Hedgehog pathway activation through conformational blockade of the Patched sterol conduit

Abstract

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix", as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.Therapeutic manipulation of the Hedgehog pathway has found its clearest application in patients with malignancies whose initiation and growth depend on pathway-activating mutations in the primary cells of the tumor, such as medulloblastoma and basal cell carcinoma 1,2. Pathway blockade, imposed by systemically administered small molecules, has provided clear benefits in such patients, albeit accompanied by unwanted but largely tolerable class effects such as muscle cramps, loss of hair, and loss of taste sensation 3. In contrast to promoting tumor growth, however, pathway activity recently has been found to suppress cancer growth and progression when it occurs in stromal cells rather than primary cells 4, particularly in cancers of endodermal

Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling

“…There is not enough free cholesterol to occupy all sites in the channel of SMO, so cholesterol may transport through the channel to the CRD. SMO is off in this state 7 , 39 . When Hedgehog binds to PTCH1, internalization of PTCH1 is triggered 10 and, thus, transport activity is abolished 6 .…”