Tracking and Inhibiting Atypical Vascular Inflammation

Mitogen‐activated protein kinase (MAPK) p38 drives the onset and progression of many clinically challenging diseases. However, three decades of research have failed to yield clinically viable, selective MAPK p38 therapeutics. The extensive and ongoing studies have principally focused on therapeutics targeting the ATP‐pocket or active site of p38. These have failed in part due to the ubiquitous expression and dichotomous role of p38, leading to blockade of both physiological and pathological signaling pathways. A promising alternative strategy is to target either the substrates of p38 or the activators that drive p38‐induced pathological signaling. Our prior studies identified an atypical mechanism for G protein‐coupled receptor mediated activation of pathological p38 signaling during vascular inflammation. Several critical problems remain: firstly, how does atypical signaling drive inflammation, and secondly how can we stop pathological p38 activity? To further define how atypical p38 signaling induces inflammation we developed a fluorescent resonance energy transfer (FRET) biosensor platform to map atypical p38 signaling. This system revealed that GPCR induced p38 signaling is initiated via an endosomal axis. Blockade of receptor internalization shifts the spatial bias to that seen for MKK3/6‐dependent signaling with enhanced nuclear p38 activity. We predict that this endosomal axis drives pathological atypical p38 activity and the progression of vasculopathic disease progression. Critically, atypical p38 activity is initiated by a direct interaction between the adaptor protein TAB1 and two binding sites on p38. Blockade of the TAB1‐p38 interaction through mutation of select residues on TAB1 can disrupt the TAB1‐p38 interaction and prevent atypical p38 signaling. Modeling studies show that small molecule inhibitors that bind to the TAB1 binding motif on p38 can block this interaction. As an alternative strategy we focused on the TAB1 peptides that directly bind to p38. Using a phage‐display library of humanized nanobodies and a yeast two‐hybrid screen we have developed an array of 40 TAB1 blocking intrabodies (IntraT‐ab’s) as the first generation of selective atypical p38 inhibitors. We show that intraT‐ab’s bind TAB1, selectively blocking GPCR induced p38 (endosomal) signaling while leaving classical MKK3/6 dependent signaling intact. IntraT‐ab’s and our FRET platform provide the first opportunity to examine in detail the specific downstream mechanisms, targets and physiological consequences of atypical p38 signaling. Intracellular delivery/expression of intraT‐ab’s could also provide a therapeutic strategy for reducing p38 signaling in multiple diseases, including acute lung injury, retinopathies and neuroinflammation.