Structural Basis for the Selective Inhibition of c-Jun N-Terminal Kinase 1 Determined by Rigid DARPin–DARPin Fusions

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.     

Differential regulation of M3/6 (DUSP8) signaling complexes in response to arsenite-induced oxidative stress

Mitogen-activated protein kinase (MAPK) cascades are involved in the regulation of cellular proliferation, differentiation, survival, apoptosis, as well as in inflammatory responses. Signal intensity and duration have been recognized as crucial parameters determining MAPK signaling output. Phosphatases play a particularly important role in this respect, by tightly controlling MAPK phosphorylation and activation. M3/6 (DUSP8) is a dual-specificity phosphatase implicated in the dephosphorylation and inactivation of JNK and, to a lesser extent, p38 MAPKs and is found in a complex with these kinases, along with other pathway components, held together by scaffold proteins. The JNK family consists of three genes, giving rise to at least ten different splice variants. Some functional differences between these gene products have been demonstrated, but the underlying molecular mechanisms and the roles of individual splice variants are still incompletely understood. We have investigated the interaction of M3/6 with JNK isoforms, as well as scaffold proteins of the JNK interacting protein (JIP) family, in order to elucidate the contribution of M3/6 to the regulation of distinct JNK signaling modules. M3/6 exhibited stronger binding towards JNK1β and JNK2α isoforms and this was reflected in higher enzymatic activity towards JNK2α2 when compared to JNK1α1 in vitro. After activation of the pathway by exposure of cells to arsenite, the interaction of M3/6 with JNK1α and JNK3 was enhanced, whereas that with JNK1β or JNK2α decreased. The modulation of binding affinities was found to be independent of JNK-mediated M3/6 phosphorylation. Furthermore, arsenite treatment resulted in an inducible recruitment of M3/6 to JNK-interacting protein 3 (JIP3) scaffold complexes, while its interaction with JIP1 or JIP2 was constitutive. The presented data suggest an isoform-specific role for the M3/6 phosphatase and the dynamic targeting of M3/6 towards distinct JNK-containing signaling complexes.     

Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders

ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.     

Correlation between chemotype-dependent binding conformations of HSP90α/β and isoform selectivity—Implications for the structure-based design of HSP90α/β selective inhibitors for treating neurodegenerative diseases

HSP90 continues to be a target of interest for neurodegeneration indications. Selective knockdown of the HSP90 cytosolic isoforms α and β is sufficient to reduce mutant huntingtin protein levels in vitro. Chemotype-dependent binding conformations of HSP90α/β appear to strongly influence isoform selectivity. The rational design of HSP90α/β inhibitors selective versus the mitochondrial (TRAP1) and endoplasmic reticulum (GRP94) isoforms offers a potential mitigating strategy for mechanism-based toxicities. Better tolerated HSP90 inhibitors would be attractive for targeting chronic neurodegenerative diseases such as Huntington’s disease.     

Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display

There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10¹⁰ individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFα, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.     

Design and synthesis of 2'-anilino-4,4'-bipyridines as selective inhibitors of c-Jun N-terminal kinase-3

The design and synthesis of a new series of c-Jun N-terminal kinase-3 (JNK3) inhibitors with selectivity against JNK1 are reported. The novel series of substituted 2'-anilino-4,4'-bipyridines were designed based on a combination of hits from high throughput screening and X-ray crystal structure information of compounds crystallized into the JNK3 ATP binding active site.     

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.     

DARPins as Bispecific Receptor Antagonists Analyzed for Immunoglobulin E Receptor Blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the α chain of the high-affinity receptor for human immunoglobulin E (IgE) (Fc?RIα). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human Fc?RIα. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-Fc?RI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule = one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.     

Balance of human choline kinase isoforms is critical for cell cycle regulation: implications for the development of choline kinase-targeted cancer therapy

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CKα1, CKα2 and CKβ that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up-regulation of the CKα genes, and specific inhibition of CKα activity has been proposed as a potential anti-cancer strategy. As a result, less attention has been given to CKβ and its interaction with CKα. With the aim of profiling the intracellular roles of CKα and CKβ, we used RNA interference (RNAi) as a molecular approach to down-regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi-based silencing of the CK α and β isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform-specific antibodies and by quantitative real-time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CKα knockdown. However, CKβ- and CKα +?CKβ-silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the α and β isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CKα silencing by simultaneous knockdown of both isoforms implies that a more effective CK-based anti-cancer strategy can be achieved by reducing cross-reactivity with CKβ.     

Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

In search of AKT kinase inhibitors as anticancer agents: structure-based design,docking, and molecular dynamics studies of 2,4,6-trisubstituted pyridines

The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian, and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435, and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.     

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38α and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.     

Selection and characterization of Her2 binding-designed ankyrin repeat proteins

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.     

The isoform-specific functions of the c-Jun N-terminal Kinases (JNKs): differences revealed by gene targeting

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family. In mammalian genomes, three genes encode the JNK family. To evaluate JNK function, mice have been created with deletions in one or more of three Jnk genes. Initial studies on jnk1(-/-) or jnk2(-/-) mice have shown roles for these JNKs in the immune system whereas studies on jnk3(-/-) mice have highlighted roles for JNK3 in the nervous system. Further studies have highlighted the contributions of JNK1 and/or JNK2 to a range of biological and pathological processes. These include bone remodelling and joint disease, inflammatory and autoimmune diseases, obesity, diabetes, cardiovascular disease, liver disease and tumorigenesis in addition to effects in neurons. These results emphasise the differences in the roles played by JNK isoforms in vivo and suggest that the design of JNK inhibitors for subsequent therapeutic uses may benefit from selective inhibition of individual JNK isoforms.     

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.     

Design and synthesis of 6-anilinoindazoles as selective inhibitors of c-Jun N-terminal kinase-3

The structure-based design and synthesis of a new series of c-Jun N-terminal kinase-3 inhibitors with selectivity against JNK1 and p38alpha is reported. The novel series of substituted 6-anilinoindazoles were designed based on a combination of hits from high throughput screening and X-ray crystal structure information of the compounds crystallized into the JNK3 ATP binding active site.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Design, synthesis, and structure-activity relationship studies of thiophene-3-carboxamide derivatives as dual inhibitors of the c-Jun N-terminal kinase

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.