For xenograft experiments body weight (grams) are plotted over time (days) for different BVD-523 treatment groups in experiment using (A) A375 (n=15 for BVD-523 treatment groups, n= 10 for vehicle group), (B) Colo205 (n=12), (D) MIAPaCa2 (n=12), and (F) ZR-75.1 (n=15). Mean tumor volume {plus minus}SEM over time (days), is shown for (C) MIAPaCA2 and (E) ZR-75.1, following BVD-523 treatment.

In SW48 colorectal cells engineered with KRAS alleles, response to paclitaxel was unaltered.

Body Weight (grams) over time (days) is shown for treatment groups in xenograft derived from a vemurafenib-relapsed patient.

Supplementary materials and methods and supplementary figure legends

Using Reverse Phase Protein Array (RPPA), effects on proteins are measured in cell lines (A375, AN3Ca, Colo205, HCT116, HT29 and MIAPaCa2) following treatment with ERK1/2 inhibitors BVD-523 (BVD), Vx11e (Vx), GDC-0994 (GDC), or SCH772984 (SCH) (n=4 for each condition).

(A) FACS data for UACC-62 cell line following 200nM and 2000nM treatment with BVD-523 for 24 hours. (B) Percentage of MIAPaCa2 cells in G1, S, or G2 phase of the cell cycle following 0, 24 and 48 hours of 5μM BVD-523 treatment.

Combination interactions between BVD-523 and vemurafenib or dabrafenib were assessed in cell line (A) G361 and (B) A375 (n=3 for each). Growth inhibition and Loewe Excess is shown for 8 x 10 dose matrices. (C) BVD-523 in combination with dabrafenib markedly delays the onset of acquired resistance in A375 BRAFV600E melanoma cells.

(A) Percent change compared to DMSO treated control for following treatment with ERK inhibitors BVD-523 (BVD), Vx11 (Vx), GDC-0994 (GDC), and SCH772984 (SCH) are shown for pAkt (S473), pP70 S6K (S371 S394), pS6 Ribo Prot (S240 244) and pS6 Ribo Prot (S240 244), and (B) pBAD (S112), n=4 for each condition. (C) Western blot assays of cellular and nuclear fractions from RKO cell line following treatment with BVD-523, trametinib, SCH772984, or dabrafenib.

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.

The endosome-associated protein 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 kinase is a Rab-5 activator, we hypothesized that inhibition of this kinase held potential as an approach to treat diseases associated with BFCN loss. Herein we report that treatment with an oral small molecule p38α kinase inhibitor reversed pathological disease progression in the basal forebrain in a mouse model that develops BFCN degeneration. Further, the preclinical results were successfully translated to the clinic, with improvement of clinical outcomes associated with cholinergic function in a clinical study in dementia with Lewy bodies (DLB), a disease in which BFCN dysfunction and degeneration is the primary driver of disease expression. The findings both advances a novel approach to treating DLB and validates the translational platform that provided the mechanistic rationale for advancing that approach.

There is unmet need for effective stroke therapies. Numerous neuroprotection attempts for acute cerebral ischemia have failed and as a result there is growing interest in developing therapies to promote functional recovery through increasing synaptic plasticity. For this research study, we hypothesized that in addition to its previously reported role in mediating cell death during the acute phase, the alpha isoform of p38 mitogen-activated protein kinase, p38α, may also contribute to interleukin-1β-mediated impairment of functional recovery during the subacute phase after acute ischemic stroke. Accordingly, an oral, brain-penetrant, small molecule p38α inhibitor, neflamapimod, was evaluated as a subacute phase stroke treatment to promote functional recovery. Neflamapimod administration to rats after transient middle cerebral artery occlusion at two dose levels was initiated outside of the previously characterized therapeutic window for neuroprotection of less than 24 hours for p38α inhibitors. Six-week administration of neflamapimod, starting at 48 hours after reperfusion, significantly improved behavioral outcomes assessed by the modified neurological severity score at Week 4 and at Week 6 post stroke in a dose-dependent manner. Neflamapimod demonstrated beneficial effects on additional measures of sensory and motor function. It also resulted in a dose-related increase in brain-derived neurotrophic factor (BDNF) protein levels, a previously reported potential marker of synaptic plasticity that was measured in brain homogenates at sacrifice. Taken together with literature evidence on the role of p38α-dependent suppression by interleukin-1β of BDNF-mediated synaptic plasticity and BDNF production, our findings support a mechanistic model in which inhibition of p38α promotes functional recovery after ischemic stroke by blocking the deleterious effects of interleukin-1β on synaptic plasticity. The dose-related in vivo efficacy of neflamapimod offers the possibility of having a therapy for stroke that could be initiated outside the short time window for neuroprotection and for improving recovery after a completed stroke.

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.

P38α MAP kinase is implicated in the pathogenesis of synaptic dysfunction in Alzheimer's disease (AD) (Colié, 2017), partially due to increased BACE1 expression resulting from p38α-mediated impairment of autophagy-lysosomal protein degradation (Schnöder, 2016). Neflamapimod, an oral p38α inhibitor, reverses spatial learning deficits in aged rats (Alam, 2015) and preliminary clinical evidence indicates it may improve episodic memory in AD (Scheltens, 2018). Neflamapimod is being evaluated in a phase 2b clinical study in Early AD. We previously showed neflamapimod reverses APP-induced endocytic dysfunction in Down Syndrome (DS) human fibroblasts (AAIC, 2017). Herein, we report on in-vivo effects of neflamapimod in Ts2 mice that model DS and develop typical AD pathology, including endocytic abnormalities and basal forebrain cholinergic degeneration (Jiang, 2016). Wild-type (WT) or Ts2 mice treated for 28 days, twice-daily, with vehicle (1%PluronicF108) or 3mg/kg neflamapimod in vehicle (n=8-10 per group; 1:1 female/male). Treatment initiated at 4.7−6.4 months of age, when endosomal pathology is evident and cholinergic neuronal loss is developing in Ts2 mice. Cortical Rab5+ endosomal number and size, and medial septal nucleus (MSN) choline-acetyltransferase (ChAT)+ neurons quantitated per Jiang, 2016 (Neurobiol Aging39:90-98). Consistent with the literature, numbers of medium (0.51−1.4 μm2)and large (>1.4 μm2) Rab5+ early-endosomes were increased and ChAT+ neurons in MSN were decreased in vehicle-treated Ts2 mice compared to vehicle-treated WT mice (p<0.001 for all comparisons). These Ts2 phenotypes were reversed by neflamapimod treatment. The number of medium (p<0.01) and large (p<0.001) Rab5+ early-endosomes declined and ChAT+ neurons in MSN were increased (p<0.001; see figure) in neflamapimod-treated Ts2 mice compared to vehicle-treated Ts2 mice. For both large Rab5+ endosomes and ChAT+ neurons, the quantitative measures in neflamapimod-treated Ts2 mice were similar to those seen in vehicle-treated WT mice, while neflamapimod treatment had no effect on WT mice.

We recently described a p38 MAP kinase-dependent synaptotoxic signaling pathway that is activated by prions (Fang et al. 2018, PLoS Pathog. 14:e1007283). Although there is evidence that p38 MAPK also plays a role in the toxicity of Aβ oligomers (Birnbaum et al, 2015, Cell Death Dis, 18;6:e1791), we reported previously that a non-selective chemical inhibitor of all four p38 isoforms (SB239063) did not prevent dendritic spine retraction caused by synthetic ADDLs (Fang et al. 2018). To further explore the relationship between p38 activation and ADDL-induced synaptotoxicity, we evaluated the effect of neflamapimod (VX-745), a selective inhibitor of the α isoform of p38 MAPK (p38α), on ADDL-induced spine degeneration in cultured hippocampal neurons. Primary mouse hippocampal neurons were treated with 500 nM ADDLs and 0, 10, 50 or 100 nM concentrations of neflamapimod for 24 hrs, and were then fixed, and spine number was quantitated after staining with Alexa 488-labeled phalloidin to visualize F-actin (which is enriched in dendritic spines). We chose neflamapimod because it is a highly selective p38α inhibitor that improved Morris water maze performance in aged rats (Alam 2015, J. Alzheimers Dis. 48:219-227); and it demonstrated potential to improve episodic memory function in early AD patients (Scheltens et al. 2018 Ann. Clin. Transl. Neurol. 5:464-473). As a positive control, we conducted a dose-response analysis of neflamapimod for its ability to prevent spine retraction induced by purified PrPSc, the infectious form of the prion protein. Neflamapimod reduced ADDL-induced spine retraction starting at the lowest concentration tested (10 nM) and fully blocked the effect at 50 nM (see Figure 1). Consistent with our previous report (Fang, et al. 2018), neflamapimod reduced dendritic spine retraction after exposure to PrPSc starting at 25 nM, and fully blocked spine retraction at 100 nM (EC50=30 nM).

Abstract 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.

ABSTRACT 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.

Abstract The MAPK (RAS-RAF-MEK-ERK) pathway is activated in many cancers, and the clinical efficacy of BRAF and MEK inhibitors in melanoma confirms that targeting the MAPK pathway has therapeutic potential. Unfortunately, intrinsic and acquired drug resistance limits use of MAPK-directed therapies, and resistance is often associated with activated ERK signaling. Here, we report characterization of BVD-523 (ulixertinib), a novel small-molecule ERK1/2 kinase inhibitor currently under investigation in Phase 1 clinical trials. BVD-523 potently and selectively inhibits ERK1 and ERK2 kinases in a reversible, ATP-competitive fashion. Consistent with its mechanism of action, BVD-523 inhibits signal transduction, cell proliferation, and cell survival, most potently in cell lines bearing mutations that activate MAPK pathway signaling. Similarly, single-agent BVD-523 inhibits tumor growth in vivo in BRAF-mutant melanoma and colorectal xenografts as well as in KRAS-mutant colorectal and pancreatic models. Combination treatment with BVD-523 and dabrafenib inhibits tumor growth in a BRAF-mutant melanoma model. Importantly, BVD-523 is effective in several models that show intrinsic or acquired resistance to other MAPK pathway inhibitors. BVD-523 inhibits with equivalent potency the growth of parental cells or those cultured for resistance to dabrafenib, trametinib, or the combination of both drugs. Additionally, BVD-523 inhibits growth in wild-type cells and a RAF/MEK cross-resistant cell line bearing a MEK1 Q56P mutation with similar potency. Lastly, single-agent BVD-523 inhibits the growth of a patient-derived tumor xenograft harboring cross-resistance to dabrafenib, trametinib, and the combination treatment following clinical progression on a MEK inhibitor. Phase 1 trials of BVD-523 are currently recruiting patients with advanced solid tumors (NCT0178429) or hematologic malignancies (NCT02296242). Eligibility criteria include diagnosis according to certain genetic features, and treatment in backgrounds including progression following prior MAPK targeted therapy. The primary objective of these studies is to identify the recommended Phase 2 dose(s) for single-agent BVD-523 treatment. Additional objectives include pharmacokinetic and pharmacodynamic assessments, and preliminary measures of efficacy. The solid tumor protocol has met its study objectives in Part 1 (defining the safety profile and maximum tolerated dose), and will be reported separately; findings appear consistent with the activity profile defined in preclinical studies. In total, preclinical and clinical studies will help elucidate how BVD-523 (ulixertinib) may be used as a novel agent in MAPK-directed therapeutic strategies, including for patients that have failed treatment due to intrinsic or acquired resistance and active signaling through ERK. Citation Format: Ursula Germann, Brinley Furey, Jeff Roix, William Markland, Russell Hoover, Alex Aronov, Michael Hale, Guanjing Chen, Gabriel Martinez-Botella, Rossitza Alargova, Bin Fan, David Sorrell, Kay Meshaw, Paul Shapiro, Michael J. Wick, Cyril Benes, Mathew Garnett, Gary DeCrescenzo, Mark Namchuk, Saurabh Saha, Dean J. Welsch. The selective ERK inhibitor BVD-523 is active in models of MAPK pathway-dependent cancers, including those with intrinsic and acquired drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2015-4693