Discovery of Conformationally Constrained ALK2 Inhibitors.

Despite decades of research on new diffuse intrinsic pontine glioma (DIPG) treatments, little or no progress has been made on improving patient outcomes. In this work, we explored novel scaffold modifications of M4K2009, a 3,5-diphenylpyridine ALK2 inhibitor previously reported by our group. Here we disclose the design, synthesis, and evaluation of a first-in-class set of 5- to 7-membered ether-linked and 7-membered amine-linked constrained inhibitors of ALK2. This rigidification strategy led us to the discovery of the ether-linked inhibitors M4K2308 and M4K2281 and the amine-linked inhibitors M4K2304 and M4K2306, each with superior potency against ALK2. Notably, M4K2304 and M4K2306 exhibit exceptional selectivity for ALK2 over ALK5, surpassing the reference compound. Preliminary studies on their in vivo pharmacokinetics, including blood-brain barrier penetration, revealed that these constrained scaffolds have favorable exposure and do open a novel chemical space for further optimization and future evaluation in orthotopic models of DIPG.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo.

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood-brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

Leveraging an Open Science Drug Discovery Model to Develop CNS-Penetrant ALK2 Inhibitors for the Treatment of Diffuse Intrinsic Pontine Glioma.

There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.

Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma.

Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009, an analogue of the previously reported ALK2 inhibitor LDN-214117. Although highly selective for ALK2 over the TGF-ßR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives (26a-c), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5.

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases, and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases, and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homologue 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.

Fostering open collaboration in drug development for paediatric brain tumours.

Brain tumours have become the leading cause of child mortality from cancer. Indeed, aggressive brainstem tumours, such as diffuse intrinsic pontine glioma (DIPG), are nearly uniformly fatal. These tumours display a unique set of driver mutations that distinguish them from adult gliomas and define new opportunity for the development of precision medicines. The specific association of ACVR1 mutations with DIPG tumours suggests a direct link to neurodevelopment and highlights the encoded bone morphogenetic protein receptor kinase ALK2 as a promising drug target. Beneficial effects of ALK2 inhibition have now been observed in two different in vivo models of DIPG. Nonetheless, such tumours present a huge challenge for traditional economic models of drug development due to their small market size, high failure rate, tumour location and paediatric population. Moreover, a toolkit of different investigational drugs may be needed to fully address the heterogeneity of these tumours in clinical trials. One new business model is suggested by M4K Pharma, a recent virtual start up that aims to align diffuse academic and industry research into a collaborative open science drug discovery programme. Fostering scientific collaboration may offer hope in rare conditions of dire unmet clinical need and provide an alternative route to affordable medicines.

The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.