Structure-guided approach to modulate small molecule binding to a promiscuous ligand-activated protein

Abstract

Ligand-binding promiscuity in detoxification systems protects the body from toxicological harm but is a roadblock to drug development due to the difficulty in optimizing small molecules to both retain target potency and avoid metabolic events. Immense effort is invested in evaluating metabolism of molecules to develop safer, more effective treatments, but engineering specificity into or out of promiscuous proteins and their ligands is a challenging task. To better understand the promiscuous nature of detoxification networks, we have used X-ray crystallography to characterize a structural feature of pregnane X receptor (PXR), a nuclear receptor that is activated by diverse molecules (with different structures and sizes) to up-regulate transcription of drug metabolism genes. We found that large ligands expand PXR’s ligand-binding pocket, and the ligand-induced expansion occurs through a specific unfavorable compound-protein clash that likely contributes to reduced binding affinity. Removing the clash by compound modification resulted in more favorable binding modes with significantly enhanced binding affinity. We then engineered the unfavorable ligand-protein clash into a potent, small PXR ligand, resulting in marked reduction in PXR binding and activation. Structural analysis showed that PXR is remodeled, and the modified ligands reposition in the binding pocket to avoid clashes, but the conformational changes result in less favorable binding modes. Thus, ligand-induced binding pocket expansion increases ligand-binding potential of PXR but is an unfavorable event; therefore, drug candidates can be engineered to expand PXR’s ligand-binding pocket and reduce their safety liability due to PXR binding.

Chemical manipulation of an activation/inhibition switch in the nuclear receptor PXR

“…1a ) as a more potent PXR antagonist than SPA70 25 . Although there are >40 reported structures of PXR LBD bound to agonists 21 , including the SPA70 analog SJB7 (Fig. 1a ) 17 , there is currently no structure of PXR with a bound antagonist, and it is unclear how binding of chemically similar compounds results in opposite transcriptional activities.…”

“…Binding of SJPYT-310 to both the π-trap and the leucine cage appears to stabilize alpha helix 2 (α2), which is disordered in PXR LBD structures with SJB7 and certain other agonists (Supplementary Fig. 1e ) 17 , 21 , 27 . The increased region B interactions with both the π-trap and leucine cage likely account for the enhanced binding affinity of the amide series compared to the sulfonyl series 24 , 25 but may not be indicative of agonist/antagonist property.…”

“…The promiscuous nature of PXR is attributed to its LBD, which is larger and more dynamic than the LBDs of other NRs 51 , 52 . It can expand its volume substantially, enabling it to accommodate large ligands or even multiple ligands simultaneously 14 , 15 , 21 , 27 . Although most PXR ligands are agonists, a few compounds have been identified that inhibit PXR’s transcriptional activation 53 .…”

“…We used a plasmid expressing PXR LBD tethered to a 33-amino acid SRC-1 peptide, as previously described 21 . A codon-optimized sequence for human PXR LBD (residues 130-434) fused to SRC-1 (residues 678-710) with SGGSGG linker was cloned into bacterial expression pET-3a vector with a His6-tag at the N-terminus (Novagen, Merck group, Darmstadt, Germany).…”

“…Because of PXR’s large, flexible ligand binding pocket that is evolutionarily tuned to activate the receptor 20 , 21 , development of selective PXR antagonists has historically been deemed a considerable challenge 22 . However, using an unbiased high-throughput screening approach, we previously identified SPA70 as a potent and selective PXR antagonist with activity in vivo 17 , 23 .…”