Computer-aided Evaluation of Polyvalent Medications' Pharmacological Potential. Multiphytoadaptogen as a Case Study.

Many human diseases including cancer, degenerative and autoimmune disorders, diabetes and others are multifactorial. Pharmaceutical agents acting on a single target do not provide their efficient curation. Multitargeted drugs exhibiting pleiotropic pharmacological effects have certain advantages due to the normalization of the complex pathological processes of different etiology. Extracts of medicinal plants (EMP) containing multiple phytocomponents are widely used in traditional medicines for multifactorial disorders' treatment. Experimental studies of pharmacological potential for multicomponent compositions are quite expensive and time-consuming. In silico evaluation of EMP the pharmacological potential may provide the basis for selecting the most promising directions of testing and for identifying potential additive/synergistic effects. Multiphytoadaptogen (MPhA) containing 70 major phytocomponents of different chemical classes from 40 medicinal plant extracts has been studied in vitro, in vivo and in clinical researches. Antiproliferative and anti-tumor activities have been shown against some tumors as well as evidence-based therapeutic effects against age-related pathologies. In addition, the neuroprotective, antioxidant, antimutagenic, radioprotective, and immunomodulatory effects of MPhA were confirmed. Analysis of the PASS profiles of the biological activity of MPhA phytocomponents showed that most of the predicted anti-tumor and anti-metastatic effects were consistent with the results of laboratory and clinical studies. Antimutagenic, immunomodulatory, radioprotective, neuroprotective and anti-Parkinsonian effects were also predicted for most of the phytocomponents. Effects associated with positive effects on the male and female reproductive systems have been identified too. Thus, PASS and PharmaExpert can be used to evaluate the pharmacological potential of complex pharmaceutical compositions containing natural products.

[Molecular Diagnostics of the Involvement of Visually Normal Mucosa in the Malignancy Process in Urothelial Bladder Cancer].

Immunofluorescent method by flow cytometry was used to quantify the expression of the tumor-associated protein ßIII-tubulin (TUBB3) in the tissue of urothelial bladder cancer and visually normal mucosa (56 samples in total). The expression of the marker was detected in 100% of cases, and heterogeneity of the TUBB3 expression level both in tumor tissue and in "normal" mucosa was revealed. The level of TUBB3 in the "normal" mucosa did not depend on the distance from the tumor (1 cm or more than 3 cm) and, on average, it was lower than in the tumor tissue (21.8 ± 10.8% and 24.9 ± 13.2% vs 35.2 ± 12.4%; p = 0.04 and 0.005, respectively). An increase of the TUBB3 expression in the tumor and in the "normal" mucosa was revealed in muscle invasive bladder cancer compared to non-muscle invasive bladder cancer. Therefore, in urothelial bladder cancer, the tumor-associated protein TUBB3 is a molecular marker of bladder mucosa involvement in the malignancy process and predicts the risk of tumor muscle invasion, which may influence indications for early cystectomy.

[Possibilities of in silico estimations for the development of pharmaceutical composition phytoladaptogene cytotoxic for bladder cancer cells]. / Vozmozhnosti otsenok in silico dlia razrabotki farmkompozitsii fitoladaptogen, tsitotoksichnoi dlia kletok raka mochevogo puzyria.

Based on the prediction of biological activity spectra for several secondary metabolites of medicinal plants using the PASS computer program and validation in vitro of the predictions results the priority direction of the pharmaceutical composition Phytoladaptogene (PLA) development was determined. PLA is a complex of structurally diverse small organic compounds including biologically active substances of phytoadaptogenes (ginsenosides from Panax ginseng, rhodionin from Rhodiola rosea and others) compiled considering previously developed pharmaceutical compositions. Two variants of the pharmaceutical composition were studied: - the major and minor variants included 22 and 13 compounds, respectively. The probability of activity exceeds the probability of inactivity for 1400 out of 1945 pharmacological effects and mechanisms predicted by PASS for the major variant of PLA. The wide range of predicted activities is mainly due to the low structural similarity of constituent compounds. An in silico prediction indicates the possibilities of antitumor properties against bladder, stomach, colon, ovarian and cervical cancers both for minor and major PLA compositions. It was found that the highest probability values of activity were predicted for three mechanisms: apoptosis agonist, caspase-3 stimulant, and transcription factor NF-κB inhibitor. According to the PharmaExpert program they are associated with the antitumor effect against bladder cancer. Experimental validation was using the human bladder cancer cell line RT-112. The results of the MTT test have shown that the cytotoxicity of the major PLA variant is higher than that of the minor PLA variant. In vitro experiments performed using two methods (double staining with annexin V and propidium iodide and detection of active caspase-3 in cells) confirmed that the death of bladder cancer cells occurred via the apoptotic mechanism. The data obtained correspond to the results of the prediction and indicate advantages of the major PLA composition. Thus, PLA can become the basis for the development of a drug with the antitumor activity against bladder cancer. The antitumor activity predicted by PASS for other cancers may be the subject of further studies.

Trastuzumab and Pertuzumab Plant Biosimilars: Modification of Asn297-linked Glycan of the mAbs Produced in a Plant with Fucosyltransferase and Xylosyltransferase Gene Knockouts.

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPB-WT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPB-ΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297-linked GnGn-type glycan contains α1,3-fucose and ß1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.

Functional Role of Carbohydrate Residues in Human Immunoglobulin G and Therapeutic Monoclonal Antibodies.

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient's serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.

Overview of Fusion Tags for Recombinant Proteins.

Virtually all recombinant proteins are now prepared using fusion domains also known as "tags". The use of tags helps to solve some serious problems: to simplify procedures of protein isolation, to increase expression and solubility of the desired protein, to simplify protein refolding and increase its efficiency, and to prevent proteolysis. In this review, advantages and disadvantages of such fusion tags are analyzed and data on both well-known and new tags are generalized. The authors own data are also presented.

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae.

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.