RESUMO
The marine metabolite diazonamide A exerts low nanomolar cytotoxicity against a range of tumor cell lines; however, its highly complex molecular architecture undermines the therapeutic potential of the natural product. We demonstrate that truncation of heteroaromatic macrocycle in natural diazonamide A, combined with the replacement of the challenging-to-synthesize tetracyclic hemiaminal subunit by oxindole moiety leads to considerably less complex analogues with improved drug-like properties and nanomolar antiproliferative potency. The structurally simplified macrocycles are accessible in 12 steps from readily available indolin-2-one and tert-leucine with excellent diastereoselectivity (99:1 dr) in the key macrocyclization step. The most potent macrocycle acts as a tubulin assembly inhibitor and exerts similar effects on A2058 cell cycle progression and induction of apoptosis as does marketed microtubule-targeting agent vinorelbine.
Assuntos
Antineoplásicos , Apoptose , Microtúbulos , Moduladores de Tubulina , Humanos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/síntese química , Linhagem Celular Tumoral , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Estereoisomerismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Indóis/química , Indóis/farmacologia , Indóis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis , OxazóisRESUMO
Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin ßIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Colchicina/metabolismo , Tubulina (Proteína)/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Microtúbulos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico , Moduladores de Tubulina/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
The taxane class of microtubule stabilizers are some of the most effective and widely used chemotherapeutics. The anticancer activity of taxanes arises from their ability to induce tubulin assembly by selectively recognizing the curved (c-) conformation in unassembled tubulin as compared to the straight (s-) conformation in assembled tubulin. We first designed and synthesized a series of 3'N-modified taxanes bearing covalent groups. Instead of discovering covalent taxanes, we found a series of non-covalent taxanes 2, in which the 3'N side chain was found to be essential for cytotoxicity due to its role in locking tubulin in the s-conformation. A representative compound bearing an acrylamide moiety (2h) exhibited increased binding affinity to the unassembled tubulin c-conformation and less cytotoxicity than paclitaxel. Further exploration of chemical space around 2h afforded a new series 3, in which derivatives such as 3l bind more tightly to both the s- and c-conformations of tubulin compared to paclitaxel, leading to more efficient promotion of tubulin polymerization and a greater persistence of in vitro efficacy against breast cancer cells after drug washout. Although 3l also had improved in vivo potency as compared to paclitaxel, it was also associated with increased systemic toxicity that required localized, intratumoral injection to observe potent and prolonged antitumor efficacy.
Assuntos
Paclitaxel , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Paclitaxel/farmacologia , Paclitaxel/química , Taxoides/farmacologia , Taxoides/química , MicrotúbulosRESUMO
We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (-)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(-)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67-95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24-71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133â nM, compared to 1-12â nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.
Assuntos
Macrolídeos , Microtúbulos , Macrolídeos/química , Relação Estrutura-Atividade , Ligação ProteicaRESUMO
Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a ßM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.
Assuntos
Taxoides , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Taxoides/farmacologia , Taxoides/química , Taxoides/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Paclitaxel/químicaRESUMO
Invited for the cover of this issue are the groups of Professors Passarella and Pieraccini at the University of Milan, in collaboration with some of the members of TubInTrain consortium. The image depicts work with the elements of nature, in particular the destabilising effect of maytansinol (the constellation) on microtubules (the trees). Read the full text of the article at 10.1002/chem.202203431.
Assuntos
Maitansina , Microtúbulos , Pesquisa , Grupo SocialRESUMO
Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.
Assuntos
Antineoplásicos , Maitansina , Tubulina (Proteína)/metabolismo , Antineoplásicos/metabolismo , MicrotúbulosRESUMO
Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.
Assuntos
Lactonas , Tubulina (Proteína) , Lactonas/farmacologia , Tubulina (Proteína)/metabolismo , Excipientes/análise , Excipientes/metabolismo , Sítios de Ligação , Microtúbulos/metabolismoRESUMO
We describe the total synthesis of the macrodiolide C(13)/C(13')-bis(desmethyl)disorazoleâ Z through double inter-/intramolecular Stille cross-coupling of a monomeric vinyl stannane/vinyl iodide precursor to form the macrocycle. The key step in the synthesis of this precursor was a stereoselective aldol reaction of a formal Evans acetate aldol product with crotonaldehyde. As demonstrated by X-ray crystallography, the binding mode of C(13)/C(13')-bis(desmethyl)disorazoleâ Z to tubulin is virtually identical with that of the natural product disorazoleâ Z. Likewise, C(13)/C(13')-bis(desmethyl)disorazoleâ Z inhibits tubulin assembly with at least the same potency as disorazoleâ Z and it appears to be a more potent cell growth inhibitor.
Assuntos
Macrolídeos , Tubulina (Proteína) , Aldeídos , EstereoisomerismoRESUMO
Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.
Assuntos
Tratamento Farmacológico da COVID-19 , Mebendazol , Animais , Antivirais/farmacologia , Mamíferos , Mebendazol/farmacologia , Microtúbulos , SARS-CoV-2 , Tubulina (Proteína)RESUMO
Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.
Assuntos
Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias Bucais , Antineoplásicos/metabolismo , Apoptose , Autofagia , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Microtúbulos , Neoplasias Bucais/tratamento farmacológicoRESUMO
Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.
Assuntos
Maitansina , Neoplasias , Humanos , Maitansina/análogos & derivados , Microtúbulos , Tubulina (Proteína) , Moduladores de TubulinaRESUMO
Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine.
Assuntos
Antineoplásicos/síntese química , Noscapina/química , Bibliotecas de Moléculas Pequenas/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzofuranos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Isoquinolinas/química , Papaver/química , Papaver/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
We prepared a series of free NH and N-substituted dibenzonthiazines with potential anti-tumor activity from N-aryl-benzenesulfonamides. A biological test of synthesized compounds (59 samples) was performed inâ vitro measuring their antiproliferative activity against a panel of six human solid tumor cell lines and its tubulin inhibitory activity. We identified 6-(phenylsulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 6-tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide as the best compounds with promising values of activity (overall range of 2-5.4â µM). Herein, we report the dibenzothiazine core as a novel building block with antiproliferative activity, targeting tubulin dynamics.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Compostos Heterocíclicos/farmacologia , Tiazinas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazinas/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.
Assuntos
Antineoplásicos/síntese química , Proteínas de Bactérias/síntese química , Produtos Biológicos/síntese química , Depsipeptídeos/síntese química , Microtúbulos/efeitos dos fármacos , Moduladores de Tubulina/síntese química , Tubulina (Proteína)/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Colchicina/química , Colchicina/farmacologia , Cristalografia por Raios X , Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Descoberta de Drogas , Células HCT116 , Humanos , Maitansina/química , Maitansina/farmacologia , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pironas/química , Pironas/farmacologia , Taxoides/química , Taxoides/farmacologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/isolamento & purificação , Moduladores de Tubulina/farmacologia , Alcaloides de Vinca/química , Alcaloides de Vinca/farmacologiaRESUMO
Studies are described toward the synthesis of an oxazole-based analog of (-)-zampanolide (2). Construction of (-)-dactylolide analog 22 was achieved via alcohol 5 and acid 4 through esterification and Horner-Wadsworth-Emmons (HWE)-based macrocyclization; however, attempts to attach (Z,E)-sorbamide to 22 proved unsuccessful. The C(8)-C(9) double bond of the macrocycle was prone to migration into conjugation with the oxazole ring, which may generally limit the usefulness of zampanolide analogs with aromatic moieties as tetrahydropyran replacements.
RESUMO
We describe the convergent synthesis of three prototypical examples of a new class of analogues of the complex, cytotoxic marine macrolide (-)-zampanolide that incorporate an embedded N-substituted morpholine moiety in place of the natural tetrahydropyran ring. The final construction of the macrolactone core was based on a high-yielding intramolecular HWE olefination, while the hemiaminal-linked side chain was elaborated through a stereoselective, BINAL-H-mediated addition of (Z,E)-sorbamide to a macrocyclic aldehyde precursor. The synthesis of the common functionalized morpholine building block involved two consecutive epoxide openings with tosylamide and the product of the first opening reaction, respectively, as nucleophiles. Of the three morpholino-zampanolides investigated, the N-acetyl and the N-benzoyl derivatives both exhibited nanomolar antiproliferative activity, thus being essentially equipotent with the natural product. In contrast, the activity of the N-tosyl derivative was significantly reduced.
Assuntos
Antineoplásicos , Produtos Biológicos , Produtos Biológicos/farmacologia , Macrolídeos/farmacologia , Estrutura Molecular , Morfolinas/farmacologia , EstereoisomerismoRESUMO
Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.
RESUMO
Optical probes operating in the second near-infrared window (NIR-II, 1,000-1,700 nm), where tissues are highly transparent, have expanded the applicability of fluorescence in the biomedical field. NIR-II fluorescence enables deep-tissue imaging with micrometric resolution in animal models, but is limited by the low brightness of NIR-II probes, which prevents imaging at low excitation intensities and fluorophore concentrations. Here, we present a new generation of probes (Ag2S superdots) derived from chemically synthesized Ag2S dots, on which a protective shell is grown by femtosecond laser irradiation. This shell reduces the structural defects, causing an 80-fold enhancement of the quantum yield. PEGylated Ag2S superdots enable deep-tissue in vivo imaging at low excitation intensities (<10 mW cm-2) and doses (<0.5 mg kg-1), emerging as unrivaled contrast agents for NIR-II preclinical bioimaging. These results establish an approach for developing superbright NIR-II contrast agents based on the synergy between chemical synthesis and ultrafast laser processing.
Assuntos
Imagem Óptica/métodos , Fotoquímica/métodos , Corantes Fluorescentes , Nanopartículas/química , Pontos QuânticosRESUMO
BACKGROUND: Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment. METHODS: Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey's post hoc test or a two-tailed paired t test was used to determine statistical significance. RESULTS: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing. CONCLUSIONS: The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.