DNA Minor Groove Binding Research Articles

Targeting pathogenic Acanthamoeba castellanii using DNA minor groove binding agents: Antiamoebic properties of DNA minor groove binders

DNA minor groove binders exhibit a high degree of sequence specificity and have a variety of biological actions including antiviral, anticancer, antibacterial, and anti-protozoal properties. Since it is the location of non-covalent interactions, the minor groove of double helical B-DNA is gaining significant interest as therapeutic targets. For the purpose of this investigation, the synthesis of five novel DNA minor groove binding agents was accomplished and antiparasitic efficacies were determined against Acanthamoeba castellanii of the T4 genotype in vitro. Using amoebicidal assays, the results revealed that all inhibitors tested showed significant killing of amoebae (P<0.05). Pre-treatment of amoebae with DNA minor groove binders inhibited parasite-mediated human cell death by measuring lactate dehydrogenase release using cytopathogenicity assays. Cytotoxicity assays revealed minimal effects on human cells. As phenotypic switching leads to infection recurrence, assays revealed that the inhibitors blocked amoebae transformation. These are promising findings and suggest that DNA minor groove binders may hold promise for further research in the effective eradication of pathogenic A. castellanii.

Metal-Based Drug-DNA Interactions and Analytical Determination Methods.

DNA structure has many potential places where endogenous compounds and xenobiotics can bind. Therefore, xenobiotics bind along the sites of the nucleic acid with the aim of changing its structure, its genetic message, and, implicitly, its functions. Currently, there are several mechanisms known to be involved in DNA binding. These mechanisms are covalent and non-covalent interactions. The covalent interaction or metal base coordination is an irreversible binding and it is represented by an intra-/interstrand cross-link. The non-covalent interaction is generally a reversible binding and it is represented by intercalation between DNA base pairs, insertion, major and/or minor groove binding, and electrostatic interactions with the sugar phosphate DNA backbone. In the present review, we focus on the types of DNA-metal complex interactions (including some representative examples) and on presenting the methods currently used to study them.

Image-Based Quantitative Single-Cell Method Showed Increase of Global Chromatin Accessibility in Tumor Compared to Normal Cells.

The phenotypic plasticity of cancer cells has recently emerged as an important factor of treatment failure. The mechanisms of phenotypic plasticity are not fully understood. One of the hypotheses is that the degree of chromatin accessibility defines the easiness of cell transitions between different phenotypes. To test this, a method to compare overall chromatin accessibility between cells in a population or between cell populations is needed. We propose to measure chromatin accessibility by fluorescence signal from nuclei of cells stained with DNA binding fluorescent molecules. This method is based on the observations that small molecules bind nucleosome-free DNA more easily than nucleosomal DNA. Thus, nuclear fluorescence is proportional to the amount of nucleosome-free DNA, serving as a measure of chromatin accessibility. We optimized the method using several DNA intercalators and minor groove binders and known chromatin-modulating agents and demonstrated that chromatin accessibility is increased upon oncogene-induced transformation and further in tumor cells.

Copper(II) and Zinc(II) Complexes with Bacterial Prodigiosin Are Targeting Site III of Bovine Serum Albumin and Acting as DNA Minor Groove Binders.

The negative environmental and social impacts of food waste accumulation can be mitigated by utilizing bio-refineries' approach where food waste is revalorized into high-value products, such as prodigiosin (PG), using microbial bioprocesses. The diverse biological activities of PG position it as a promising compound, but its high production cost and promiscuous bioactivity hinder its wide application. Metal ions can modulate the electronic properties of organic molecules, leading to novel mechanisms of action and increased target potency, while metal complex formation can improve the stability, solubility and bioavailability of the parent compound. The objectives of this study were optimizing PG production through bacterial fermentation using food waste, allowing good quantities of the pure natural product for further synthesizing and evaluating copper(II) and zinc(II) complexes with it. Their antimicrobial and anticancer activities were assessed, and their binding affinity toward biologically important molecules, bovine serum albumin (BSA) and DNA was investigated by fluorescence emission spectroscopy and molecular docking. The yield of 83.1 mg/L of pure PG was obtained when processed meat waste at 18 g/L was utilized as the sole fermentation substrate. The obtained complexes CuPG and ZnPG showed high binding affinity towards target site III of BSA, and molecular docking simulations highlighted the affinity of the compounds for DNA minor grooves.

Transcriptomic analysis of MCF7 breast cancer cells treated with MGBs reveals a profound inhibition of estrogen receptor genes

Breast cancer poses a significant health risk worldwide. However, the effectiveness of current chemotherapy is limited due to increasing drug resistance and side effects, making it crucial to develop new compounds with novel mechanism of action that can surpass these limitations. As a consequence of their reversible and targeted mechanism, DNA minor groove binders (MGBs) are considered as a relatively safer and more effective alternative. In this study, transcriptomic analysis was conducted to reveal the dysregulated genes and signaling pathways in MCF7 cancer cells following treatment with novel MGB ligands to gain insights into the mechanism of action of MGBs at the molecular level. The transcriptomic results were validated using real-time PCR. The findings of this study indicate that the investigated MGBs primarily inhibit the genes associated with the estrogen receptor. Remarkably, ligand 5 showed downregulation of 34 out of the 35 genes regulated by estrogen receptor, highlighting its potential as a promising candidate for breast cancer therapy.

Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5′-WGWWCW-3′ motif

Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5′-d(CGCACTAGTGCG)-3′ and 5′-d(CGCAGTACTGCG)-3′. The ligands were carefully designed to target the DNA response element, 5′-WGWWCW-3′, the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.

Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties

Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.

Effect of Silver-Graphene Oxide-Cobalt Oxide Nanocomposite on Cytotoxic Levels in MRC-5 and HepG2 Cell Lines and Molecular Docking Studies

The cytotoxic properties of cobalt oxide (Co3O4) nanoparticles (NPs), in addition to graphene oxide (GO)-Co3O4 and silver (Ag)-GO-Co3O4 nanocomposites (NCs), were evaluated against both human healthy lung fibroblast (MRC-5) and hepatocellular carcinoma (HepG2) cell lines utilizing the XTT assay. The investigation revealed that synthesized Co3O4 NPs and NCs (GO-Co3O4 and Ag-GO-Co3O4) elicited significant cytotoxic responses in MRC-5 and HepG2 cell lines in a concentration-dependent manner. Through molecular docking analyses, it was observed that all fabricated nanomaterials exhibited DNA recognition via minor groove binding, with molecular affinities ranging from − 4.82 to -11.66 kcal/mol. Furthermore, the docking outcomes illustrated that the angular conformations of GO-Co3O4 and Ag-GO-Co3O4 conferred ‘shape-selective’ characteristics as DNA minor groove binders, leading to heightened cytotoxicity, particularly in the HepG2 cell line compared to the normal MRC-5 cell line.

Abstract 5805: A novel platform of diversified payloads to drive ADC innovation

Abstract First generation of antibody drug conjugates (ADCs) for anticancer therapy has been described more than two decades ago with gemtuzumab-ozogamicin receiving the first approval in 2000. Over the last 4 years, 8 new ADCs got FDA approval essentially due to the innovation on the linker improving the disposition in patients and the manufacturability together with the technical advantages in the engineering of biologics and the introduction of new cytotoxic payloads such as deruxtecan. In the future, next generation ADCs will be developed exploring payloads beyond the currently used cytotoxins to improve safety, efficacy and durability of responses and therefore the short- and long-term therapeutic benefit to patients. Nerviano Medical Sciences (NMS) generated a portfolio of novel and diversified payload linkers leveraging on its proprietary chemical collection that includes cytotoxic and targeted payloads. A rigorous screening funnel has been applied considering diversification in terms of MoAs, pharmacokinetic properties, mechanism of resistance and combinability with other therapies to identify the optimal match with specific tumor types and targets, generating a new class of ADCs with improved selectivity and efficacy for hard-to-treat cancers even in chemoresistant settings. NMS ADC platform incorporates 1) our patent protected payload linker NMS-P945, a duocarmycin like DNA minor groove binder and alkylating agent inducing DNA damage, acting in chemoresistant cells with bystander effect and immunogenic cell death properties and generating ADCs highly efficacious in mouse xenograft models and safe when administered in NHP, 2) novel anthracyclines (PNU-682 derivatives) with an improved stability and safety profile and 3) targeted molecules with different mechanism of action including payloads active in synthetic lethality contexts. Payload linkers with different functionalization generated from each payload have been evaluated for chemical stability, sensitivity to lysosomal protease cleavage, and conjugability to monoclonal antibodies. The ADCs, generated with proof-of-concept antibodies (e.g. Trastuzumab), were characterized in terms of Drug to Antibody Ratio (DAR), aggregation level, plasma stability, antigen binding and differential antiproliferative activity in target positive vs target negative tumor cells. The best products resulting from the screening funnel have been subjected to a multidimensional analysis taking into consideration payload properties, tumor features and antigen expression in selected populations with the aim of identifying tumor types particularly sensitive to the payload. Our multidimensional approach coupling new payload linker with innovative MoA to careful selection of patients will allow to reach unprecedented efficacy and safety profile. Citation Format: Paolo Orsini, Matteo Salsa, Simona Rizzi, Ulisse Cucchi, Daniela Faiardi, Alessia Burocchi, Antonella Ciavolella, Rosita Lupi, Fabio Gasparri, Barbara Valsasina. A novel platform of diversified payloads to drive ADC innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5805.

Evaluating the substitution effects of bis(β-iminoenolate)copper(ii) complexes on their photophysical, DNA binding/photocleavage, and cytotoxic activities

Substituent in a ligand modulate the bioactivity of the bis(β-iminoenolate)copper(ii) complexes.

Iridium(III)-based minor groove binding complexes as DNA photocleavage agents.

Transition metal complexes containing the qtpy ligand (2':4,4'':4',4'''-quaterpyridyl) are known to be DNA intercalators or minor groove binders. In this study, new tricationic iridium(III) complexes of qtpy are reported. Both [Ir(bpy)2(qtpy)]3+1 and [Ir(phen)2(qtpy)]3+2 display good water solubility as chloride salts. The complexes possess high-energy excited states, which are quenched in the presence of duplex DNA and even by the mononucleotides guanosine monophosphate and adenosine monophosphate. Further studies reveal that although the complexes bind to quadruplex DNA, they display a preference for duplex structures, which are bound with an order of magnitude higher affinities than their isostructural dicationic RuII-analogues. Detailed molecular dynamics simulations confirm that the complexes are groove binders through the insertion of, predominantly, the qtpy ligand into the minor groove. Photoirradiation of 1 in the presence of plasmid DNA confirms that this class of complexes can function as synthetic photonucleases by cleaving DNA.

Probing the role of the protonation state of a minor groove-linker histidine in Exd-Hox–DNA binding

DNA recognition and targeting by transcription factors (TFs) through specific binding are fundamental in biological processes. Furthermore, the histidine protonation state at the TF-DNA binding interface can significantly influence the binding mechanism of TF-DNA complexes. Nevertheless, the role of histidine in TF-DNA complexes remains underexplored. Here, we employed all-atom molecular dynamics simulations using AlphaFold2-modeled complexes based on previously solved co-crystal structures to probe the role of the His-12 residue in the Extradenticle (Exd)-Sex combs reduced (Scr)-DNA complex when binding to Scr and Ultrabithorax (Ubx) target sites. Our results demonstrate that the protonation state of histidine notably affected the DNA minor-groove width profile and binding free energy. Examining flanking sequences of various binding affinities derived from SELEX-seq experiments, we analyzed the relationship between binding affinity and specificity. We uncovered how histidine protonation leads to increased binding affinity but can lower specificity. Our findings provide new mechanistic insights into the role of histidine in modulating TF-DNA binding.

Synthesis and Biophysical and Biological Studies of N-Phenylbenzamide Derivatives Targeting Kinetoplastid Parasites.

The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.

Structural insights and cytotoxicity evaluation of benz[e]indole pyrazolyl-substituted amides.

Five new compounds of benz[e]indole pyrazolyl-substituted amides (2a-e) were synthesised in low to good yields via the direct amide-coupling reaction between a pyrazolyl derivative containing a carboxylic acid and several amine substrates. The molecular structures were determined by various spectroscopic methods, such as NMR (1H, 13C and 19F), FT-IR and high-resolution mass spectrometry (HRMS). X-ray crystallographic analysis on the 4-fluorobenzyl derivative (2d) reveals the amide-O atom to reside to the opposite side of the molecule to the pyrazolyl-N and pyrrolyl-N atoms; in the molecular packing, helical chains feature amide-N‒H⋯N(pyrrolyl) hydrogen bonds. Density-functional theory (DFT) at the geometry-optimisation B3LYP/6-31G(d) level on the full series shows general agreement with the experimental structures. While the LUMO in each case is spread over the benz[e]indole pyrazolyl moiety, the HOMO spreads over the halogenated benzo-substituted amide moieties or is localised near the benz[e]indole pyrazolyl moieties. The MTT assay showed that 2e, exhibited the highest toxicity against a human colorectal carcinoma (HCT 116 cell line) without appreciable toxicity towards the normal human colon fibroblast (CCD-18Co cell line). Based on molecular docking calculations, the probable cytotoxic mechanism of 2e is through the DNA minor groove binding.

Targeting genomic DNAs and oligonucleotide on base specificity: A comparative spectroscopic, computational and in vitro study

Drug discovery in targeted nucleic acid therapeutics encompass several stages and rigorous challenges owing to less specificity of the DNA binders and high failure rate in different stages of clinical trials. In this perspective, we report newly synthesized ethyl 4-(pyrrolo[1,2-a]quinolin-4-yl)benzoate (PQN) with minor groove A-T base pair binding selectivity and encouraging in cell results. This pyrrolo quinolin derivative has shown excellent groove binding ability with three of our inspected genomic DNAs (cpDNA 73 % AT, ctDNA58% AT and mlDNA 28 % AT) with varying A-T and G-C content. Notably in spite of similar binding patterns PQN have strong binding preference with A-T rich groove of genomic cpDNA over the ctDNA and mlDNA. Spectroscopic experiments like steady state absorption and emission results have established the relative binding strengths (Kabs = 6.3 × 105 M−1, 5.6 × 104 M−1, 4.3 × 104 M−1 and Kemiss = 6.1 × 105 M−1, 5.7 × 104 M−1 and 3.5 × 104 M−1 for PQN-cpDNA, PQN-ctDNA and PQN-mlDNA respectively) whereas circular dichroism and thermal melting studies have unveiled the groove binding mechanism. Specific A-T base pair attachment with van der Waals interaction and quantitative hydrogen bonding assessment were characterized by computational modeling. In addition to genomic DNAs, preferential A-T base pair binding in minor groove was also observed with our designed and synthesized deca-nucleotide (primer sequences 5/-GCGAATTCGC-3/ and 3/-CGCTTAAGCG-5/). Cell viability assays (86.13 % in 6.58 μM and 84.01 % in 9.88 μM concentrations) and confocal microscopy revealed low cytotoxicity (IC50 25.86 μM) and efficient perinuclear localization of PQN. We propose PQN with excellent DNA-minor groove binding capacity and intracellular permeation properties, as a lead for further studies encompassing nucleic acid therapeutics.

Preparation of graphene-based nanocomposites with spinel ferrite nanoparticles: Their cytotoxic levels in different human cell lines and molecular docking studies

Nanocomposite-modified structures based on reduced graphene oxide (rGO) are preferred because of their excellent advantages. Here we assessed the comparative interaction of graphene oxide (GO), chemically reduced GO (rGO), copper and nickel spinel ferrite nanoparticles (CuFe2O4 NPs and NiFe2O4 NPs) and their nanocomposites with reduced graphene oxide (rGOCuFe2O4, rGONiFe2O4 NCs). The synthesized nanomaterials were characterized by X-ray diffraction (XRD) and transmission electron microscope (TEM) analysis. The cytotoxic analysis revealed a nontoxic behavior at concentrations below 50 µg/mL in human fetal lung fibroblast (MRC-5), human hepatocellular carcinoma (HepG2), and human glioblastoma (TG98) cell lines. The IC50 values were found to be lower in rGO than that of GO in MRC-5 and T98G cell lines. 50 µg/mL and higher concentrations of rGOCuFe2O4 NCs displayed more potent cytotoxic effects in MRC-5 and HepG2 cell lines compared with the CuFe2O4 NPs. Cytotoxicity was decreased in HepG2 and T98G cell lines treated with rGONiFe2O4 NCs compared to NiFe2O4 NPs but increased in MRC-5. According to the molecular docking results, DNA binding modes of all nanomaterials were found to be minor groove recognition, while their DNA binding affinities ranged between -4.78 and -12.89 kcal/mol. Docking results showed that, based on their sharp-edged conformations, GO and rGO, and rGO-MFe2O4 (M: Cu, Ni) composites may be promising shape-selective DNA minor groove binders, with the potential to cause specific DNA damage in cancer cells.

The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic.

Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.

Investigation of the effect of structure modification of furamidine on the DNA minor groove binding and antiprotozoal activity

New analogs of the antiprotozoal agent Furamidine were prepared utilizing Stille coupling reactions and amidation of the bisnitrile intermediate using lithium bis-trimethylsilylamide. Both the phenyl groups and the furan moiety of furamidine were replaced by heterocycles including thiophene, selenophene, indole or benzimidazole. Based upon the ΔTm and the CD results, the new compounds showed strong binding to the DNA minor groove. The new analogues are also more active both in vitro and in vivo than furamidine. Compounds 7a, 7b, and 7f showed the highest activity in vivo by curing 75% of animals, and this merits further evaluation.

Methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate: A new reagent for the synthesis of N-phenylpyridinecarboxamidine (“arylimidamide”) DNA-minor groove binders from poorly nucleophilic amines

We report the synthesis and use of methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate as new reagent for the preparation of N-phenylpyridinecarboxamidines (“arylimidamides”), a class of DNA minor groove binding molecules with antiprotozoal activity. This versatile reagent allowed the access to electron-deficient halogen-containing bis(arylimidamides) that could not be obtained with the classical methods reported in the literature. With this two-step protocol, the N-Boc-protected arylimidamide intermediate, which is soluble in organic solvents, can be purified by centrifugal preparative thin layer chromatography on silica and/or by reverse-phase (C-18) chromatography. The target N-phenylpyridinecarboxamidines are obtained as salts by smooth hydrolysis of the Boc-protecting group with TFA. This methodology allows the synthesis of a pharmaceutically important class of antiparasitic compounds otherwise inaccessible.

Synthesis and Evaluation of Novel DNA Minor Groove Binders as Antiamoebic Agents.

The free-living amoeba Acanthamoeba castellanii is responsible for the central nervous infection granulomatous amoebic encephalitis and sight-threatening infection Acanthamoeba keratitis. Moreover, no effective treatment is currently present, and a combination drug therapy is used. In this study, twelve DNA minor groove binders (MGBs) were synthesized and tested for their antiamoebic activity via amoebicidal, encystation, excystation, and cytopathogenicity assays. It was found that the compounds MGB3, MGB6, MGB22, MGB24, and MGB16 significantly reduce amoeba viability to 76.20%, 59.45%, 66.5%, 39.32%, and 43.21%, respectively, in amoebicidal assays. Moreover, the compounds MGB6, MGB20, MGB22, MGB28, MGB30, MGB32, and MGB16 significantly inhibit Acanthamoeba cysts, leading to the development of only 46.3%, 39%, 30.3%, 29.6%, 27.8%, 41.5%, and 45.6% cysts. Additionally, the compounds MGB3, MGB4, MGB6, MGB22, MGB24, MGB28, MGB32, and MGB16 significantly reduce the re-emergence of cysts to trophozoites, with viable trophozoites being only 64.3%, 47.3%, 41.4%, 52.9%, 55.4%, 40.6%, 62.1%, and 51.7%. Moreover, the compounds MGB3, MGB4, and MGB6 exhibited the greatest reduction in amoeba-mediated host-cell death, with cell death reduced to 41.5%, 49.4%, and 49.5%. With the following determined, future in vivo studies can be carried out to understand the effect of the compounds on animal models such as mice.