Dopaminergic neurons express mixed lineage kinases which regulate the expression of cell death genes. In Parkinson's disease, cell death via apoptosis is prevalent, and previous work testing mixed lineage kinase inhibitors in animal models suggested the inhibitors had some neuroprotective potential. CLFB-1134 is a new, brain-penetrant inhibitor specific for MLK3, tested here in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of dopaminergic depletion and nigral neuron death in mice. After ensuring that treatment with CLFB-1134 did not alter conversion of MPTP to MPP+, we demonstrated CLFB-1134's inhibition of MLK3 and neuroprotective efficacy. Specifically we evaluated the integrity of the nigrostriatal dopamine system following MPTP by assessing protein expression, high performance liquid chromatography, and immunohistology with stereology. We found that CLFB-1134 achieves protection of striatal dopaminergic terminals and nigral cell bodies when dosed simultaneously or following MPTP treatment. By preventing phosphorylation of JNK and other downstream targets of MLK3, CLFB-1134 protects against the neurotoxin MPTP. Inhibition of MLK3 may be a valid target for future work investigating treatment of Parkinson's disease.

During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2R gamma c-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance.From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future. (C) 2015 The Authors. Published by Elsevier Inc.

Backgrounds and aims: We have recently demonstrated that genetic deletion of mixed lineage kinase 3 (MLK3) reduces inflammation in a murine model of NASH. However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are not fully elucidated. Our hypothesis is that MLK3 mediates the release of chemokine rich extracellular vehicles (EVs), which induce macrophage activation & trafficking to the liver in NASH. Methods: Two MLK3 inhibitors CLFB-1134 and URMC099 were kindly provided by Califia Bio, Inc. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with lysophosphatidylcholine (LPC), a toxic metabolite of saturated fatty acid with and without one of the MlK3 inhibitors. Released EVs were isolated by differential ultracentrifugation, quantified by nanoparticle tracking analysis, and employed for macrophage treatment. EVs protein contents were profiled by mass spectrometry (MSP). Results: LPC treatment of PMH & Huh7 cells induced a 3.6-fold and 140-fold increase in release of EVs, respectively, which was prevented by either genetic knock down or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs. CXCL10 was markedly enriched in EVs isolated from LPC treated PMH versus untreated cells, as assessed by immuno-gold electron microscopy and immunoblot analysis. Unexpectedly, either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EV (corrected for the number of EV secreted). Treatment of mouse bone marrow-derived macrophages with physiologically relevant concentrations of lipotoxic hepatocyte-derived EVs induced macrophage activation and chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antiserum. Finally, activating phospho-MLK3 expression was increased in liver biopsies of patients with fatty liver compared to the normal control. In Conclusion: during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes which are proinflammatory for macrophages. We speculate that these EVs mediate hepatic inflammation in vivo by inducing macrophages trafficking to the liver and that inhibition of MLK3-dependent EV release from hepatocytes could be salutary in human NASH.

INTRODUCTION:Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP. METHODS:The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice. RESULTS:The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration. CONCLUSIONS:Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Mixed lineage kinase 3 (MLK3) is a ubiquitously expressed pro-inflammatory, pro-apoptotic mitogen activated protein kinase kinase kinase (MAP3K). MLK3 is a key regulator of the p38 and c-jun terminal kinase (JNK) pathways and has been studied in cancer and neurodegenerative disease. Although the p38 and JNK pathways have been studied in the cardiac function and disease, little is known regarding the role of MLK3 in the heart. Studies in our laboratory have indicated that MLK3 RNA is highly expressed in macrophages, cardiomyocytes and cardiac fibroblasts, suggesting an important role in cardiac function. Recently published work has indicated that MLK3 plays an important role in inflammatory cell motility, leading us to hypothesize that MLK3 is involved in inflammatory cell-fibroblast communication during cardiac disease. Knockout (KO) or inhibition of MLK3 using the novel small molecule inhibitor URMC-099 does not significantly affect heart rate, mean arterial pressure, systolic pressure, minimum or maximum dp/dt compared to wild type (WT) controls as measured by invasive hemodynamics at 3 months of age. Echocardiographic analysis indicates that MLK3 KO or inhibition does not affect cardiac architecture or cardiac function, indicated by fractional shortening or ejection fraction. However, upon transaortic constriction (TAC), MLK3 KO and URMC-099 treatment results in decreases in Mac-3 positive staining at 3 and 7 days post-TAC as well as decreases in CD-45 staining 7 days post-TAC compared to WT TAC operated, vehicle treated controls, suggesting that MLK3 KO and drug treatment may attenuate the early inflammatory response after TAC. Studies examining the relationship between the MLK3 mediated inflammatory response and subsequent fibrosis and cardiac dysfunction post-TAC are currently ongoing.

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.

Cardiovascular disease (CVD) and the final clinical iteration, heart failure (HF), affect more than 82 million Americans yearly, affects >5.7 million). Although the cardiac myocyte has long been the focus of cardiac cellular research, our lab and others have demonstrated that pathological activation of cardiac fibroblasts (CF) after onset of disease or cardiac injury is also a key player in HF pathogenesis. Pathologic activation of CFs causes a release of various paracrine and autocrine factors that target cardiomyocytes (CM), local inflammatory cells and the CFs themselves. Elucidating the mechanisms involved in pathological ‘support cell (CF)’- ‘functional cell (CM)’ communication may hold therapeutic promise. Mixed lineage kinase 3 (MLK3) is a stress activated mitogen-activated protein kinase kinase kinase (MAPKKK) involved in pro-apoptotic pathways. Inhibition of MLK3 in HAND results in the attenuation of microglial cell activation, preservation of neuronal function and synaptic structures. The parity between microglia-neuron communication and CF-CM communication and the effects of pathological activation on the respective support cells for each tissue type suggested a role for MLK3 in aberrant CF-CM cross-talk associated with the development and progression of HF. Preliminary data in mice subjected to a pharmacological model of HF (chronic isoproterenol) that were treated with a small molecule inhibitor (URMC-099) of MLK3 demonstrated a reduction in the development of myocardial fibrosis compared to vehicle treated animals. Additionally, small molecule MLK3 inhibition attenuated the development of myocardial hypertrophy as measured by heart weight: body weight and heart weight: tibia length ratios. Studies in cultured neonatal rat ventricular fibroblasts (NRVF) demonstrated that small molecule MLK3 inhibition attenuated CF activation and transition to a myofibroblast phenotype, including reductions of pathologic CF markers such as α-SMA, IL-6, IL-1β, and others. Our data demonstrate that MLK3 plays an important role in pathologic CF-CM communication and myocardial hypertrophy, and suggest that small molecule inhibition of MLK3 holds therapeutic promise for HF.

Heart failure (HF) is a manifestation of most cardiovascular diseases whose increasing prevalence highlights the need for novel therapeutics. The extent of pathologic cardiac remodeling is correlated with clinical outcome. Importantly, cardiac injury enhances cardiac fibroblast (CF) activation, producing myofibroblasts that release pro-fibrotic/inflammatory mediators which target cardiomyocytes (CM), CFs, and local inflammatory cells to exacerbate remodeling. Mixed Lineage Kinases (MLKs) are a family of stress-activated MAPKKKs whose functional role(s) in the heart remain largely unknown. MLK3 has been implicated in HIV-associated neurocognitive disorder (HAND), where it mediates deleterious cross-talk between microglia and neurons, suggesting an analogous mechanism of pathologic intercellular CF-CM communication in HF. We hypothesize that MLK3 exacerbates cardiac remodeling through enhanced CF activation that contributes to pathologic cardiac intercellular communication. We have synthesized a series of MLK3-specific small molecule inhibitors, one of which (URMC-099) was found to attenuate microglial-mediated neurotoxicity in murine models of HAND. To investigate the role of MLK3 in CF activation, neonatal rat ventricular CFs were stimulated with isoproterenol (Iso) or angiotensin II (AngII). Concurrent treatment with URMC-099 attenuated both α-smooth muscle actin expression, indicative of myofibroblast transition, and proinflammatory cytokine production. Further, therapeutic efficacy and specificity of URMC-099 were tested in iso-infused and myocardial infarction (MI) models of HF using wild-type (WT) and MLK3-/- mice (shown to have no overt cardiac phenotype). URMC-099 significantly attenuated cardiac hypertrophy (HW:BW) and reduced interstitial fibrosis (assessed by Masson's Trichrome staining) in an acute iso-pump model of HF in WT mice. Current echocardiographic data post-MI suggest cardioprotection in the MLK3-/- and URMC-099 treated mice. In conclusion, our collaborative data not only suggest a role for MLK3 in cardiac remodeling through pathologic CF activation but indicate a possibly novel paradigm of pathologic intercellular communication in multiple disease states.

La presente invention concerne des composes ayant un effet inhibiteur sur des kinases de lignage mixte. La presente invention concerne en outre des compositions pharmaceutiques, des procedes de preparation des composes, des intermediaires synthetiques, et des procedes d'utilisation des composes, independamment ou en combinaison avec d'autres agents therapeutiques, pour traiter des maladies et affections qui sont affectees par l'inhibition de kinase de lignage mixte. La presente invention concerne en outre des procedes de traitement de troubles neuropsychiatriques qui comprennent l'inhibition de kinases de lignage mixte.

A series of thienopyrimidinone bis-aminopyrrolidine ureas were designed, synthesized, and evaluated for their ability to bind melanin-concentrating hormone receptor-1. These compounds exhibit potent binding affinity (K(i)=3 nM) and good in vitro metabolic stability.

The design, synthesis, and SAR of a series of substituted spirohydantoins are described. Optimization of an in-house screening hit gave compounds that exhibited potent binding affinity and functional activity at MCH-R1.

Derivatives of 1-(4-amino-phenyl)-pyrrolidin-3-yl-amine and 6-(3-amino-pyrrolidin-1-yl)-pyridin-3-yl-amine were identified as potent and functionally active MCH-R1 antagonists. One compound with Ki = 2.3 nM demonstrated good oral bioavailability (32%) and in vivo efficacy in rats.

Melanin-concentrating hormone receptor antagonists containing thieno- and a benzopyridazinone cores were designed and tested as potential anorectic agents. These ligands showed high affinity for the receptor, potent functional activity in vitro, and good oral bioavailabilty in rats. The thiophene analogue exhibited low iv clearance, long half-life, and high brain penetration. In obese rats, the thienopyridazinone demonstrated a dose-dependent reduction in feeding and body weight with doses between 1 and 10 mg kg-1.