Background Primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri is a highly fatal central nervous system infection for which effective treatment options remain limited. This study explored CDK-related pathways in N. fowleri using a focused CDK inhibitor library and structure-based analysis of a selected CDK-like protein. Methods A library of 126 CDK inhibitors was screened against N. fowleri trophozoites using a luminescence-based ATP viability assay, and active compounds were further evaluated in dose–response assays. Human CDK1–9 sequences were used for BLASTp searches against the N. fowleri proteome, followed by comparative sequence analysis with Trypanosoma brucei cdc2-related kinases (CRKs). Nf_CDK-like protein1 (FDP41_013684) was selected for homology modeling, molecular docking, and 100-ns molecular dynamics simulations. Results Screening identified 23 active CDK inhibitors with IC50 values ≤ 10 μM against N. fowleri, including five prioritized compounds with submicromolar activity: AZD5438, GSK-3 inhibitor IX, milciclib, roniciclib, and SU9516 (IC50, 0.30–0.88 μM). Sequence analysis identified five CDK-like proteins in the N. fowleri proteome, among which Nf_CDK-like protein1 showed the highest similarity to T. brucei CRK3 and shared approximately 58–61% sequence identity with human CDK1/2. Conserved kinase features, including the glycine-rich loop, αC-helix, hinge region, DFG motif, and activation loop, were retained in Nf_CDK-like protein1. Docking analysis placed all five prioritized compounds within the predicted ATP-binding cleft, with shared interactions around Lys60, Tyr42, Asp113, and the hinge-proximal residues Ile110 and Asp111. Molecular dynamics simulations showed generally stable protein backbones and ligand poses over 100 ns. Conclusions This study identified multiple CDK inhibitor scaffolds with potent in vitro activity against N. fowleri and supports Nf_CDK-like protein1 as a plausible CDK-like kinase target candidate through integrated phenotypic and structure-based analyses, with direct biochemical validation needed in future studies. These findings suggest that CDK-like pathways may represent a relevant molecular axis in N. fowleri and provide a basis for future structure-guided optimization of anti-amoebic candidates. Graphical Abstract

Cyclin-dependent kinase-inhibitor screening and structural analysis identify a CDK-like kinase candidate in Naegleria fowleri

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2026年6月24日

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Min-Jeong Kim,
Joo Hwan No

摘要

Background

Primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri is a highly fatal central nervous system infection for which effective treatment options remain limited. This study explored CDK-related pathways in N. fowleri using a focused CDK inhibitor library and structure-based analysis of a selected CDK-like protein.

Methods

A library of 126 CDK inhibitors was screened against N. fowleri trophozoites using a luminescence-based ATP viability assay, and active compounds were further evaluated in dose–response assays. Human CDK1–9 sequences were used for BLASTp searches against the N. fowleri proteome, followed by comparative sequence analysis with Trypanosoma brucei cdc2-related kinases (CRKs). Nf_CDK-like protein1 (FDP41_013684) was selected for homology modeling, molecular docking, and 100-ns molecular dynamics simulations.

Results

Screening identified 23 active CDK inhibitors with IC₅₀ values ≤ 10 μM against N. fowleri, including five prioritized compounds with submicromolar activity: AZD5438, GSK-3 inhibitor IX, milciclib, roniciclib, and SU9516 (IC₅₀, 0.30–0.88 μM). Sequence analysis identified five CDK-like proteins in the N. fowleri proteome, among which Nf_CDK-like protein1 showed the highest similarity to T. brucei CRK3 and shared approximately 58–61% sequence identity with human CDK1/2. Conserved kinase features, including the glycine-rich loop, αC-helix, hinge region, DFG motif, and activation loop, were retained in Nf_CDK-like protein1. Docking analysis placed all five prioritized compounds within the predicted ATP-binding cleft, with shared interactions around Lys60, Tyr42, Asp113, and the hinge-proximal residues Ile110 and Asp111. Molecular dynamics simulations showed generally stable protein backbones and ligand poses over 100 ns.

Conclusions

This study identified multiple CDK inhibitor scaffolds with potent in vitro activity against N. fowleri and supports Nf_CDK-like protein1 as a plausible CDK-like kinase target candidate through integrated phenotypic and structure-based analyses, with direct biochemical validation needed in future studies. These findings suggest that CDK-like pathways may represent a relevant molecular axis in N. fowleri and provide a basis for future structure-guided optimization of anti-amoebic candidates.

Graphical Abstract