A small molecule bidentate-binding dual inhibitor probe of the LRRK2 and JNK kinases.

Both JNK and LRRK2 are associated with Parkinson's disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. Compound 6 also exhibited good cell potency, inhibited LRRK2:G2019S-induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD.

Structural mechanisms of allostery and autoinhibition in JNK family kinases.

c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations.

Convergent synthesis and discovery of a natural product-inspired paralog-selective Hsp90 inhibitor.

A convergent synthesis of benzoquinone ansamycin analogs is described that proceeds by a sequence of metallacycle-mediated alkyne-alkyne coupling, followed by site- and stereoselective dihydroxylation and global carbamate formation. These studies have led to (1) validation of alkyne-alkyne coupling to produce geldanamycin analogs that lack the problematic quinone, (2) the discovery that C6-C7 bis-carbamate functionality is compatible with Hsp90 inhibition, and (3) the identification of 1 as a nonquinone geldanamycin-inspired paralog-selective Hsp90 inhibitor.

Small Molecule c-jun-N-terminal Kinase (JNK) Inhibitors Protect Dopaminergic Neurons in a Model of Parkinson's Disease.

There are currently no drugs to treat neurodegeneration in Parkinson's disease (PD) and all existing medications only treat symptoms, lose efficacy over time, and produce untoward side effects. In the current work, we report the first highly selective, orally bioavailable, c-jun-N-terminal kinase (JNK) inhibitor for protection of dopaminergic neurons in vitro and in vivo. At 300 nM this compound showed statistically significant protection of primary dopaminergic neurons exposed to 1-methyl-4-phenylpyridinium (MPP(+)), had pharmacokinetic properties in rodents consistent with twice daily (b.i.d.) dosing, and was orally efficacious at 30 mg/kg in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Moreover, a dose-dependent target modulation of c-jun phosphorylation served as a biomarker for demonstrating on-target inhibition of JNK as the mechanism of action for this compound. Collectively these results suggest that this JNK inhibitor could be a promising therapeutic neuroprotective agent in the treatment of Parkinson's disease.

Selective inhibition of mitochondrial JNK signaling achieved using peptide mimicry of the Sab kinase interacting motif-1 (KIM1).

The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNK interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell-permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-Sab(KIM1) selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-Sab(KIM1) prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

RhoH is required to maintain the integrin LFA-1 in a nonadhesive state on lymphocytes.

Lymphocyte function-associated antigen 1 (LFA-1) is relatively nonadhesive on resting lymphocytes; however, the mechanisms underlying changes in its adhesiveness are poorly understood. In this study, we generated a Jurkat T cell clone, J+hi1.14, that contained low amounts of mRNA for RhoH, a leukocyte-specific inhibitory Rho family member. J+hi1.14 cells expressed constitutively adhesive LFA-1 and the cells bound spontaneously to intracellular adhesion molecules 1, 2 and 3. Reconstitution of RhoH mRNA expression in J+hi1.14 cells reverted the adhesion phenotype to that of wild-type. We obtained similar results using RNA interference in peripheral blood lymphocytes. These data demonstrate that RhoH is required for maintenance of lymphocyte LFA-1 in a nonadhesive state.

Inactivation of TNF signaling by rationally designed dominant-negative TNF variants.

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.