Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran; 2Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 3Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain; 4Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; 5Quality Control Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, IranBackground: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr- cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.Keywords: nanoPEGylated EPO, cysteine PEGylation, pharmacokinetic propert

Effect of anti-HIV activity of novel compounds 8-phenyl-4-quinolone containing different substituents at position 3

Background and Objective: HIV treatment influences the global health and finding new compounds against HIV virus is increased. This study was done to evaluate anti-HIV activity of 8-phenyl-4-quinolone derivatives containing different substituents at position 3. Methods: In this descriptive study, single cycle replicable (SCR) HIV Virions were produced by co-transfecting HEK 293T cells with pmzNL4-3, pSPAX.2, pMD2.G plasmids. HeLa cells were infected with the SCR virions and then inhibit of virus replication by compounds were measured by p24 Antigen with ELISA kit. The cytotoxicity of these compounds on HeLa cells were measured by XTT method. Results: All compounds including NPZ_4F, NPZ-2F, NPZ-4CL and NPZ-2CL had the best inhibitory effect at a concentration of 100µM with the inhibition rate of respectively 51%, 48%, 33%, and 25%, respectively. The compounds of NPZ-4F and NPZ-2CL had negligible cellular toxicity and have inhibited HIV replication at the highest concentration. This issue can make them a valuable compound since they are better compounds in therapeutic terms, which at a suitable concentration, they have the lowest rate of cellular toxicity and highest power to inhibit HIV replication. Conclusion: Novel compounds derived from 8-phenyl-4-quinolone containing different substituents at position 3 can prevent HIV replication which is capable of high anti-viral and low cellular toxicity and suitable candidates for further investigation in antiviral studies

Anti-viral effect of methanolic extract of Sea cucumber on HIV-1 virus

Background and Objective: Sea cucumber (Holothuria leucospilota) is used for food purposes and traditional medicine in the South East and East Asia. This study was done to determine the antiviral effect of methanolic extract, of Holothuria leucospilota species against HIV-1 virus. Methods: In this laboratory study, sea cucumbers were collected from Larak Island, Persian Gulf, Iran at depths of 10-30 m. Methanol solvent was used for extraction process. Extract was concentrated by rotary evaporator at 40-45 degree C, and subsequently was prepared in the form of dry powder using vacuum freeze dryer lyophilization. Results: The extract in 100 and 1000 µg/ml of concentrations inhibited by 94% and 92.5% the replication of HIV-1, respectively. 10 µg/ml of extract had not specific antiviral effect. Approximately the half of concentration of extract (35.89 µg/ml) prevents 50% of proliferation of HIV-1, which was 50% toxic of on host cells (P<0.05). Conclusion: Sea cucumber methanolic body wall extract of Holothuria leucospilota species had no antiviral effect against HIV-1 virus. It can be due to cytotoxic effect of extract on the host cells

Solvent effects on structural and thermochemical properties of p53 tumor-suppressor gene: a molecular modeling approach in drug design

Shiva Irani1, Seyed Mohammad Atyabi2, Houri Mivehchi3, Seyed Davar Siadat2, Mohammad Reza Aghasadeghi2, Ali Farhangi21Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran; 2Department of Pilot Biotechnology, Pasteur Institute of Iran, Tehran, Iran; 3Department of Novel Drug Delivery System, Iran Polymer and Petrochemical Institute, Tehran, IranAbstract: The p53 tumor-suppressor protein is a cellular phosphoprotein and a negative regulator of cell growth. Most p53 mutations occur in exons 5&amp;ndash;8 within the DNA-binding domain. Therefore, p53 can potentially be targeted with novel drugs designed to bind to a mutation and restore its stability or wild-type conformation. For the current study, Hartree&amp;ndash;Fock calculations were used to investigate the solvent-induced effects of five different solvent media (acetone, ethanol, methanol, dimethyl sulfoxide, and water) on the thermochemical parameters and relative energies, and on the multinuclear nuclear magnetic resonance shielding tensors of oxygen, nitrogen, and phosphorus nuclei, of GAT. To understand how the solvent affects the mutation region (the &amp;ldquo;hot spot&amp;rdquo;) of p53, the relative energies of GAT in selected solvent media were determined. Some biological evidence suggested the structural stabilities of hot spots of GAT have the optimum temperature and solvent type for mutation. All the authors&amp;rsquo; findings are in accordance with common biological phenomena. Another important objective of this study was to compare the hydration Gibbs free energies of CUA and GAT in water using two different approaches where the solvent was treated as a continuum of the constant at different levels of Hartree&amp;ndash;Fock theory. The Gibbs hydration energy values obtained in water with the polarized continuum model directly applied on the isolated CUA and GAT sequences were compared with those determined from the hydrated models with four, six, and eight water molecule clusters around the hot spots uracil and adenine. The clustered structures of water molecules around the hot spots of GAT (in DNA level) and CUA (in transcriptional level) were found to be energetically favored. The results of this study provide a reliable insight into the nature of mutation processes, which is of utmost importance for the study of biochemical structures, and provide a basis for drug design.Keywords: polarized continuum model (PCM), nuclear magnetic resonance (NMR), Hartree&amp;ndash;Fock theor

Evaluation of a Native Preparation of HCV Core Protein (2-122) For Potential Applications in Immunization, Diagnosis and Mab Production

Infection with hepatitis C virus (HCV) is a worldwide problem. Among HCV proteins, core antigen (Ag), besides its importance for diagnostic application is a prime candidate for component of a vaccine. Herein, we report results of studies on production of the hydrophilic domain of core Ag (2-122) in native conformation by an arabinose induction system in E.coli and the primary characterization of this recombinant protein for applications in diagnosis, immunization and mAb production. Recombinant core (r-Core) was able to detect anti-core antibodies in HCV positive serum samples in a dilution rate of 1/3200. It was also capable to elicit a potent anti-HCV humoral immune response in BALB/c mice. Finally, we established two stable clones of hybridoma which shown to produce specific and sensitive mAbs against the core protein. HCV core was able to elicit a broad range of antibody specificities depending on the immunogen conformation. Therefore, it may be possible to get new mAbs with higher affinities towards native conformation of core Ag

Novel chlorambucil-conjugated anionic linear-globular PEG-based second-generation dendrimer: in vitro/in vivo improved anticancer activity

Artin Assadi,1 Vahideh Sharifi Najafabadi,1 Seyed Ataollah Sadat Shandiz,2 Azadah Shayeq Boroujeni,1 Sepehr Ashrafi,1 Ali Zaman Vaziri,1 Seyedeh Masoumeh Ghoreishi,1 Mohammad Reza Aghasadeghi,3 Seyed Esmaeil Sadat Ebrahimi,4 Morteza Pirali-Hamedani,4 Mehdi Shafiee Ardestani1 1Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, 2Young Researchers and Elite Club, East Tehran Branch, Islamic Azad University, 3Department of Hepatitis and AIDS, Pasteur Institute of Iran, 4Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Abstract: Evaluating the efficacy of anticancer drugs is an evolving and research-oriented issue. The objective of this study was to reduce the insolubility of chlorambucil (CBL) in water and improve the anticancer activity of CBL in vitro and in vivo through the conjugation of CBL with anionic linear-globular dendrimer (second generation, G2). In the current study, the anticancer activity among three groups that include CBL, CBL&ndash;G2 dendrimer, and control was measured in vitro and in vivo. In vitro studies showed that G2 anionic linear-globular polyethylene-glycol-based dendrimer, which conjugated to the CBL exterior through an ester linkage, was able to significantly improve the treatment efficacy over clinical CBL alone with respect to proliferation assay, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; half maximal inhibitory concentration (IC50) was calculated to be 141&nbsp;&micro;g/mL for CBL alone and 27.7&nbsp;&micro;g/mL for CBL&ndash;G2 dendrimer; P&lt;0.05. In addition, CBL&ndash;G2 dendrimer conjugate forestalled the growth of MCF-7 cancerous cells in addition to enhancing the number of apoptotic and necrotic cells as demonstrated by an annexin V-fluorescein isothiocyanate assay. CBL&ndash;G2 dendrimer conjugate was able to checkmate antiapoptotic Bcl-2 expression and Bcl-2/Bax ratio in a large scale compared with the control group and CBL alone (P&lt;0.005). In vivo studies showed that tumor treatment by CBL&ndash;G2 dendrimer conjugate outstrips the efficacy of treatment compared with CBL alone. The evaluation was based on reduction in tumor volume and tumor growth inhibition of murine 4T1 mammary tumor cells. Tumor volume of 140%&plusmn;8% was measured in the treatment with CBL&ndash;G2 dendrimer, whereas 152%&plusmn;13.5% was calculated in the treatment with free CBL (P&lt;0.05). However, there were no significant differences in histological assay among the three groups. In conclusion, tumor growth suppression potential of CBL&ndash;G2 dendrimer, which was assessed in both in vitro and in vivo experiments, has provided empirical evidence to buttress the fact that this compound could be considered for functional cancer treatment with low side effects. Keywords: anionic linear-globular dendrimer, G2, chlorambucil, CBL, in vitro cytotoxicity, in vivo efficac