Pyrido-pyrimidinone Derivatives as RIPK2 Kinase and NOD Signaling Inhibitors



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Receptor interacting protein kinase-2 (RIPK2) is an enzyme that transduces pro-inflammatory signaling from the nucleotide binding oligomerization containing proteins (NOD1/2) during bacterial invasion in immune cells. However, NOD 1/2 mutations cause uncontrolled activation of RIPK2, resulted in excessive signaling and cytokine production. Such dysregulated signaling has been associated with inflammatory bowel (Crohn’s disease) and neuro-inflammatory diseases (Multiple Sclerosis), making RIPK2 an important target. Although, several RIPK2 kinase inhibitors have been reported in the literature, they possess limitations including off-target effects, reduced cellular potency, compromised in vivo efficacy and hERG/CYP inhibition. Thus, novel types of RIPK2 inhibitors are needed to address such problems. To this end we have identified a pyrido-pyrimidine based inhibitor (UH15) that has shown potent RIPK2 inhibition in enzymes and cells, but also showed non-selective inhibition of structurally similar activin like kinase-2 (ALK2). Nevertheless, molecular docking studies of UH15 in RIPK2 and ALK2 identified key differences that have been used to optimize the compound towards potent and selective RIPK2 inhibitors. The docking model-based optimization of UH15 identified two sets of analogs that differentiate RIPK2 inhibition in cells based upon size and position of substituents at a specific site on the inhibitor. Compounds that occupy the shallow hydrophobic pocket between the β3/β5 strands and the α-C helix in the kinase domain, showed effective RIPK2 cellular inhibition. The behavior of this subset of UH15 inhibitors correlates to the recently identified CSLP compounds that have shown RIPK2 cellular potency by interfering with RIPK2-XIAP interaction after occupying the hydrophobic pocket.
The most potent and selective RIPK2 inhibitor (UH15-15) has shown optimal absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic (PK) properties, making it a suitable probe for delineating the role of RIPK2 in disease models. Furthermore, modifications of the central pyrido-pyrimidine scaffold of UH15 series resulted in several other distinct structural classes of RIPK2 inhibitors. Overall, the identified RIPK2 inhibitors show mechanism based cellular potency, selectivity verses the structurally similar ALK2 kinase and promising pharmacokinetic properties. These compounds will allow investigation of RIPK2 function in disease models and will provide the basis for further optimization and advancement of RIPK2 inhibitors.



RIPK2, Kinase inhibitors, Drug design, NOD2 signaling