Leveraging Chemotype-Specific Resistance for Drug Target Identification and Chemical Biology

Abstract

Identifying the direct physiological targets of drugs and chemical probes remains challenging. Here we describe how resistance can be used to achieve 'gold-standard' validation of a chemical inhibitor's direct target in human cells. This involves demonstrating that a silent mutation in the target that suppresses inhibitor activity in cell-based assays can also reduce inhibitor potency in biochemical assays. Further, phenotypes due to target inhibition can be identified as those observed in the inhibitor-sensitive cells, across a range of inhibitor concentrations, but not in genetically matched cells with a silent resistance-conferring mutation in the target. We propose that chemotype-specific resistance, which is generally considered to be a limitation of molecularly targeted agents, can be leveraged to deconvolve the mechanism of action of drugs and to properly use chemical probes.

Keywords: chemical biology; chemical probes; drug resistance; target identification.

FIGURE 1

Common methods for methods for elucidation of the cellular targets of chemical inhibitors. (A) When compounds are active in genetically tractable organisms such as budding yeast, the mutations that confer resistance to the inhibitor can be analyzed. If the identified mutation is sufficient to confer chemotype-specific resistance, it can guide further studies that help establish the chemical inhibitor’s direct target. (B) A ‘handle’ can be introduced into the chemical inhibitor for ‘pull-down’ experiments. In the example shown, an alkyne is introduced into the parent compound. The modified analog should cause phenotypes that are similar to those due to treatments with the parent compound. Addition of the modified analog to cells is generally followed by the preparation of lysates. CLICK chemistry can then be used to attach a tag (e.g. biotin) for affinity-based isolation of the compound and associated proteins. Control experiments typically involve adding the unmodified parent compound to the pull-downs so that specific and non-specific interactions can be distinguished.

FIGURE 2

Schematic for DrugTargetSeqR. A genetically heterogeneous population of HCT116 cells is treated with the chemical inhibitor of interest. Clones that have reduced sensitivity to the inhibitor are isolated and expanded separately. Clones resistant to multidrug resistance (MDR) substrates are excluded from further analyses. Transcriptomes of six to eight clones and the parental cells are sequenced. Genetic differences between each clone and the parental cell population are identified. Genes that are altered (e.g. mutated) in multiple independent clones are selected. These mutations are introduced in other drug sensitive cell lines and those mutations sufficient to confer resistance are considered to be in the gene likely encoding the physiological target of the inhibitor. Biochemical assays are then used to test inhibition of the target protein’s activity or direct inhibitor binding. ‘Gold standard’ proof of target is established when the same mutation reduces the chemical inhibitor sensitivity in both cell-based and biochemical assays.

FIGURE 3

Using chemotype-specific resistance for chemical biology. (A) Dose-dependent effects of inhibitors are typically analyzed. As the inhibitor concentration increases, the more complete inhibition of the target’s activity in the cellular context is expected. (B) As inhibitor concentration increases the number of potential targets is also likely to be higher. Inhibition of different targets can make interpretation of phenotypes using chemical inhibitors difficult. (C) One solution is to examine dose-dependent responses to chemical inhibitors using two matched cell lines. One cell line is drug sensitive and the other is genetically identical but has a drug-resistance-conferring mutation in the target protein. It is critical that this mutation alone does not cause a phenotype (i.e., is a silent mutation). DrugTargetSeqR and related methods readily yield these genetically matched cell line pairs.