p38α MAP Kinase C-Terminal Domain Binding Pocket Characterized by Crystallographic and Computational Analyses

The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38α acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest. Here, we present the crystal structure of human p38α MAP kinase, which has a lead compound bound both in the active site and in the lipid-binding site of the C-terminal cap. This C-terminal cap is formed from an extension to the kinase fold, unique to the MAP kinase and cyclin-dependent kinase families and glycogen synthase kinase 3. Binding of this lead, 4-[3-(4-fluorophenyl)-1H-pyrazol-4-yl]pyridine, to wild-type p38α induces movement of the C-terminal cap region, creating a hydrophobic pocket centered around residue Trp197. Computational analysis of this C-terminal domain pocket indicates notable flexibility for potentially binding different-shaped compounds, including lipids, oxidized arachidonic acid species such as leukotrienes, and small-molecule effectors. Furthermore, our structural results defining the open p38α C-lobe pocket provide a detailed framework for the design of novel small molecules with affinities comparable to active-site binders: to bind and potentially modulate the shape and activity of p38α in predetermined ways. Moreover, these results and analyses of p38α suggest strategies for designing specific binding compounds applicable to other MAP kinases, as well as the cyclin-dependent kinase family and glycogen synthase kinase 3β that also utilize the C-terminal insert in their interactions.     

The design and synthesis of novel alpha-ketoamide-based p38 MAP kinase inhibitors

We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory conditions.     

Optimization of α-ketoamide based p38 inhibitors through modifications to the region that binds to the allosteric site

We have optimized a novel series of potent p38 MAP kinase inhibitors based on an α-ketoamide scaffold through structure based design that due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME, in vivo PK and efficacy studies show these compounds to have drug-like characteristics and have resulted in the nomination of a development candidate which is currently in phase II clinical trials for the oral treatment of inflammatory conditions.     

Two classes of p38alpha MAP kinase inhibitors having a common diphenylether core but exhibiting divergent binding modes

Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.     

Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.     

Prevention of MKK6-dependent activation by binding to p38alpha MAP kinase

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.     

Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.     

Human p38α mitogen-activated protein kinase in the Asp168-Phe169-Gly170-in (DFG-in) state can bind allosteric inhibitor Doramapimod

Doramapimod (BIRB-796) is widely recognized as one of the most potent and selective type II inhibitors of human p38α mitogen-activated protein kinase (MAPK); however, the understanding of its binding mechanism remains incomplete. Previous studies indicated high affinity of the ligand to a so-called allosteric pocket revealed only in the ‘out’ state of the DFG motif (i.e. Asp168-Phe169-Gly170) when Phe169 becomes fully exposed to the solvent. The possibility of alternative binding in the DFG-in state was hypothesized, but the molecular mechanism was not known. Methods of bioinformatics, docking and long-time scale classical and accelerated molecular dynamics have been applied to study the interaction of Doramapimod with the human p38α MAPK. It was shown that Doramapimod can bind to the protein even when the Phe169 is fully buried inside the allosteric pocket and the kinase activation loop is in the DFG-in state. Orientation of the inhibitor in such a complex is significantly different from that in the known crystallographic complex formed by the kinase in the DFG-out state; however, the Doramapimod’s binding is followed by the ligand-induced conformational changes, which finally improve accommodation of the inhibitor. Molecular modelling has confirmed that Doramapimod combines the features of type I and II inhibitors of p38α MAPK, i.e. can directly and indirectly compete with the ATP binding. It can be concluded that optimization of the initial binding in the DFG-in state and the final accommodation in the DFG-out state should be both considered at designing novel efficient type II inhibitors of MAPK and homologous proteins.

Communicated by Ramaswamy H. Sarma     

New modifications to the area of pyrazole-naphthyl urea based p38 MAP kinase inhibitors that bind to the adenine/ATP site

Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.     

Targeting Conformational Activation of CDK2 Kinase

Cyclin‐dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP‐binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T‐loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP‐competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T‐loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T‐loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.     

Utilization of a nitrogen–sulfur nonbonding interaction in the design of new 2-aminothiazol-5-yl-pyrimidines as p38α MAP kinase inhibitors

The design, synthesis, and structure–activity relationships (SAR) of a series of 2-aminothiazol-5-yl-pyrimidines as novel p38α MAP kinase inhibitors are described. These efforts led to the identification of 41 as a potent p38α inhibitor that utilizes a unique nitrogen–sulfur intramolecular nonbonding interaction to stabilize the conformation required for binding to the p38α active site. X-ray crystallographic studies that confirm the proposed binding mode of this class of inhibitors in p38α and provide evidence for the proposed intramolecular nitrogen–sulfur interaction are discussed.     

Allosteric interactions between the myristate- and ATP-site of the Abl kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.     

Discovery and design of benzimidazolone based inhibitors of p38 MAP kinase

A new class of benzimidazolone p38 MAP kinase inhibitors was discovered through high-throughput screening. X-ray crystallographic data of the lead molecule with p38 were used to design analogues with improved binding affinity and potency in a cell assay of LPS-induced TNF production. Herein, we report the SAR of this new class of p38 inhibitors.     

Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.     

Modeling of Arylamide Helix Mimetics in the p53 Peptide Binding Site of hDM2 Suggests Parallel and Anti-Parallel Conformations Are Both Stable

The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix.     

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.     

Substituting c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their p38 counterparts affects binding of JNK- and p38-selective inhibitors

c-Jun N-terminal kinase (JNK) activation is linked to the aberrant cell death in several neurodegenerative disorders, including Parkinson's and Alzheimer's disease. The sequence similarity among the JNK isoforms and fellow MAP kinase family member p38 has rendered the challenge of producing JNK3-specific inhibitors difficult. Using the crystal structure of JNK3 complexed with JNK inhibitors, potential compound-interacting amino acid residues were mutated to the corresponding residues in p38. The effects of these mutations on the kinetic parameters with three compounds were examined: a JNK3- (vs. p38-) selective inhibitor (SP 600125); a p38-selective inhibitor (Merck Z); and a potent combined JNK3 and p38 inhibitor (Merck Y). The data confirm the role of the JNK3 residues Ile-70 and Val-196 in both inhibitor and ATP-binding. Remarkably, the Ile-70-Val and Val-196-Ala mutations caused an increase and decrease, respectively, in the binding affinity of the p38-specific compound, Merck Z, of 10-fold. The Ile-70-Val effect may be due to the increased capacity of the active site to accommodate Merck Z, whereas the Val-196-Ala mutant may induce an unfavourable conformational change. Conservative mutations of the Asn-152 and Gln-155 residues inactivated the JNK3 enzyme, possibly interfering with protein folding in a critical hinge region of the protein.     

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Break CDK2/Cyclin E1 Interface Allosterically with Small Peptides

Most inhibitors of Cyclin-dependent kinase 2 (CDK2) target its ATP-binding pocket. It is difficult, however, to use this pocket to design very specific inhibitors because this catalytic pocket is highly conserved in the protein family of CDKs. Here we report some short peptides targeting a noncatalytic pocket near the interface of the CDK2/Cyclin complex. Docking and molecular dynamics simulations were used to select the peptides, and detailed dynamical network analysis revealed that these peptides weaken the complex formation via allosteric interactions. Our experiments showed that upon binding to the noncatalytic pocket, these peptides break the CDK2/Cyclin complex partially and diminish its kinase activity in vitro. The binding affinity of these peptides measured by Surface Plasmon Resonance can reach as low as 0.5 µM.