Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a.

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

Do Small Molecules Activate the TrkB Receptor in the Same Manner as BDNF? Limitations of Published TrkB Low Molecular Agonists and Screening for Novel TrkB Orthosteric Agonists.

TrkB is a tyrosine kinase receptor that is activated upon binding to brain-derived neurotrophic factor (BDNF). To date, the search for low-molecular-weight molecules mimicking BDNF's action has been unsuccessful. Several molecules exerting antidepressive effects in vivo, such as 7,8-DHF, have been suggested to be TrkB agonists. However, more recent publications question this hypothesis. In this study, we developed a set of experimental procedures including the evaluation of direct interactions, dimerization, downstream signaling, and cytoprotection in parallel with physicochemical and ADME methods to verify the pharmacology of 7,8-DHF and other potential reference compounds, and perform screening for novel TrkB agonists. 7,8 DHF bound to TrkB with Kd = 1.3 µM; however, we were not able to observe any other activity against the TrkB receptor in SN56 T48 and differentiated SH-SY5Y cell lines. Moreover, the pharmacokinetic and pharmacodynamic effects of 7,8-DHF at doses of 1 and 50 mg/kg were examined in mice after i.v and oral administration, respectively. The poor pharmacokinetic properties and lack of observed activation of TrkB-dependent signaling in the brain confirmed that 7,8-DHF is not a relevant tool for studying TrkB activation in vivo. The binding profile for 133 molecular targets revealed a significant lack of selectivity of 7,8-DHF, suggesting a distinct functional profile independent of interaction with TrkB. Additionally, a compound library was screened in search of novel low-molecular-weight orthosteric TrkB agonists; however, we were not able to identify reliable drug candidates. Our results suggest that published reference compounds including 7,8-DHF do not activate TrkB, consistent with canonical dogma, which indicates that the reported pharmacological activity of these compounds should be interpreted carefully in a broad functional context.

Case Study and Attempt of Treatment of Mycobacteriosis Caused by Mycobacterium avium in a Parental Flock of Meat-Breed Pigeons.

Mycobacteriosis caused by Mycobacterium avium subsp. avium was observed in a parental loft of 70 meat-breed pigeons. It was decided to undertake treatment as the birds represented a substantial value to the owner. A multiagent therapy using azithromycin, marbofloxacin, and ethambutol was administered. After 4 mo of therapy, the desired results were not obtained. At the end of treatment, the birds were in poor general condition, with white blood cells above 20 g/L, and after clutching, 2-yr-old and older birds were euthanatized. Overall, postmortem lesions were found in 17 out of 49 necropsied individuals. Slide agglutination tests with a M. avium subsp. avium lysate were conducted in all examined pigeons. In 28 pigeons, blood count was conducted once a month during therapy, while in 24 pigeons, a tuberculin sensitivity test was conducted before the planned euthanatization. The tuberculin sensitivity test did not prove useful in the diagnosis of ill individuals. Slide agglutination yielded positive results in only four birds, all of which also had postmortem lesions. Blood count in a large number of cases allowed distinguishing between ill and healthy individuals, which was used for subsequent selection. The comparison of cultured strains with the (CCG)4-based PCR method showed the variation of M. avium isolates up to a maximum of 30%. The described case proves that the treatment of mycobacteriosis in pigeon flocks is not effective, mainly due to the high resistance to M. avium subsp. avium. In addition, therapy may contribute to an even greater increase in mycobacterial resistance to antibiotics, which may pose a potential risk to public health.

Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins.

The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.

Drug Susceptibility of Non-tuberculous Strains of Mycobacterium Isolated from Birds from Poland.

Mycobacterioses are a constant problem in backyard poultry, as well as pet birds. To date, no evidence of direct transmission of atypical bacilli between humans has been demonstrated, but it cannot be ruled out that sick animals can be a source of infection for people in their environment. The aim of the study was to identify mycobacteria isolated from birds with diagnosed mycobacteriosis and to determine the susceptibility of mycobacterial isolates from these animals to antituberculous drugs most commonly used in the treatment of mycobacterial infections in humans. For drug susceptibility tests, drugs such as isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, capreomycin, cycloserine and ethionamide were used. A high degree of drug resistance was demonstrated, particularly in Mycobacterium avium . Isolates of Mycobacterium xenopi showed a relatively good susceptibility to the drugs tested. The drug resistance of Mycobacterium genavense has not been determined, but this mycobacterium was identified in ten cases, which is the second most frequent occurrence in the cases studied.Mycobacterioses are a constant problem in backyard poultry, as well as pet birds. To date, no evidence of direct transmission of atypical bacilli between humans has been demonstrated, but it cannot be ruled out that sick animals can be a source of infection for people in their environment. The aim of the study was to identify mycobacteria isolated from birds with diagnosed mycobacteriosis and to determine the susceptibility of mycobacterial isolates from these animals to antituberculous drugs most commonly used in the treatment of mycobacterial infections in humans. For drug susceptibility tests, drugs such as isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, capreomycin, cycloserine and ethionamide were used. A high degree of drug resistance was demonstrated, particularly in Mycobacterium avium. Isolates of Mycobacterium xenopi showed a relatively good susceptibility to the drugs tested. The drug resistance of Mycobacterium genavense has not been determined, but this mycobacterium was identified in ten cases, which is the second most frequent occurrence in the cases studied.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

Synthesis and Tuberculostatic Activity Evaluation of Novel Benzazoles with Alkyl, Cycloalkyl or Pyridine Moiety.

Compounds possessing benzimidazole system exhibit significant antituberculous activity. In order to examine how structure modifications affect tuberculostatic activity, a series of benzazole derivatives were synthesized and screened for their antitubercular activity. The compounds 1⁻20 were obtained by the reaction between o-diamine, o-aminophenol, or o-aminothiophenol with carboxylic acids or thioamides. The newly synthesized compounds were characterized by IR, ¹H-NMR, 13C-NMR spectra, and elemental analysis. Synthesized benzazoles were evaluated for their tuberculostatic activity toward Mycobacterium tuberculosis strains. Quantum chemical calculations were performed to study the molecular geometry and the electronic structure of benzimidazoles GK-151B, 4, 6, and benzoxazole 11, using the Gaussian 03W software (Gaussian, Inc., Wallingford, CT, USA). Three-dimensional structure of benzimidazoles 1⁻3, MC-9, and GK-151B was determined by ab initio calculation using Gamess-US software. The activity of the received benzimidazoles was moderate or good. All of the benzoxazoles and benzothiazoles demonstrated much lower activity. Benzoxazoles were less active by about 50 times, and benzothiazole by 100 times than the benzimidazole analogs. Quantum chemical calculations showed differences in the distribution of electrostatic potential in the benzazole system of benzimidazoles and benzoxazoles. Three-dimensional structure calculations revealed how the parity of the alkyl substituent at the C2 position impacts the activity. Benzimidazole system is essential for the antituberculosis activity that is associated with the presence of the imine nitrogen atom in N-1 position. Its replacement by an oxygen or sulfur atom results in a decrease of the activity. The parity of the alkyl substituent at the C-2 position also modifies the activity.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, ß-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

Lipase-catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives.

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug-resistant Mycobacterium tuberculosis strains (M. tuberculosis H37 Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435-catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert-butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert-butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88-99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)-4-(1,3-benzoxazol-2-ylsulfanyl)butan-2-ol ((±)-3a), (±)-4-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3b), and (±)-4-[(5,7-dibromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3c), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug-Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50).

RNA polymerase motions during promoter melting.

All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources.

From Latent Tuberculosis Infection to Tuberculosis. News in Diagnostics (QuantiFERON-Plus).

It is estimated that one third of the world's population have latent tuberculosis infection and that this is a significant reservoir for future tuberculosis cases. Most cases occur within two years following initial infection. The identification of individuals with latent tuberculosis infection is difficult due to the lack of an ideal diagnostic assay and incomplete understanding of latent infection. Currently, there are three tests: the oldest tuberculin skin test, T-SPOT.TB and the latest QuantiFERON-Plus for the detection of Mycobacterium tuberculosis infection. The interpretation of the test results must be used in the conjunction with a patient's epidemiological history, risk assessment, current clinical status, radiography and microbiological methods to ensure accurate diagnosis.

Synthesis and Antimicrobial Activity of 4-Chloro-3-Nitrophenylthiourea Derivatives Targeting Bacterial Type II Topoisomerases.

A series of novel 4-chloro-3-nitrophenylthiourea derivatives were synthesized and evaluated for their antimicrobial, antibiofilm and tuberculostatic activities. Most of compounds exhibited high antibacterial activity against both standard and hospital strains (MIC values 0.5-2 µg/mL), as compared to Ciprofloxacin. Derivatives with 3,4-dichlorophenyl (11) and 3-chloro-4-methylphenyl (13) substituents were the most promising towards Gram-positive pathogens. Both of them exhibited antibiofilm potency and effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Two N-alkylthioureas (20, 21) showed twofold to fourfold increase in in vitro potency against isolates of Mycobacterium tuberculosis, as compared to Isoniazid. An action of 7, 10, 11, 13, 20 and 21 against activity of topoisomerases isolated from Staphylococcus aureus was studied. Synthesized compounds were found as non-genotoxic.

Real-Time Observation of Backtracking by Bacterial RNA Polymerase.

RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA-RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking.

Second-line anti-tuberculosis drug resistance and its genetic determinants in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

BACKGROUND: Mutations in several genetic loci have been implicated in the development of resistance to second-line anti-tuberculosis (TB) drugs (SLDs). The purpose of this study was to investigate the prevalence of resistance to SLDs and its association with specific mutations in multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates. MATERIALS AND METHODS: The study included 46 MDR-TB isolates. Mutation profiling was performed by amplifying and sequencing the following six genes: gyrA/gyrB, rrs, tlyA, and ethA/ethR, in which mutations are implicated in resistance of tubercle bacilli to ofloxacin (OFX), amikacin (AMK), capreomycin, and ethionamide (ETH), respectively. RESULTS: Of the strains analyzed, 14 (30.4%) showed resistance to at least one of the four SLDs tested. Mutations in the gyrA gene occurred in 34 (73.9%) strains, with the most common amino acid change being Ser95Thr. The Asp94Asn and Ala90Val substitutions in the gyrA were present exclusively in OFX-resistant strains, yet represented only 40% of all OFX-resistant strains. The only mutation in the gyrB gene was substitution Ser447Phe, detected in one OFX-resistant isolate. None of the AMK-resistant strains carried a mutation in the rrs gene. Mutations in the ethA/ethR loci were found in one ETH-resistant and 11 ETH-susceptible strains. CONCLUSIONS: The results of this study challenge the usefulness of sequence analyses of tested genes (except gyrA) for the prediction of SLD resistance patterns and highlight the need for searching other genetic loci for detection of mutations conferring resistance to SLDs in M. tuberculosis.

Structure of a bacterial RNA polymerase holoenzyme open promoter complex.

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.

Mutation profiling for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Progress in the detection of drug-resistant TB has been underpinned by the development and implementation of new, reliable and rapid diagnostic tools. These rely mostly on the detection of specific mutations conferring resistance to anti-TB drugs. The aim of this study was to search for mutations associated with isoniazid resistance among Mycobacterium tuberculosis clinical isolates. METHODS: A collection of 150 M. tuberculosis strains, including 50 MDR, 50 isoniazid-monoresistant and 50 pan-susceptible strains, was used. For all the strains, seven structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and two regulatory regions (mabA-inhA promoter and oxyR-ahpC intergenic region) were PCR amplified and sequenced in their entirety. RESULTS: Sixty-six distinct mutations were detected at all nine loci investigated, accounting for 109 (72.7%) of the strains tested. The number of strains with any mutation among the MDR, isoniazid-monoresistant and pan-susceptible groups was 49 (98%), 37 (74%) and 23 (46%), respectively. Mutations in the katG gene predominated, with 29 different types distributed among 46 (92%) MDR, 31 (62%) isoniazid-monoresistant and 2 (4%) pan-susceptible strains. Twenty-nine and 19 mutations were found exclusively in MDR and isoniazid-monoresistant strains, respectively. CONCLUSIONS: This study revealed 17 mutations, previously unreported, that might be of potential use as new surrogate markers of isoniazid resistance. Their diagnostic accuracy needs to be confirmed on larger strain samples and from different geographical settings. For isoniazid resistance detection, molecular approaches should still be a complement to rather than a replacement for conventional drug susceptibility testing. This is supported by the lack of mutations in any of the nine genetic loci investigated in 18 isoniazid-resistant strains from this study.

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety.

A total of 31 of thiourea derivatives was prepared reacting 3-(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yields varied from 35% to 82%. All compounds were evaluated in vitro for antimicrobial activity. Derivatives 3, 5, 6, 9, 15, 24 and 27 showed the highest inhibition against Gram-positive cocci (standard and hospital strains). The observed MIC values were in the range of 0.25-16 µg/ml. Inhibitory activity of thioureas 5 and 15 against topoisomerase IV isolated from Staphylococcus aureus was studied. Products 5 and 15 effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Moreover, all obtained thioureas were evaluated for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses. Compounds 5, 6, 8-12, 15 resulted cytotoxic against MT-4 cells (CC50 ≤ 10 µM).

Novel 2-(2-phenalkyl)-1H-benzo[d]imidazoles as antitubercular agents. Synthesis, biological evaluation and structure-activity relationship.

A series of novel 2-(2-phenalkyl)-1H-benzo[d]imidazole derivatives and analogues (2a-3l) have been synthesized and evaluated for tuberculostatic activity. Benzimidazoles substituted at the C-2 position with phenethyl, styryl and 3,5-dichlorophenethyl moiety were obtained. Compounds 2g, 2h and 2i bearing methyl groups at the benzimidazole system and phenalkyl substituent at the C-2 position showed high tuberculostatic activity against Mycobacterium tuberculosis strains with MIC values ranging from 0.8 to 6.2 µg/mL (2.5-25 µM). More importantly, derivatives 2g (5,6-dimethyl-2-phenethyl-1H-benzo[d]imidazole) and 2i (2-(3,5-dichlorophenethyl)-5,6-dimethyl-1H-benzo[d]imidazole) appeared selective for M. tuberculosis as compared with eukaryotic cells: non-malignant (neonatal human dermal fibroblasts) and malignant (mouse melanoma B16-F10 cell line). These compounds may thus represent a novel, selective class of anti-tubercular agents. SAR studies resulted in interesting conclusions on structural factors affecting tuberculostatic activity.

Antimicrobial and anti-biofilm activity of thiourea derivatives incorporating a 2-aminothiazole scaffold.

A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds were also tested for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain, as well as two 'wild' strains isolated from tuberculosis patients. Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital strain). The range of MIC values is 2-32 µg/mL. Products 3 and 9 effectively inhibited the biofilm formation of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3rd position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4 showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against MT-4 cells (CC50<10 µM).