Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn-porphyrins.

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

Determination of alkaloid-inspired molecule vindeburnol in rabbit plasma by UPLC-HRMS and its application to pharmacokinetic studies and preliminary metabolite identification.

The eburnamine-vincamine alkaloids exhibit a range of pharmacological activities. There is a limited understanding of the pharmacokinetics and pharmacodynamics of vindeburnol, a synthetic derivative of this chemical class of alkaloids. A fast and reliable UPLC-HRMS method was developed and validated to quantify vindeburnol in Soviet Chinchilla rabbit plasma from pharmacokinetics studies. An ultra-performance liquid chromatography system equipped with a Waters Acquity UPLC HSS T3 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and acetonitrile. An Impact II QqTOF high-resolution mass spectrometer equipped with an Apollo II electrospray ionization source was used for analysis in positive mode; the ions [M+H]+m/z 269.1648 ± 0.003 and m/z 351.2067 ± 0.003 were monitored for vindeburnol and internal standard (vinpocetine), respectively. Preliminary metabolite profiling was also performed, and the pharmacokinetics of the identified metabolites were evaluated. The mean retention times for vindeburnol and vinpocetine were 2.0 and 3.5 min. The UPLC-HRMS method was validated with accuracy and precision within the 15% acceptance limit (8.2% and 11.0%, respectively). The mean extraction recovery value of vindeburnol from rabbit plasma was 77%. Pharmacokinetic evaluation of vindeburnol revealed that the compound is distributed rapidly with a short elimination half-life. Vindeburnol undergoes extensive first-pass metabolism and is metabolized into hydroxyvindeburnol and vindeburnol glucuronide.

Pyrrolo[2,3-e]indazole as a novel chemotype for both influenza A virus and pneumococcal neuraminidase inhibitors.

Influenza infections are often exacerbated by secondary bacterial infections, primarily caused by Streptococcus pneumoniae. Both respiratory pathogens have neuraminidases that support infection. Therefore, we hypothesized that dual inhibitors of viral and bacterial neuraminidases might be an advantageous strategy for treating seasonal and pandemic influenza pneumonia complicated by bacterial infections. By screening our in-house chemical library, we discovered a new chemotype that may be of interest for a further campaign to find small molecules against influenza. Our exploration of the pyrrolo[2,3-e]indazole space led to the identification of two hit compounds, 6h and 12. These molecules were well-tolerated by MDCK cells and inhibited the replication of H3N2 and H1N1 influenza A virus strains. Moreover, both compounds suppress viral and pneumococcal neuraminidases indicating their dual activity. Given its antiviral activity, pyrrolo[2,3-e]indazole has been identified as a promising scaffold for the development of novel neuraminidase inhibitors that are active against influenza A virus and S. pneumoniae.

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities.

Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 µM while the standard one had 0.0180 µM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.

N-Phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine as a New Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitor.

Highly active antiretroviral therapy (HAART) has revolutionized human immunodeficiency virus (HIV) healthcare, turning it from a terminal to a potentially chronic disease, although some patients can develop severe comorbidities. These include neurological complications, such as HIV-associated neurocognitive disorders (HAND), which result in cognitive and/or motor function symptoms. We now describe the discovery, synthesis, and evaluation of a new class of N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) aimed at avoiding HAND. The most promising molecule, 12126065, exhibited antiviral activity against wild-type HIV-1 in TZM cells (EC50 = 0.24 nM) with low in vitro cytotoxicity (CC50 = 4.8 µM) as well as retained activity against clinically relevant HIV mutants. 12126065 also demonstrated no in vivo acute or subacute toxicity, good in vivo brain penetration, and minimal neurotoxicity in mouse neurons up to 10 µM, with a 50% toxicity concentration (TC50) of >100 µM, well below its EC50.

Enfuvirtide biosynthesis in thermostable chaperone-based fusion.

Synthetic peptides are in high demand as biologically active substances. Solid phase synthesis is the primary method of peptide production. However, it has drawbacks: large amount of chemical waste and rapid increase in price with peptide length. Biosynthesis is intended as method to bypass these flaws. Direct biosynthesis is usually not effective and among other approaches for improving quality and quantity of target product fusion partners are widely used. In this study we used a thermostable chaperon-based fusion partner developed by us to produce enfuvirtide in Escherichia coli expression system. Fusion partner's thermal stability provided additional purification mode by thermal denaturation of host proteins in lysate. Fusion protein was purified by ion exchange chromatography after lysate heating step and was then hydrolyzed with cyanogen bromide to release enfuvirtide. Enfuvirtide was isolated by RP-HPLC up to 94% purity with total yield of 2.86-3.31 mg per 1 L of low-density culture. The data demonstrate the posibility of thermostable chaperone-based fusion partner GroEL use for effective peptide biosynthesis.

Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli.

BACKGROUND: Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. RESULTS: Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. CONCLUSIONS: A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.

Addressing Reversibility of R-NHC Coupling on Palladium: Is Nano-to-Molecular Transition Possible for the Pd/NHC System?

It has recently been shown that palladium-catalyzed reactions with N-heterocyclic carbene (NHC) ligands involve R-NHC coupling accompanied by transformation of the molecular catalytic system into the nanoscale catalytic system. An important question appeared in this regard is whether such a change in the catalytic system is irreversible. More specifically, is the reverse nano-to-molecular transformation possible? In view of the paramount significance of this question to the area of catalyst design, we studied the capability of 2-substituted azolium salts to undergo the breakage of C-C bond and exchange substituents on the carbene carbon with corresponding aryl halides in the presence of Pd nanoparticles. The study provides important experimental evidence of possibility of the reversible R-NHC coupling. The observed behavior indicates that the nanosized metal species are capable of reverse transition to molecular species. Such an option, known for phosphine ligands, was previously unexplored for NHC ligands. The present study for the first time demonstrates bidirectional dynamic transitions between the molecular and nanostructured states in Pd/NHC systems. As a unique feature, surprisingly small activation barriers (<18 kcal/mol) and noticeable thermodynamic driving force (-5 to -7 kcal/mol) were calculated for C-C bond oxidative addition to Pd(0) centers in the studied system. The first example of NHC-mediated Pd leaching from metal nanoparticles to solution was observed and formation of Pd/NHC complex in solution was detected by ESI-MS.