Naphthoindole-2-carboxamides as a lipophilic chemotype of hetarene-anthraquinones potent against P-gp resistant tumor cells.

The acquisition of multidrug resistance (MDR) to chemotherapy is a major obstacle to successful cancer treatment. Aiming to improve the potency of anthraquinone-derived antitumor compounds against MDR cancer cells, we employed a rational design approach to develop new heteroarene-fused anthraquinones. Shifting the carboxamide group in the naphtho[2,3-f]indole scaffold from the 3-position to 2 increased the lipophilicity and P-glycoprotein (P-gp) binding of the derivatives, potentially enhancing their ability to circumvent P-gp-mediated MDR. To validate the computations, we developed a scheme for heterocyclization into esters of naphtho[2,3-f]indole-2-carboxylic acid, based on the 5-endo-dig cyclization of 2-alkynyl-3-amino-1,4-dimethoxyanthraquinone under mild basic conditions using tetra-n-butylammonium fluoride (TBAF). The synthesized naphthoindole-2-carboxamides, particularly compound 1a bearing (S)-3-aminopyrrolidine in the carboxamide fragment, demonstrated the highest antiproliferative activity. Most importantly, 1a suppressed the growth of the P-gp-positive K562/4 leukemia tumor cell line (resistance index = 2.4), while its 3-isomer LCTA-2640 and Dox did not (RI = 125 and 140, respectively). Studies of intracellular uptake and distribution showed that 1a, unlike its 3-substituted isomer, effectively accumulated in resistant tumor cells, confirming the correlation between in silico and experimental data. The lead compound 1a interacts with DNA duplex and inhibits topoisomerase 1 but does not induce oxidative stress. Treatment with 1a increases the population of apoptotic cells in both K562 and K562/4 sublines, regardless of the cell cycle phase. Taken together, this work provides an interesting example of how a little modification in chemical structure can lead to striking differences in antitumor properties. In conclusion, we have identified a potent class of compounds that offer distinct advantages in combating resistant tumor cells.

How the immune mousetrap works: Structural evidence for the immunomodulatory action of a peptide from influenza NS1 protein.

One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules.

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

Anticancer activity of G4-targeting phenoxazine derivatives in vitro.

G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.

Heterocyclic ring expansion yields anthraquinone derivatives potent against multidrug resistant tumor cells.

Chemical modifications of anthraquiones are aimed at novel derivatives with improved antitumor properties. Emergence of multidrug resistance (MDR) due to overexpression of transmembrane ATP binding cassette transporters, in particular, MDR1/P-glycoprotein (Pgp), can limit the use of anthraquinone based drugs. Previously we have demonstrated that annelation of modified five-membered heterocyclic rings with the anthraquinone core yielded a series of compounds with optimized antitumor properties. In the present study we synthesized a series of anthraquinone derivatives with six-membered heterocycles. Selected new compounds showed the ability to kill parental and MDR tumor cell lines at low micromolar concentrations. Molecular docking into the human Pgp model revealed a stronger interaction of 2-methylnaphtho[2,3-g]quinoline-3-carboxamide 17 compared to naphtho[2,3-f]indole-3-carboxamide 3. The time course of intracellular accumulation of compound 17 in parental K562 leukemia cells and in Pgp-positive K562/4 subline was similar. In contrast, compound 3 was readily effluxed from K562/4 cells and was significantly less potent for this subline than for K562 cells. Together with reported strategies of drug optimization of the anthracycline core, these results add ring expansion to the list of perspective modifications of heteroarene-fused anthraquinones.

Anticoagulant Oligonucleotide-Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics.

Oligonucleotide-peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3-Ser-Phe-Asn (SFN) or T3-Tyr-Trp-Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

Photochemical synthesis, intercalation with DNA and antitumor evaluation in vitro of benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives.

A new anticancer benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives were synthesized and characterized. Anticancer evaluation in vitro against four cancer cell lines including adenocarcinomic human alveolar basal epithelial cells (A549), hepatocellular carcinoma (HepG2), prostate cancer (PC3) and breast cancer (MCF7) indicated that some of prepared compounds shows higher selectivity in comparison with doxorubicin. DNA interaction studies by optical, CD, NMR spectroscopies showed the high affinity of benzothiazole ligands towards the dsDNA. The ligand-DNA interaction occurs through the intercalation of benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives with nucleic acid. The investigation of formed ligand - DNA complexes by docking and molecular dynamic calculations was applied for analysis of the relationship between structure and anticancer activity. The results suggested that benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives might serve as a novel scaffold for the future development to new antitumor agents.

Phenoxazine-based scaffold for designing G4-interacting agents.

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. Herein, we probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers ('arms') of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one single-armed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

Amides of pyrrole- and thiophene-fused anthraquinone derivatives: A role of the heterocyclic core in antitumor properties.

Heteroarene-fused anthraquinone derivatives represent a class of perspective anticancer drug candidates capable of targeting multiple vital processes including drug resistance. Taking advantage of previously demonstrated potential of amide derivatives of heteroarene-fused anthraquinones, we herein dissected the role of the heterocyclic core in antitumor properties. A new series of naphtho[2,3-f]indole-3- and anthra[2,3-b]thiophene-3-carboxamides was synthesized via coupling the respective acids with cyclic diamines. New compounds demonstrated a submicromolar antiproliferative potency close to doxorubicin (Dox) against five tumor cell lines of various tissue origin. In contrast to Dox, the new compounds were similarly cytotoxic for HCT116 colon carcinoma cells (wild type p53) and their isogenic p53 knockout counterparts. Modification of the heterocyclic core changed the targeting properties: the best-in-series naphtho[2,3-f]indole-3-carboxamide 8 formed more affine complexes with DNA duplex than furan and thiophene analogs, a property that can be translated into a stronger inhibition of topoisomerase 1 mediated DNA unwinding. At tolerable doses the water soluble derivative 8 significantly inhibited tumor growth (up to 79%) and increased the lifespan (153%) of mice bearing P388 lymphoma transplants. Together with better solubility for parenteral administration and well tolerance by animals of the indole derivative 8 indicates prospects for further search of new antitumor drug candidates among the heteroarene-fused anthraquinones.

Transcription-facilitating histone chaperons interact with genomic and synthetic G4 structures.

Affinity for G-quadruplex (G4) structures may be a common feature of transcription-facilitating histone chaperons (HCs). This assumption is based on previous unmatched studies of HCs FACT, nucleolin (NCL), BRD3, and ATRX. We verified this assumption and considered its implications for the therapeutic applications of synthetic (exogenous) G4s and the biological significance of genomic G4s. First, we questioned whether exogenous G4s that recognize cell-surface NCL and could trap other HCs in the nucleus are usable as anticancer agents. We performed in vitro binding assays and selected leading multi-targeted G4s. They exhibited minor effects on cell viability. The presumed NCL-regulated intracellular transport of G4s was inefficient or insufficient for tumor-specific G4 delivery. Next, to clarify whether G4s in the human genome could recruit HCs, we compared available HC ChIP-seq data with G4-seq/G4-ChIP-seq data. Several G4s, including the well-known c-Myc quadruplex structure, were found to be colocalized with HC occupancy sites in cancer cell lines. As evidenced by our molecular modeling data, c-Myc G4 might interfere with the HC function of BRD3 but is unlikely to prevent the BRD3-driven assembly of the chromatin remodeling complex. The c-Myc case illustrates the intricate role of genomic G4s in chromatin remodeling, nucleosome remodeling, and transcription.

Benzothiazole-based cyanines as fluorescent "light-up" probes for duplex and quadruplex DNA.

Analogs of benzothiazole orange (BO) with one, two or three methylbenzothiazolylmethylidene substituents in the 1-methylpyridinium ring were obtained from the respective picolinium, lutidinium or collidinium salts. Fluorescence parameters of the known and new dyes in complexes with various DNA structures, including G-quadruplexes (G4s) and i-motifs (IMs), were analyzed. All dyes efficiently distinguished G4s and ss-DNA. The bi- and tri-substituted derivatives had basically similar distributions of relative fluorescence intensities. The mono-substituted derivatives exhibited enhanced sensitivity to parallel G4s. All dyes were particularly sensitive to a G4 structure with an additional duplex module (the thrombin-binding aptamer TBA31), presumably due to a distinctive binding mode (interaction with the junction between the two modules). In particular, BO showed a strong (160-fold) enhancement in fluorescence quantum yield in complex with TBA31 compared to the free dye. The fluorescence quantum yields of the 2,4-bisubstituted derivative in complex with well-characterized G4s from oncogene promoters were in the range of 0.04-0.28, i.e. comparable to those of ThT. The mono/bi-substituted derivatives should be considered as possible light-up probes for G4 formation.

New anthra[2,3-b]furancarboxamides: A role of positioning of the carboxamide moiety in antitumor properties.

Derivatives of the anthraquinone (anthracene-9,10-dione) such as doxorubicin, mitoxantrone and others have proved great clinical efficacy for decades. Currently the search in this exceptionally productive chemical class is aimed at optimization of antitumor properties including circumvention of drug resistance. Previously we have reported that heteroarene-fused anthraquinones fused to a 5-membered heterocyclic ring are advantageous in killing drug resistant tumor cells. Herein we present the synthesis and antitumor properties of a series of new anthra[2,3-b]furan-2-carboxamides. Vast majority of new derivatives were similarly cytotoxic to wild type tumor cell lines and their isogenic sublines with P-glycoprotein overexpression and/or p53 inactivation. Comparison of structurally close derivatives varying in their position relative to the furan moiety, that is, furan-3-carboxamide 1vs furan-2-carboxamides 5 and 6, revealed fundamental differences in the cytotoxicity profiles, formation of drug-DNA complexes, efficacy of topoisomerase 1 inhibition and mechanisms of tumor cell death. Together with previous SAR data on the role of individual substituents, these results provide evidence that regioisomerization of anthra[2,3-b]furancarboxamides generates the practically perspective derivatives whose properties may vary significantly.

Tri-armed ligands of G-quadruplex on heteroarene-fused anthraquinone scaffolds: Design, synthesis and pre-screening of biological properties.

Here, a combined molecular modelling methodology was used to identify the binding mode of 4,11-bis((2-guanidinoethyl)amino)anthra[2,3-b]thiophene-5,10-dione (1), a previously reported G4 ligand. After calculating the optimal interaction parameters 1 with the target, two series of tri-armed ligands based on furan- or thiophene-fused anthraquinone scaffolds were designed and synthesized. The new compounds bearing an additional side chain at the 2-position of the heterocycle and the 4,11-side chains with different spacer lengths and structures of terminal groups demonstrated much stronger affinity for telomeric G4 (4-15 times) versus the parental ligand. Moreover, the specificity to the quadruplex over duplex DNA was significantly improved (up to 75 times) when the 3-guanidinopropyl side chain was introduced at the 2-position of the heterocycle ring. All tri-armed ligands demonstrated modest antiproliferative potency, which is likely due to low intracellular penetration. Nevertheless, this work shows how computer-aided rational design of new potent compounds can be used for targeted anticancer therapy.

Verification of oligomycin A structure: synthesis and biological evaluation of 33-dehydrooligomycin A.

Although, the structure of oligomycin A (1) was confirmed by spectroscopic and chemical evaluations, some crystallographic data cast doubt on the originally adopted structure of the side 2-hydroxypropyl moiety of this antibiotic. It was suggested that the side chain of the oligomycin is enol-related (2-hydroxy-1-propenyl). To clarify this matter we synthesized and evaluated 33-dehydrooligomycin A (2) prepared by the Kornblum oxidation of 33-O-mesyloligomycin A (3) by dimethyl sulfoxide. NMR data for 33-dehydrooligomycin (2) and results of quantum chemical calculations have shown that this derivative exists in the keto rather than in the enol tautomer 2a. The in vitro antimicrobial activity of 2 was approximately two times weaker in comparison with oligomycin A against Streptomyces fradiae ATCC-19609 and reference Candida spp. strains and similar activity against certain filamentous fungi. The docking binding estimate of 2 with FOF1ATP synthase showed a slight decrease in binding affinity for 2 when compared with oligomycin A; that correlated with its activity against S. fradiae ATCC 19609 that is supersensitive to oligomycin A. The in vitro antiproliferative activities of 2 are also discussed.

Optical properties of animal tissues in the wavelength range from 350 to 2600 nm.

The optical properties of different cow and pig biological tissues such as skeletal muscle, adipose, spinal cord, and dura mater of the spinal cord were investigated in the spectral range of 350 to 2600 nm. The measurements were carried out by a commercially available spectrophotometer SHIMADZU UV 3101PC. The wavelength dependence on the scattering coefficient has been observed to follow a power-law decay for skeletal muscle and dura mater of spinal cord. The influence of time delay between the sample preparation and measuring of transmittance spectra on the data reasonableness was reviewed. The conclusion about the benefits of 2 - ? m lasers application in surgery is given for the tissue types listed above.

The Oxime Derivatives of 1-R-1H-Naphtho[2,3-d][1,2,3]triazole-4,9-dione 2-oxides: Synthesis and Properties.

OBJECTIVE: To synthesize a novel chemotype based on the naphthoquinone scaffold with retained cytotoxicity and provisionally low intracellular oxidation potential. BACKGROUND: Derivatives of naphthoquinone, although potent anticancer agents, can exert heart toxicity due to generation of free oxygen species. METHODS: In this study, we modified the scaffold by replacing one carbonyl group with the oxime moiety. Interestingly, only one carbonyl group in 1-R-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione 2-oxides reacted with hydroxylamine. The spatial structure was determined by X-ray analysis. New compounds were tested for the ability to form stable complexes with double stranded DNA by spectroscopy and molecular docking and to induce death of tumor cell lines and non-malignant counterparts. RESULTS: The resulting 1-R-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione 4-oxime 2-oxides were further acylated to produce a series of 1-R-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione 4-(O-acyloxime) 2-oxides. Newly synthesized compounds demonstrated a higher (in submicromolar or low micromolar range) cytotoxic potency against human colon and breast adenocarcinoma cell lines than to non-malignant skin fibroblasts. Spectroscopic measurements revealed that, unlike other classes of quinone derivatives, new naphthotriazoledione oxides did not form stable complexes with double stranded DNA regardless of their fitting to the DNA minor groove (as determined by molecular modeling). CONCLUSION: Thus, our chemical modifications yielded a new chemotype with good cytotoxic properties and yet-to-be-identified intracellular target(s).

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

Anti-HIV Activities of Intramolecular G4 and Non-G4 Oligonucleotides.

New natural and chemically modified DNA aptamers that inhibit HIV-1 activity at submicromolar concentrations (presumably via preventing viral entry into target cells) are reported. The new DNA aptamers were developed based on known intramolecular G-quadruplexes (G4s) that were functionally unrelated to HIV inhibition [the thrombin-binding aptamer and the fragment of the human oncogene promoter (Bcl2)]. The majority of previously described DNA inhibitors of HIV infection adopt intermolecular structures, and thus their folding variability represents an obvious disadvantage. Intramolecular architectures refold correctly after denaturation and are generally easier to handle. However, whether the G4 topology or other factors account for the anti-HIV activity of our aptamers is unknown. The impact of chemical modification (thiophosphoryl internucleotide linkages) on aptamer activity is discussed. The exact secondary structures of the active compounds and further elucidation of their mechanisms of action hopefully will be the subjects of future studies.

Quantitative parameters of complexes of tris(1-alkylindol-3-yl)methylium salts with serum albumin: Relevance for the design of drug candidates.

Triarylmethane derivatives are extensively investigated as antitumor and antibacterial drug candidates alone and as photoactivatable compounds. In the series of tris(1-alkylindol-3-yl)methylium salts (TIMs) these two activities differed depending on the length of N-alkyl chain, with C4-5 derivatives being the most potent compared to the shorter or longer chain analogs and to the natural compound turbomycin A (no N-substituent). Given that the human serum albumin (HSA) is a major transporter protein with which TIMs can form stable complexes, and that the formation of these complexes might be advantageous for phototoxicity of TIMs we determined the quantitative parameters of TIMs-HSA binding using spectroscopic methods and molecular docking. TIMs bound to HSA (1:1 stoichiometry) altered the protein's secondary structure by changing the α-helix/ß-turn ratio. The IIa subdomain (Sudlow site I) is the preferred TIM binding site in HSA as determined in competition experiments with reference drugs ibuprofen and warfarin. The values of binding constants increased with the number of CH2 groups from 0 to 6 and then dropped down for C10 compound, a dependence similar to the one observed for cytocidal potency of TIMs. We tend to attribute this non-linear dependence to an interplay between hydrophobicity and steric hindrance, the two key characteristics of TIMs-HSA complexes calculated in the molecular docking procedure. These structure-activity relationships provide evidence for rational design of TIMs-based antitumor and antimicrobial drugs.