Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors.

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

Development of Macrocycle Kinase Inhibitors for ALK2 Using Fibrodysplasia Ossificans Progressiva-Derived Endothelial Cells.

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to activins, thereby turning them into bone-inducing agents. To date, there is no cure for FOP. The further development of FOP patient-derived models may contribute to the discovery of novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO. We have characterized peripheral blood-derived endothelial colony-forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo endothelial-to-mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

In vivo inhibition of RIPK2 kinase alleviates inflammatory disease.

The RIPK2 kinase transduces signaling downstream of the intracellular peptidoglycan sensors NOD1 and NOD2 to promote a productive inflammatory response. However, excessive NOD2 signaling has been associated with numerous diseases, including inflammatory bowel disease (IBD), sarcoidosis and inflammatory arthritis, making pharmacologic inhibition of RIPK2 an appealing strategy. In this work, we report the generation, identification, and evaluation of novel RIPK2 specific inhibitors. These compounds potently inhibit the RIPK2 tyrosine kinase activity in in vitro biochemical assays and cellular assays, as well as effectively reduce RIPK2-mediated effects in an in vivo peritonitis model. In conjunction with the development of these inhibitors, we have also defined a panel of genes whose expression is regulated by RIPK2 kinase activity. Such RIPK2 activation markers may serve as a useful tool for predicting settings likely to benefit from RIPK2 inhibition. Using these markers and the FDA-approved RIPK2 inhibitor Gefitinib, we show that pharmacologic RIPK2 inhibition drastically improves disease in a spontaneous model of Crohn Disease-like ileitis. Furthermore, using novel RIPK2-specific inhibitors, we show that cellular recruitment is inhibited in an in vivo peritonitis model. Altogether, the data presented in this work provides a strong rationale for further development and optimization of RIPK2-targeted pharmaceuticals and diagnostics.

REALISIS: a medicinal chemistry-oriented reagent selection, library design, and profiling platform.

REALISIS is a software system for reagent selection, library design, and profiling, developed to fit the workflow of bench chemists and medicinal chemists. Designed to be portable, the software offers a comprehensive graphical user interface and rapid, integrated functionalities required for reagent retrieval and filtering, product enumeration, and library profiling. REALISIS is component-based, consisting of four main modules: reagent searching; reagent filtering; library enumeration; and library profiling. Each module allows the chemist to access specific functionalities and diverse filtering and profiling mechanisms. By implementing the entire process of reagent selection, library design, and profiling and by integrating all the necessary functionalities for this process, REALISIS cuts the time required to design combinatorial and noncombinatorial libraries from several days to a few hours.