In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.

Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofolinteracting proteins.

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Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofolinteracting proteins.

Aberrant post-translational modifications (PTMs) on arginine residues, such as methylation and citrullination, are closely linked to oncogenic processes. Detection of these PTMs is challenging with current technology and can be hampered by low abundance and mass differences that are difficult to resolve by mass spectrometry. To overcome these challenges, we developed methods for PTM detection using Quantum-Si's Platinum single-molecule protein sequencing platform. Proteins are sequenced on Quantum-Si's Platinum instrument using our library prep protocol. Briefly, proteins are digested into peptide fragments, conjugated to linkers, then immobilized at the bottom of nanoscale reaction chambers on a semiconductor chip, resulting in exposed N-termini for sequencing. During sequencing, the immobilized peptides are exposed to a solution of dye-labeled N-terminal amino acid (NAA) recognizers that reversibly bind to their cognate NAAs with distinct kinetic properties. Aminopeptidases sequentially remove NAAs to expose subsequent amino acids for recognition. Fluorescence lifetime, intensity, and kinetic data are collected in real time and analyzed to determine primary sequence and PTM content. The data output consists of distinct pulsing regions called recognition segments (RSs). Each RS corresponds to a period of time between aminopeptidase cleavage events, during which an NAA recognizer binds on/off to its exposed target NAA. Chemical modification to a NAA or to a nearby downstream amino acid can modulate recognizer affinity, resulting in a characteristic change in the average pulse duration (PD) during an RS. To demonstrate the detection and differentiation of arginine PTMs, we applied our platform to distinguish between asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) and between citrulline and native arginine residues. Sequencing traces revealed that arginine and ADMA bind the arginine recognizer (PS621) with similar PD, whereas SDMA exhibited no binding. These results indicate that SDMA, in contrast to ADMA, has reduced binding affinity with PS621, providing a clear kinetic difference between these isomeric arginine PTMs. Moreover, citrulline and arginine side chains also exhibited distinguishable kinetic signatures. Citrullination eliminated the N-terminal arginine recognized by PS621 and resulted in a large increase in the median PD of preceding amino acid residues. Taken together, single-molecule protein sequencing offers an alternative approach to detection of arginine PTMs that is not based on m/z, but rather on the kinetic signature of binding between recognizers and N-terminal amino acids. The ability to directly detect arginine PTMs offers potential for biomedical research. We envisage applications using Quantum-Si's platform to key areas of cancer research including biomarker development and drug discovery. Citation Format: Kenneth Skinner, David Kamber, Brian Reed. Detection of arginine posttranslational modifications by single-molecule protein sequencing on the Quantum-Si platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5301.