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Sarka Salajkova,1,2 Filip Havel,1,3 Michal Sramek,1 Filip Novotny,1,4 David Malinak,2,5 Rafael Dolezal,2,5 Lukas Prchal,2 Marketa Benkova,2 Ondrej Soukup,2 Kamil Musilek,2,5 Kamil Kuca,2,5 Jiri Bartek,1,6,7 Jan Proska,3 Monika Zarska,1 Zdenek Hodny1 1Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic; 2Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic; 3Department of Physical Electronics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic; 4Center for Advanced Functional Nanorobots, Department of Inorganic Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology Prague, Prague, Czech Republic; 5Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic; 6Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark; 7Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Division of Genome Biology, Karolinska Institute, Stockholm, SwedenCorrespondence: Monika ZarskaDepartment of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, Prague 4, Prague, CZ 142 20, Czech RepublicTel +420- 24106 3151Fax + 420-24106 2289Email monika.zarska@img.cas.czKamil KucaBiomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove, CZ 500 05, Czech RepublicTel +420-495 833 447Email kamil.kuca@fnhk.czPurpose: Plasmonic photothermal cancer therapy by gold nanorods (GNRs) emerges as a promising tool for cancer treatment. The goal of this study was to design cationic oligoethylene glycol (OEG) compounds varying in hydrophobicity and molecular electrostatic potential as ligand shells of GNRs. Three series of ligands with different length of OEG chain (ethylene glycol units = 3, 4, 5) and variants of quaternary ammonium salts (QAS) as terminal functional group were synthesized and compared to a prototypical quaternary ammonium ligand with alkyl chain – (16-mercaptohexadecyl)trimethylammonium bromide (MTAB).Methods: Step-by-step research approach starting with the preparation of compounds characterized by NMR and HRMS spectra, GNRs ligand exchange evaluation through characterization of cytotoxicity and GNRs cellular uptake was used. A method quantifying the reshaping of GNRs was applied to determine the effect of ligand structure on the heat transport from GNRs under fs-laser irradiation.Results: Fourteen out of 18 synthesized OEG compounds successfully stabilized GNRs in the water. The colloidal stability of prepared GNRs in the cell culture medium decreased with the number of OEG units. In contrast, the cellular uptake of OEG+GNRs by HeLa cells increased with the length of OEG chain while the structure of the QAS group showed a minor role. Compared to MTAB, more hydrophilic OEG compounds exhibited nearly two order of magnitude lower cytotoxicity in free state and provided efficient cellular uptake of GNRs close to the level of MTAB. Regarding photothermal properties, OEG compounds evoked the photothermal reshaping of GNRs at lower peak fluence (14.8 mJ/cm2) of femtosecond laser irradiation than the alkanethiol MTAB.Conclusion: OEG+GNRs appear to be optimal for clinical applications with systemic administration of NPs not-requiring irradiation at high laser intensity such as drug delivery and photothermal therapy inducing apoptosis.Keywords: gold nanorods, quaternary ammonium salts, oligoethylene glycol, cellular uptake, photothermal stability

Nosocomial infections, which greatly increase morbidity among hospitalized patients, together with growing antibiotic resistance still encourage many researchers to search for novel antimicrobial compounds. Picolinium salts with different lengths of alkyl chains (C12, C14, C16) were prepared by Menshutkin-like reaction and evaluated with respect to their biological activity, i.e., lipophilicity and critical micellar concentration. Picolinium salts with C14 and C16 side chains achieved similar or even better results when in terms of antimicrobial efficacy than benzalkoniums, notably, their fungicidal efficiency was substantially more potent. The position of the methyl substituent on the aromatic ring does not seem to affect antimicrobial activity, in contrast to the effect of length of the N-alkyl chain. Concurrently, picolinium salts exhibited satisfactory low cytotoxicity against mammalian cells, i.e., lower than that of benzalkonium compounds, which are considered as safe.

Quaternary ammonium salts (QASs) have been widely used for disinfection purposes because of their low price, high efficacy and low human toxicity for decades. However, precise mechanisms of action nor the powerful versatile agent against all antimicrobial species are known. In this study we have prepared 43 novel N-alkyl monoquaternary ammonium salts including 7 N,N-dialkyl monoquaternary ammonium salts differing bearing alkyl chain either of 12, 14 or 16 carbons. Together with 15 already published QASs we have studied the antimicrobial efficacy of all water-soluble compounds together with standard benzalkonium salts against Gram-positive (G+) and Gram-negative (G-) bacteria, anaerobic spore-forming Cl. difficile, yeasts, filamentous fungi and enveloped Varicella zoster virus (VZV). To address the mechanism of action, lipophilicity seems to be a key parameter which determines antimicrobial efficacy, however, exceptions are likely to occur and therefore QSAR analysis on the efficacy against G+ and G- bacteria was applied. We showed that antibacterial activity is higher when the molecule is larger, more lipophilic, less polar, and contains fewer oxygen atoms, fewer methyl groups bound to heteroatoms or fewer hydrogen atoms bound to polarized carbon atoms. In addition, from an application point of view, we have formulated mixtures, on the basis of obtained efficiency of individual compounds, in order to receive wide-spectrum agent. All formulated mixtures completely eradicated tested G+ and G- strains, including the multidrug-resistant P. aeruginosa as well as in case of yeasts. However, effect on A. fumigatus, Cl. difficile and VZV the exposition towards mixture resulted in significant reduction only. Finally, 3 out of 4 formulated mixtures were safer than reference commercial agent based on benzalkonium salts only in the skin irritation test using reconstructed human epidermidis.

Berberine, a naturally occurring compound, possesses an interesting multipotent pharmacological profile potentially applicable for Alzheimer’s disease (AD) treatment. In this study, a series of novel 22 berberine derivatives was developed and tested in vitro. Berberine core was substituted at position 9-O of its aromatic ring region. All the hybrids under the study revealed multi-targeted profile inhibiting prolyl oligopeptidase, acetylcholinesterase and butyrylcholinesterase highlighting 4a, 4g, 4j, 4l and 4s possessing balanced activities in the micromolar range. The top-ranked candidates in terms of the most pronounced potency against POP, AChE and BChE can be classified as 4d, 4u and 4v, bearing 4-methylbenzyl, (naphthalen-2-yl)methylene and 1-phenoxyethyl moieties, respectively. In vitro data were corroborated by detailed kinetic analysis of the selected lead molecules. 4d, 4u and 4v were also inspected for their potential to inhibit aggregation of two abberant proteins in AD, namely amyloid beta and tau, indicating their potential disease-modifying properties. To explain the results of our study, we carried out docking simulation to the active sites of the respective enzyme with the best berberine derivatives, along with QSAR study. We also investigated compounds’ potential permeability through blood-brain barrier by applying parallel artificial membrane permeation assay and addressed their cytotoxicity profile.

YNT-185 is the first known small molecule acting as orexin 2 receptor (OX2R) agonist with implication to narcolepsy treatment, served as a template scaffold in generating a small set of seven compounds with predictive affinity to OX2R. The design of the new small molecules was driven mostly by improving physicochemical properties of the parent drug YNT-185 in parallel with in silico studies, later suggesting their favorable binding modes within the active site of OX2R. We obtained seven new potential OX2R binders that were evaluated in vitro for their CNS availability, cytotoxicity, and behavior pattern on OX2R. Out of them, 15 emerged as the most potent modulator of OX2R, which, contrary to YNT-185, displayed inverse mode of action, i.e. antagonist profile. 15 was also submitted to an in vivo experiment revealing its ability to permeate through BBB into the brain with a short half-life.

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aβ42 self-aggregation (58.6 ± 5.1% at 50 μM) as well as hAChE-induced Aβ40 aggregation (48.3 ± 6.3% at 100 μM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.

The neuropeptides, orexin A and orexin B (also known as hypocretins), are produced in hypothalamic neurons and belong to ligands for orphan G protein-coupled receptors. Generally, the primary role of orexins is to act as excitatory neurotransmitters and regulate the sleep process. Lack of orexins may lead to sleep disorder narcolepsy in mice, dogs, and humans. Narcolepsy is a neurological disorder of alertness characterized by a decrease of ability to manage sleep-wake cycles, excessive daytime sleepiness, and other symptoms, such as cataplexy, vivid hallucinations, and paralysis. Thus, the discovery of orexin receptors, modulators, and their causal implication in narcolepsy is the most important advance in sleep-research. The presented work is focused on the evaluation of compounds L1&ndash, L11 selected by structure-based virtual screening for their ability to modulate orexin receptor type 2 (OX2R) in comparison with standard agonist orexin-A together with their blood-brain barrier permeability and cytotoxicity. We can conclude that the studied compounds possess an affinity towards the OX2R. However, the compounds do not have intrinsic activity and act as the antagonists of this receptor. It was shown that L4 was the most potent antagonistic ligand to orexin A and displayed an IC50 of 2.2 &micro, M, offering some promise mainly for the treatment of insomnia.

In order to identify novel lead structures for human toll-like receptor 4 (hTLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the hTLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate hTLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

The purpose of this study was to identify new small molecules that possess activity on human toll-like receptor 4 associated with the myeloid differentiation protein 2 (hTLR4/MD2). Following current rational drug design principles, we firstly performed a ligand and structure based virtual screening of more than 130 000 compounds to discover until now unknown class of hTLR4/MD2 modulators that could be used as novel type of immunologic adjuvants. The core of the in silico study was molecular docking of flexible ligands in a partially flexible hTLR4/MD2 receptor model using a peta-flops-scale supercomputer. The most promising substances resulting from this study, related to anthracene-succimide hybrids, were synthesized and tested. The best prepared candidate exhibited 80% of Monophosphoryl Lipid A in vitro agonistic activity in cell lines expressing hTLR4/MD2.

In this study, we described the structure-activity relationships of substituted 3,5-dinitrophenyl tetrazoles as potent antitubercular agents. These simple and readily accessible compounds possessed high in vitro antimycobacterial activities against Mycobacterium tuberculosis, including clinically isolated multidrug (MDR) and extensively drug-resistant (XDR) strains, with submicromolar minimum inhibitory concentrations (MICs). The most promising compounds showed low in vitro cytotoxicity and negligible antibacterial and antifungal activities, highlighting their highly selective antimycobacterial effects. 2-Substituted 5-(3,5-dinitrophenyl)-2H-tetrazole regioisomers, which are the dominant products of 5-(3,5-dinitrophenyl)-1H-tetrazole alkylation, showed better properties with respect to antimycobacterial activity and cytotoxicity than their 1-substituted counterparts. The 2-substituent of 5-(3,5-dinitrophenyl)-2H-tetrazole can be easily modified and can thus be used for the structure optimization of these promising antitubercular agents. The introduction of a tetrazole-5-thioalkyl moiety at position 2 of the tetrazole further increased the antimycobacterial activity. These compounds showed outstanding in vitro activity against M. tuberculosis (MIC values as low as 0.03 μM) and high activity against non-tuberculous mycobacterial strains.