Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies

Background: The elastase inhibitor \u3b1-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-\u3b2-sheets, nine-\u3b1-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into \u3b2-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris. Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region

Encapsulation of Alpha-1 antitrypsin in PLGA nanoparticles: In Vitro characterization as an effective aerosol formulation in pulmonary diseases

Abstract Background Alpha 1- antitrypsin (α1AT) belongs to the superfamily of serpins and inhibits different proteases. α1AT protects the lung from cellular inflammatory enzymes. In the absence of α1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized α1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation. Results Nonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line. Nanoparticles were spherical with an average size in the range of 100 nm to 1μ. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that α1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of α1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells. Conclusion In summary, α1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.</p

Biological Evaluation of New Oxadiazole-Based Synthetic α-glycosidase Inhibitors for Hyperglycemia Management: A Research Study: New Oxadiazoles act as a-glycosidase inhibitor

The present study was conducted to investigate the hypoglycemic activity of 3,4,5-triphenyl-oxadiazole derivatives and its effects on liver, lung, and kidney function in streptozotocin (STZ)-induced diabetic rats. For this purpose, male Wistar rats were divided into four groups (n = 4). Diabetes was induced in four groups by a single dose of STZ at 65 mg/kg body weight, administrated intraperitoneal. After 28 days of treatment, fasting blood sugar (FBS) levels and other biochemical parameters such as cholesterol, triglycerides phosphorous, urea creatinine, etc. were measured. Also, the markers of liver and kidney function, such as urea, serum creatinine, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase levels were determined. The study showed that the 3,4,5-triphenyl-oxadiazole derivatives at 100 mg/kg body weight had a significant antidiabetic activity after 28 days of treatment as the FBS levels decreased significantly while the serum insulin levels increased. Moreover, a significant decrease in the liver and kidney function markers in treated rats indicated the protective effect of the 3,4,5-triphenyl-oxadiazole derivatives against liver and kidney damage. The serum concentrations were normal in the control and the healthy group treated with these derivatives. The results of this study showed that both derivatives can regulate hyperglycemia and complications of diabetes

The Construction of Chimeric T-Cell Receptor with Spacer Base of Modeling Study of VHH and MUC1 Interaction

Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, VHH and CD8α, and FcgIIα are used as signaling moieties, costimulating domain, antibody, and spacers, respectively. To investigate the influence of the ligation of spacers on the conformational structure of VHH, models of VHH were constructed. Molecular dynamics simulation was run to study the influence of the presence of spacers on the conformational changes in the binding sites of VHH. Root mean square deviation and root mean square fluctuation of critical segments in the binding site showed no noticeable differences with those in the native VHH. Results from molecular docking revealed that the presence of spacer FcgIIα causes an increasing effect on VHH with MUC1 interaction. Each of the constructs was transformed into the Jurkat E6.1. Expression analysis and evaluation of their functions were examined. The results showed good expression and function

Radiosynthesis, Biological Evaluation, and Preclinical Study of a 68Ga-Labeled Cyclic RGD Peptide as an Early Diagnostic Agent for Overexpressed αvβ3 Integrin Receptors in Non-Small-Cell Lung Cancer

The αvβ3 integrin receptors have high expression on proliferating growing tumor cells of different origins including non-small-cell lung cancer. RGD-containing peptides target the extracellular domain of integrin receptors. This specific targeting makes these short sequences a suitable nominee for theranostic application. DOTA-E(cRGDfK)2 was radiolabeled with 68Ga efficiently. The in vivo and in vitro stability was examined in different buffer systems. Metabolic stability was assessed in mice urine. In vitro specific binding, cellular uptake, and internalization were determined. The tumor-targeting potential of [68Ga]Ga-DOTA-E(cRGDfK)2 in a lung cancer mouse model was studied. Besides, the very early diagnostic potential of the 68Ga-labeled RGD peptide was evaluated. The acquisition and reconstruction of the PET-CT image data were also carried out. Radiochemical and radionuclide purity for [68Ga]Ga-DOTA-E(cRGDfK)2 was >%98 and >%99, respectively. Radiotracer showed high in vivo, in vitro, and metabolic stability which was determined by ITLC. The dissociation constant (Kd) of [68Ga]Ga-DOTA-E(cRGDfK)2 was 15.28 nM. On average, more than 95% of the radioactivity was specific binding (internalized + surface-bound) to A549 cells. Biodistribution data showed that radiolabeled peptides were accumulated significantly in A549 tumor and excreted rapidly by the renal system. Tumor uptake peaks were at 1-hour postinjection for [68Ga]Ga-DOTA-E(cRGDfK)2. The tumor was clearly visualized in all images. [68Ga]Ga-DOTA-E(cRGDfK)2 can be used as a peptide-based imaging agent allowing very early detection of different cancers overexpressing αvβ3 integrin receptors and can be a potential candidate in clinical peptide-based imaging for lung cancer