Discovery of Cyclopentane-Based Phospholipids as Miltefosine Analogs with Superior Potency and Enhanced Selectivity Against Naegleria fowleri

Abstract

Background/Objectives:Naegleria fowleri is a free-living amoeba that invades brain tissues causing fatal primary amoebic meningoencephalitis (PAM). An effective and tolerable therapeutic agent is still lacking. Methods: A series of conformationally restricted analogs of miltefosine with varied restriction positions, stereochemical configuration and lengths of alkyl chain was investigated to discover more effective and less toxic agents than miltefosine. Results: Among tested compounds, derivatives 2a, 3b and 3d featuring 1,2- or 2,3-positional restriction with trans-configuration and tridecyl or behenyl alkyl chains were discovered as more potent and less cytotoxic agents. Compounds 2a, 3b and 3d elicited 3.49-, 3.58- and 6.03-fold relative potencies to miltefosine and 7.53, 3.90 and 3.49 selectivity indices, respectively. Furthermore, compounds 2a and 3b showed IC₉₀ values for N. fowleri lower than CC₅₀ against glial C6 cells. Compounds 2a, 3b and 3d induced morphological changes and programmed cell death of N. fowleri via the apoptosis-like pathway. The induced death of N. fowleri involved DNA fragmentation along with the loss of mitochondrial membrane potential. Conclusions: The current research presents compounds 2a and 3b as more potent, selective and effective agents than miltefosine against N. fowleri for further development.

Keywords: Naegleria fowleri; miltefosine analogs; potential drug; primary amoebic meningoencephalitis.

Figure 1

Currently used therapeutic agents for the treatment of PAM.

Figure 2

Rational underlying repurposing of conformationally restricted cyclopentane-based miltefosine analogues, where derivatives 1 are conformationally restricted at positions 1/2 of glycerol moiety of lysophosphatidylchoine in a cis configuration, derivatives 2 are conformationally restricted at positions 1/2 in a trans configuration, while derivatives 3 are conformationally restricted at positions 2/3 in a trans configuration.

Figure 3

Structures of investigated compounds.

Figure 4

Anti-amoebic activities of compounds 2a, 3b and 3d against N. fowleri trophozoites. (A) Microscopic analysis. Images represented the cell populations in three individual experiments. Miltefosine (MF: 150 μM) was employed as a positive control drug. NC, negative controls with 0.1% DMSO treatment. (B) Viability assay: the viabilities of amoebae and C6 glial cells are presented as a percentage relative to the untreated negative control. Results are shown as mean and standard deviation (error bar) of each assay obtained from three independent assays. (C) Summary. IC₅₀, IC₉₀, CC₅₀ and SI values were calculated from three independent assays.

Figure 5

Apoptosis/necrosis assay. A fluorescence staining assay was performed using N. fowleri trophozoites treated with compounds 2a, 3b, 3d, and miltefosine (MF) as a reference; NC, negative controls with 0.1% DMSO treatment. Blue fluorescence representing living cells stained with cytocalcein (DAPI channel, Ex/Em = 405/450 nm). Green apopxin stains apoptotic cells to show green fluorescence (GFP channel, Ex/Em = 490/525 nm). 7-Amino Actinomycin D (7-AAD) stains late apoptotic cells and necrotic cells to show red fluorescence (RFP channel, Ex/Em = 550/650 nm). Images were representatives of cell populations in three individual experiments. Size bar: 10 µm.

Figure 6

TUNEL assay. DNA fragmentation implying apoptosis was detected by TUNEL (GFP channel, (Ex/Em = 490/525 nm) in the amoebae treated with tested compounds. These amoebae were also counterstained with PI (RFP channel, Ex/Em = 550/650 nm). Miltefosine (MF) was included as a positive reference control. NC, negative controls with 0.1% DMSO treatment. Images were representatives of the cell population in three individual experiments. Size bar: 10 µm.

Figure 7

Disruption of mitochondrial functions in N. fowleri. Mitochondrial membrane potential changes. Aggregate form (RFP channel, Ex/Em = 550/650 nm), implying healthy mitochondria, was found in N. fowleri trophozoites treated with 0.1% DMSO (NC), while monomer (GFP channel, Ex/Em = 490/525 nm) indicating the collapse of mitochondrial membrane potential was increased in treated amoebae. Miltefosine (MF) was included as a positive control drug. Images were representatives of the cell population in three individual experiments. Size bar: 10 µm.