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Small-Molecule Mimicry Intercepts PEX5 Recognition by Trypanosomal PEX14 as Revealed by Molecular Dynamics

This study utilizes molecular dynamics simulations to elucidate the structural determinants of the essential PEX5–PEX14 interaction in trypanosomes and identifies SuFEx-derived naphthoquinones as promising small-molecule inhibitors that effectively mimic the native recognition motif to disrupt glycosomal protein import.

Original authors: Amanda R. Guimarães, Esther R. S. Paz, Allison Serrano-Trejos, Chonny Herrera-Acevedo, Iván Rivilla, Oscar R. Ballesteros, Caroline R. Kwawu, Guilherme A. M. Jardim, Eufrânio N. da Silva Júnior, Felip

Published 2026-06-25

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CC BY 4.0

Original authors: Amanda R. Guimarães, Esther R. S. Paz, Allison Serrano-Trejos, Chonny Herrera-Acevedo, Iván Rivilla, Oscar R. Ballesteros, Caroline R. Kwawu, Guilherme A. M. Jardim, Eufrânio N. da Silva Júnior, Felipe Fantuzzi

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Picture: A Broken Delivery System

Imagine a city (the parasite) that relies on a specialized delivery service to move important packages (enzymes) from the street (the cell's main body) into a secure warehouse (a tiny organelle called a glycosome). Without these packages inside the warehouse, the city's power grid fails, and the city shuts down.

In these parasites, the delivery system works like this:

The Truck (PEX5): A protein called PEX5 picks up the packages in the street.
The Docking Bay (PEX14): The truck drives to the warehouse and must lock into a specific docking bay called PEX14 to unload the cargo.
The Connection: The truck has a specific "hook" (a pattern of amino acids) that fits perfectly into the docking bay's "keyhole."

The Goal: The scientists wanted to build a fake key or a sticky obstacle that jams the docking bay. If they can stop the truck from locking in, the packages never get delivered, the parasite's power grid fails, and the parasite dies. This is a potential new way to treat diseases like Chagas disease and Sleeping Sickness.

How They Did It: The Digital Simulation Lab

Instead of mixing chemicals in a test tube immediately, the researchers used powerful computers to simulate this process in a "digital lab." They used three main tools:

Molecular Docking: Like trying different keys in a lock on a computer to see which one fits best.
Molecular Dynamics (MD): A high-speed movie simulation showing how the key and lock wiggle, shake, and hold hands over time.
Energy Calculations: A way to measure how "sticky" the connection is.

What They Found

1. How the Real Truck Locks In

First, they studied how the real PEX5 truck locks into the PEX14 docking bay. They found it wasn't just one simple click; it was a complex handshake involving several types of "glue":

Aromatic Stacking: Imagine two flat plates (like pancakes) stacking or leaning against each other. The truck uses specific flat, ring-shaped parts of its body to lean against flat rings on the docking bay.
Electrostatics: Like a magnet, a positive part of the truck is attracted to a negative part of the bay.
Hydrogen Bonds: Tiny, temporary Velcro strips holding them together.

The most important "glue" came from two specific ring-shaped parts on the docking bay (called Phe12 and Phe29) and a positive magnet (Lys33).

2. Testing a Known Key (ET7)

They tested a known inhibitor called ET7 (a drug candidate found in previous studies) to see if it could jam the lock.

The Result: It worked well! In their computer movie, ET7 stayed stuck in the docking bay for the entire simulation. It successfully mimicked the truck's "hook," leaning against the right rings and sticking to the right magnet. This confirmed that blocking this specific spot is a viable strategy.

3. Testing New Keys (The Naphthoquinones)

Next, they looked at a new family of compounds called SuFEx-functionalised naphthoquinones. Think of these as new keys being forged in a lab. They have a special "handle" (a sulfur-fluoride group) that allows scientists to easily attach different decorations to the end of the key.

Attempt 1: The Basic Key (3L)
They took a basic version of this new key.
- The Result: It looked like it fit at first, but in the computer movie, it was wobbly. It slipped out of the docking bay after about 50 seconds (in simulation time). It didn't have enough "glue" to stay stuck.
Attempt 2: The Upgraded Key (3L-OAr)
The researchers used the special "handle" to attach an extra aromatic ring (a decoration) to the key. This is like adding a second prong to a key to make it fit deeper into the lock.
- The Result: This upgrade worked much better. The new key (3L-OAr) stayed stuck in the docking bay for the entire movie. It leaned against the right rings and stuck to the magnet just like the real truck and the successful ET7 drug. The computer calculations showed it was much "stickier" than the basic version.

The Conclusion

The paper concludes that to successfully jam this parasite's delivery system, a drug candidate needs to be able to:

Lean against specific ring-shaped parts of the docking bay (like the T-shaped interactions).
Stick to the magnetic part of the bay.

The study shows that SuFEx-functionalised naphthoquinones are promising candidates because, when you add the right "decoration" (the extra aromatic ring), they can mimic the natural truck's hook very well. This gives scientists a clear blueprint for designing better drugs to treat these neglected tropical diseases.

Technical Summary: Small-Molecule Mimicry Intercepts PEX5 Recognition by Trypanosomal PEX14

Problem Statement
Human trypanosomiases, including Chagas disease (Trypanosoma cruzi) and Human African Trypanosomiasis (Trypanosoma brucei), remain significant neglected tropical diseases with limited therapeutic options and high toxicity profiles. A distinctive feature of trypanosomatid metabolism is the compartmentalization of glycolysis within glycosomes, peroxisome-like organelles essential for parasite viability. The biogenesis of glycosomes relies on the protein import machinery mediated by peroxins, specifically the interaction between the soluble receptor PEX5 and the membrane docking protein PEX14. This protein–protein interaction (PPI) is critical for the translocation of cargo proteins into the organelle. Disrupting the PEX5–PEX14 interface offers a promising strategy for developing new trypanocidal agents, yet the structural dynamics of this interface and the requirements for small-molecule inhibition remain incompletely resolved.

Methodology
The study employed an integrated computational workflow to characterize the PEX5–PEX14 interface and evaluate small-molecule inhibitors.

Structural Basis: The analysis utilized the crystal structure of the T. cruzi peroxisomal import assembly (PDB ID: 8GI0), focusing on the PEX5–PEX14 subcomplex. The PEX14 receptor was isolated for ligand docking studies.
Molecular Docking: AutoDock Vina was used to dock ligands into the PEX14 recognition groove, defined by the binding site of the canonical PEX5 Wxxx(F/Y) motif. The protocol was validated by redocking the known inhibitor ET7 (PDB ID: 7QRC).
Molecular Dynamics (MD): Three independent 500 ns production MD simulations were performed using the Amber 24 suite with the ff14SB force field. Simulations were conducted for the native PEX5–PEX14 complex, the ET7–PEX14 complex, and complexes involving SuFEx-functionalized naphthoquinones.
Free Energy Calculations: Binding free energies were estimated using the MM–PBSA method on the final trajectory frames to provide comparative stability estimates.
Ligand Series: The study evaluated the reference inhibitor ET7 and a series of SuFEx-functionalized naphthoquinones, specifically comparing a parent compound (3L) with an in silico derivatized analogue (3L-OAr) where the sulfonyl fluoride was converted to an aryl sulfonate.

Key Contributions and Results

Characterization of the Native Interface: MD simulations confirmed that the PEX5–PEX14 complex is stabilized by a dynamic, multivalent interaction network. The canonical 60–WVREY–64 motif of PEX5 is central, with Trp60 and Tyr64 engaging PEX14 residues Phe12, Phe29, Lys33, and Thr21. Key stabilizing forces include:
- Aromatic Interactions: T-shaped edge-to-face contacts between PEX5 Trp60/Tyr64 and PEX14 Phe12/Phe29.
- Electrostatics: A $\pi$ –cation interaction between PEX5 Trp60 and PEX14 Lys33, and a salt bridge between PEX5 Glu63 and Lys33.
- Hydrogen Bonding: Persistent hydrogen bonds, notably between PEX5 Tyr64 and PEX14 Thr21.
- A secondary non-canonical region (residues 86–90) also contributes transient contacts, reinforcing the multivalent nature of the binding.
Validation of ET7: The reference inhibitor ET7 successfully mimicked the native PEX5 recognition mode. Throughout the MD simulations, ET7 remained associated with the PEX14 groove, occupying the Trp60 and Tyr64 pockets. It maintained T-shaped aromatic interactions with Phe12/Phe29 and a $\pi$ –cation contact with Lys33, yielding an average MM–PBSA binding free energy of $-5.45 \pm 1.36$ kcal mol $^{-1}$ .
Evaluation of SuFEx-Functionalized Naphthoquinones:
- Compound 3L: The initial sulfonyl fluoride derivative showed a favorable docking pose but failed to maintain stable binding during MD simulations, dissociating from the groove. MM–PBSA calculations on the initial bound phase yielded a weak binding free energy of $-1.43 \pm 2.05$ kcal mol $^{-1}$ .
- Compound 3L-OAr: An in silico derivative of 3L, modified via SuFEx chemistry to introduce an additional aromatic phenoxy substituent (forming an aryl sulfonate), demonstrated significantly improved behavior. This modification increased the residence time within the PEX14 groove and improved the MM–PBSA binding free energy to $-4.04 \pm 1.16$ kcal mol $^{-1}$ . The added aromatic group facilitated recurrent T-shaped interactions with Phe12 and $\pi$ –cation contacts with Lys33, effectively mimicking the native PEX5 motif.

Significance and Claims
The paper claims that aromatic and electrostatic contacts are the primary determinants for ligand stabilization within the PEX14 groove. The study establishes that successful inhibition requires small molecules to reproduce the specific spatial arrangement of the native PEX5 Wxxx(F/Y) motif, particularly the engagement of Phe12, Phe29, and Lys33.

The authors conclude that SuFEx-derived naphthoquinones represent promising scaffolds for targeting glycosomal protein import. Specifically, the results support the strategy of using SuFEx chemistry to introduce additional aromatic substituents to enhance complementarity with the PEX14 binding site. While the current derivatives show improved binding characteristics, the authors modestly suggest that future optimization should focus on incorporating hydrogen-bond donor groups and carefully controlling ligand size to further improve binding strength and residence time. This work provides a structural framework for the rational design of new inhibitors against Chagas disease and Human African Trypanosomiasis by targeting the essential PEX5–PEX14 interface.