Understanding the molecular basis of VEGFR1 autoinhibition and targeting downstream signaling molecule

Chakraborty, Manas Pratim (2023) Understanding the molecular basis of VEGFR1 autoinhibition and targeting downstream signaling molecule. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Manas Pratim Chakraborty (17RS008)) 17RS008.pdf - Submitted Version Restricted to Repository staff only Download (13MB)

Abstract

Ligand-independent activation of VEGFR is a hallmark in diabetes and several cancers. Like most RTKs, the VEGFR2, the primary VEGF receptor, is activated spontaneously at higher receptor concentrations. An exception is VEGFR1, which remains constitutively inactive in the basal state. Ligand stimulation transiently phosphorylates VEGFR1 and induces weak kinase activation in endothelial cells. Recent studies, however, suggest that VEGFR1 signaling is indispensable in regulating many physiological or pathological events, which is puzzling. Why VEGFR1 is differentially regulated is an open question. Here we elucidate a mechanism of JM inhibition that prevents ligand-independent VEGFR1 activation, possibly by shifting the equilibrium to the inactive state, rendering it an inefficient kinase. Our data suggest that a combination of tyrosine phosphatase activity and JM inhibition suppress the basal phosphorylation of VEGFR1. In diabetic patients, ROS generation due to hyperglycemia activates Src kinase, which inturn activates VEGFR2 in the absence of a ligand. Increasing evidence showed that Src kinase can also act as a modulator of VEGFR signaling. Apart from that, Src family kinases act as a key player in cell signaling, which makes them an attractive therapeutic target. There are still very few FDA-approved drugs for SFKs. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). Notably, the compound preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Our attempt to inhibit three other kinases (DYRK2, Src and Abl) with the compound showed no inhibitory effect. We demonstrate that the cytotoxicity is mediated via inhibition of the SFK signalling pathway in triple-negative breast cancer and murine macrophage cells. In summary, this thesis addresses the long-standing question of why VEGFR1 is differentially regulated than VEGFR2 and proposes a way to regulate VEGFR-mediated signaling by targeting downstream Src family kinases.

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