# The Nature of Nanodisc Lipids Influences Fragment‐Based Drug Discovery Results

## TL;DR

This study shows that the type of lipid in nanodiscs affects how well drug fragments bind to membrane proteins, influencing drug discovery outcomes.

## Contribution

The study reveals that nanodisc lipid composition critically affects fragment-based drug discovery results for membrane proteins.

## Key findings

- Fragment binding to nanodiscs correlates with analyte hydrophobicity and lipid composition.
- POPC nanodiscs showed higher fragment binding than DMPC and DPhPC, reducing CYP3A4 hit rates.
- Some fragments only bound to CYP3A4 in the presence of the nanodisc membrane.

## Abstract

Membrane proteins (MPs) are important yet challenging targets for drug discovery. MPs can be reconstituted in protein‐lipid Nanodiscs (NDs), which resemble the native membrane environment. Drug‐membrane interactions can affect the apparent binding stoichiometry and affinity, as well as the kinetics of ligands for a particular target, which is important for the extrapolation to pharmacokinetic studies. To investigate the role of the membrane, we have applied fragment‐based drug discovery (FBDD) methods to cytochrome P450 3A4 (CYP3A4), reconstituted in NDs composed of different phosphocholine lipids: 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC), 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or 1,2‐diphytanoyl‐sn‐glycero‐3‐phosphocholine (DPhPC). Surface plasmon resonance screening of fragments and marketed drugs revealed extensive binding to the empty ND, correlating with analyte hydrophobicity, and the binding was critically dependent on ND lipid composition. POPC NDs showed much higher binding of fragments than DMPC and DPhPC NDs, resulting in a lower hit rate for CYP3A4 in POPC NDs, which demonstrated that the choice of the ND lipid is crucial to the outcome of a screen. The number of binders that were rejected based on atypical binding kinetics was lower for monomeric CYP3A4 in NDs than for non‐native oligomeric CYP3A4 without the ND. Several fragments were exclusively identified as hits for CYP3A4 in the presence of the ND membrane. It is concluded that the nature of the ND is a critical factor for fragment screening of membrane proteins.

Fragment‐based drug discovery (FBDD) was applied to cytochrome P450 3A4 reconstituted in Nanodiscs (NDs) with various lipid compositions. The choice of ND lipid influenced drug –membrane interactions and fragment hit rates, demonstrating the critical role of the membrane environment in fragment screening for membrane proteins.