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Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.

International audience; Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs.

In this work we aimed to develop parasite-selective histone deacetylase inhibitors (HDAC) inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis, and biological testing of a series of 13 terephthalic acid-based HDAC inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC50: 8–>51 μm), with 11 also having sub-micromolar in vitro activity against drug-sensitive (3D7) and multidrug-resistant (Dd2) asexual blood-stage P. falciparum parasites (IC50≈0.1–0.5 μm). A subset of compounds were examined for activity against early- and late-stage P. falciparum gametocytes and P. berghei exo-erythrocytic-stage parasites. While only moderate activity was observed against gametocytes (IC50>2 μm), the most active compound (N1-((3,5-dimethylbenzyl)oxy)-N4-hydroxyterephthalamide, 1 f) showed sub-micromolar activity against P. berghei exo-erythrocytic stages (IC50 0.18 μm) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual-stage in vitro potency (IC50≈0.1 μm) and selectivity of this compound versus human cells (SI>450), suggests that 1 f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity.

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM), with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM) or S. mansoni schistosomula (IC50 > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P, Graphical abstract Image 1, Highlights • HDAC inhibitor cancer drugs have in vitro activity against P. knowlesi. • HDAC inhibitor drugs cause hyperacetylation of P. knowlesi histone H4. • HDAC inhibitor drugs are not active against Leishmania amastigotes or promastigotes. • Romidepsin inhibited S. mansoni adult worm parings and egg production. • Oral Panobinostat has better activity in P. berghei infected mice than Vorinostat.

Malaria is one of the most significant tropical diseases and remains a major challenge due to the lack of a broadly effective vaccine and parasite resistance to current drugs. This means there is a need for new drug candidates with novel modes of action. Aromatic bisamidines, such as furamidine (DB75), were initially developed as anti-Trypanosoma agents however as clinical trials with furamidine highlighted potential side effects they were not pursued further in that setting. Despite apparent cytotoxicity liabilities the potency of furamidine against Plasmodium falciparum makes it a promising scaffold for the development of new anti-Plasmodium agents with improved selectivity. In this study a bisamidine compound series based on furamidine was synthesized by introducing modifications at the furan core structure and terminal amidine groups. The activity of the derived compounds was tested in vitro against drug sensitive and resistant P. falciparum lines and a human cell line (HEK293 cells) to generate anti-Plasmodium structure-activity relationships and to provide preliminary selectivity data.

The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [ 3 H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC 50 s) consistent with those obtained with P. falciparum . This assay provides a platform to use P. knowlesi in vitro for drug discovery.

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. RNA interference (RNAi), a powerful tool in functional genomics to selectively silence messenger RNA (mRNA) expression, can be harnessed to rapidly develop novel drugs against any disease target. The ability of synthetic small interfering RNA (siRNA) to effectively silence genes in vitro and in vivo, has made them particularly well suited as a drug therapeutic. However, since naked siRNA is unable to passively diffuse through cellular membranes, delivery of siRNA remains the major hurdle to fully exploit the potential of siRNA technology. Here pH-sensitive carbonate apatite has been developed to efficiently deliver siRNA into the mammalian cells by virtue of its high affinity interactions with the siRNA and the desirable size of the resulting siRNA/apatite complex for effective cellular endocytosis. Moreover, following internalization by cells, siRNA was found to be escaped from the endosomes in a time-dependent manner and finally, more efficiently silenced reporter genes at a low dose than commercially available lipofectamine. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite resulted in the significant death of cancer cells, suggesting that the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy.

Yong Tsuey Li,1 Ming Jang Chua,1 Anil Philip Kunnath,1 Ezharul Hoque Chowdhury,1,21Faculty of Medicine and Health Science, International Medical University (IMU), No 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia; 2Jeffrey Cheah School of Medicine and Health Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University Kuala Lumpur, MalaysiaBackground: Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs.Methods and results: We report that carbonate apatite-mediated delivery of the siRNAs targeting ABCG2 and ABCB1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose-dependently enhanced chemosensitivity to doxorubicin, paclitaxel and cisplatin, the traditionally used chemotherapeutic agents. Moreover, codelivery of two specific siRNAs targeting ABCB1 and ABCG2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells, indicating the reversal of ABC transporter-mediated multidrug resistance.Conclusion: The delivery concept of multiple siRNAs against ABC transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer.Keywords: carbonate apatite, siRNA, gene expression, transfection, breast cancer, ABC transporter, multidrug resistance, chemosensitivity

Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a ‘protein aggregate’ using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the ‘protein aggregate’ for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to properly treat critical human diseases like cancer. The ability of synthetically designed small interfering RNA (siRNA) to effectively silence genes at post-transcriptional level has made them attractive options in targeted therapeutics. However, naked siRNA being unable to passively diffuse through cellular membranes, poses difficulty in fully exploiting the potential of the technology. pH-sensitive carbonate apatite has been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and effective cellular endocytosis. Moreover, internalized siRNA has been found to escape from the endosomes in a time-dependent manner and effectively silenced reporter gene expression. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite has resulted in significant death of cervical cancer cells. Moreover, delivery of siRNA against cyclin B1 gene has led to the sensitization of the cancer cells to both cisplatin and doxorubicin at a particulat drug concentration. Thus, the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy using siRNA therapeutics.