New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

Pharmacological characterisation of 5-hydroxytryptamine-induced contractile effects in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda

The indolealkylamine 5-hydroxytryptamine (5-HT, 0.1 nM-1 μM) caused dose-dependent increases in the number of contractions observed in guts isolated from the caterpillar Spodoptera frugiperda. Of the 5-HT analogues tested for agonist action, 2-methyl-5-HT (0.1-10 μM) was a full agonist with reduced potency while α-methyl-5-HT (0.1-100 μM), 5-carboxamidotryptamine (0.1-100 μM), 5-methoxytryptamine (5-MeOT) (10 nM-10 μM), and tryptamine (1-100 μM) were partial agonists. Incubation of isolated guts with proven mammalian 5-HT receptor antagonists showed that cyproheptadine (10 nM-1 μM), MDL 72222 (1-10 μM), tropisetron (1-10 μM) and 5-benzoyloxygramine (1-10 μM) were potent non-competitive antagonists of 5-HT-induced tissue contraction. In comparison, ketanserin (0.1-1 μM) was a competitive antagonist. The mammalian selective serotonin reuptake inhibitors, clomipramine (10 nM-10 μM) and fluoxetine (10 nM-10 μM) also caused non-competitive inhibition of 5-HT-induced contraction while fluvoxamine (10 nM-10 μM) was a weak competitive antagonist. Low doses of clomipramine (0.1 μM) caused potentiation of 5-HT-induced gut contraction thereby suggesting the presence of 5-HT reuptake systems in this tissue. The contractile effects of 5-HT were inhibited by verapamil, Li⁺ and H7 and potentiated by theophylline thereby indicating that L-type Ca²⁺ channels, phosphatidylinositol second messengers and cAMP, respectively, are involved in 5-HT-induced tissue contraction. The 5-HT receptors mediating contractility in the gut of S. frugiperda have properties in common with mammalian 5-HT₂ and Drosophila 5-HT_dro2A/2B receptors. In addition, these data suggest that the tissue also contains receptors that are similar to mammalian 5-ht₆ and 5-HT₇ as well as Drosophila_dro1 receptors. However, the primary amino acid sequence of these lepidopteran 5-HT receptors will have to be elucidated before full comparisons can be made.     

Design of novel N-phenylnicotinamides as selective cyclooxygenase-1 inhibitors

A series of N-phenylnicotinamides (1-40) were designed and evaluated in vitro for their COX inhibitory activities. Most of the synthesized compounds were proved to be potent and selective inhibitors of COX-1. Compound 28 showed the most potent COX-1 inhibitory activity (COX-1 IC₅₀ = 0.68 ± 0.07 μM) and good selectivity (COX-2 IC₅₀ >100 μM). This compound may be useful as a lead compound for superior COX-1 inhibitors. On the basis of the biological results, structure-activity relationships for the COX-1-inhibitory activities of the synthesized N-phenylnicotinamides were discussed concisely.     

In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K_m ∼ 5.5 and 1.2 μM, respectively), and also oxidized norspermidine (K_m ∼ 64.0 μM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K_i value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K_i values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K_i value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited (∼85%) S. sclerotiorum mycelial growth on Czapek-Dox medium.Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Ammonium uptake kinetics in root and leaf cells of Zostera marina L.

Ammonium uptake rates and the mechanism for ammonium transport into the cells have been analysed in Zostera marina L. In the cells of this species, a proton pump is present in the plasmalemma, which maintains the membrane potential. However, this seagrass shows a high-affinity transport mechanism both for nitrate and phosphate which is dependent on sodium and is unique among angiosperms. We have then analysed if the transport of another N form, ammonium, is also dependent of sodium. First, we have studied ammonium transport at the cellular level by measurements of membrane potentials, both in epidermal root cells and mesophyll cells. And second, we have monitored uptake rates in whole leaves and roots by depletion experiments. The results showed that ammonium is taken up by a high-affinity transport system both in root and leaf cells, although two different of kinetics could be discerned in mesophyll cells (with affinity constants of 2.2 ± 1.1 μM NH₄⁺, in the range 0.01-10 μM NH₄⁺, and 23.2 ± 7.1 μM NH₄⁺, at concentrations between 10 and 500 μM NH₄⁺). However, only one kinetic could be observed in epidermal root cells, which showed a K_m = 11.2 ± 1.0 μM NH₄⁺, considering the whole ammonium concentration range assayed (0.01-500 μM NH₄⁺). The higher affinity of leaf cells for ammonium was consistent with the higher uptake rates observed in leaves, with respect to roots, in depletion experiments at 10 μM NH₄⁺ initial concentration. However, when an initial concentration of 100 μM was assayed, the difference between uptake rates was reduced, but still being higher in leaves. Variations in proton or sodium-electrochemical gradient did not affect ammonium uptake, suggesting that the transport of this nutrient is not driven by these ions and that the ammonium transport mechanism could be different to the transport of nitrate and phosphate in this species.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20 μM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K_i value of 14.2 μM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K_i value of 0.32 μM. In human CYP2C9 isoform, the K_i value of PSP was 29.5 μM and the K_i value of sulfaphenazole was 0.04 μM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K_i values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction.     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC₅₀ values from 2.33 × 10⁻⁷ M to 36.0 × 10⁻⁷ M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.     

Structure–activity relationships of analogs of 3,4,5-trimethylfuran-2(5H)-one with germination inhibitory activities

Smoke-derived butenolide compounds have, in recent years, been shown to be important germination signaling molecules, which also affect seedling growth. The butenolide 3,4,5-trimethylfuran-2(5H)-one was previously isolated from plant-derived smoke and was found to significantly reduce the effect on germination by the highly active promotor karrikinolide (KAR₁, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. In this study, 11 analogs of 3,4,5-trimethylfuran-2(5H)-one were synthesized and their effect on the germination of light-sensitive ‘Grand Rapids’ lettuce seeds (Lactua sativa cv. ‘Grand Rapids’) were evaluated. A concentration series (1 mM–1 μM) of the analogs were tested alone, or in combination with 0.01 μM KAR₁. Only two compounds were found to reduce the germination promotory effect of 0.01 μM KAR₁ in a similar manner as observed with 3,4,5-trimethylfuran-2(5H)-one, with activity ranging from 1 mM to 10 μM. Four compounds were found to have inhibitory activity at 1 mM and 100 μM. The retention of activity by some of the analogs may be useful for designing novel compounds with improved activity. Furthermore, understanding the structure–activity relationships of these compounds may be helpful in synthesizing molecular probes that can be used to further investigate the mechanism of action of these compounds in regulating seed germination.     

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E₂ is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE₂ biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE₂ biosynthesis, namely cytosolic phospholipase (cPL)A₂, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E₂ synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA₂ up to 30 μM and moderately blocked isolated COX-1 (IC₅₀ > 30 μM). However, EGCG efficiently inhibited the transformation of PGH₂ to PGE₂ catalyzed by mPGES-1 (IC₅₀ = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE₂ generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF_1α) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE₂ biosynthesis by EGCG.     

Activity of elaeochytrin A from Ferula elaeochytris on leukemia cell lines

Phytochemical investigation of the roots of Ferula elaeochytris made it possible to isolate two sesquiterpene esters, 6-anthraniloyljaeschkeanadiol (elaeochytrin A) and 4β-hydroxy-6α-(p-hydroxybenzoyloxy)dauc-9-ene (elaeochytrin B), as well as eight known compounds: 6-angeloyljaeschkeanadiol, teferidin, ferutinin, 6-(p-hydroxybenzoyl)epoxyjaeschkeanadiol, 6-(p-hydroxybenzoyl)lancerotriol, 5-caffeoylquinic acid, 1,5-dicaffeoylquinic acid and sandrosaponin IX. The cytotoxic activities of all compounds were investigated on K562R (imatinib-resistant) human chronic myeloid leukaemia and DA1-3b/M2^BCR-ABL (dasatinib-resistant) mouse leukemia cell line. Elaeochytrin A was the most active compound on both cell lines (IC₅₀ = 12.4 and 7.8 μM, respectively). It was also tested on non-resistant human promyelocytic leukemia cells (HL60, IC₅₀ = 13.1 μM) and was not toxic to normal peripheral blood mononuclear cells up to 100 μM.