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Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the D4Z4 locus leading to aberrant double homeobox 4 (DUX4) expression in skeletal muscle. Activation of this early embryonic transcription factor results in the expression of its target genes causing muscle fiber death. Although progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent, and specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity, and cell death across patient-derived FSHD1 and FSHD2 lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective therapeutics to treat FSHD at its root cause. SIGNIFICANCE STATEMENT: Using patient-derived facioscapulohumeral muscular dystrophy (FSHD) myotubes, we characterize the pharmacological relationships between p38α/β inhibition, double homeobox 4 (DUX4) expression, its downstream transcriptional program, and muscle cell death. p38α/β inhibition results in potent and specific DUX4 downregulation across multiple genotypes without significant effects in the process of myogenesis in vitro. These findings highlight the potential of p38α/β inhibitors for the treatment of FSHD, a condition that today has no approved therapies.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the

FSHD is caused by the loss of repression at the D4Z4 locus leading to DUX4 expression in skeletal muscle, activation of its early embryonic transcriptional program and muscle fiber death. While progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent and specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity and cell death across FSHD1 and FSHD2 patient-derived lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective therapeutics to treat FSHD at its root cause.VISUAL ABSTRACT

Red blood cell disorders like Sickle Cell Disease (SCD) and β-thalassemias are caused by mutations within the gene for the hemoglobin β (HBβ) subunit. A fetal ortholog of HBβ, hemoglobin γ (HBγ) can prevent or reduce disease-related pathophysiology in these disorders by forming nonpathogenic complexes with the required hemoglobin α-subunit. Globin expression is developmentally regulated, with a reduction in production of the fetal ortholog (γ)occurring shortly after birth and a concomitant increase in the levels of the adult ortholog (β). It has been postulated that maintaining expression of the anti-sickling γ ortholog may be of therapeutic benefit in children and adults with SCD. Indeed, individuals with the SCD mutation who also have genetic variants that maintain HBγ expression at clinically meaningful levels do not present with SCD-related symptoms. Parallel target identification efforts using CRISPR and the Fulcrum proprietary, annotated chemical probe screening set in HUDEP2 cells identified a protein complex as a key regulator of HbF expression. Structure-guided medicinal chemistry optimization led to the design of FTX-6058, a novel, potent and selective small molecule with desirable DMPK properties suitable for clinical testing. FTX-6058 treatment of differentiated primary CD34+ cells from multiple healthy donors demonstrated target engagement and potent upregulation of HBG1/2 mRNA and HbF protein. Across multiple healthy and SCD donors, FTX-6058 treatment resulted in a clinically desirable globin profile (e.g., up to 30% absolute HbF) accompanied by pancellular HbF expression, resembling the phenotype of SCD mutation carriers with hereditary persistence of fetal hemoglobin. FTX-6058 demonstrated a superior pharmacological profile relative to hydroxyurea and other small molecule compounds whose putative mechanism of action is to induce HbF. FTX-6058 treatment resulted in robust target engagement and subsequent elevation of the endogenous mouse Hbb-bh1 mRNA in wildtype CD-1 mice and, importantly, also elevation of the human HBG1 mRNA and HbF protein in the Townes SCD mouse model. Preclinical studies using a variety of in vitro and in vivo models have demonstrated the potential of FTX-6058 as a novel HbF-inducing small molecule that could be beneficial to patients with SCD and β-thalassemias. FTX-6058 was shown to be potent and selective in vitro, was well tolerated and elicited a desirable exposure-response relationship in multiple preclinical rodent models with once-a-day oral dosing and at plasma concentrations predicted to be achievable in patients. IND enabling studies for FTX-6058 have been completed. Disclosures Rahl: Fulcrum Therapeutics: Ended employment in the past 24 months. Efremov:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Stuart:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Xie:Fulcrum Therapeutics: Current Employment. Roth:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Barnes:Fulcrum Therapeutics: Ended employment in the past 24 months. Appiah:Fulcrum Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Peters:Fulcrum Therapeutics: Current Employment. Li:Fulcrum Therapeutics: Ended employment in the past 24 months. Kazmirski:Fulcrum Therapeutics: Ended employment in the past 24 months. Bruno:Fulcrum Therapeutics: Current Employment. Stickland:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Ronco:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Cadavid:Fulcrum Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thompson:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Wallace:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company. Moxham:Fulcrum Therepeutics: Current Employment, Current equity holder in publicly-traded company.

By targeting an extended region of the conventional 'DFG-out' pocket of p38alpha, while minimizing interactions with the specificity pocket and eliminating interactions with the adenine binding site, we are able to design and synthesize a number of pyrazole-urea based DFG-out p38alpha inhibitors with good potencies, and excellent selectivity.

Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.

An approach and preliminary results for utilizing legacy MEK inhibitors as templates for a reiterative structural based design and synthesis of novel, type III NCKIs (non-classical kinase inhibitors) is described. Evidence is provided that the MEK-pocket or pockets closely related to it may exist in kinases other than MEK.