Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule

Yasser Tabana; Chih-Hsuan Lin; Dinesh Babu; Ramanaguru Siva-Piragasam; Ashley A Ponich; Tae Chul Moon; Arno G Siraki; Shokrollah Elahi; Richard Fahlman; Frederick G West; Khaled Barakat

doi:10.1016/j.heliyon.2023.e21408

Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule

Heliyon. 2023 Oct 29;9(11):e21408. doi: 10.1016/j.heliyon.2023.e21408. eCollection 2023 Nov.

Authors

Yasser Tabana^{1

2}, Chih-Hsuan Lin³, Dinesh Babu⁴, Ramanaguru Siva-Piragasam², Ashley A Ponich³, Tae Chul Moon¹, Arno G Siraki¹, Shokrollah Elahi^{5

6

7}, Richard Fahlman², Frederick G West^{3

6}, Khaled Barakat^{1

7}

Affiliations

¹ Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, T6G 2H1, Canada.
² Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
³ Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada.
⁴ Li Ka Shing Applied Virology Institute, University of Alberta, Edmonton, AB, T6G 2E1, Canada.
⁵ School of Dentistry, University of Alberta, Edmonton, AB, T6G 1C9, Canada.
⁶ Department of Oncology, University of Alberta, Edmonton, AB, T6G 1Z2, Canada.
⁷ Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, T6G 2E1, Canada.

Abstract

In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.