Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

The enzymes in COVID-19: A review.

COVID-19 brought a scientific revolution since its emergence in Wuhan, China, in December 2019. Initially, the SARS-CoV-2 virus came to attention through its effects on the respiratory system. However, its actions in many other organs also have been discovered almost daily. As enzymes are indispensable to uncountable biochemical reactions in the human body, it is not surprising that some enzymes are of relevance to COVID-19 pathophysiology. Past evidence from SARS-CoV and MERS-CoV outbreaks provided hints about the role of enzymes in SARS-CoV-2 infection. In this setting, ACE-2 is an enzyme of great importance since it is the cell entry receptor for SARS-CoV-2. Clinical data elucidate patterns of enzymatic alterations in COVID-19, which could be associated with organ damage, prognosis, and clinical complications. Further, viral mutations can create new disease behaviors, and these effects are related to the activity of enzymes. This review will discuss the main enzymes related to COVID-19, summarizing the findings on their role in viral entry mechanism, the consequences of their dysregulation, and the effects of SARS-CoV-2 mutations on them.

Magnetite-levan nanoparticles for lectin purification: a single-step strategy for protein isolation from the seeds extract of the plant Cratylia mollis

The ability to reversibly bind carbohydrates is an incredible property from lectins. Such characteristic has led these molecules to be employed in several applications involving medical research and biotechnology. Generally, these proteins follow several steps towards purification. Here, the synthesis, physical characterization, and use of levan-coated magnetite nanoparticles (MNPs-levan) for lectin isolation is described. Canavalia ensiformis and Cratylia mollis were used as sources of Concanavalin A and Cramoll, respectively, that were purified by using MNPs-levan. Mass spectrometry, SDS-PAGE, and hemagglutinating activity were employed to assess the efficiency of the process. Moreover, by using mass spectrometry approaches, a novel lectin, similar to Canavalin, was also identified for C. mollis, corroborating the advantages of using nanoparticles over microparticles. MNPs-levan could also be recycled, making this a low-cost, scalable process that can be efficiently employed over crude samples.

A simple method for obtaining human albumin and its use for in vitro interaction assays with indole-thiazole and indole-thiazolidinone derivatives.

This work aimed to develop a simple and low-cost method to obtain human serum albumin (HSA) and its consequent application for in vitro drug interaction assays. The HSA was purified by classic principles of plasma precipitation and thermocoagulation, using a multiple-stage fractionation. The quality of the final product was assessed by electrophoresis, protein dosage by the Lowry method and the pharmacopeial thermal stability. At the end, an isotonic solution of HSA with a total protein concentration of 2.7 mg·mL-1 was obtained, which was visualized as a single band corresponding to the molecular weight of 66 kDa. After the thermal stability test, there was no indication of turbidity or color change of the solution. Finally, the HSA was useful for interaction assays with indole-thiazole and indole-thiazolidinone derivatives through UV-vis absorption and fluorescence spectroscopic studies, as well as by docking molecular analysis. Derivatives quenched the intrinsic fluorescence of HSA, disrupted the tryptophan residues microenvironment, and probably bind at Sudlow's site I. Therefore, the simplified methodology developed in this work proved to be effective in obtaining HSA that can be applied to research goals including drug interaction assays.

Novel indole-thiazole and indole-thiazolidinone derivatives as DNA groove binders.

In this study, we report the synthesis of eight novel indole-thiazole and indole-thiazolidinone derivatives, as well as their ability to interact with DNA, analysed through the UV-vis absorption, fluorescence, circular dichroism (CD), viscosity techniques and molecular docking. The ctDNA interaction analysis demonstrated different spectroscopic effects and the affinity constants (Kb) calculated by the UV-vis absorption method were between 2.08 × 105 and 6.99 × 106 M-1, whereas in the fluorescence suppression constants (Ksv) ranged between 0.38 and 0.77 × 104 M-1 and 0.60-7.59 × 104 M-1 using Ethidium Bromide (EB) and 4',6-Diamidino-2-phenylindole (DAPI) as fluorescent probes, respectively. Most derivatives did not alter significantly the secondary structure of the ctDNA according to the CD results. None of the compounds was able to change the relative viscosity of the ctDNA. These results prove that compounds interact with ctDNA via groove binding, which was confirmed by A-T rich oligonucleotide sequence assay with compound JF-252, suggesting the importance of both the phenyl ring coupled to C-4 thiazole ring and the bromo-unsubstituted indole nucleus.

COVID-19 therapy: What weapons do we bring into battle?

Urgent treatments, in any modality, to fight SARS-CoV-2 infections are desired by society in general, by health professionals, by Estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding COVID-19: knowledge is the means to discover or to produce an effective treatment against this global disease. Scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of 2020 with over 25,000 published articles related to the new coronavirus. Three great lines of publications related to COVID-19 were identified for building this article: The first refers to knowledge production concerning the virus and pathophysiology of COVID-19; the second regards efforts to produce vaccines against SARS-CoV-2 at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat COVID-19 by drug repurposing. In this review, the drugs that have been repurposed so far are grouped according to their chemical class. Their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions. This can help identifying anti-SARS-CoV-2 promising therapeutic agents.

Identification of blood plasma proteins using heparin-coated magnetic chitosan particles.

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe2+/Fe3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe3O4 in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 µm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability.

Novel 4-quinoline-thiosemicarbazone derivatives: Synthesis, antiproliferative activity, in vitro and in silico biomacromolecule interaction studies and topoisomerase inhibition.

Twelve 2-(quinolin-4-ylmethylene) hydrazinecarbothioamide derivatives were synthetized and their biological properties were investigated, among which, the ability to interact with DNA and BSA through UV-Vis absorption, fluorescence, Circular Dichroism, molecular docking and relative viscosity, antiproliferative activity against MCF-7 and T-47D mammary tumor cells and RAW-264.7 macrophages and inhibitory capacity of the enzyme topoisomerase IIα. In the binding study with DNA and BSA, all the compounds displayed affinity for interaction with both biomolecules, especially JF-92 (p-ethyl-substituted), with binding constant of 1.62 × 106 and 1.43 × 105, respectively, and DNA binding mode by intercalation. The IC50 values were obtained between 0.81 and 1.48 µM and topoisomerase inhibition results in 10 µM. Thus, we conclude that the reduction of the acridine to quinoline ring did not disrupt the antitumor action and that substitution patterns are important for biomolecule interaction affinity as they demonstrate the potential of these compounds for anticancer therapy.

Topoisomerase inhibition and albumin interaction studies of acridine-thiosemicarbazone derivatives.

In the present study, acridine-thiosemicarbazones (ATD) derivatives were tested for their interaction properties with BSA through UV-Vis absorption and fluorescence spectroscopic studies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated after the derivatives were added to the BSA. Values for the binding constant (Kb) ranged from 1.62 × 104 to 8.71 × 105 M-1 and quenching constant (KSV) from 3.46 × 102 to 7.83 × 103 M-1 indicating a good affinity to BSA protein. Complementary, two compounds were selected to assess their inhibition activity against topoisomerase IIα enzyme, of which derivative 3a presented the best result. Moreover, to evaluate protein-ligand interactions, as well as the antitopoisomerase potential of these compounds, tests of molecular modeling were performed between all compounds using the albumin and Topoisomerase IIα/DNA complex. Finally, in silico studies showed that all derivatives used in this research displayed good oral bioavailability potential.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.

Lectin-carbohydrate complex evaluation by chemiluminescence.

In order to characterize the affinity between specific carbohydrate-binding proteins such as lectins, a model is proposed to study these interactions using a polysaccharide membrane to simulate such adsorption. Here, lectin-carbohydrate interactions were chemiluminescently investigated using lectins conjugated to acridinium ester (AE) and polysaccharides composed of their respective specific carbohydrates. The lectin-AE conjugates were incubated with discs (0.0314-0.6358 cm2) of phytagel, chitosan and carrageenan. The complex formation chemiluminescently detected followed the Langmuir isotherm from which constants were estimated. The association constant (Ka) and maximum binding sites on the membranes were 2.4 × 10-7 M-1 ±â€¯0.8 × 10-7 M-1 and 1.3 × 10-3 mol. mg-1 ± 0.3 × 10-3 mol. mg-1 (Con A); 0.9 × 10-6 M-1 ±â€¯0.4 × 10-6 M-1 and 0.021 × 10-3 mol. mg-1 ± 0.003 × 10-3 mol. mg-1 (WGA) and 2.0 × 10-6 M-1 ±â€¯0.9 × 10-6 M-1 and 0.069 × 10-3 mol. mg-1 ± 0.010 × 10-3 mol. mg-1 (PNA). The proposed model might be useful to study binding affinity and estimate the amount of binding not limited by the sugar content in the membrane.

Synthesis of novel indole derivatives as promising DNA-binding agents and evaluation of antitumor and antitopoisomerase I activities.

Molecules bearing indole nucleus present diverse biological properties such as antitumor and anti-inflammatory activities that can be associated both to DNA and protein interactions. This study focused on the synthesis of new indole derivatives with thiazolidines and imidazolidine rings condensed as side chains as well as the evaluation of their ability to interact with the DNA and antitumor and topoisomerase inhibition activities. All derivatives were successfully synthesized and their structures were elucidated by mass spectrometry (MS), infrared (IR), spectroscopy 1H NMR, 13C NMR, COSY 1H-1H and HSQC 1H-13C. The antitumor activity was evaluated against different cancer cell lines using the antiproliferative MTT assay. DNA binding ability was analyzed by absorption spectroscopy and fluorescence technique using ethidium bromide (EB) as a fluorescent probe. Changes were observed in spectroscopic properties of the compounds after interacting with ctDNA (calf thymus DNA), with hypochromic and hyperchromic effects, besides blue or red shifts in the maxima of spectra. The indole derivative 5-(1H-Indol-3-ylmethylene)-thiazolidin-2,4-dione (4c) presented the best results in antitumor assay against the breast line tested (T47D), with IC50 value lower than the positive control, doxorubicin (1.93 and 4.61 µM, respectively). On the other hand, the compound 3-amino-5-(1H-indol-3-ylmethylene)-2-thioxo-thiazolidin-4-one (4a) was active against leukemia cell lines (HL60 and K562) with the high value of the DNA binding constant, Kb of 5.69 × 104. However, this compound (4a) did not inhibit the topoisomerase-I activity evaluated by relaxation assay. These results show that the indole nucleus contribute to the incorporation of molecules into the DNA. Moreover, it was highlighted that basic side chains, such as thiazolidines and imidazolidines, and free amino group, are relevant for design of promising antitumor and DNA binding compounds.

Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides' obtainment.

This work reports an optimization of protease from Penicillium aurantiogriseum immobilization on polyaniline-coated magnetic nanoparticles for antioxidant peptides' obtainment derived from bovine casein. Immobilization process was optimized using a full two-level factorial design (24) followed by a response surface methodology. Using the derivative, casein was hydrolyzed uncovering its peptides that were sequenced and had antioxidant properties tested through (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) radical scavenging and hydrogen peroxide scavenging assays. Optimal conditions for immobilization were 2 hr of immobilization, offered protein amount of 200 µg/mL, immobilization pH of 6.3 and 7.3 hr of activation. Derivative keeps over 74% of its original activity after reused five times. Free and immobilized enzyme casein hydrolysates presented similar peptide mass fingerprints, and prevalent peptides could be sequenced. Hydrolysates presented more than 2.5× higher ROS scavenging activity than nonhydrolyzed casein, which validates the immobilized protease capacity to develop casein-derived natural ingredients with potential for functional foods.

Dimethyl-2-[(acridin-9-yl)methylidene]-malonate as fluorescent probe for histochemical analysis.

Fluorescent compounds have been widely used for biomolecule labeling in cytochemistry and histochemistry analysis. Here, it is described the optical properties of dimethyl 2-[(acridin-9-yl)methylidene]-malonate (LPSF/IP-81), an acridine derivative. This compound was conjugated to Concanavalin A (Con A) lectin and applied as sugar probe in lectin histochemistry. Evaluation of luminescent properties showed that LPSF/IP-81 is photoluminescent with excitation at 360 nm and emission at 428 nm. Con A hemagglutinating activity and LPSF/IP81 photoluminescence were unaltered after conjugation. Circular dichroism of Con A-LPSF/IP81 conjugate showed the maintenance of the Con A structure. Lectin histochemistry with Con A-LPSF/IP81 conjugate demonstrated different pattern recognition studying normal, fibroadenoma, and invasive ductal carcinoma of human breast. These findings indicate that LPSF/IP-81 can be proposed as an alternative probe for histochemical analysis.

Chemiluminescent detection of glycocode alterations in hepatic granulomatous lesions of experimental schistosomiasis.

This work describes the glycophenotype evaluation of mice liver granulomatous lesion caused by infection of Schistosoma mansoni by using lectins labeled to acridinium ester (AE). The employed lectins were Concanavalin A (Con A), wheat germ agglutinin (WGA) and Sambucus nigra agglutinin (SNA) that specifically recognize α-D-glucose/mannose, N-acetyl-D-glucosamine and α-NeuNAc-[2→6]-Gal/GalNAc. The chemiluminescence expressed in relative light unit (RLU) obtained from the hepatic granuloma tissues (0.25 cm2) treated with the lectins-AE was compared with control tissues. Con A-AE infected tissues showed higher statistically significant values (1,501,182 ± 163,450 RLU) compared with the control tissue (575,280 ± 97,216 RLU). WGA-AE results also showed higher values (189,654 ± 20,686 RLU) than that found for the controls (82,878 ± 24,411). SNA-AE results did not present statistical difference between granulomatous tissues (198,990 ± 15,131) and controls (167,290 ± 25,194). There is a significant increase in glucose/mannose residues and N-acetyl-D-glucosamine in hepatic granuloma caused by S. mansoni, while the sialic acid remains virtually unchanged. The understanding of schistosome glycophenotype is relevant for the development of new diagnostic methods for schistosomiasis, design of new drug targets and preparation of glycan-based vaccines.

Synthesis and characterisation of magnetised Dacron-heparin composite employed for antithrombin affinity purification.

Human antithrombin is a blood derivative widely used in the treatment of coagulation dysfunction. Affinity chromatography using heparin (HEP) derivatives is usually used for antithrombin purification. In this study, an affinity procedure based on a magnetic Dacron-HEP composite is proposed. Dacron was firstly converted to Dacron-hydrazide and magnetised by co-precipitation with of Fe2+/Fe3+ (mDAC). HEP was activated by carbodiimide and N-hydroxysuccinimide and covalently linked to mDAC (mDAC-HEP). EDX and infrared spectra analyses confirmed each synthesis step of mDAC-HEP. This composite exhibited superparamagnetism behaviour. Human plasma was incubated with mDAC-HEP (fresh and stored over a long period) and washed with phosphate buffer containing increasing concentrations of NaCl. Human plasma antithrombin activity was reduced by approximately 20% in the presence of the 1.0M NaCl fraction, and this eluate was able to prolong coagulation time (aPTT) using both preparations. Electrophoresis of the eluates revealed bands corresponding to the expected size of antithrombin (58kDa). The mDAC-HEP particles are reusable. This method presents the following advantages: easy, low-cost synthesis of the composite, magnet-based affinity purification steps, and reusability.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60µM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

New spiro-acridines: DNA interaction, antiproliferative activity and inhibition of human DNA topoisomerases.

Two new spiro-acridines were synthesized by introducing cyano-N-acylhydrazone between the acridine and phenyl rings followed by spontaneous cyclization. The final compounds (E)-1'-(benzylideneamino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-01) and (E)-1'-((4-methoxybenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-02) were evaluated for their interactions with calf thymus DNA, antiproliferative and human topoisomerase I and IIα inhibitory activities. Both compounds presented ability to bind DNA. The binding constant determined by UV-vis spectroscopy was found to be 104M-1. Antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. The compound did not present Topo I inhibitory activity. However, both derivatives displayed topoisomerase IIα inhibitory activity comparable to amsacrine, and AMTAC-02 was more potent than AMTAC-01 with methoxy substituent group on phenyl ring. This study demonstrates that the new derivatives are promising molecules with topoisomerase IIα inhibitory and antiproliferative activities.

PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography / PVA-Glutaraldeído como suporte para imobilização de lectina e cromatografia de afinidade

Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA) and glutaraldehyde (GA) were used as a support for Concanavalin A (Con A) covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG). Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL) . These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

Lectinas imobilizadas são uma poderosa ferramenta biotecnológica para a separação e isolamento de glicoconjugados. No presente trabalho álcool polivinílico (PVA) e glutaraldeído (GA) foram utilizados como um suporte para a imobilização covalente da Concanavalina A (Con A) e para aprisionamento da goma de semente de Parkia pendula (PpeG). A eficiência da imobilização da Con A foi aproximadamente 30 % e a concentração mínima de glucose capaz de eluir a fetuína da coluna foi 0,6 M. Coluna de PVA - GA - PpeG foi eficientemente reconhecida pela lectina de P. pendula (PpeL) pura. Estes resultados indicam que a rede interpenetrada de PVA-GA mostrou-se um suporte eficiente para a imobilização covalente de lectina e como matriz de cromatografia de afinidade após aprisionamento de PpeG.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.