# Probing Passive Permeation of Tetracycline: Are Simulations Ready for beyond-Rule-of-Five Drug Permeability Calculation?

## TL;DR

The study uses simulations to understand how tetracycline, a drug that breaks traditional rules, permeates lipid membranes, revealing insights into drug design.

## Contribution

The study introduces a method combining pH-partitioning and tautomer effects to calculate permeability of bRo5 drugs like tetracycline.

## Key findings

- Tetracycline's neutral tautomer contributes most to permeability despite low abundance.
- Membrane patch size significantly affects permeability estimates for tetracycline.
- Hydrogen-bond interactions with lipids influence permeation barriers and free-energy calculations.

## Abstract

Passive permeation
across lipid membranes is a key determinant
of drug bioavailability and efficacy. Accurate computational estimation
of drug permeability is essential for rational drug design, yet remains
challenging, particularly for ionizable and beyond-Rule-of-Five (bRo5)
compounds. In this study, we employ advanced molecular simulations
and the inhomogeneous solubility-diffusion model to calculate the
effective permeability and elucidate the membrane permeation mechanism
of the antibiotic tetracycline (TC), the six hydrogen-bond donors
of which violates one of Lipinski’s Rule-of-Five. By integrating
the pH-partitioning and Boltzmann-weighted average potential schemes
and accounting for both its neutral (TCN) and zwitterionic
(TCZ) tautomers, we show that the dominant contribution
to the effective permeability of TC arises from TCN, despite
its low abundance. This result is attributed to the relatively small
permeation barrier of TCN, explaining why the antibiotic
exhibits moderate effective permeability even though it predominantly
exists in the zwitterionic form at neutral pH. A further systematic
investigation reveals that membrane patch size significantly impacts
permeability estimates for TC, in contrast to the relative insensitivity
observed for three other permeants. This unique sensitivity can be
attributed to the hydrogen-bond network formed between TC and its
lipid environment, with the smallest 32-POPC patch artificially raising
the drug molecule’s permeation barrier and the largest 256-POPC
patch exhibiting significant hysteresis that compromises the quality
of the one-dimensional free-energy calculation. Overall, our results
suggest that while probing the passive permeation of bRo5 drugs by
molecular simulations appears increasingly feasible, the protonation
and tautomeric states of the permeants, the uncertainty in their microscopic
acid dissociation constants, as well as the potential impact of membrane
patch-size effects need to be fully considered in permeability predictions
for these complex molecules.