BPS2026 – De novo design of functional mini-binders for TRPV1 modulation

The capsaicin receptor TRPV1 is a nociceptive ion channel and an attractive target for developing non-addictive analgesics. Although small-molecule antagonists that block the capsaicin-binding pocket can effectively regulate channel activity, they often induce acute hyperthermia in animal models, limiting their clinical potential. In our previous work, we de novo designed protein mini-binders that bind intracellular ankyrin repeat domain (ARD) hotspots and regulate TRPV1 activity both in vitro and in vivo without triggering hyperthermia. However, this strategy relies on predefined binding hotspots, which restricts its generalizability. In this study, we employed RosettaFold diffusion to generate new mini-binders that modulate TRPV1 activity without requiring hotspot engagement, and functional experiments confirmed their efficacy. To further broaden the modulation landscape, we also developed mini-binders that specifically target the extracellular pore domain of TRPV1. This region plays a central role in ion permeation and gating, yet has rarely been explored as a mini-binder-binding site. Notably, this extracellular approach provides intrinsic advantages for therapeutic delivery, enabling direct channel modulation without the need for intracellular access. These pore-binding mini-binders exhibit strong and promising modulatory effects in cellular tests. Overall, our study leverages advanced design approaches to establish a domain-targeting mini-binder design framework, enabling multi-domain control of TRPV1 and opening new avenues for the development of safe and effective analgesics.