New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma.

Pharmacologically active salicylanilides (2-hydroxy-N-phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG]n, n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein.

Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH-Peptide Conjugates.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide-drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative-daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH2). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display.

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

Quantitative Analysis of a Pilot Transwell Barrier Model with Automated Sampling and Mathematical Modeling.

In the preclinical phase of drug development, it is necessary to determine how the active compound can pass through the biological barriers surrounding the target tissue. In vitro barrier models provide a reliable, low-cost, high-throughput solution for screening substances early in the drug candidate development process, thus reducing more complex and costly animal studies. In this pilot study, the transport properties of TB501, an antimycobacterial drug candidate, were characterized using an in vitro barrier model of VERO E6 kidney cells. The compound was delivered into the apical chamber of the transwell insert, and its concentration passing through the barrier layer was measured through the automated sampling of the basolateral compartment, where media were replaced every 30 min for 6 h, and the collected samples were stored for further spectroscopic analysis. The kinetics of TB501 concentration obtained from VERO E6 transwell cultures and transwell membranes saturated with serum proteins reveal the extent to which the cell layer functions as a diffusion barrier. The large number of samples collected allows us to fit a detailed mathematical model of the passive diffusive currents to the measured concentration profiles. This approach enables the determination of the diffusive permeability, the diffusivity of the compound in the cell layer, the affinity of the compound binding to the cell membrane as well as the rate by which the cells metabolize the compound. The proposed approach goes beyond the determination of the permeability coefficient and offers a more detailed pharmacokinetic characterization of the transwell barrier model. We expect the presented method to be fruitful in evaluating other compounds with different chemical features on simple in vitro barrier models. The proposed mathematical model can also be extended to include various forms of active transport.

Evaluation of the Chemical Stability, Membrane Permeability and Antiproliferative Activity of Cyclic Diarylheptanoids from European Hornbeam (Carpinus betulus L.).

Four cyclic diarylheptanoids-carpinontriols A (1) and B (2), giffonin X (3) and 3,12,17-trihydroxytricyclo [12.3.1.12,6]nonadeca-1(18),2(19),3,5,14,16-hexaene-8,11-dione (4)-were isolated from Carpinus betulus (Betulaceae). Chemical stability of the isolated diarylheptanoids was evaluated as a function of storage temperature (-15, 5, 22 °C) and time (12 and 23 weeks). The effect of the solvent and the pH (1.2, 6.8, 7.4) on the stability of these diarylheptanoids was also investigated. Compounds 2 and 4 showed good stability both in aqueous and methanolic solutions at all investigated temperatures. Only 2 was stable at all three studied biorelevant pH values. Degradation products of 1 and 3 were formed by the elimination of a water molecule from the parent compounds, as confirmed by ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HR-MS). The permeability of the compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Compound 3 possesses a logPe value of -5.92 ± 0.04 in the blood-brain barrier-specific PAMPA-BBB study, indicating that it may be able to cross the blood-brain barrier via passive diffusion. The in vitro antiproliferative activity of the compounds was investigated against five human cancer cell lines, confirming that 1 inhibits cell proliferation in A2058 human metastatic melanoma cells.

Synthesis and Anticancer Activity of Phosphinoylated and Phosphonoylated N-Heterocycles Obtained by the Microwave-Assisted Palladium Acetate-Catalyzed Hirao Reaction.

A literature survey showed that different derivatives with the 9-phenyl-9H-carbazole or the dihydroindoline scaffold may be of biological activity including cytotoxic effect. Driven by this experience, P-functionalized derivatives of these N-heterocycles were synthesized. Three N-heterocycles, 9-(4-bromophenyl)-9H-carbazole, 3-bromo-9-phenyl-9H-carbazole and 1-(5-bromoindolin-1-yl)ethan-1-one, were coupled with dialkyl phosphites and diarylphosphine oxides using Pd(OAc)2 (10 %) as the catalyst precursor and triethylamine as the base in ethanol under microwave irradiation. The excess of the Y2 P(O)H reagent (Y=alkoxy, aryl) (30 %) served as the P-ligand in its trivalent tautomeric form (Y2 POH), hence there was no need for the usual P-ligands meaning cost and environmental burden. Hence, the presented method is a "green" approach that proved to be more efficient than the preparation by the traditional method. The products, dialkyl phosphonates and tertiary phosphine oxides obtained in 58-84 % yields were characterized, one of them also by single crystal X-ray analysis, and were subjected to in vitro biological activity evaluation. A (carbazol)yl-phenylphosphonate, an N-phenyl-(carbazol)yl-phosphonate, a (carbazol)yl-phenylphosphine oxide and an N-phenyl-(carbazol)ylphosphine oxide revealed a significant cytotoxic activity on A549 human non-small-cell lung carcinoma and MonoMac-6 acute monocytic leukemia cancer cells. The cytotoxic effect was significant as compared to that of the reference compounds.

Design and Characterization of a Multistage Peptide-Based Vaccine Platform to Target Mycobacterium tuberculosis Infection.

The complex immunopathology ofMycobacterium tuberculosis(Mtb) is one of the main challenges in developing a novel vaccine against this pathogen, particularly regarding eliciting protection against both active and latent stages. Multistage vaccines, which contain antigens expressed in both phases, represent a promising strategy for addressing this issue, as testified by the tuberculosis vaccine clinical pipeline. Given this approach, we designed and characterized a multistage peptide-based vaccine platform containing CD4+ and CD8+ T cell epitopes previously validated for inducing a relevant T cell response against Mtb. After preliminary screening, CFP10 (32-39), GlfT2 (4-12), HBHA (185-194), and PPE15 (1-15) were selected as promising candidates, and we proved that the PM1 pool of these peptides triggered a T cell response in Mtb-sensitized human peripheral blood mononuclear cells (PBMCs). Taking advantage of the use of thiol-maleimide chemoselective ligation, we synthesized a multiepitope conjugate (Ac-CGHP). Our results showed a structure-activity relationship between the conjugation and a higher tendency to fold and assume an ordered secondary structure. Moreover, the palmitoylated conjugate (Pal-CGHP) comprising the same peptide antigens was associated with an enhanced cellular uptake in human and murine antigen-presenting cells and a better immunogenicity profile. Immunization study, conducted in BALB/c mice, showed that Pal-CGHP induced a significantly higher T cell proliferation and production of IFNγ and TNFα over PM1 formulated in the Sigma Adjuvant System.

A Study of the Bisphosphonic Derivatives from the Pudovik Reaction of Dialkyl α-Oxophosphonates and >P(O)H Reagents: X-ray Structure and Bioactivity.

New hydroxy-methylenebisphosphonic derivatives were prepared with different P-functions. The outcome of the reaction of α-oxophosphonates (YC(O)P(O)(OR)2) and dialkyl phosphites or diarylphosphine oxides depended on the Y substituent of the oxo-compound, the nature of the P-reagent and the amount of the diethylamine catalyst. Starting from dimethyl α-oxoethylphosphonate, in the presence of 5% of diethylamine, the corresponding Pudovik adduct was the single product. While using 40% of the catalyst, the rearranged species with the >P(O)-O-CH-P(O)< skeleton was the exclusive component. A similar reaction of α-oxobenzylphosphonate followed the rearrangement protocol. X-ray crystallography revealed not only the spatial structures of the three products, but also an intricate pattern evolving from the interplay of slight chemical differences, solvent inclusion and disorder as well as H-bridge patterns, which invite further investigation. In vitro activity of the compounds was assessed on different tumor cell cultures using end-point-type cell tetrazolium-based measurements. These structure-activity studies revealed a cytostatic effect for four rearranged derivatives containing aromatic units. One of them had a pronounced effect on MDA-MB 231 and Ebc-1 cells, showing IC50 = 37.8 and 25.9 µM, respectively.

2,6-Disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines as a new class of potent antitubercular agents inhibiting DprE1.

Phenotypic screening of an in-house library of small molecule purine derivatives against Mycobacterium tuberculosis (Mtb) led to the identification of 2-morpholino-7-(naphthalen-2-ylmethyl)-1,7-dihydro-6H-purin-6-one 10 as a potent antimycobacterial agent with MIC99 of 4 µM. Thorough structure-activity relationship studies revealed the importance of 7-(naphthalen-2-ylmethyl) substitution for antimycobacterial activity, yet opened the possibility of structural modifications at positions 2 and 6 of the purine core. As the result, optimized analogues with 6-amino or ethylamino substitution 56 and 64, respectively, were developed. These compounds showed strong in vitro antimycobacterial activity with MIC of 1 µM against Mtb H37Rv and against several clinically isolated drug-resistant strains, had limited toxicity to mammalian cell lines, medium clearance with respect to phase I metabolic deactivation (27 and 16.8 µL/min/mg), sufficient aqueous solubility (>90 µM) and high plasma stability. Interestingly, investigated purines, including compounds 56 and 64, lacked activity against a panel of Gram-negative and Gram-positive bacterial strains, indicating a specific mycobacterial molecular target. To investigate the mechanism of action, Mtb mutants resistant to hit compound 10 were isolated and their genomes were sequenced. Mutations were found in dprE1 (Rv3790), which encodes decaprenylphosphoryl-ß-d-ribose oxidase DprE1, enzyme essential for the biosynthesis of arabinose, a vital component of the mycobacterial cell wall. Inhibition of DprE1 by 2,6-disubstituted 7-(naphthalen-2-ylmethyl)-7H-purines was proved using radiolabelling experiments in Mtb H37Rv in vitro. Finally, structure-binding relationships between selected purines and DprE1 using molecular modeling studies in tandem with molecular dynamic simulations revealed the key structural features for effective drug-target interaction.

Insight into the Antibacterial Action of Iodinated Imine, an Analogue of Rafoxanide: a Comprehensive Study of Its Antistaphylococcal Activity.

In this study, we have focused on a multiparametric microbiological analysis of the antistaphylococcal action of the iodinated imine BH77, designed as an analogue of rafoxanide. Its antibacterial activity against five reference strains and eight clinical isolates of Gram-positive cocci of the genera Staphylococcus and Enterococcus was evaluated. The most clinically significant multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant Enterococcus faecium, were also included. The bactericidal and bacteriostatic actions, the dynamics leading to a loss of bacterial viability, antibiofilm activity, BH77 activity in combination with selected conventional antibiotics, the mechanism of action, in vitro cytotoxicity, and in vivo toxicity in an alternative animal model, Galleria mellonella, were analyzed. The antistaphylococcal activity (MIC) ranged from 15.625 to 62.5 µM, and the antienterococcal activity ranged from 62.5 to 125 µM. Its bactericidal action; promising antibiofilm activity; interference with nucleic acid, protein, and peptidoglycan synthesis pathways; and nontoxicity/low toxicity in vitro and in vivo in the Galleria mellonella model were found to be activity attributes of this newly synthesized compound. In conclusion, BH77 could be rightfully minimally considered at least as the structural pattern for future adjuvants for selected antibiotic drugs. IMPORTANCE Antibiotic resistance is among the largest threats to global health, with a potentially serious socioeconomic impact. One of the strategies to deal with the predicted catastrophic future scenarios associated with the rapid emergence of resistant infectious agents lies in the discovery and research of new anti-infectives. In our study, we have introduced a rafoxanide analogue, a newly synthesized and described polyhalogenated 3,5-diiodosalicylaldehyde-based imine, that effectively acts against Gram-positive cocci of the genera Staphylococcus and Enterococcus. The inclusion of an extensive and comprehensive analysis for providing a detailed description of candidate compound-microbe interactions allows the valorization of the beneficial attributes linked to anti-infective action conclusively. In addition, this study can help with making rational decisions about the possible involvement of this molecule in advanced studies or may merit the support of studies focused on related or derived chemical structures to discover more effective new anti-infective drug candidates.

Cytotoxic Activity of α-Aminophosphonic Derivatives Coming from the Tandem Kabachnik-Fields Reaction and Acylation.

Encouraged by the significant cytotoxic activity of simple α-aminophosphonates, a molecular library comprising phosphonoylmethyl- and phosphinoylmethyl-α-aminophosphonates, a tris derivative, and N-acylated species was established. The promising aminophosphonate derivatives were subjected to a comparative structure-activity analysis. We evaluated 12 new aminophosphonate derivatives on tumor cell cultures of different tissue origins (skin, lung, breast, and prostate). Several derivatives showed pronounced, even selective cytostatic effects. According to IC50 values, phosphinoylmethyl-aminophosphonate derivative 2e elicited a significant cytostatic effect on breast adenocarcinoma cells, but it was even more effective against prostatic carcinoma cells. Based on our data, these new compounds exhibited promising antitumor activity on different tumor types, and they might represent a new group of alternative chemotherapeutic agents.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Label-free biosensing of lignans for therapeutics using engineered model surfaces.

The label-free interaction analysis of macromolecules and small molecules has increasing importance nowadays, both in diagnostics and therapeutics. In the blood vascular system, human serum albumin (HSA) is a vital globular transport protein with potential multiple ligand binding sites. Characterizing the binding affinity of compounds to HSA is essential in pharmaceutics and in developing new compounds for clinical application. Aryltetralin lignans from the roots of Anthriscus sylvestris are potential antitumor therapeutic candidates, but their molecular scale interactions with specific biomolecules are unrevealed. Here, we applied the label-free grating-coupled interferometry (GCI) biosensing method with a polycarboxylate-based hydrogel layer with immobilized HSA on top of it. With this engineered model surface, we could determine the binding parameters of two novel aryltetralin lignans, deoxypodophyllotoxin (DPT), and angeloyl podophyllotoxin (APT) to HSA. Exploiting the multi-channel referencing ability, the unique surface sensitivity, and the throughput of GCI, we first revealed the specific biomolecular interactions. Traditional label-free kinetic measurements were also compared with a novel, fast way of measuring affinity kinetics using less sample material (repeated analyte pulses of increasing duration (RAPID)). Experiments with well-characterized molecular interactions (furosemide to carbonic-anhydrase (CAII) and warfarin, norfloxacin to HSA) were performed to prove the reliability of the RAPID method. In all investigated cases, the RAPID and traditional measurement gave similar affinity values. In the case of DPT, the measurements and relevant modeling suggested two binding sites on HSA, with dissociation constant values of Kd1 = 1.8 ± 0.01 µM, Kd2 = 3 ± 0.02 µM. In the case of APT, the experiments resulted in Kd1 = 9 ± 1.7 µM, Kd2 = 28 ± 0.3 µM. The obtained binding values might suggest the potential medical application of DPT and APT without further optimization of their binding affinity to HSA. These results could be also adapted to other biomolecules and applications where sample consumption and the rapidity of the measurements are critical.

Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide-Drug Conjugates.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide-drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.

Synthesis and SAR Analysis of Novel 4-Hydroxytamoxifen Analogues Based on Their Cytotoxic Activity and Electron-Donor Character.

Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Review of Label-Free Monitoring of Bacteria: From Challenging Practical Applications to Basic Research Perspectives.

Novel biosensors already provide a fast way to detect the adhesion of whole bacteria (or parts of them), biofilm formation, and the effect of antibiotics. Moreover, the detection sensitivities of recent sensor technologies are large enough to investigate molecular-scale biological processes. Usually, these measurements can be performed in real time without using labeling. Despite these excellent capabilities summarized in the present work, the application of novel, label-free sensor technologies in basic biological research is still rare; the literature is dominated by heuristic work, mostly monitoring the presence and amount of a given analyte. The aims of this review are (i) to give an overview of the present status of label-free biosensors in bacteria monitoring, and (ii) to summarize potential novel directions with biological relevancies to initiate future development. Optical, mechanical, and electrical sensing technologies are all discussed with their detailed capabilities in bacteria monitoring. In order to review potential future applications of the outlined techniques in bacteria research, we summarize the most important kinetic processes relevant to the adhesion and survival of bacterial cells. These processes are potential targets of kinetic investigations employing modern label-free technologies in order to reveal new fundamental aspects. Resistance to antibacterials and to other antimicrobial agents, the most important biological mechanisms in bacterial adhesion and strategies to control adhesion, as well as bacteria-mammalian host cell interactions are all discussed with key relevancies to the future development and applications of biosensors.

Efficient Synthesis of Acylated, Dialkyl α-Hydroxy-Benzylphosphonates and Their Anticancer Activity.

An efficient method applying acyl chlorides as reagents was developed for the acylation of the hindered hydroxy group of dialkyl α-hydroxy-benzylphosphonates. The procedure did not require any catalyst. A few acylations were also performed with the SC-enantiomer of dimethyl α-hydroxy-benzylphosphonate, and the optical purity was retained. A part of the acyloxyphosphonates was tested against eight tumor cell lines of different tissue origin at c = 50 µM concentration. The compounds elicited moderate cytostatic effect against breast, skin, prostate, colon, and lung carcinomas; a melanoma cell line; and against Kaposi's sarcoma cell lines. Then, dose-dependent cytotoxicity was assayed, and benzoylation of the α-hydroxy group was identified as a moiety that increases anticancer cytotoxicity across all cell lines. Surprisingly, a few analogues were more toxic to multidrug resistant cancer cell lines, thus evading P-glycoprotein mediated drug extrusion.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides.

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.

Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation.

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%-10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation.

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.