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The current “gold standard” therapy is levodopa (L-DOPA) due to its effectiveness and ability to permeate the blood-brain barrier (BBB). However, long-term treatment with L-DOPA can lead to serious side effects and increase oxidative stress, thereby worsening the disease [2]. The oxidative stress caused by either L-dopa or dopamine could be lessened by the combination of antioxidants – one that promises is selenium (Se) – an essential trace element that can protect from oxidative damage through selenoproteins [3]. Direct use of Se as an antioxidant supplement is toxic due to a narrow therapeutic index, so Se should be applied in other therapeutical forms. The use of Se in the form of nanoparticles (SeNPs) may elicit the same beneficial effects with reduced toxicity [4]. This study tested the safety of SeNPs as L-DOPA carrier using differentiated neuroblastoma (SH- SY5Y) cells as in vitro model of Parkinson`s disease [5]. SeNPs were prepared via reduction of sodium selenite by L-ascorbic acid and functionalization by polysorbate 20 (SeTWEEN) and poly(vinylpyrrolidone) (SePVP). The shape, size, and surface charge of SeNPs were determined with transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements, respectively. Cell viability and apoptosis induction test were performed using flow cytometry, while oxidative stress was determined by measuring the concentration od reactive oxygen species, glutathione (GSH) and mitochondrial membrane potential. In all experiments L-DOPA loaded SeNPs were compared with SeNPs and L-DOPA alone. L- DOPA loaded SeNPs (1ppm) were non-toxic to differentiated SH-SY5Y cells after 24-hour treatment, while L-DOPA reduced cell viability at the 100 μM, but not at 50 μM concentration. L-DOPA induced oxidative stress at the concentration of 100 µM which was effectively reduced after combining with SeNPs (1 ppm). Results suggest that SeNPs could be a promising approach for the reduction of toxicity caused by L-DOPA.

Silver nanoparticles (AgNPs) show a plethora of possible applications due to their antimicrobial properties. Different coatings of AgNPs are used in order to increase stability, availability and activity. However, the question about the toxicity after prolonged exposure still remains. Here, we show that different surface coatings affect in vitro toxicity and internalisation of AgNPs in porcine kidney (PK15) cells. AgNPs coated with cetyltrimethylammonium bromide (CTAB), poly (vinylpyrrolidone) (PVP), sodium bis(2- ethylhexyl)-sulfosuccinate (AOT), poly-L-lysine (PLL), and bovine serum albumin (BSA) were toxic at the concentration of 10 mg Ag/L and higher. The toxicity increased in the following manner: PVP- AgNPs < CTAB-AgNPs < PLL-AgNPs < AOT-AgNPs < BSA- AgNPs. All types of AgNPs were internalised by the PK15 cells in a dose-dependent manner with greater internalisation of AgNPs bearing positive surface charge. Transmission electron microscopy (TEM) experiments showed that AgNPs were located in the lysosomal compartments, while the co-treatment with known inhibitors of endocytosis pathways suggested macropinocytosis as the preferred internalisation pathway. When inside the cell, all types of AgNPs induced the formation of reactive oxygen species while decreasing the concentration of the cell’s endogenous antioxidant glutathione. The comet assay indicated possible genotoxicity of tested AgNPs starting at the concentration of 2 mg Ag/L or higher, depending on the surface functionalisation. This study demonstrates the toxicity of AgNPs pointing to the importance of biosafety evaluation when developing novel AgNPs- containing materials.

Graphical abstract In the present study, we report the design and synthesis of novel amide-type hybrid molecules based on anthranilic acid and quinoline or β-carboline heterocyclic scaffolds. Three types of biological screenings were performed: (i) in vitro antiproliferative screening against a panel of solid tumor and leukemia cell lines, (ii) antiviral screening against several RNA viruses, and (iii) anti-quorum sensing screening using gram-negative Chromobacterium violaceum as the reporter strain. Antiproliferative screening revealed a high activity of several compounds. Anthranilamides 12 and 13 with chloroquine core and halogenated anthranilic acid were the most active agents toward diverse cancer cell lines such as glioblastoma, pancreatic adenocarcinoma, colorectal carcinoma, lung carcinoma, acute lymphoblastic, acute myeloid, chronic myeloid leukemia, and non-Hodgkin lymphoma, but also against noncancerous cell lines. Boc-protected analogs 2 and 3 showed moderate activities against the tested cancer cells without toxic effects against noncancerous cells. A nonhalogenated quinoline derivative 10 with N-benzylanthranilic acid residue was equally active as 12 and 13 and selective toward tumor cells. Chloroquine and quinoline anthranilamides 10–13 exerted pronounced antiviral effect against human coronaviruses 229E and OC43, whereas 12 and 13 against coronavirus OC43 (EC50 values in low micromolar range; selectivity indices from 4.6 to > 10.4). Anthranilamides 14 and 16 with PQ core inhibited HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively. Compound 13 displayed significant anti-quorum/biofilm effect against the quorum sensing reporter strain (IC50 of 3.7 μM) with no apparent bactericidal effect. Supplementary Information The online version contains supplementary material available at 10.1007/s11030-021-10347-8.

Drug safety assessment in the early phases of drug discovery is critical to facilitate the rapid development of novel therapeutics. Recently, teleost zebrafish (Danio rerio) has emerged as a promising vertebrate model for the assessment of drug safety. Zebrafish is a convenient model because of its small size, high fecundity, embryo transparency, and ex utero development. In this study, we developed a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) method applied to zebrafish larvae to investigate safety and metabolism of sahaquine (Sq), an anticancer agent inhibiting histone deacetylase 6. This technique improves on prior studies using liquid chromatography-mass spectrometry (LC-MS) by adding analysis of the drug spatial distribution. Using this method, it was determined that Sq dissolved in fish water (1-2000 μM) did not reach the larval body and was mainly distributed throughout the yolk. High Sq concentration (800 μM) administered intravenously allowed the compound to reach the larval body but did not induce phenotypic abnormalities. Sq was metabolized into its glucuronidated form within 24 h and was excreted within 72 h. MALDI MSI was instrumental in showing that Sq-glucuronide was mainly formed in the gut and slightly in yolk syncytial layer, and provided valuable insights into xenobiotics elimination in zebrafish larvae. This study indicates that Sq has a good safety profile and merits further investigations in other disease models. In addition, the optimized MALDI MSI protocol provided here can be widely applied to study distribution and metabolic fate of other structurally related molecules.

Drug safety assessment in the early phases of drug discovery is critical to facilitate the rapid development of novel therapeutics. Recently, teleost zebrafish (Danio rerio) has emerged as a promising vertebrate model for the assessment of drug safety. Zebrafish is a convenient model because of its small size, high fecundity, embryo transparency, and ex utero development. In this study, we developed a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) method applied to zebrafish larvae to investigate safety and metabolism of sahaquine (Sq), an anticancer agent inhibiting histone deacetylase 6. This technique improves on prior studies using liquid chromatography-mass spectrometry (LC-MS) by adding analysis of the drug spatial distribution. Using this method, it was determined that Sq dissolved in fish water (1–2000 μM) did not reach the larval body and was mainly distributed throughout the yolk. High Sq concentration (800 μM) administered intravenously allowed the compound to reach the larval body but did not induce phenotypic abnormalities. Sq was metabolized into its glucuronidated form within 24 h and was excreted within 72 h. MALDI MSI was instrumental in showing that Sq-glucuronide was mainly formed in the gut and slightly in yolk syncytial layer, and provided valuable insights into xenobiotics elimination in zebrafish larvae. This study indicates that Sq has a good safety profile and merits further investigations in other disease models. In addition, the optimized MALDI MSI protocol provided here can be widely applied to study distribution and metabolic fate of other structurally related molecules.

In this paper, we report the synthesis of novel hybrids 2–14 based on itaconic acid and fluoroaniline, pyridine, indole and quinoline scaffolds. Itaconic acid is a naturally occurring compound with a Michael acceptor moiety, a key structural feature in several anticancer and antiviral drugs, responsible for the covalent binding of a drug to the cysteine residue of a specific protein. Aromatic parts of the hybrids also come from the substances reported as anticancer or antiviral agents. The synthetic route employed to access the amido-ester hybrids 2–13 used monomethyl itaconate or monomethyl itaconyl chloride and corresponding amines as the starting materials. Dimers 14 and 15 with two aminoindole or mefloquine moieties were prepared from itaconic acid and corresponding amino derivative, using standard coupling conditions (HATU/DIEA). All hybrids exerted anticancer effects in vitro against almost all the tumour cell lines that were evaluated (MCF-7, HCT 116, H460, LN-229, Capan-1, DND-41, HL-60, K-562, Z-138). Solid tumour cells were, in general, more responsive than the haematological cancer cells. The MCF-7 breast adenocarcinoma cell line appeared the most sensitive. Amido-ester 12 with chloroquine core and mefloquine homodimer 15 showed the highest activity with GI50 values between 0.7 and 8.6 µM. In addition, compound 15 also exerted antiviral activity against Zika virus and Coxsackievirus B4 in low micromolar concentrations. Graphic abstract Electronic supplementary material The online version of this article (10.1007/s11030-020-10147-6) contains supplementary material, which is available to authorized users.

Quorum sensing inhibitors (QSIs) that specifically interfere with bacterial cell-to-cell communication are considered as an alternative approach to conventional antibacterial therapy. In our study, a set of twenty-six fumardiamides with a quinoline head-group were evaluated as potential QSIs. Two strains of Gram-negative Chromobacterium violaceum (violacein-producing strain ATCC31532 and violacein-negative, mini-Tn5 mutant derivative CV026) were used as QS reporters for testing anti-QS and bactericidal activity of various quinoline fumardiamides. The initial screening of eighteen fumardiamides with primaquine, mefloquine and chloroquine scaffolds identified chloroquine derivatives as the most promising QSIs. Tail-group optimization of chloroquine fumardiamides led to the most active compounds 27, 29 and 30 bearing aminoethyl or piperidine moieties. At 400 mu M concentration, these compounds inhibited the QS of C. violaceum strains in a manner similar to quercetin (the model QSI), while at the 40 mu M concentration their inhibitory effect was twice less than that of quercetin. As none of the compounds displayed a bactericidal effect and that the QS inhibition was specific to the CV026 strain, our findings indicate that the structurally optimized chloroquine derivatives could function as quorum quenching (QQ) agents with a potential to block the signaling without entering the cell. In conclusion, our finding provides an important step toward the further design of agents targeting cell-to-cell communication.

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1&ndash, 6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11&ndash, 16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1&ndash, 6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11&ndash, 16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents.

Support for this study by Croatian Science Foundation (project number IP-09-2014-1501), Canadian Institute of Health Research (CIHR; project number MOP-119425), and National Sciences and Engineering Research Council of Canada (NSERC; project number RGPIN 04994-15) is acknowledged., We report the synthesis of SAHAquines and related primaquine (PQ) derivatives. SAHAquines are novel hybrid compounds that combine moieties of suberoylanilide hydroxamic acid (SAHA), an anticancer agent with weak antiplasmodial activity, and PQ, an antimalarial drug with low antiproliferative activity. The preparation of SAHAquines is simple, cheap, and high yielding. It includes the following steps: coupling reaction between primaquine and a dicarboxylic acid monoester, hydrolysis, a new coupling reaction with O- protected hydroxylamine, and deprotection.SAHAquines 5a-d showed significant reduction in cell viability. Among the three human cancer cell lines (U2OS, HepG2, and MCF-7), the most responsive were the MCF-7 cells. The antibodies against acetylated histone H3K9/H3K14 in MCF7 cells revealed a significant enhancement following treatment with N-hydroxy-N'-{4-[(6-methoxyquinolin-8-yl) amino] pentyl}pentanediamide (5b). Ethyl (2E)-3-({4-[(6-methoxyquinolin-8yl) amino] pentyl} carbamoyl) prop-2-enoate (2b) and SAHAquines were the most active compounds against both the hepatic and erythrocytic stages of Plasmodium parasites, some of them at sub-micromolar concentrations. The results of our research suggest that SAHAquines are promising leads for new anticancer and antimalarial agents., Croatian Science Foundation [IP-09-2014-1501]; Canadian Institute of Health Research (CIHR) [MOP-119425]; National Sciences and Engineering Research Council of Canada (NSERC) [RGPIN 04994-15]

Novel primaquine (PQ) and halogenaniline asymmetric fumardiamides 4a&ndash, f, potential Michael acceptors, and their reduced analogues succindiamides 5a&ndash, f were prepared by simple three-step reactions: coupling reaction between PQ and mono-ethyl fumarate (1a) or mono-methyl succinate (1b), hydrolysis of PQ-dicarboxylic acid mono-ester conjugates 2a,b to corresponding acids 3a,b, and a coupling reaction with halogenanilines. 1-[bis(Dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was used as a coupling reagent along with H&uuml, nig&prime, s base. Compounds 4 and 5 were evaluated against a panel of bacteria, several Mycobacterium strains, fungi, a set of viruses, and nine different human tumor cell lines. p-Chlorofumardiamide 4d showed significant activity against Staphylococcus aureus,Streptococcus pneumoniae and Acinetobacter baumannii, but also against Candida albicans (minimum inhibitory concentration (MIC) 6.1&ndash, 12.5 &micro, g/mL). Together with p-fluoro and p-CF3 fumardiamides 4b,f, compound 4d showed activity against Mycobacterium marinum and 4b,f against M. tuberculosis. In biofilm eradication assay, most of the bacteria, particularly S. aureus, showed susceptibility to fumardiamides. m-CF3 and m-chloroaniline fumardiamides 4e and 4c showed significant antiviral activity against reovirus-1, sindbis virus and Punta Toro virus (EC50 = 3.1&ndash, 5.5 &micro, M), while 4e was active against coxsackie virus B4 (EC50 = 3.1 &micro, M). m-Fluoro derivative 4a exerted significant cytostatic activity (IC50 = 5.7&ndash, 31.2 &mu, M). Acute lymphoblastic leukemia cells were highly susceptible towards m-substituted derivatives 4a,c,e (IC50 = 6.7&ndash, 8.9 &mu, M). Biological evaluations revealed that fumardiamides 4 were more active than succindiamides 5 indicating importance of Michael conjugated system.

Glioblastoma multiforme is one of the most aggressive brain tumors and current therapies with temozolomide or suberoylanilide hydroxamic acid (SAHA, vorinostat) show considerable limitations. SAHA is a histone deacetylase (HDAC) inhibitor that can cause undesirable side effects due to the lack of selectivity. We show here properties of a novel hybrid molecule, sahaquine, which selectively inhibits cytoplasmic HDAC6 at nanomolar concentrations without markedly suppressing class I HDACs. Inhibition of HDAC6 leads to significant α-tubulin acetylation, thereby impairing cytoskeletal organization in glioblastoma cells. The primaquine moiety of sahaquine reduced the activity of P-glycoprotein, which contributes to glioblastoma multiforme drug resistance. We propose the mechanism of action of sahaquine to implicate HDAC6 inhibition together with suppression of epidermal growth factor receptor and downstream kinase activity, which are prominent therapeutic targets in glioblastoma multiforme. Sahaquine significantly reduces the viability and invasiveness of glioblastoma tumoroids, as well as brain tumor stem cells, which are key to tumor survival and recurrence. These effects are augmented with the combination of sahaquine with temozolomide, the natural compound quercetin or buthionine sulfoximine, an inhibitor of glutathione biosynthesis. Thus, a combination of agents disrupting glioblastoma and brain tumor stem cell homeostasis provides an effective anti–cancer intervention.

Numerous modifications of the well-known antimalarial drug primaquine, both at the quinoline ring and at the primary amino group, have been reported, mostly to obtain antimalarial agents with improved bioavailability, reduced toxicity and/or prolonged activity. Modifications of the terminal amino group were made with the main idea to prevent the metabolic pathway leading to inactive and toxic carboxyprimaquine (follow-on strategy), but also to get compounds with different activity (repurposing strategy). The modifications undertaken until 2009 were included in a review published in the same year. The present review covers various classes of primaquine N-derivatives with diverse biological profiles, prepared in the last decade by our research group as well as the others. We have summarized the synthetic procedures applied for their preparation and discussed the main biological results. Several hits for the development of novel antiplasmodial, anticancer, antimycobacterial and antibiofilm agents were identified., Graphical abstract Image 1, Highlights • This review is focused on primaquine modifications at terminal amino group. • Synthetic pathways leading to more than 200 primaquine derivatives are presented. • Follow-on and repurposing strategies yielded novel hits for the discovery of antiplasmodial and anticancer drugs. • Compounds with antimycobacterial and antibiofilm potential were identified as well. • The scaffold attached to primaquine has a significant impact both on activity and selectivity.

Ribosomal protein biosynthesis is a key cellular process because proteins are the main cellular building blocks on all scales of life. Ensuring substrates for protein biosynthesis, evolutionarily conserved aminoacyl-tRNA synthetases (aaRSs) covalently couple amino acids to cognate tRNAs. This makes aaRSs important targets for natural and man-made antibiotics. A good example are isoleucyl-tRNA-synthetases (IleRS) which are susceptible to inhibition with mupirocin, a naturally produced antibiotic from the bacterium Pseudomonas fluorescens. Bacterial IleRSs cluster into two clades differing in susceptibility to mupirocin inhibition. IleRS1, present in most pathogenic bacteria, are up to 9 orders of magnitude more susceptible to mupirocin inhibition than bacterial IleRS2, which share similarities with eucaryotic IleRS, thus making IleRS1 favorable pharmaceutical targets. To investigate the origin of mupirocin insensitivity in IleRS2, crystal structures of IleRS2, and IleRS1 in a complex with mupirocin and a nonhydrolyzable analog of the reaction intermediate, Ile-AMS, were solved. Bacillus megaterium was chosen because it has genomic copies of both IleRS1 and IleRS2. By analyzing the solved structures we have shown that mupirocin binding to the two IleRS types, although similar, differs in the number of established mupirocin interactions and steric rearrangements which coincide with mupirocin binding. In IleRS2, mupirocin lacks two active site interactions compared to IleRS1 and the binding coincides with steric rearrangements of key active site parts. In contrast, the IleRS1 active site seems preformed for high-affinity mupirocin binding, with only a little steric rearrangement taking place upon the mupirocin binding. Thus, we propose a mechanism wherein the differences between mupirocin resistance of IleRS1 and IleRS2 are not only correlated to the differences in the interactions mupirocin establishes in the active site, but also to the steric rearrangements (or lack thereof) of the IleRS active site that accompany mupirocin binding.