Identifying Promising Novel Compounds Against Free-Living Amoebae: A Systematic Review of In Vitro and In Vivo Studies

Abstract

The increasing global incidence of infections caused by free-living amoebae (FLA) and the lack of effective, safe, and approved treatments highlight the urgent need for novel amoebicidal compounds with pharmacological potential. Despite a growing body of literature on the anti-FLA properties of various compounds, comprehensive reviews summarizing this progress remain scarce. This study aimed to identify the most promising compounds tested in vitro and/or in vivo for anti-FLA activity. A systematic review was conducted, analyzing 108 studies published between 1986 and 2024, selected from an initial pool of 23,653 database results. A total of 537 compounds were evaluated for their in vitro anti-FLA activity. Compounds exhibiting ≥50% reduction in amoeba viability relative to untreated controls were classified as promising if they showed low toxicity in mammalian cell models, particularly when active at concentrations ≤ 10 µM, consistent with predicted favorable pharmacokinetic and pharmacodynamic profiles. The most promising compounds for drug and disinfectant development include ten trophocidal agents against B. mandrillaris, thirty-two trophocidal and four cysticidal agents against N. fowleri, and sixty-two trophocidal and nineteen cysticidal agents against Acanthamoeba spp. Compounds active at low concentrations (≤10 µM or <0.014 mg/mL) prioritized for in vivo drug development studies include: against Balamuthia mandrillaris, trophocidal 515, 531, 533; against Naegleria fowleri, trophocidal 421, 416, 518, 46, 254, 522, 111–120 and cysticidal 16; and against Acanthamoeba spp., trophocidal 498, 499, 500, 535, 107, 347, 348, and 340. Future studies should evaluate their efficacy, safety, pharmacokinetics, and pharmacodynamics toward developing effective drugs, antiseptics, and disinfectants.

1. Introduction

Free-living amoebae (FLA), particularly Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Vermamoeba vermiformis, and Sapinia pedata, are known to cause severe opportunistic infections in humans and other animals. Among these infections is amoebic keratitis, an ocular infection primarily caused by Acanthamoeba spp. and, more rarely, by V. vermiformis [1,2]. Brain infections linked to these amoebae include primary amoebic meningoencephalitis (PAM), caused by N. fowleri [3], and granulomatous amoebic encephalitis (GAE), caused by Acanthamoeba spp., B. mandrillaris, and S. pedata [4]. PAM is a rapidly progressive infection with an extremely high mortality rate of 95–98% [3]. GAE, although typically characterized by a more insidious onset and slower progression, is likewise highly lethal, with mortality estimates of approximately 90% for cases caused by B. mandrillaris and 94% for those due to Acanthamoeba spp. [4]. FLA can also lead to disseminated infections, especially in immunocompromised or transplant patients, affecting various tissues such as the skin, lungs, liver, bones, kidneys, lymph nodes, breast, heart, spleen, testicles, thyroid, and urethra [5,6,7,8,9].

The ubiquity of FLA in natural and anthropogenic environments [10,11,12,13,14], including extreme and hospital environments [15,16], makes human exposure to these microorganisms almost inevitable. FLA’s marked resistance to various physical and chemical biocidal agents [17,18,19] makes its environmental control and treatment of related infections challenging. Beyond their own pathogenicity, FLA are clinically important for their role in protecting, proliferating, and promoting antimicrobial pseudo-resistance of various viral, bacterial, and fungal pathogens [16,17,18,19,20,21,22].

Despite recent approvals, such as polihexanide 0.08% for Acanthamoeba keratitis (in 2024) and corifungin for PAM (in 2025), effective therapies for FLA infections remain generally unavailable. Current treatment relies on combinations of antifungals (amphotericin B, fluconazole, flucytosine, pentamidine) and antibacterials (azithromycin, rifampin, sulfadiazine), yet evidence of their efficacy is limited and these regimens are often poorly tolerated [3,4,23]. This underscores the value and pressing need for identifying molecules with antiamoebic activity that could potentially be developed into specific treatments for FLA infections. Although considerable research has been conducted over the years to discover new compounds with anti-FLA activity [24,25,26,27,28,29,30], to our knowledge, no literature has yet summarized these findings to highlight the most promising compounds for prioritization in future in vitro or in vivo studies. This study aimed to answer the question, “Which are the most promising compounds tested in vitro and in vivo against FLA?” through a systematic review. This review identifies the most promising compounds based on their amoebicidal potency and toxicological profiles in non-target mammalian cells, and offers recommendations for future research aimed at developing drugs, antiseptics, and/or disinfectants.

2. Materials and Methods

2.1. Objectives of the Review

This review was conducted to identify the most promising new chemical compounds (those not yet established as drugs) with potential pharmacological applications that have been tested in in vitro or in vivo studies against FLA. It aims to provide insights relevant to the field of antimicrobial drug development, particularly for the treatment of infections caused by FLA.

Additionally, this review seeks to identify compounds that, beyond their potential as therapeutic drugs, may also be suitable for clinical use as antiseptic agents or environmental disinfectants.

Furthermore, the review aims to highlight molecules that, although not yet fully optimized for clinical or environmental applications, may serve as valuable scaffolds or starting points for future chemical engineering efforts aimed at enhancing their biological activity, selectivity, or physicochemical properties, thus improving their suitability for therapeutic, prophylactic, or sanitation purposes.

The specific questions guiding this review are as follows:

-

Which novel chemical compounds (those not yet established as drugs) have been tested in vitro and/or in vivo against FLA?

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Which tested compounds demonstrate promising performance in terms of amoebicidal activity, effective concentration, and mammalian toxicological profile?

2.2. Data Collection

The planning, conduct, and reporting of this review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines (Table S1. PRISMA 2020 checklist) [31]. The review protocol was registered in the Protocols.io under registration ID number 227843, (https://dx.doi.org/10.17504/protocols.io.4r3l213y3g1y/v1). Primary studies were identified through a comprehensive search strategy applied across PubMed, EMBASE, ProQuest, and Web of Science, using the following search strategy “Acanthamoeba* OR Balamuthia OR Naegleria* OR Vermamoeba”. Two authors validated the performance of the search strategy, and one author conducted the formal search for articles in the databases on 11 June 2024.

Inclusion Criteria: Studies were selected based on the following criteria: (1) full texts were accessible online, including those provided by authors upon request; (2) written in English, Portuguese, Spanish, or another language translatable with online tools; and (3) reporting in vitro or in vivo evaluation of a novel compound against FLA, specifically Acanthamoeba, Balamuthia, Naegleria, or Vermamoeba (Figure 1).

Exclusion Criteria: Studies were excluded if they were (1) non-primary research, (2) had unclear or unreliable methodology or data, (3) tested a complex mixture of chemical compounds, (4) did not report quantifiable amoebicidal effects, (5) tested approved drugs or commercial disinfectants, or (6) if they presented only the chemical structures of the compounds without corresponding names, or if the names (whether common or IUPAC—International Union of Pure and Applied Chemistry) were unavailable or inaccessible. Studies involving pure compounds extracted from plants were also not included, as these have already been addressed in a specific review [32].

The references of the selected articles were evaluated in search of additional relevant studies that may not have been identified through the primary database search.

2.3. Screening Stages

The screening of the identified records was carried out in three main stages:

(1) Removal of duplicates—Duplicated titles were removed using semi-automated tools available in Mendeley Reference Manager. (2) Title screening—The titles were read and those clearly unrelated to the scope of the study were excluded. Titles that did not allow a clear judgment about their relevance were retained for further evaluation. (3) Abstract screening—Abstracts were reviewed to identify and exclude studies whose content, based on the abstract, was evidently outside the scope of the review.

All screening stages were conducted independently by at least three authors. Discrepancies were resolved through consensus meetings. Studies selected by consensus were then included in the next steps of full-text retrieval and data extraction.

The quality of the included studies was evaluated based on methodological robustness, clarity, and reproducibility of procedures, as conventional risk-of-bias tools designed for clinical trials were not applicable to the laboratory-based nature of these studies. Studies with low quality, limited reliability, or insufficient methodological reproducibility were not selected for inclusion.

2.4. Data Extraction Quantification of Compound Efficacy

Data extraction was initially carried out by one author, followed by verification for accuracy by two or more independent authors.

The extracted data from the articles included: reference, country (of all study authors), compound name, FLA (genus or species), amoebae form (cyst or trophozoite), density of FLA used in the tests, effective concentration (exhibiting the most relevant effect), exposure time, FLA mortality, concentration causing 50% FLA mortality (IC₅₀), highest concentration tested for toxicity in mammalian cells (CT-toxicity), cytotoxicity in mammalian cells, and the cell line used in the toxicity test.

For each compound identified in the included publications, biocidal efficacy data were extracted as reported by the original study authors. Whenever available, values for the MIC, IC₅₀, or the concentration corresponding to the maximum observed effect (E_max) were collected.

When multiple measurements were available, the value indicating the greatest efficacy against amoebae (according to the source article) was selected. These measurements were used as comparative indicators of the compounds’ amoebicidal activity. No extrapolation or artificial standardization was applied to homogenize the data across studies, due to methodological heterogeneity among them. The compounds were then grouped according to the highest and lowest reported effects, aiming to provide a useful comparative reference for future studies investigating these molecules in greater depth.

2.5. Data Analysis Procedure

Prior to analysis, compounds were ordered alphabetically and assigned numeric codes starting from 0 using the label encoder function in Scikit-learn. The full list of compounds and their corresponding codes is provided in Table S2 (Names and compounds codes).

Compounds were subsequently ranked according to their Effective Concentration and Mortality values. Ranking was based on the observed value ranges (minimum and maximum) and the relative distances between each compound’s Effective Concentration or Mortality. Rankings started at 1, with compounds exhibiting lower Effective Concentration values and higher Mortality values receiving the top ranks (i.e., a rank of 1 or the closest possible value).

In other words, compounds demonstrating the highest amoebicidal activity (e.g., 100% mortality) or lowest concentrations (e.g., sub-micromolar levels) were assigned ranks equal to or nearest to 1. An average ranking value was calculated for each compound based on its Effective Concentration and Mortality rankings. These mean rank values (rankAvg) were visualized using scatterplots. For interactive visualization, refer to Figure S1. (Ranking of all compounds by code), Figure S2. (Mortality of all compounds by code), and Figure S3. (Effective Concentration of all compounds by code).

Mean values and standard deviations (with 95% confidence intervals) were calculated for the variables: effective concentration, exposure time, FLA mortality, IC₅₀, CT-toxicity, and cytotoxicity. These calculations focused on compounds that exhibited mortality rates ≥50%. Mean values were further grouped and analyzed by compound, species, and amoebae form.

The primary data and analyses are summarized in Table S3 (Data and analysis). All analyses were conducted using Python 3 and GraphPad Prism 8.02 software.

Compounds mentioned more than once in the same or different graphs (Figure S1.1–S1.28) for the same amoeba genus mean that they were tested in more than one species.

3. Results

A total of 108 studies were selected from an initial pool of 23,653 document titles retrieved from scientific databases (Figure 1). These studies involved authors from 35 countries across five continents. The distribution of studies by country, as well as the number of studies published per year, are shown in Figure 2 [33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140] (Table S3).

3.1. Ranking of Tested Compounds Based on Their Amoebicidal Potency

A total of 537 different chemical compounds were evaluated for their anti-FLA activity in vitro (Figure 3). These compounds exhibited amoebicidal activity (0–100%) across a wide concentration range, from 0.015 to 5600 µM (S1). Among the tested compounds, 375 exhibited a mean rank position <200, indicating a significant amoebicidal effect (≥50%). These compounds include those coded as 0, 2, 13–18, 20, 21, 25, 27, 28, 30, 31, 32, 34, 36, 37, 39, 40–55, 57, 58, 60, 61, 64–79, 81–83, 88–103, 107, 111, 113–120, 123–125, 127–130, 132–134, 137, 138, 140, 143, 148, 150, 152, 155–157, 159, 164, 167, 170–189, 192, 200, 205, 206, 208–211, 214, 218, 219–229, 237–244, 246–250, 252–258, 260–266, 268–269, 272–282, 284, 286–295, 297, 299, 302, 304–312, 315–334, 337–344, 346–349, 361, 363–372, 374–381, 383–387, 389, 391–399, 403–404, 408, 411, 412, 414, 416–419, 421–428, 431, 432, 434–466, 468–504, 513, and 514. The compounds with the lowest average ranking values are those that present a relatively greater amoebicidal effect at relatively lower concentrations (Figure 3, refer to Figures S1 and S2 for the interactive version).

3.2. In Vitro Amoebicidal Activity of Compounds

The amoebicidal activity of all tested compounds is presented in the Supplementary Materials (Figures S2 and S3 and Table S3), while the names of the compounds corresponding to the codes reported throughout the manuscript are provided in Table S2.

Detailed performance data for compounds showing ≥50% amoebicidal activity in vitro are provided in Supplementary Materials S1. Compounds with anti-FLA activity (≥50%) at concentrations ≤10 µM or <0.2 mg/mL are highlighted in the subsequent sections (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9). The structural chemical formulas of the most promising compounds are presented in Table S4.

3.2.1. Amebicidal Activity Against Balamuthia mandrillaris

Twenty-seven compounds showed ≥50% amoebicidal activity against B. mandrillaris trophozoites (Figure S1.1). Of these, 13 were active at concentrations <10 µM (Figure 4). Except for compound 534, all displayed notable activity; compounds 217, 243, 363, 364, 515, 528, 529, 530, 531, 532, and 533 also showed low cytotoxicity to non-target mammalian cells (Figure 4).

3.2.2. Compounds Exhibiting Trophocidal Activity Against Naegleria fowleri

Among the compounds tested against N. fowleri, 108 demonstrated the ability to inactivate ≥50% of trophozoites at concentrations ranging from 0.02 to 5489.38 µM (S1, Figures S1.2–S1.8). The compounds exhibiting trophocidal activity at concentrations below 10 µM are listed below.

Anti-N. fowleri Activity from 0.02 to 10 µM

A total of 27 compounds demonstrated the capacity to inactivate ≥50% of N. fowleri trophozoites at low concentrations (0.02–10 µM), as presented in Figure 5, Figure 6 and Figure 7. These compounds exhibited pronounced amoebicidal activity, achieving 50–100% trophozoite inactivation while generally displaying low cytotoxicity toward mammalian cells.

Figure 5 depicts the compounds capable of inactivating ≥50% of N. fowleri trophozoites at concentrations ranging from 0.02 to 3.3 µM. With the exception of compounds 272, 273, 248, and 281 (whose cytotoxicity has not yet been assessed) all demonstrated potent anti-Naegleria activity combined with minimal toxicity to non-target mammalian cells. It is noteworthy that compounds 46, 217, 254, 281, 364, 416, 421, and 518 exhibited amoebicidal activity at sub-micromolar concentrations. Among them, compounds 281 and 421 were particularly prominent, achieving 100% and 94% trophozoite inactivation within 36 and 24 h, respectively, at a concentration of only 0.02 µM (Figure 5).

The compounds that exhibited ≥50% trophocidal activity against N. fowleri at concentrations ranging from 3.5 to 8.2 µM are presented in Figure 6. Notably, for all compounds shown, potency is expressed as the concentration required to inhibit (kill) 50% of the parasites (IC₅₀). All demonstrated pronounced anti-N. fowleri activity, and (excepting the compound 225, which also displayed cytotoxicity) they exhibited remarkably low toxicity in mammalian cell models.

Thirteen compounds exhibited substantial trophocidal activity against N. fowleri (≥50%) at concentrations of 8.5–10.0 µM. Among those depicted in Figure 7, several displayed strong anti-N. fowleri effects. Notably, compounds 54, 111, 113–119, and 229 stood out for their potent amoebicidal activity combined with low toxicity toward mammalian cell models (Figure 7).

Compounds Exhibiting ≥50% Cysticidal Activity Against N. fowleri

Eight compounds exhibited considerable cysticidal activity (≥50%) against N. fowleri at concentrations ranging from 7 to 75 µM, except for compound 391, 513, and 514 which showed activity at 2551, 10,440, and 9410 µM, respectively (S1, Figure S1.8). Notably, compounds 15, 34, 25, 226, 513, and 514 stood out for their favorable toxicological profiles in mammalian cells.

As shown in Figure 8, no compound exhibited substantial cysticidal activity (≥50%) against N. fowleri at concentrations ≤10 µM. Only compound 34 displayed the lowest cysticidal IC₅₀ (14.46 µM) while maintaining an acceptable toxicity profile in mammalian cell models.

3.2.3. Compounds Exhibiting Trophocidal Activity Against Acanthamoeba spp.

A total of 246 compounds exhibited significant acanthamoebicidal activity (≥50%) against Acanthamoeba spp. trophozoites across a wide concentration span, ranging from 0.02 to 3536 µM (S1).

The compounds identified as most promising, based on their acanthamoebicidal potency and low toxicity are presented. The following paragraphs focus on those exhibiting ≥50% anti-Acanthamoeba activity at concentrations ranging from 0.02 to 10 µM (Figure 9, Figure 10, Figure 11, Figure 12 and Figure 13).

Compounds Exhibiting Trophocidal Activity Against Acanthamoeba spp. at Concentrations ≤10 μM

Figure 9 presents compounds exhibiting ≥50% trophocidal activity against Acanthamoeba spp. at concentrations of 0.02–3.5 µM. Notably, compound 421 demonstrated rapid activity (24 h) with low toxicity to mammalian cell models, while compounds 217, 363, 522, 535, and 536 also showed minimal toxicity. Compound 305 displayed rapid trophocidal action (12 h) but lacks a characterized toxicity profile in non-target mammalian cells. Similarly, compound 281 achieved 100% trophozoite inactivation at 0.02 µM after 48 h of exposure (Figure 9).

Figure 10 shows compounds achieving ≥50% trophocidal activity against Acanthamoeba spp. at 3.7–5.26 µM. Compound 377 was notable for its rapid action (24 h) and low toxicity, while compounds 54 and 537 also displayed minimal cytotoxicity. For the remaining compounds, although their trophocidal activity was significant, their toxicity profiles in non-target mammalian cells remain unreported.

Figure 11 illustrates twenty-one compounds that inactivated ≥50% of Acanthamoeba spp. trophozoites at concentrations ranging from 5.4 to 8.7 µM. Among these, compounds 107, 174, 340, 341, 342, 343, 425, and 426 exhibited pronounced acanthamoebicidal activity while maintaining low cytotoxicity in mammalian cell models. The remaining compounds also demonstrated substantial trophocidal effects; however, their toxicological profiles in mammalian cells remain uncharacterized, except for compound 395, which showed marked toxicity to mammalian cells (Figure 11).

Ten compounds showed ≥50% trophocidal activity against Acanthamoeba spp. at 9–10 µM (Figure 12). Compounds 348 and 427 combined high activity with low mammalian cytotoxicity; notably, 348 induced ≥50% trophozoite death within 2 h. Compounds 346, 347, and 349 also displayed favorable toxicological profiles, while 186 and 280 still require cytotoxicity assessment (Figure 12).

Compounds with Anti-Acanthamoeba spp. Activity at Concentrations of 0.004–0.05 mg/mL

Seventeen compounds exhibited ≥50% trophocidal activity against Acanthamoeba spp. at 0.004–0.05 mg/mL (Figure 13). Compounds 477, 480, and 497–501 showed low cytotoxicity toward mammalian cells (HeLa or HaCaT). Among these, compound 477 achieved complete trophozoite inactivation within 4 h, demonstrating exceptional potency. Compounds 492 and 494 also exhibited strong trophocidal activity with favorable cytotoxicity profiles.

Figure 14 presents fourteen compounds that inactivated ≥50% of Acanthamoeba spp. trophozoites at concentrations ranging from 0.06 to 0.51 mg/mL. Among these, only compound 489 exhibited low toxicity toward mammalian cells. Similarly, compounds 479 and 486 showed a favorable balance between trophozoite inactivation and cytotoxicity in non-target mammalian cells (Figure 14).

3.2.4. Compounds That Displayed Cysticidal Activity Against Acanthamoeba spp.

Among all the different compounds evaluated for their cysticidal activity, 33 inactivated ≥50% of Acanthamoeba spp. cysts at concentrations ranging from 3.5 to 2243.10 µM, these results are presented in detail in Supplementary Materials S1. The paragraphs below present compounds that exerted proven cysticidal activity (≥50%) at concentrations ≤10 µM and 0.05 to 0.5 mg/mL (Figure 15).

Fifteen compounds inactivated ≥50% of Acanthamoeba spp. cysts at concentrations ranging from 3.5 to 10 µM (Figure 15). Among these, compounds 338, 340, and 342 demonstrated low toxicity to non-target mammalian cells. While the remaining compounds showed high cysticidal potency, their cytotoxic profiles in mammalian cells have yet to be characterized (Figure 15).

Seventeen compounds inactivated ≥50% of Acanthamoeba spp. cysts at concentrations ranging from 0.05 to 0.5 mg/mL (Figure 16). Among these, all compounds evaluated for their selectivity potential (479, 480, 486, 489, and 492) were cytotoxic to non-target mammalian cells. The remaining compounds demonstrated high cysticidal potency, but their toxicity in mammalian cells has yet to be determined.

3.2.5. Photochemical Inactivation of Free-Living Amoebae

All photobioactive chemical compounds that had their anti-FLA activity evaluated targeted Acanthamoeba spp. Twelve different compounds (474, 475, 495, 503, 504–512) were tested against trophozoites, and three (495, 504, and 505) against cysts (

Table 1. Photochemical inactivation efficacy of various compounds against Acanthamoeba spp.

Cod	Concentration (µM)	Light λ (nm)	Light Dose (J/cm2)	Irradiation Time (h)	Mortality (%)	IC50 (µM)	CT-Toxicity	Cytotoxicity (%)	Cell Line
Trophozoites
475	250	570–585	-	2	57.5 (±7.5)	-	-	-
495	0.0019 *	>470	90	0.5	100	0.00044 *	0.3	100	ESRC
503	100	400–700	-	24	12.7	-	-	-
505	10 *	660	30	3	79	-	-	-
506	0.25 **	365	5.4	0.16	11	-	-	-
507	0.25 **	523	5.4	0.16	66	-	-	-
508	50	465–480	-	1	20	-	-	-
509	50	465–480	-	1	10	-	-	-
510	50	465–480	-	1	75	-	-	-
511	500	650–670	10.8	0.5	87.3 (±7.4)	-	-	-
512	1	600–700	30	0.08	98	0.2
Cysts
495	0.037 *	>470	90	0.5	100	7.2 *	0.3	100	ESRC
504	5	600–700	60	0.3	90	0.5	-	-
505	10 *	660	30	3	23	-	-	-

(*)—Concentration in mg/L. (**)—Concentration in percentage. ESRC—epithelial and stromal rabbit cells.

). Among these, the trophocidal (474, 475, 505, 507, 510–512) and cysticidal (504) compounds appear promising; however, their cytotoxicological profile in non-target cells needs to be established. It is important to highlight that although compound 495 shows strong trophocidal and cysticidal potency, it is extremely cytotoxic in non-target cells.

4. Discussion

The treatment of infections caused by FLA remains a significant challenge, as no approved drugs currently offer both effectiveness and safety. The existing treatment options rely on nonspecific drugs, often leading to unfavorable outcomes and frequent adverse effects. Amidst a rising incidence of FLA infections [3,23,141,142] and projections of an epidemiological worsening partly driven by climate change [143], the search for amoebicidal compounds for drug development is increasingly critical. This study reviews new compounds tested in vitro for their anti-FLA activity, aiming to identify the most promising candidates based on amoebicidal potency and potential selectivity.

Our findings (

Table 2. Most promising compounds with anti-Balamuthia mandrillaris effects.

Code	Concentration (µM)	Mortality (%)	IC50 µM	CT-Toxicity (µM)	Toxicity (%)	Exposure Time (h)	Cell Line
515	0.38	50	0.38	10	50	72	A549
531	0.58	50	0.58	10	50	72	A549
529	0.64	50	0.64	10	50	72	A549
533	1.01	50	1.01	10	50	72	A549
53	39.60	50	39.60	100	3	24	HaCaT
218	50	53	-	50	20	24	HeLa
223	50	65	-	50	11	24	HeLa
83	62.50	50	62.50	100	6	24	HaCaT
358	99.80	50	99.80	100	4	24	HaCaT
277	106	50	106	100	0	24	HaCaT
Miltefosine (troph.)	250	100				48
Miltefosine (cysts)	510	100				48
Chlorhexidine (troph.)	20	64				72

CT-toxicity—highest concentration tested for toxicity in mammalian cells. HaCaT—human keratinocytes. A549—lung carcinoma cells.

) indicate that compounds 53, 83, 218, 223, 277, 358, 515, 529, 531, and 533 exhibit the most potent trophocidal activity against B. mandrillaris, while also demonstrating notable selectivity. B. mandrillaris is a FLA with well-characterized virulence mechanisms toward animal cells, including the capacity to phagocytose neural cells [144]. It has been implicated in numerous cases of encephalitis in immunocompetent individuals [145,146] as well as in skin infections [147].

Collectively, these results highlight ten compounds as promising leads for the future development of therapeutic agents and disinfectants targeting B. mandrillaris. Notably, compounds 515, 531, 529, and 533 emerge as the most promising candidates for drug development, exhibiting potent biocidal activity at sub-10 µM concentrations, consistent with the range predicted to confer favorable ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiles in biological systems.

Compounds 515 (1,1-dioxide 1-thioflavone, MMV1006203), 531 (MMV1582495), 529 (MMV1580844), and 533 (MMV1634399) belong to the thioflavone class, in which a sulfur atom substitutes the oxygen in the canonical flavone scaffold [148]. Included in the MMV Pandemic Response Box, these compounds exhibit activity against Nematocida parisii [149], multiple developmental stages of Plasmodium [150], and B. mandrillaris. Although their precise molecular mechanisms remain to be elucidated, current evidence suggests pleiotropic effects potentially mediated through redox pathways or oxidative enzymes, such as NADH oxidase [149]. To date, no validated biochemical target has been identified in amoebae.

The promising activity of these compounds represents a significant advance in the search for targeted treatments against B. mandrillaris. However, further studies are required to confirm their in vitro efficacy and, crucially, to assess their safety, pharmacokinetics, mechanisms of action, and in vivo effectiveness, both as monotherapies and in combination with other candidate compounds or approved drugs. Of particular interest is the exceptionally high selectivity of compound 277 (cellulase). Its potential for combinatorial use with other compounds, as well as strategies to enhance its bioavailability given its high active concentration, warrants further investigation.

Regarding N. fowleri, our findings (

Table 3. Most promising compounds with anti-Naegleria fowleri activity.

Code	Concentration (µM)	Mortality (%)	IC50 µM	CT-Toxicity (µM)	Toxicity (%)	Exposure Time (h)	Cell Line
Trophozoites
421 *	0.02	94	-	0.01	17	24	HeLa
416	0.08	50	0.08	8.74	50	48	J774A.1
518	0.08	50	0.08	10	50	72	A549
46	0.23	50	0.23	1.07	50	48	J774A.1
254	1.18	50	1.18	5.20	50	48	J774A.1
522	2.40	50	2.40	10	50	72	A549
83	7.50	50	7.50	100	6	24	HaCaT
111	10	98.80	6	20	45	48	SH-SY5Y
113	10	99.50	-	20	26	48	SH-SY5Y
114	10	100	0.92	20	0.50	48	SH-SY5Y
115	10	100	1.40	20	2	48	SH-SY5Y
116	10	52.80	11.50	20	24	48	SH-SY5Y
117	10	99.30	10	20	17	48	SH-SY5Y
118	10	99.20	3	20	10	48	SH-SY5Y
119	10	99.80	3.30	20	0	48	SH-SY5Y
120	10	100	1.10	20	41	48	SH-SY5Y
229	10	84.40	7.20	20	15	48	SH-SY5Y
34	20.30	50	20.30	85.21	50	24	J774A.1
16	23.67	50	23.67	262.57	50	24	J774A.1
226	29.19	50	29.19	118.06	50	24	J774A.1
15	37.56	50	37.56	319.53	50	24	J774A.1
13	50	72		50	4	24	HeLa
79	50	53	-	50	18	24	HeLa
206	50	88	23.34	23.34	16	24	HBEC-5i
214	50	58	62.91	62.91	16.30	24	HBEC-5i
218	50	69	-	50	20	24	HeLa
329	56.08	90	27.67	129.40	50	48	J774A.1
333	89.07	50	89.07	304.60	50	48	J774A.1
334	95.34	90	29.28	141	50	48	C6, CHO-K1
18	100	72	9.40	100	1	24	HaCaT
53	100	66	24.20	100	3	24	HaCaT
358	100	60	53.70	100	4	24	HaCaT
103	102.10	50	102.10	100	5	24	HaCaT
325	103.06	90	17.42	509.68	50	48	J774A.1
386	171.30	80	-	171.30	21	24	HBEC-5i
306	397.70	59	-	397.70	15.80	24	HBEC-5i
47	447.91	51.39	-	447.91	21	24	HaCaT
96	447.91	95		447.91	31	24	HaCaT
393	515.10	54	-	515.10	11	24	pHCEC, HaCaT
514	17,070	90	4440	68,500	50	72	J774A.1
513	20,630	90	9310	48,760	50	72	J774A.1
Amphotericin B	0.48	50				72
Azithromycin	0.03	50				24
Fluconazole	13.9	50				48
Miltefosine	91.5	90				72
Nitroxoline	1.16	50				24
Pentamidine	10	50				120
Rifampicin	320	90				72
Cysts
16	7.15	50	7.15	262.57	50	24	J774A.1
34	14.46	50	14.46	85.21	50	24	J774A.1
15	39.96	50	39.96	319.53	50	24	J774A.1
226	43.13	50	43.13	118.06	50	24	J774A.1
Amphotericin B	10.82	100				48
Miltefosine	21.6	50				24
Nitroxoline	1.26	50				24

(*)—Concentration in mg/mL. CT-toxicity—highest concentration tested for toxicity in mammalian cells. A549—lung carcinoma cells. C6—rat glial cells. CHO-K1—Chinese hamster ovary cells. HaCaT—human keratinocytes. HBEC-5i—human brain microvascular endothelial cell. J774A.1—murine macrophages. RBCs—Horse Red Blood Cell. SH-SY5Y—human neuroblastoma. pHCEC—primary corneal epithelial cell. HCEC—human corneal epithelial cells.

) identify compounds 13, 15, 16, 18, 34, 47, 53, 79, 83, 96, 103, 111, 113–120, 206, 214, 218, 229, 306, 325, 358, 393, 416, 421, 513, 514, and 518 as the most promising trophocidal candidates for prioritization in future studies aimed at developing therapeutic agents and disinfectants, owing to their trophocidal activity and potentially high selectivity. For cysticidal activity, compounds 15, 34, and 226 are particularly notable. It is important to note that compounds 15, 16, and 34 exhibit both anti-N. fowleri trophocidal and cysticidal activity. Compounds 53, 83, and 354 also demonstrate significant anti-B. mandrillaris activity.

These compounds, particularly those exhibiting anti-Naegleria activity at concentrations ≤10 µM and 0.02 mg/mL, represent high-priority candidates for future research, encompassing confirmatory in vitro studies and in vivo evaluation. It is crucial to note that infections caused by N. fowleri are increasing worldwide. Although they are relatively rare compared to other FLA infections, they are by far the most lethal [151,152]. The rarity, high lethality, and rapid health deterioration in patients affected by PAM make clinical trials unfeasible. Consequently, research using various animal models of PAM is both urgent and essential. Future studies should focus on evaluating the isolated and combined effects of these promising compounds, including their safety and pharmacokinetics.

Among the most promising compounds for anti-Naegleria fowleri activity and selectivity, trophocides 421, 416, 518, 46, 254, 522, 111, 113–120, 229, and cysticide 16 should be prioritized for future studies, exhibiting substantial activity at concentrations ≤10 µM, compatible with favorable pharmacokinetic and pharmacodynamic profiles.

Compound 421 (Tyrocidine-derived peptide) is a synthetic antimicrobial based on the amphipathic cyclic decapeptide Tyrocidine A from Bacillus brevis. Its amphipathic nature allows insertion into microbial membranes, forming ionic pores that disrupt integrity and induce cell death. Tyrocidine-derived peptides retain this mechanism and are active against various pathogens [153].

Compound 416 (staurosporine) an indole alkaloid from Streptomyces spp., is a potent inhibitor of serine/threonine kinases, including PKC, CDKs, and MAPKs [154]. The presence and critical roles of these kinases in FLA, including N. fowleri [155,156], likely underlie its observed trophocidal activity against N. fowleri.

Compound 518 (MMV1578884 or REP3123) is a promising antimalarial candidate identified by Medicines for Malaria Venture (MMV), functioning as an inhibitor of methionyl-tRNA synthetase (MetRS) from Plasmodium falciparum [157]. The presence of MetRS in free-living amoebae, such as A. polyphaga [158], may partly account for the anti-N. fowleri activity and mechanism of action of compound 518.

Compound 46 (K252a), an indole alkaloid from Nocardiopsis, is a kinase inhibitor that disrupts proliferation, differentiation, and cell death [159,160]. Its mechanism likely affects similar processes in amoebae, accounting for its observed naeglericidal activity.

Compound 254 (7-oxostaurosporine), an indole alkaloid derived from staurosporine, originates from Streptomyces platensis subsp. malvinus RK-1409. It is a potent PKC inhibitor that disrupts the cell cycle and induces apoptosis in various human tumor cells [161,162]. Its trophocidal effect against A. castellanii trophozoites has been attributed to increased membrane permeability and mitochondrial damage [163], mechanisms that may likewise underlie its activity against N. fowleri.

Compound 522 ((2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol) belongs to the azole class and is identified as oteseconazole, a selective inhibitor of the enzyme lanosterol 14α-demethylase (CYP51A1), which is essential for sterol biosynthesis in free-living amoebae (FLA), including Acanthamoeba spp. and Naegleria spp. This enzyme catalyzes the removal of the methyl group at the 14α position of lanosterol, a critical step in the synthesis of ergosterol, a key structural component of the cell membranes of protozoa such as Acanthamoeba and fungi [164]. The inhibition of CYP51A1 by compound 522 may therefore account for its amoebicidal effect against both Acanthamoeba and N. fowleri.

Compounds 111–120 (various 3-((4-(tert-butyl)benzyl)amino)-substituted picolinic acids) are benzylamine picolinate derivatives exhibiting potent trophocidal activity against N. fowleri, likely through interference with essential energy and signaling pathways, as well as mitochondrial or redox enzymes. These compounds were predicted to have good blood–brain barrier permeability by parallel artificial membrane permeability assay (PAMPA) [107]. Further studies are needed to clarify the precise anti-FLA mechanisms of these compounds.

Compound 16 ((E)-3-cyano-N-(4-methoxyphenyl)acrylamide) is a cyanoacrylamide belonging to the subclass of α,β-unsaturated amides. This chemical scaffold functions as a Michael acceptor, in which the electrophilic β-carbon is highly susceptible to nucleophilic attack, particularly by thiol groups of cysteine residues. Such interactions enable covalent, and in some cases reversible, inhibition of enzymes containing catalytic cysteines, including transforming growth factor-β–activated kinase 1 (TAK1) [165,166]. The behavior of molecules within this class may, therefore, partially account for the anti-N. fowleri activity observed for compound 16; nonetheless, further studies are required to elucidate its precise mechanism of action in FLA.

The top-ranked compounds active against Acanthamoeba spp. trophozoites and cysts are summarized in

Table 4. Most promising compounds with anti-Acanthamoeba spp. Activity.

Code	Concentration (µM)	Mortality (%)	IC50 µM	CT-Toxicity (µM)	Toxicity (%)	Exposure Time (h)	Cell Line
Trophozoites
498 *	0.00405	50	0.00405	0.05308	50	168	HaCaT
499 *	0.0042	50	0.0042	0.0572	50	168	HaCaT
500 *	0.0067	50	0.0067	0.129	50	168	HaCaT
501 *	0.0132	50	0.0132	0.144	50	168	HaCaT
522	0.16	50	0.16	10	50	72	A549
535	0.61	50	0.61	10	50	72	A549
377	5	76	-	10	18	24	HeLa
107	5.70	50	5.90	38.50	50	72	MRC5, LC
347	10	80	-	10	24	2	HeLa
348	10	85	-	10	18	2	HeLa
294	15.60	100	6.45	51	50	24	SRB
394	30	100	--	15	8	24	HaCaT
275	30.37	100	-	91.14	0	12	L929
299	30.77	100	-	266.16	50	6	NB1RGB, SRB
302	33.56	53	-	503.36	50	6	NB1RGB, SRB
60	50	52.70		100	19	24	HeLa
125	50	68.75	-	99.99	2	24	HaCaT
398	60.13	67.70	-	120	8	24	HaCaT
374	65.51	100	-	131	0	72	J774A.1
240	99.94	62.25	-	99.94	19	24	HaCaT
148	99.96	57.50	-	99.96	1	24	HaCaT
239	99.97	68.75	-	99.97	2	24	HaCaT
237	99.98	82.50	-	99.98	0	24	HaCaT
132	99.99	74.50	-	99.99	2	24	HaCaT
152	99.99	68.75	-	99.99	0	24	HaCaT
155	99.99	75	-	99.99	4	24	HaCaT
123	100	62.50	-	100	3	24	HaCaT
127	100	86.25	-	100	1	24	HaCaT
164	100	50.40	-	100	1.20	24	HaCaT
241	100	81.25	-	100	6.50	24	HaCaT
286	100	65	-	272.70	15	24	HeLa
128	100.01	60	-	100.01	15	24	HaCaT
133	100.01	77.50	-	100.01	0	24	HaCaT
238	100.02	75	-	100.02	8	24	HaCaT
137	100.03	68.75	-	100.03	3	24	HaCaT
130	100.06	75	-	100.06	6	24	HaCaT
138	100.06	78.75	-	100.06	15	24	HaCaT
150	100.07	68.75	-	100.07	19	24	HaCaT
279	131	83.5 (±0.5)	-	131	0	72	J774A.1
157	138.10	63	-	276.10	0	24	HaCaT
397	162.40	58	-	162.40	5	24	HaCaT
167	187.74	56	-	925.80	50	24	HaCaT
284	195.70	65	-	195.70	15	24	HaCaT
318	149 (±112)	50	149 (±112)	255	50	24	J774A.1
134	276.10	75	-	276.10	6	24	HaCaT
200	278.20	71	53.92	278.20	15	24	HBEC-5i
385	301	53	-	301	5		HBEC-5i
141	317.30	60	-	317.30	11	24	HaCaT
209	320.10	100	27.21	320.10	16	24	HBEC-5i
211	335.20	100	30.65	335.20	15	24	HBEC-5i
208	336.30	95	44.13	336.30	20	24	HBEC-5i
129	337.50	50	-	337.50	0	24	HaCaT
256	354.20	60	-	354.20	0	24	HaCaT
159	375.50	50	-	375.50	0	24	HaCaT
143	393.30	86	-	393.30	0	24	HaCaT
156	393.30	84	--	393.30	0	24	HaCaT
306	397.70	61	-	397.70	15.80	24	HBEC-5i
257	399.50	65	-	399.50	0	24	HaCaT
258	399.50	60	-	399.50	0	24
69	500	100	14.73	104.77	50	24	3T3, HeLa, MCF-7
396	745	56	-	745	4	24	HaCaT
392	3536.40	55	-	3536	2	24	HaCaT
Amphotericin B	8.65	50				72
Azithromycin	6.68	88				120
Chlorhexidine	4.28	50				96
Corifungin	200	80				120
Fluconazole	121	90				24
Miltefosine	168	50				48
Pentamidine	1.11	50				72
PHB	16.6	98				24
Propamidine	3201	100				48
Rifampicin	110	50				72
Cysts
340	7.25 (±3)	50	7.25 (±3)	550	50	96	J774A.1
338	8.25 (±2)	50	8.25 (±2)	550	50	96	J774A.1
343	11 (±0.3)	50	11 (±0.3)	550	50	96	J774A.1
342	15 (±5)	50	15 (±5)	550	50	96	J774A.1
341	22 (±4)	50	22 (±4)	550	50	96	J774A.1
339	41 (±15)	50	41 (±15)	550	50	96	J774A.1
344	57 (±5.5)	50	57 (±5.5)	550	50	96	J774A.1
384	69.95	60	-	69.95	15	24	pHCEC
275	91.14	100	-	91.14	0	24	L929
374	131	97.44	-	131	0	72	J774A.1
Chlorhexidine	642	100				96
Fluconazole	1671	100				24
Miltefosine	2460	50				48
Propamidine	885	100				48
PHB	7.87	100				24

(*)—Concentration in mg/mL. CT-toxicity—highest concentration tested for toxicity in mammalian cells. 3T3—Murine fibroblasts. A549—lung carcinoma cells. HaCaT—Human keratinocyte cells. HBEC-5i—human brain microvascular endothelial cell. HeLa—human cervical adenocarcinoma. J774A.1—murine macrophage cell line. L929: mouse fibroblasts. L6—Rat skeletal muscle cell line. MCF-7—human breast adenocarcinoma. MRC-5—Epithelial lung cells. NB1RGB—Human neonate dermal fibroblast. pHCEC—Primary human corneal epithelial cells. SRB—Sheep red blood cells. PHB—Polyhexamethylene biguanide.

. Compounds with notable trophocidal activity include 60, 69, 107, 123, 125, 127–130, 132–134, 137, 138, 141, 143, 148, 150, 152, 155–157, 159, 164, 167, 200, 208, 209, 211, 237–241, 256–258, 275, 279, 284, 286, 294, 299, 302, 306, 318, 347, 348, 374, 377, 385, 392, 394, 396–398, 498–501, 522, and 535. Those with cysticidal activity include 275, 338–344, and 374–384.

These candidates warrant prioritization for confirmatory studies as potential therapeutics or disinfectants. Compounds active at ≤10 µM are particularly promising for drug development, including assessment of administration, safety, and pharmacokinetics/pharmacodynamics in animal models of FLA infection.

Notably, compounds 275 and 374 exhibit exceptional potency, showing both trophocidal and cysticidal activity against Acanthamoeba spp. with no detectable cytotoxicity. However, because their activity occurs at concentrations above 10 µM, further studies are needed to address predictable bioavailability challenges.

Acanthamoeba infections are among the most prevalent FLA infections. These primarily include Acanthamoeba keratitis (AK), which predominantly occurs in contact lens wearers (86%) and individuals with ocular trauma (27%). Granulomatous amoebic encephalitis, another form of acanthamoebiasis, carries a mortality rate exceeding 90% [167,168]. Additionally, disseminated infections affecting various organs have been documented in the literature [1,2,3,4,5].

The absence of approved drugs that are both effective and safe for the treatment of Acanthamoeba infections underscores the urgency and importance of developing targeted therapeutics. Among the promising candidates, particular attention should be given to trophocidal compounds 498, 499, 500, 501, 522, 535, 377, 107, 347, and 348, as well as the cysticidal compounds 340 and 338.

Compounds 498, 499, 500, and 501 belong to the class of amino acid-based rhamnolipids, derived from arginine (RLMIX_Arg) or lysine (RLMIX_Lys). Owing to the cationic nature of these amino acids, these molecules can strongly interact with negatively charged components of the Acanthamoeba membrane, including lipoproteins, thereby disrupting their structural and functional integrity at the concentrations reported [169]. This membrane-targeting mechanism likely accounts for their potent amebicidal activity [62]. Assessing the potential synergistic effects of these compounds in combination with drugs acting on non-membrane targets, such as nitroxoline, could represent a promising strategy for enhancing therapeutic efficacy.

Compound 535 ([4-(benzylamino)-5-chloro-2,6-difluorobenzene-1,3-dicarbonitrile]) is included in the Pandemic Response Box and corresponds to OSU-03012 (also known as AR-12), a compound classically recognized as an inhibitor of 3-phosphoinositide-dependent kinase-1 (PDK1) [170]. OSU-03012/AR-12 has also been reported as a potent inhibitor of fungal and parasitic acetyl-CoA synthetase, while exhibiting only weak inhibition of the human isoform ACCS2 [171]. Additional mechanisms of action, including inhibition of dihydroorotate dehydrogenase (DHODH) and equilibrative nucleoside transporter 1 (ENT1) (both involved in nucleotide metabolism) have also been proposed [172].

Although there is currently no experimental evidence confirming the presence of PDK1, DHODH, or ENT1 in FLA, there is also no basis to associate the amoebicidal activity of compound 535 with the inhibition of these proteins. Conversely, the documented presence of acetyl-CoA synthetase in Naegleria gruberi [173], suggests that inhibition of this key enzyme could explain the amoebicidal activity of compound 535, further indicating its potential activity against N. fowleri.

The amoebicidal activity of compound 377 (oleic acid-AgNPs) likely results from the combined biocidal effects of oleic acid and silver nanoparticles (AgNPs). Oleic acid has been reported to exert a strong inhibitory effect on glucosyltransferase [174] and to disrupt the integrity of the plasma membrane, consistent with its well-documented bactericidal properties [175]. Silver nanoparticles, in turn, have been extensively characterized for their potent antimicrobial activity, exhibiting fungicidal [176], bactericidal [177], and antiviral effects [178]. In human colorectal carcinoma cells (HCT-116), AgNPs have been shown to induce cell death through deterioration of the cytoskeletal structure and membrane nanostructure [179]. In parasitic protozoa, AgNPs have been associated with a wide range of deleterious effects, including plasma membrane disruption, DNA damage mediated by reactive oxygen species (ROS), and inhibition of ATP synthesis due to the release of silver ions.

Although direct evidence of oleic acid-mediated inhibition of amoebic glucosyltransferase is lacking, the documented presence of this enzyme in Entamoeba histolytica [180] and A. castellanii [181], combined with evidence suggesting galactose as a molecular target in FLA [182], strongly supports the hypothesis that the acanthamoebicidal effect of compound 377 is associated with the inhibition of glucosyltransferase, synergistically enhanced by the multifaceted cytotoxic effects of AgNPs on eukaryotic cells.

Compound 107 (3-((4-(2-Cyclopropyl-6-morpholinopyrazolo[1,5-b]-pyridazin-3-yl)pyrimidin-2-yl)amino)benzonitrile) is an aminopyrimidine derivative containing a pyrazolo[1,5-b]pyridazine core. Molecules of this class have been identified as inhibitors of cyclin-dependent kinases (CDKs), particularly CDK2 and CDK4, and exhibit antiproliferative activity against Trypanosoma brucei [183]. Considering the presence of these essential regulatory proteins in FLA, including Acanthamoeba [184], it is plausible that the acanthamoebicidal activity of compound 107 arises from its inhibitory effect on CDKs.

Compound 347 is a hybrid formulation consisting of two distinct components: methyltrioctylammonium chloride and ethylene glycol. Ethylene glycol, commonly used as an antifreeze agent, is known to be toxic but exhibits lower cytotoxicity than diethylene glycol and propylene glycol in human oral squamous cell carcinoma cells [185]. Methyltrioctylammonium chloride, on the other hand, is a quaternary ammonium compound (QAC) widely employed as an environmental disinfectant. QACs possess broad-spectrum antimicrobial activity primarily associated with their ability to disrupt cell membranes through mechanisms such as pore formation, lysis, and protein denaturation [186]. Therefore, the acanthamoebicidal activity of compound 347 likely results from the combined action of both constituents, with a predominant contribution from methyltrioctylammonium chloride, whose anti-FLA activity has been previously reported [187].

Compound 348 is structurally analogous to 347, differing only by the substitution of ethylene glycol with fructose. Given that fructose functions primarily as a metabolic substrate and lacks reported amoebicidal properties, it is reasonable to infer that methyltrioctylammonium chloride is the principal active component responsible for the amoebicidal activity observed in compound 348.

Compounds 338 (methyl 4-chloro-1H-indole-3-carboxylate) and 340 (methyl 6-chloro-1H-indole-3-carboxylate) are methyl indole-3-carboxylate derivatives featuring a chlorine atom at positions 4 and 6 of the indole ring, respectively. Scaffolds of this class have been recognized as promising in drug design, exhibiting antimicrobial, antiparasitic, and antitumor activities [188]. Although both compounds remain underexplored, compound 340 has been reported to induce programmed cell death in Acanthamoeba spp. via mitochondrial dysfunction [131].

The data presented in Table 1 indicates that, among the thirteen compounds tested for their photoactive biocidal effects, eight showed promising acanthamoebicidal activity, comprising seven trophocidal and one cysticidal compound. Nonetheless, further studies are required to evaluate their selectivity and determine safe conditions for potential therapeutic applications.

Photoactivated chromophore compounds used in corneal crosslinking therapy have emerged as a promising adjuvant treatment for AK [189]. However, additional research is essential to fully realize and enhance their clinical potential.

Compound 511 (0.5 mM + 10.8 J/cm², visible light) demonstrated significant in vitro trophocidal activity and exhibited a synergistic acanthamoebicidal effect when combined with amphotericin B (1 ppm) and polyhexamethylene biguanide (100 µg/mL). However, no synergistic effect was observed when combined with voriconazole (400–1000 µg/mL) [100]. Similarly, compound 505 showed promising results in in vivo studies with rabbits, achieving complete recovery from AK in 58% of animals. However, the authors highlighted the need for further research to optimize conditions for maximum therapeutic efficacy [144]. Finally, compound 507 is one of the most extensively studied photoactive compounds, with evidence supporting its potential as an adjunctive therapy to reduce the need for therapeutic penetrating keratoplasty in AK cases [190,191,192,193]. Nonetheless, additional studies, including clinical trials are required.

Overall, the compounds highlighted throughout the article represent a significant achievement in the search for effective drugs to treat FLA infections. Developing formulations based on these compounds that incorporate nanomaterials could be a promising strategy to enhance biocidal potency and/or reduce cytotoxic effects on non-target cells, as previously demonstrated [45,76,100,129].

It has been suggested that the shape of nanomaterials influences their amoebicidal activity. For example, silver nanoparticles exhibited trophocidal and cysticidal effects, reaching inactivation rates ranging from 40% to 100%. Among these, silver nanowires (AgNWs) exhibited the highest amoebicidal efficacy, outperforming silver nanorings (AgNRs), which in turn were significantly more effective than silver nanospheres (AgNSs) [71]. Therefore, it is desirable that the shape of nanoparticles be considered in studies seeking to develop nanobased formulations of the promising compounds discussed.

Among the 537 compounds tested, 123 were generally selected as suitable candidates for the development of drugs and disinfectants based on their efficacy and low cytotoxicity. The most active against Balamuthia mandrillaris were ten trophocidal compounds (53, 83, 218, 223, 277, 358, 515, 529, 531, and 533). For N. fowleri, thirty trophocidal (13, 15, 18, 34, 47, 53, 79, 83, 96, 103, 111, 113–120, 206, 214, 218, 229, 306, 325, 358, 393, 416, 421, and 518) and four cysticidal (15, 16, 34, and 226) compounds were identified. Against Acanthamoeba spp., sixty-two trophocidal (60, 69, 107, 123, 125, 127–130, 132–134, 137, 138, 141, 143, 148, 150, 152, 155–157, 159, 164, 167, 200, 208, 209, 211, 237–241, 256–258, 275, 279, 284, 286, 294, 299, 302, 306, 318, 347, 348, 374, 377, 385, 392, 394, 396–398, 498–501, 522, and 535) and nineteen cysticidal compounds (275, 338–344, 374, and 384) were highlighted.

Limitations

The findings of this study were obtained under certain limiting factors that may have influenced the strength and direction of the results: (1) The anti-FLA potential of many compounds was based on findings from a single study (and/or tests performed on a single FLA strain) available in the literature. (2) Compounds that showed <50% anti-FLA activity in vitro were classified as unpromising; however, this does not necessarily preclude their potential to demonstrate significant anti-FLA activity and selectivity in in vivo studies. (3) Compounds with uncharacterized cytotoxicological profiles in non-target cells could not be included in the list of top-ranked compounds, despite their high anti-FLA potency.

We acknowledge that E_max, MIC, and IC₅₀ values may not fully represent a compound’s intrinsic potency, as they can be influenced by physicochemical properties like solubility and permeability. Moreover, the highest tested concentrations may be limited by toxicity or compound stability.

5. Conclusions

This study aimed to identify the most promising chemical compounds with anti-FLA activity reported in the indexed literature, recommending them for future in vitro confirmation and primarily in vivo studies to evaluate their efficacy, mechanisms of action, safety, pharmacokinetics, and pharmacodynamics (

Table A1. Codes and Names of the Most Promising Compounds with Anti-FLA Activity.

Code	Name
13	(E)-2-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-4ylidene)hydrazine-1-carbothioamide
15	(E)-3-Cyano-N-(4-methoxybenzyl)acrylamide
16	(E)-3-Cyano-N-(4-methoxyphenyl)acrylamide
18	(E)-3-phenylprop-2-enoic acid
34	1-Benzyl-5-Imino-1H-Pyrrol-2(5H)-One
46	2,3,9S,10R,11,12R-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, methyl ester
47	2,3-dimethylacridin-9(10H)-one
53	2,6-dichlorobenzonitrile
60	2-(3-Cyano-6-methyl-4,6-dip-tolyl-5,6-dihydropyridin-2(1H)-ylidene)malono-nitrile
69	2-(N-methylazepanio-1-yl)ethyl pentadecyloxycarbonylphosphonate
79	2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol
83	2-N-tert-butyl-6-chloro-4-N-ethyl-1,3,5-triazine-2,4-diamine
96	2-ethylacridin-9(10H)-one
103	3,7-Dichloro-8-quinolinecarboxylic acid
107	3-((4-(2-Cyclopropyl-6-morpholinopyrazolo[1,5-b]- pyridazin-3-yl)pyrimidin-2-yl)amino)benzonitrile
111	3-((4-(tert-butyl)benzyl)amino)-5-chloropicolinic acid
113	3-((4-(tert-butyl)benzyl)amino)-5-fluoropicolinic acid
114	3-((4-(tert-butyl)benzyl)amino)-5-methoxypicolinic acid
115	3-((4-(tert-butyl)benzyl)amino)-5-methylpicolinic acid
116	3-((4-(tert-butyl)benzyl)amino)-5-morpholinopicolinic acid
117	3-((4-(tert-butyl)benzyl)amino)-5-phenylpicolinic acid
118	3-((4-(tert-butyl)benzyl)amino)picolinic acid
119	3-((4-(tert-butyl)phenethyl)amino)-5-chloropicolinic acid
120	3-((4-(tert-butyl)phenethyl)amino)-5-methoxypicolinic acid
123	3-(2,3-Dichlorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
125	3-(2,4-Difluorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
127	3-(2,4-Dimethylphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
128	3-(2,5-Dimethoxyphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
129	3-(2,5-Dimethoxyphenyl)-8-methylquinazolin-4(3H)-one
130	3-(2,6-Diethylphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
132	3-(2-Bromophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
133	3-(2-Chlorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
134	3-(2-Iodophenyl)-8-methylquinazolin-4(3H)-one
137	3-(3,4-Dichlorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
138	3-(3,5-Dimethylphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
141	3-(3-Bromophenyl)-8-methylquinazolin-4(3H)-one
143	3-(3-Fluorophenyl)-8-methylquinazolin-4(3H)-one
148	3-(4-Bromophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
150	3-(4-Butylphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
152	3-(4-Chlorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
155	3-(4-Fluorophenyl)-6,7-dimethoxyquinazolin-4(3H)-one
156	3-(4-Fluorophenyl)-8-methylquinazolin-4(3H)-one
157	3-(4-Iodophenyl)-8-methylquinazolin-4(3H)-one
159	3-(4-Methoxyphenyl)-8-methylquinazolin-4(3H)-one
164	3-([1,1′-Biphenyl]-4-yl)-2H-benzo[b] [1,4]thiazine
167	3-Phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one
200	4-(Cycloheptanecarboxamido)phenyl benzenesulfonate
206	4-(Cyclohexanecarboxamido)phenyl dimethylsulfamate
208	4-(Cyclohexanecarboxamido)phenyl methanesulfonate
209	4-(Cyclohexanecarboxamido)phenyl methylsulfamate
211	4-(Cyclohexanecarboxamido)phenyl sulfamate
214	4-(Cyclopentanecarboxamido)phenyl 4-methylbenzenesulfonate
218	4-(biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole
223	5-(3-Chlorobenzyl)-1H-tetrazole
226	5-Imino-1-Phenyl-1H-Pyrrol-2(5H)-One
229	5-bromo-3-((4-(tert-butyl)benzyl)amino)picolinic acid
237	6,7-Dimethoxy-3-(4-(methylthio)phenyl)quinazolin-4(3H)-one
238	6,7-Dimethoxy-3-(4-methoxyphenyl)quinazolin-4(3H)-one
239	6,7-Dimethoxy-3-(m-tolyl)quinazolin-4(3H)-one
240	6,7-Dimethoxy-3-(o-tolyl)quinazolin-4(3H)-one
241	6,7-Dimethoxy-3-(p-tolyl)quinazolin-4(3H)-one
254	7-oxostaurosporine
256	8-Methyl-3-(3-(Methylthio)Phenyl)QUINAZOLIN-4(3H)-One
257	8-Methyl-3-(o-tolyl)quinazolin-4(3H)-one
258	8-Methyl-3-(p-tolyl)quinazolin-4(3H)-one
275	Cationic Steroid Antibiotic—13
277	Cellulase: (2S,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6S)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol
279	Chitosan
284	Co3(PO4)2•8H2O
286	Cobalt phosphate octahydrate (Co3(PO4)2⋅8H2O)
294	Dodecyltriphenylphosphonium Bromide
299	Ethyl(5-decyl-2-amino-1,3-thiazol-4-yl)acetate
302	Ethyl(5-octyl-2-amino-1,3-thiazol-4-yl)acetate
306	Gallic acid-ZnO
318	Isobenzofuran-1(3H)-one QOET-3
325	Isobenzofuran-1(3H)-one QOET-1
338	Methyl 4-chloro-1H-indole-3-carboxylate
339	Methyl 5-methyl-1H-indole-3-carboxylate
340	Methyl 6-chloro-1H-indole-3-carboxylate
341	Methyl 6-methyl-1H-indole-3-carboxylate
342	Methyl 7-chloro-4-fluoro-1H-indole-3-carboxylate
343	Methyl 7-fluoro-1H-indole-3-carboxylate
344	Methyl Indol-3-carboxylate
347	Methyltrioctylammonium chloride: Ethylene Glycol (1:1)
348	Methyltrioctylammonium chloride: Fructose (1:1.25)
358	N-(2,3-dihydro-2,6-dimethyl-1H-inden-1-yl)-6-(1-fluoroethyl)-1,3,5-triazine-2,4-diamine
374	Nano-chitosan
377	Oleic acid-AgNPs
384	PANI/MoS2 (1:5)
385	Patuletin
392	Polyaniline—hexagonal boron nitride (1:5)
394	Polyhomoarginine (10 homoarginines)
396	Polypyrrole
397	Polypyrrole-Co3O4
398	Polypyrrole-Co3O4-AgNPs
416	Staurosporine
421	Tyrocidine-derived peptide
498	Mono-rhamnolipids-Arginine
499	Mono-rhamnolipids-Arginine + Di-rhamnolipids-Arginine
500	Mono-rhamnolipids-Lysine
501	Mono-rhamnolipids-Lysine + Di-rhamnolipids-Lysine
515	1,1-dioxide 1-thioflavone
518	MMV1578884
522	(2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol
529	MMV1580844
531	MMV1582495
533	MMV1634399
535	MMV1582496 (4-(Benzylamino)-5-chloro-2,6-difluoro-benzene-1,3-dicarbonitrile)

). Future studies focused on drug development should prioritize the following compounds:

Against B. mandrillaris: trophocidal compounds 515 (1,1-dioxide 1-thioflavone, MMV1006203), 531 (MMV1582495), 529 (MMV1580844), and 533 (MMV1634399).

Against N. fowleri: trophocidal compounds 421 (tyrocidine-derived peptide), 416 (staurosporine), 518 (MMV1578884 or REP3123), 46 (K252a), 254 (7-oxostaurosporine), 522 ((2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol), and compounds 111–120 (a series of 3-((4-(tert-butyl)benzyl)amino)-substituted picolinic acids). The cysticidal compound 16 ((E)-3-cyano-N-(4-methoxyphenyl)acrylamide) also deserves special attention.

Against Acanthamoeba spp.: trophocidal compounds 498 (mono-rhamnolipids–arginine), 499 (mono-rhamnolipids–arginine + di-rhamnolipids–arginine), 500 (mono-rhamnolipids–lysine), 535 ([4-(benzylamino)-5-chloro-2,6-difluorobenzene-1,3-dicarbonitrile]), 377 (oleic acid–AgNPs), 107 (3-((4-(2-cyclopropyl-6-morpholinopyrazolo [1,5-b]-pyridazin-3-yl)pyrimidin-2-yl)amino)benzonitrile), 347 (methyltrioctylammonium chloride + ethylene glycol, 1:1), and 348 (methyltrioctylammonium chloride + fructose, 1:1.25), as well as cysticidal compounds 4-chloro-1H-indole-3-carboxylate and 340 (methyl 6-chloro-1H-indole-3-carboxylate).

To the best of our knowledge, this is the first comprehensive review to systematically compile and analyze in vitro studies aimed at identifying bioactive compounds with anti-FLA activity, thereby contributing to the acceleration of efforts toward the development of safe and effective therapeutic agents and disinfectants against FLA infections.