RESUMO
Photoaffinity probes are routinely utilized to identify proteins that interact with small molecules. However, despite this common usage, resolving the specific sites of these interactions remains a challenge. Here we developed a chemoproteomic workflow to determine precise protein binding sites of photoaffinity probes in cells. Deconvolution of features unique to probe-modified peptides, such as their tendency to produce chimeric spectra, facilitated the development of predictive models to confidently determine labeled sites. This yielded an expansive map of small-molecule binding sites on endogenous proteins and enabled the integration with multiplexed quantitation, increasing the throughput and dimensionality of experiments. Finally, using structural information, we characterized diverse binding sites across the proteome, providing direct evidence of their tractability to small molecules. Together, our findings reveal new knowledge for the analysis of photoaffinity probes and provide a robust method for high-resolution mapping of reversible small-molecule interactions en masse in native systems.
Assuntos
Marcadores de Fotoafinidade , Bibliotecas de Moléculas Pequenas , Sítios de Ligação , Humanos , Marcadores de Fotoafinidade/química , Bibliotecas de Moléculas Pequenas/química , Ligação Proteica , Proteômica/métodos , Proteoma/metabolismo , Proteínas/química , Proteínas/metabolismo , Peptídeos/química , Peptídeos/metabolismoRESUMO
SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.
Assuntos
Proteômica , Animais , Humanos , Camundongos , Proteômica/métodos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Proteínas de Transporte de Nucleosídeos/metabolismo , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Feminino , Proteínas do Tecido Nervoso , Proteínas de Membrana TransportadorasRESUMO
Chemical probes are invaluable tools to investigate biological processes and can serve as lead molecules for the development of new therapies. However, despite their utility, only a fraction of human proteins have selective chemical probes, and more generally, our knowledge of the "chemically-tractable" proteome is limited, leaving many potential therapeutic targets unexploited. To help address these challenges, powerful chemical proteomic approaches have recently been developed to globally survey the ability of proteins to bind small molecules (i. e., ligandability) directly in native systems. In this review, we discuss the utility of such approaches, with a focus on the integration of chemoproteomic methods with fragment-based ligand discovery (FBLD), to facilitate the broad mapping of the ligandable proteome while also providing starting points for progression into lead chemical probes.