Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.

UNLABELLED: Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage-associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone-marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10-neutralizing antisera. MLK3-deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild-type mice. CONCLUSIONS: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.

The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy.

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/IL2Rγc-/- 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.

The new small-molecule mixed-lineage kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflammatory in models of human immunodeficiency virus-associated neurocognitive disorders.

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.

The second generation mixed lineage kinase-3 (MLK3) inhibitor CLFB-1134 protects against neurotoxin-induced nigral dopaminergic neuron loss.

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.

A thienopyridazinone-based melanin-concentrating hormone receptor 1 antagonist with potent in vivo anorectic properties.

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.

Manipulation of small-molecule inhibitory kinetics modulates MCH-R1 function.

The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.

The melanocortin system and its role in obesity and cachexia.

Melanocortin receptors (MC-R) activated by one of several peptides derived from the pro-opiomelanocortin (POMC) precursor have become leading contenders for a pivotal role in controlling food intake. Evidence has emerged over the last decade to implicate primarily the MC4-R and, to a lesser extent, MC3-R as the key sub-types involved and both are strategically located in those regions within the hypothalamus known to be associated with feeding. The receptors are within class A of the GPCR superfamily and the key electrostatic interaction with the positively charged peptide (Arg) has been mapped to one or more Asp or Glu residues located on helices II and III of the seven helical bundle characteristic of this class of receptor. Sites for secondary interactions from which sub-type selectivity may be derived have also been located in the extracellular and helical domains. Unique amongst GPCRs is the presence of endogenous antagonist peptides, Agouti and Agouti-related peptide (AGRP), which confer an extra level of control on the system. Recently, several reports of potent and selective non-peptide ligands have been published and these are seen as prototypic molecules from which drugs may emerge to treat obesity (agonists) and cachexia (antagonists). The role played by the melanocortin system is the subject of this review and advances in our understanding of the structure of the endogenous ligand(s), non-peptide, small molecule ligands and the receptors at which they interact will be discussed.

Body weight regulation by selective MC4 receptor agonists and antagonists.

There has been great interest in melanocortin (MC) receptors as targets for the design of novel therapeutics to treat disorders of body weight, such as obesity and cachexia. Both genetic and pharmacological evidence points toward central MC4 receptors as the principal target. Using highly selective peptide tools for the MC4 receptor, which have become available recently, we have provided pharmacological confirmation that central MC4 receptors are the prime mediators of the anorexic and orexigenic effects reported for melanocortin agonists and antagonists, respectively. The current progress with receptor-selective small molecule agonist and antagonist drugs should enable the therapeutic potential of MC4 receptor activation and inhibition to be assessed in the clinic in the near future.

Assessment of a small molecule melanocortin-4 receptor-specific agonist on energy homeostasis.

The central melanocortin system has been demonstrated to play an important role in regulating different aspects of energy homeostasis. Understanding the specific contributions of MC3 and MC4 receptors, however, requires specific agonists and antagonists for each of the predominant forms of brain melanocortin receptors, MC3-R and MC4-R. We report here the characterization of a small peptide mimetic MC4-R-specific agonist that possesses both high affinity (K(i)=11.3 nM) and potency (EC(50)=1.62 nM) in vitro and is capable of inhibiting feeding behavior in mice when administered intracerebroventricularly (i.c.v.). Depending on the paradigm, acute (1 h following an overnight fast) or long-term (greater than 6 h under normal nocturnal feeding conditions) feeding inhibition was observed following icv injection. No effect on long-term feeding inhibition was observed with this compound in MC4-R knockout mice, and central administration of this compound had no effect on either metabolic rate or insulin release.

MLK3 regulates fMLP-stimulated neutrophil motility.

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.

Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3.

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.

Thienopyrimidinone bis-aminopyrrolidine ureas as potent melanin-concentrating hormone receptor-1 (MCH-R1) antagonists.

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.

Synthesis and structure-activity relationships of spirohydantoin-derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1).

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.

Substituted chromones and quinolones as potent melanin-concentrating hormone receptor 1 antagonists.

A series of substituted chromones were designed, synthesized, and evaluated for their ability to bind melanin-concentrating hormone receptor 1. Compounds with subnanomolar binding affinity and 66% oral bioavailability in rats were discovered.

Synthesis and structure-activity relationships of retro bis-aminopyrrolidine urea (rAPU) derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1). Part 1.

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.

Synthesis and structure-activity relationships of retro bis-aminopyrrolidine urea (rAPU) derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1). Part 2.

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited considerable binding affinity (Ki = 1 nM) and functional activity at MCH-R1, acceptable CYP2D6 inhibition, and good rat brain exposure.

Bis(aminopyrrolidine)-derived ureas (APUs) as potent MCH1 receptor antagonists.

Ureas derived from two substituted 3-aminopyrrolidine subunits were prepared as constrained analogs of a linear lead compound and tested as antagonists of the MCH(1) receptor. The series was optimized for substitution and stereochemistry to generate a functional antagonist with a K(i) of 3.3 nM and IC(50) of 12 nM (GTPgammaS).

Synthesis and structure-activity relationships of biarylcarboxamide bis-aminopyrrolidine urea derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1).

A novel series of bis-aminopyrrolidine ureas containing either a 4-biphenylcarboxmide or 5-phenyl-2-thiophenecarboxamide group have been identified as potent and functional antagonists of the melanin-concentrating hormone receptor-1. Syntheses and SAR are described, which led to the discovery of compounds with high binding affinity (Ki = 1 nM) for the receptor. Preliminary in vitro metabolic stability data are also reported for key compounds.

1-(4-Amino-phenyl)-pyrrolidin-3-yl-amine and 6-(3-amino-pyrrolidin-1-yl)-pyridin-3-yl-amine derivatives as melanin-concentrating hormone receptor-1 antagonists.

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.

Potent and orally active non-peptide antagonists of the human melanocortin-4 receptor based on a series of trans-2-disubstituted cyclohexylpiperazines.

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of energy homeostasis. Recent studies have shown that blockade of the MC4R reverses tumor-induced weight loss in mice. Herein, we describe the synthesis and identification of potent and selective non-peptide antagonists of the human MC4R from a series of 2-ethoxycarbonylcyclohexyl-piperazines. Compound 12i was found to possess low nanomolar affinity for the MC4R, and exhibit oral bioavailability in rats. More importantly, when administered orally to mice (10 mg/kg), it led to statistically significant increases in food intake over a 24-h period.