P38α MAPK Signaling-A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease.

Multifactorial pathologies, involving one or more aggregated protein(s) and neuroinflammation are common in major neurodegenerative diseases, such as Alzheimer's disease and dementia with Lewy bodies. This complexity of multiple pathogenic drivers is one potential explanation for the lack of success or, at best, the partial therapeutic effects, respectively, with approaches that have targeted one specific driver, e.g., amyloid-beta, in Alzheimer's disease. Since the endosome-associated protein Rab5 appears to be a convergence point for many, if not all the most prominent pathogenic drivers, it has emerged as a major therapeutic target for neurodegenerative disease. Further, since the alpha isoform of p38 mitogen-activated protein kinase (p38α) is a major regulator of Rab5 activity and its effectors, a biology that is distinct from the classical nuclear targets of p38 signaling, brain-penetrant selective p38α kinase inhibitors provide the opportunity for significant therapeutic advances in neurogenerative disease through normalizing dysregulated Rab5 activity. In this review, we provide a brief summary of the role of Rab5 in the cell and its association with neurodegenerative disease pathogenesis. We then discuss the connection between Rab5 and p38α and summarize the evidence that through modulating Rab5 activity there are therapeutic opportunities in neurodegenerative diseases for p38α kinase inhibitors.

Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors.

Through antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellent in vitro and in vivo properties have been identified. To evaluate the in vivo efficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration.

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

Flipped out: structure-guided design of selective pyrazolylpyrrole ERK inhibitors.

The Ras/Raf/MEK/ERK signal transduction is a key oncogenic pathway implicated in a variety of human cancers. We have identified a novel series of pyrazolylpyrroles as inhibitors of ERK. Aided by the discovery of two distinct binding modes for the pyrazolylpyrrole scaffold, structure-guided optimization culminated in the discovery of 6p, a potent and selective inhibitor of ERK.

Targeting the MAPK Signaling Pathway in Cancer: Promising Preclinical Activity with the Novel Selective ERK1/2 Inhibitor BVD-523 (Ulixertinib).

Aberrant activation of signaling through the RAS-RAF-MEK-ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK-targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351-63. ©2017 AACR.

Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human lymphocytes.

The enzyme inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides. Inhibition of IMPDH leads to immunosuppression by decreasing guanine nucleotides that are required for the proliferation of lymphocytes. IMPDH activity is mediated by two highly conserved isoforms, type I and type II. We have characterized the mRNA and protein expression of the two isoforms in a variety of human tissues, peripheral blood mononuclear cells (PBMCs), and selected cell lines to investigate their regulation. Type I mRNA was expressed in most tissues with high expression in PBMCs and low expression in thymus. IMPDH type II transcript was also detected in most tissues with low expression in spleen and PBMCs. In PBMCs, induction of both type I and type II mRNAs was observed within 12 hr of mitogenic stimulation. Using type-selective IMPDH antibodies, an increase in the levels of type I and type II proteins was observed after mitogenic stimulation. The effect of two IMPDH inhibitors, MPA and VX-497, was investigated on the expression of type I and type II isoforms. VX-497 is an orally bioavailable, potent and reversible inhibitor of IMPDH, with broad applicability in many viral and immune system-mediated diseases. MPA and VX-497 inhibit both isoforms of IMPDH in vitro. Prolonged treatment of lymphocytes with either VX-497 or MPA did not lead to an increase in type I or type II IMPDH protein levels. These results are discussed in the context of IMPDH being a target for immunosuppressive, anti-viral and anti-cancer therapy.

The Discovery of VX-745: A Novel and Selective p38α Kinase Inhibitor.

The synthesis of novel, selective, orally active 2,5-disubstituted 6H-pyrimido[1,6-b]pyridazin-6-one p38α inhibitors is described. Application of structural information from enzyme-ligand complexes guided the selection of screening compounds, leading to the identification of a novel class of p38α inhibitors containing a previously unreported bicyclic heterocycle core. Advancing the SAR of this series led to the eventual discovery of 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one (VX-745). VX-745 displays excellent enzyme activity and selectivity, has a favorable pharmacokinetic profile, and demonstrates good in vivo activity in models of inflammation.

Structure-guided design of potent and selective pyrimidylpyrrole inhibitors of extracellular signal-regulated kinase (ERK) using conformational control.

The Ras/Raf/MEK/ERK signal transduction, an oncogenic pathway implicated in a variety of human cancers, is a key target in anticancer drug design. A novel series of pyrimidylpyrrole ERK inhibitors has been identified. Discovery of a conformational change for lead compound 2, when bound to ERK2 relative to antitarget GSK3, enabled structure-guided selectivity optimization, which led to the discovery of 11e, a potent, selective, and orally bioavailable inhibitor of ERK.

Janus kinase 2 inhibitors. Synthesis and characterization of a novel polycyclic azaindole.

The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18.

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation.

The extracellular signal-regulated kinase (ERK) is a component of the mitogen-activated protein kinase cascade. Exon 2 of erk2 was deleted by homologous recombination and resulted in embryonic lethality at embryonic day 6.5. erk2 mutant embryos did not form mesoderm and showed increased apoptosis but comparable levels of BrdUrd incorporation, indicating a defect in differentiation. erk2 null embryonic stem (ES) cells exhibited reduced total ERK activity upon serum stimulation, augmented ERK1 phosphorylation, and decreased downstream p90Rsk phosphorylation and activity; yet ES cell proliferation was unaffected. Mutant ES cells were capable of forming mesoderm; however, treatment of mutant ES cells with the mitogen-activated protein kinase kinase inhibitor PD184352 decreased total ERK activity and expression of the mesodermal marker brachyury, suggesting that ERK1 can compensate for ERK2 in vitro. Normal embryos at embryonic day 6.5 expressed activated ERK1/2 in the extraembryonic ectoderm, whereas erk2 mutant embryos had no detectable activated ERK1/2 in this region, suggesting that activated ERK1 was not expressed, and therefore cannot compensate for loss of ERK2 in vivo. These data indicate that ERK2 plays an essential role in mesoderm differentiation during embryonic development.