Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Summary

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Abstract

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

Introduction

Bioactive small molecules fundamentally work by interacting with and altering the function of one or more "target" molecules, most commonly proteins, in the cell. In drug discovery, when an active compound is discovered through phenotypic screening, identification of the molecular target(s) of that compound is crucial, not just for understanding the mechanism of action and potential side-effects of the compound, but also for potentially discovering new biology underlying the disease model and paving the way for development of new mechanistic classes of therapeutics¹. Although target identification is not required for a drug to be used therapeuticall....

Protocol

NOTE: This protocol was adapted from MacKinnon and Taunton¹⁰ for use in live cells.

1. Preparation of Cultured Cells

1. Prepare sterile 6-cm cell culture dishes for the number of samples desired (see below).
   NOTE: One dish of cells is used per treatment condition, but if more protein is required 2 or 3 dishes can be prepared per condition and combined after UV irradiation.
   1. Prepare at least 3 dishes of cells; Negative control with DMSO only (D), Probe treatment only (P), and probe + competitor (C).
   2. Optionally, prepare a 4^th dish as a no UV control, to be treated with probe but not ....

Results

The results shown here were obtained with a photo-affinity probe of the antifungal drug itraconazole, the use of which has been previously published¹⁶. These results demonstrate the use of the live-cell photoaffinity labeling technique to successfully identify a major itraconazole-binding protein as the 35 kDa membrane protein Voltage-Dependent Anion Channel 1 (VDAC1).

The above protocol was performed in HEK293T cells.......

Discussion

Different approaches to identifying the targets of small molecules can be broadly grouped into two categories: top-down, where the cellular phenotype of the drug is used to narrow down its potential targets based on their known functions, or bottom-up, where the target is identified directly by chemical or genetic means³. Top-down or phenotypic studies can identify certain cellular processes affected by the drug (e.g., transcription/translation/DNA synthesis, cell cycle block, signaling pathway activa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris(2-carboxyethyl)phosphine (TCEP)	Life Technologies	20490	Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)	AnaSpec	63360-50	Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20 °C.
Copper Sulfate (CuSO4•5H2O)	LabChem, Inc.	LC13440-1	Prepare 50 mM stock in water, store at room temperature.
Biotin-azide	Click Chemistry Tools	AZ104-100	Prepare 10 mM stock in DMSO, store at -20 °C.
Alexa Fluor 647-azide	Life Technologies	A10277	Prepare 1 mM stock in DMSO, store at -20 °C.
365 nm UV lamp	Spectroline	FC100	UV-blocking glasses should be worn while operating.
Protease inhibitor tablets, EDTA-free	Roche Life Science	11873580001	Prepare 50x solution in water and store at -20 °C.
Sonicator	Branson	Sonifier 250	Set to output 1, duty 30%.
Fluorescent gel scanner	GE Healthcare Life Sciences	FLA 9500	Use red laser to detect Alexa-fluor 647.
Detergent-compatible Dc protein assay kit	Bio-Rad	5000112
High Capacity Streptavidin Agarose beads	Life Technologies	20359
Dulbecco's Modified Eagles Medium, low glucose	ThermoFisher Scientific	11885092
Fetal Bovine Serum, qualified	ThermoFisher Scientific	26140079
Penicillin/Streptomycin solution	ThermoFisher Scientific	15140122
SDS Sample Buffer (2x)	ThermoFisher Scientific	LC2676