexible region and the C-terminal extension bound to the N-terminal region of the adjacent molecules. The latter insight is likely to promote the production of allosteric MAP2K7 inhibitors.

The juxtamembrane region of TrkA kinase is critical for inhibitor selectivity.

Although numerous crystal structures for protein kinases have been reported, many include only the kinase domain but not the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases (RTKs). In this study, we determined the X-ray crystal structure of the tropomyosin receptor kinase (Trk) A selective inhibitor A1 complexed with the TrkA kinase domain and the JM region. This structure revealed that the unique inhibitor-binding pocket created by a novel JM configuration yields significant potency and high selectivity against TrkB and TrkC. Moreover, we validated the importance of the JM region for the potency of A1 using in vitro assays. The introduction of moieties that interact with the JM region will be one of the most effective strategies for producing highly selective RTK inhibitors.

A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-Jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site. This closed form was configured by the n-σ* interaction of Cys218, a non-conserved residue among the MAP2K family kinases, with Gly145 in the glycine-rich loop. The interaction was unaltered in the presence of an ATP analog, whereas the C218S mutation precluded the closed configuration. These structural insights are potentially valuable for drug discovery of highly selective MAP2K7 inhibitors.

Identification of allosteric ERK2 inhibitors through in silico biased screening and competitive binding assay.

Extracellular signal-regulated kinase 2 (ERK2) is a drug target for type 2 diabetes mellitus. A peptide-type ERK2 inhibitor (PEP) was discovered in the previous study through the knowledge-based method and showed physiological effects on the db/db mice model of type 2 diabetes. Here, the crystal structure showed that PEP bound to the allosteric site without the interruption of the ATP competitive inhibitor binding to ERK2. An in silico biased-screening using the focused library rendered three compounds with inhibitory activity of IC50 <100 µM. Among them, two compounds revealed the concentration-dependent competition with PEP and could be lead compounds for antidiabetic medicine.

Structure-activity relationship study of 4-(thiazol-5-yl)benzoic acid derivatives as potent protein kinase CK2 inhibitors.

Two classes of modified analogs of 4-(thiazol-5-yl)benzoic acid-type CK2 inhibitors were designed. The azabenzene analogs, pyridine- and pyridazine-carboxylic acid derivatives, showed potent protein kinase CK2 inhibitory activities [IC50 (CK2α)=0.014-0.017µM; IC50 (CK2α')=0.0046-0.010µM]. Introduction of a 2-halo- or 2-methoxy-benzyloxy group at the 3-position of the benzoic acid moiety maintained the potent CK2 inhibitory activities [IC50 (CK2α)=0.014-0.016µM; IC50 (CK2α')=0.0088-0.014µM] and led to antiproliferative activities [CC50 (A549)=1.5-3.3µM] three to six times higher than those of the parent compound.

5Z-7-Oxozeaenol covalently binds to MAP2K7 at Cys218 in an unprecedented manner.

5Z-7-Oxozeaenol (5Z7O) is a covalent bonding inhibitor against the several protein kinases (e.g., ERK2 and TAK1) that possess a free cysteine at the gatekeeper-2 position. In addition to this cysteine, MAP2K7 has three other cysteine residues that are candidate for covalent bonding by the inhibitor 5Z7O. The crystal structure of the MAP2K7/5Z7O complex revealed that the inhibitor binds to MAP2K7 at a cysteine residue located at the end of the hinge region and not at the gatekeeper-2 residue. The structural insights into the interaction of 5Z7O with MAP2K7 should aid the development of 5Z7O derivatives with improved potency and selectivity.

Enhanced inhibitory activity of compounds containing purine scaffolds compared to protein kinase CK2α considering crystalline water.

We recently reported novel purine-based CK2α inhibitors using the solvent ordering-based method as virtual screening. Among these, the X-ray crystal structure of a complex with CK2α was determined. The results showed that the crystalline water molecules observed in many previously reported complex structures of CK2α and its inhibitors had been eliminated. We then proposed a structure-based drug design. Since the removal of water molecules would be detrimental to inhibitor binding, new groups of compounds were designed by changing the position of the carboxy group located at the point where a water molecule would be present so as not to eliminate it. Compounds with (E)-2-carboxyethenyl and 3-carboxyphenyl substituted at the 2-position on the purine scaffold showed much higher inhibitory potency than 4-carboxyphenyl derivatives. Furthermore, in the presence of a 4-fluorophenyl group at the 9-position on the purine scaffold, the inhibitory activity of the 3-carboxyphenyl derivative against CK2α was 0.18 µM, a 167-fold improvement compared to the 4-carboxyphenyl derivative. The strategy of leaving crystalline water can significantly increase inhibitory activity.

Crystal structure of human CK2α at 1.06 Å resolution.

The Ser/Thr kinase CK2 consists of two catalytic subunits (CK2α) and a dimer of the regulatory subunits (CK2ß), and is a ubiquitous enzyme that regulates growth, proliferation and the survival of cells. CK2 is a remarkable drug target for potentially treating a wide variety of tumours and glomerulonephritis. The purified CK2α protein was crystallized using ethylene glycol as a precipitant. The crystal structure of CK2α with 21 loci of alternative conformations, including a niacin, 19 ethylene glycols and 346 waters, was determined at 1.06 Å resolution to an Rwork of 14.0% (Rfree = 16.5%). The alternative ensemble in the internal hydrophobic core underpins the plasticity of the αD-helix responsible for the regulation of ATP/GTP binding. The clear density map indicates that a niacin molecule, contained in the Escherichia coli culture medium, binds to the ATP binding site. An ethylene glycol molecule binds in the hydrophobic pocket lateral to the αD-helix forming the rim of the active site. The other ethylene glycol molecules occupy physiologically significant sites, including the CK2ß binding interface and substrate binding site, as well as the gap in the crystal packing. Together with water molecules in the active site, these structural insights should facilitate drug discovery.

Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1.

Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.