With the expeditious development ofstructural biology techniques represented by single-particle cryo-electronmicroscopy, the 3D structures of an increasing number of important proteinshave been revealed. Taking ion channels as an example, at present, thestructure of at least one member of virtually every major ion channel familyhas been successfully resolved. It is thus one major research direction topromote the development of drugs targeting important proteins by utilizingnumerous high-resolution 3D structures.

In collaboration with researchers at UCDavis and Qingdao University, the research team led by Prof. YANG Fan from theDepartment of Biophysics at the Zhejiang University School of Medicine designedpeptidic positive allosteric modulators (PAMs) based on the high-resolutionstructure of the transient receptor potential vanilloid 1 (TRPV1) ion channel.They exhibit long-lasting in vivo analgesic effects in rats. The researchfindings are published in an article entitled “De novo design of peptidicpositive allosteric modulators targeting TRPV1 with analgesic effects” in thejournal Advanced Science.

Severe pain not only drastically lowers thequality of life in patients but also incurs enormous socioeconomic costs.Therefore, there is an urgent need to study the molecular mechanisms of painand develop safe and effective analgesic drugs. As a prototypical sensorinvolved in pain sensation, the TRPV1 ion channel is an effective target foranalgesic drugs by controlling the flow of ions across cell membranes and convertingpain and stimulus signals into neural electrical signals.

Computational design of protein binders tothe ARD of TRPV1

The TRPV1 ion channel is closely bound upwith nociception. Interestingly, both the agonists and antagonists of TRPV1 caneffectively alleviate pain. In recent years, TRPV1 has been regarded as one ofthe most promising targets for the development of analgesic drugs. However,since the TRPV1 channel is a polymodal receptor activated by heat and involvedin body temperature regulation, blockade of this channel may incur substantialhyperthermia in clinical trials, thereby impeding further drug development.Side effects should thus be avoided in the development of analgesic drugstargeting the TRPV1 channel. On the strength of previous studies, researchersdesigned several positive allosteric modulators to increase the flow of calciumthrough the TRPV1 channel and reversibly inactivate the TRPV1-expressing nerveterminus, thus exerting analgesic effects.

TRPV1 is a calcium-dependent desensitizedchannel, and desensitization is closely related to the ankyrin repeat-likedomain (ARD) of TRPV1. Researchers first improved the optimized hotspot centricapproach (OHCA) protein design strategy to increase the success rate ofobtaining robust designed binders. They then applied the improved Rosettaprotein design approach to precisely target the ARD of TRPV1 to achievepositive allosteric modulation. Moreover, both the crystal structure of the ARDand the cryo-electron microscopy structures of TRPV1 were determined. With acombination of OHCA design, fluorescence resonance energy transfer (FRET)imaging, protein chemistry, surface plasmon resonance (SPR), patch-clamprecordings, and animal behavioral tests, they revealed that the designed PAMs couldpositively modulate the TRPV1 channel. Furthermore, they observed noticeableanalgesic effects in rats.

The advent of high-resolution 3D structuresopens the door to the rational designing of regulatory molecules based on thestructure of target proteins, which is more time-saving, less costly, andsignificantly more successful compared with the conventional large-scalescreening. “In this study, we made an attempt to design regulatory moleculestargeting specific structural domains, which will provide theoretical supportand new ideas for the development of analgesic drugs targeting the TRPV1channel,” said Yang. “The OHCA computational design strategy can also be widelyapplied to the development of regulatory molecules for various protein drug targetswith known 3D structures.”