Hidden modes of DNA binding by human nuclear receptors.

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA-protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

Target Identification Using Cell Permeable and Cleavable Chloroalkane Derivatized Small Molecules.

An important aspect for gaining functional insight into the activity of small molecules revealed through phenotypic screening is the identification of their interacting proteins. Yet, isolating and validating these interacting proteins remains difficult. Here, we present a new approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules to isolate their respective protein targets. Derivatization of small molecules with the CA moiety has been shown to not significantly impact their cell permeability or potency, allowing for phenotypic validation of the derivatized small molecule prior to capture. The retention of cell permeability also allows for treatment of live cells with the derivatized small molecule and the CA moiety enables rapid covalent capture onto HaloTag coated magnetic beads. Additionally, several options are available for the elution of interacting proteins, including chemical cleavage of the CA moiety, competitive elution using excess unmodified small molecule, or sodium dodecyl sulfate (SDS) elution. These features taken together yield a highly robust and efficient process for target identification, including capture of weak or low abundance interactors.