Publications by authors named "Hamzeh Rahimi"

34 Publications

Potential aggregation hot spots in recombinant human keratinocyte growth factor: a computational study.

J Biomol Struct Dyn 2021 Apr 10:1-16. Epub 2021 Apr 10.

Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1908912DOI Listing
April 2021

Expansion of a Subset Within the C2 Subclade of Sequence Type 131 (ST131) Is Driving the Increasing Rates of Aminoglycoside Resistance.

Open Forum Infect Dis 2020 Nov 8;7(11):ofaa410. Epub 2020 Sep 8.

Section for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Lyngby, Denmark.

Background: Sequence type 131 (ST131) of is a pandemic clone that drives the increasing rates of antibiotic resistance. While the pervasiveness of ST131 clade C, especially subclades C2 and C1-M27, has been demonstrated in numerous global surveys, no report about the ST131 clades and their virotypes has been published from Iran so far.

Methods: A collection of 73 consecutive ST131 isolates from extraintestinal specimens was investigated for determination of virotypes, antibiotic susceptibility patterns, resistance/virulence determinants, and clade subsets.

Results: Most of the isolates belonged to subclade C2 (33/73; 45.2%), which had the highest virulence factor (VF) scores and resistance rates, followed by C1-M27 (18; 24.6%), C1-non-M27 (14; 19.1%), and A (8; 10.9%). The distinctive profiles of subclade C2 virulence genes were revealed by principle coordinates analysis testing. The distribution of the virulence gene among subclade C2 was not uniform, so that positive strains (21; 63.6%) showed significantly higher rates of resistance (, , , , ) and virulence (, , , , , ) markers and gentamicin/tobramycin resistance. Virotype C as the most common virotype (34; 46.5%) was predominant among the subclade C1 population, while virotypes E and F (21; 28.7%) were detected among subclade C2, which had the highest VF scores and aminoglycoside resistance rates.

Conclusions: The appearance of virotypes E and F among subclade C2 strains with higher rates of aminoglycoside resistance/virulence gene content shows the shifting dynamics of this pandemic clone in response to antibiotic selection pressure by establishing subsets with higher survival potential.
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http://dx.doi.org/10.1093/ofid/ofaa410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691798PMC
November 2020

Novel Small Molecules against Two Binding Sites of Wnt2 Protein as potential Drug Candidates for Colorectal Cancer: A Structure Based Virtual Screening Approach.

Iran J Pharm Res 2020 ;19(2):160-174

Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran.

Wnts are the major ligands responsible for activating Wnt signaling pathway through binding to Frizzled proteins (Fzd) as the receptors. Among these ligands, Wnt2 plays the main role in the tumorigenesis of several human cancers especially colorectal cancer (). Therefore, it can be considered as a potential drug target. The aim of this study was to identify potential drug candidates against two binding sites of Wnt2. Structure-based virtual screening approaches were applied to identify compounds against binding sites of Wnt2 for inhibiting the interaction Wnt2 and Fzd receptors. The best hit compounds from molecular docking of National Cancer Institute diversity set II database were used for structural similarity search on ZINC database, obtaining large hit compounds query to perform a virtual screening and retrieving potential lead compounds. Eight lead compounds were selected while their binding affinity, binding modes interactions, and molecular dynamics simulations studies were assessed. Molecular docking studies showed that eight selected lead compounds can bind to the desired binding sites of Wnt2 in a high affinity manner. Bioavailability analysis of the selected lead compounds indicated that they possessed significant drug like properties. Thus, these lead compounds were considered as potential drug candidates for inhibiting Wnt signaling pathway through combining with the binding sites of Wnt2 and hindering the interaction of Wnt2 and Fzd receptors. Our findings suggest that Wnt2 binding sites may be a useful target for treatment for CRC fueling the future efforts for developing new compounds against Wnt signaling pathway.
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http://dx.doi.org/10.22037/ijpr.2019.15297.13037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667561PMC
January 2020

Analysis of Single Nucleotide Polymorphisms in , and Genes in Multiple Sclerosis.

Rep Biochem Mol Biol 2020 Jul;9(2):198-208

Zoonoses Research Center, North Research Center, Pasteur Institute of Iran, Amol, Iran.

Background: Multiple sclerosis (MS) is a common demyelinating neurodegenerative disorder with significant heritability. Previous studies have associated genetic variants in human leukocyte antigen complex, , and genes with the pathophysiology of MS.

Methods: In order to investigate the gene association in the Iranian population, we performed a genotyping study of 36 variants in the mentioned genes using Sanger sequencing in 102 MS patients and 113 healthy controls.

Results: Our results identified significant associations as well as significant allele frequency differences in some of the studied single-nucleotide polymorphisms including rs4935356, rs3177928, and rs7197 from gene, and rs12722489 and rs12722490 variants from gene (p< 0.05). Moreover, the strong linkage disequilibrium of two common haplotypes was estimated from the gene.

Conclusion: This association study may suggest the role of these polymorphisms in the genetic susceptibility of MS in the Iranian population and would facilitate the recognition of causative variants in this disease.
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http://dx.doi.org/10.29252/rbmb.9.2.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603253PMC
July 2020

Computational design of antagonist peptides based on the structure of secreted frizzled-related protein-1 (SFRP1) aiming to inhibit Wnt signaling pathway.

J Biomol Struct Dyn 2020 Nov 6:1-20. Epub 2020 Nov 6.

Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Aberrant activation of Wnt/β-catenin signaling pathway, due to the genetic or epigenetic changes, is responsible for tumorigenesis in epithelial cells of different types of cancer such as colorectal cancer. Secreted Frizzled-Related Protein-1 (SFRP1), as one of the antagonist proteins of this pathway, is hyper-methylated in colorectal cancer leading to the formation of Wnt-Fz-LRP and activation of Wnt/β-catenin signaling pathway. We aimed to design antagonist peptides based on SFRP1 structure against wingless-type 2 (Wnt2), a highly expressed ligand in different cancers like colorectal cancer, to inhibit the formation of the initial triple complex of Wnt-Fz-LRP. After homology modeling of SFRP1, molecular docking showed that Wnt2 and SFRP1 interact in the same mode of xWnt8-mFz8 and hWnt3-mFz8 through the thumb and finger binding sites. These binding sites were selected for designing peptides using either substitution or deep learning-based approaches. The efficiency of each designed peptide in interacting with Wnt2 was evaluated by molecular docking. Stability assessment of Wnt2-peptide complexes via molecular dynamic (MD) revealed that the designed peptides could effectively interact with Wnt2 binding sites during the simulation. However, the designed peptides against the thumb site had higher binding affinity and hydrogen bonds compared to the initial sequence. The secondary structure of the designed peptides indicated an alpha-helix structure which is a favorable structure for peptide drugs. Computing the physicochemical properties of peptides predicted a fairly acceptable structure which made them promising candidates in the treatment of cancers like CRC.
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http://dx.doi.org/10.1080/07391102.2020.1835718DOI Listing
November 2020

Repurposing of the approved small molecule drugs in order to inhibit SARS-CoV-2 S protein and human ACE2 interaction through virtual screening approaches.

J Biomol Struct Dyn 2020 Sep 24:1-16. Epub 2020 Sep 24.

Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Most recently, the new coronavirus (SARS-CoV-2) has been recognized as a pandemic by the World Health Organization (WHO) while this virus shares substantial similarity with SARS-CoV. So far, no definitive vaccine or drug has been developed to cure Covid-19 disease, since many important aspects about Covid-19 such as pathogenesis and proliferation pathways are still unclear. It was proven that human ACE2 is the main receptor for the entry of Covid-19 into lower respiratory tract epithelial cells through interaction with SARS-CoV-2 S protein. Based on this observation, it is expected that the virus infection can be inhibited if protein-protein interaction is prevented. In this study, using structure-based virtual screening of FDA databases, several lead drugs were discovered based on the ACE2-binding pocket of SARS-CoV-2 S protein. Then, binding affinity, binding modes, critical interactions, and pharmaceutical properties of the lead drugs were evaluated. Among the previously approved drugs, Diammonium Glycyrrhizinate, Digitoxin, Ivermectin, Rapamycin, Rifaximin, and Amphotericin B represented the most desirable features, and can be possible candidates for Covid-19 therapies. Furthermore, molecular dynamics (MD) simulation was accomplished for three S protein/drug complexes with the highest binding affinity and best conformation and binding free energies were also computed with the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. Results demonstrated the stable binding of these compounds to the S protein; however, in order to confirm the curative effect of these drugs, clinical trials must be done.
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http://dx.doi.org/10.1080/07391102.2020.1824816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576931PMC
September 2020

Merkel Cell Polyomavirus Gene Expression and Mutational Analysis of Large Tumor Antigen in Non-Merkel Cell Carcinoma Tumors of Iranian Patients.

Public Health Genomics 2020 23;23(5-6):210-217. Epub 2020 Sep 23.

Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran.

Introduction: The presence of Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma (MCC). However, there was sparse information on the link of other common nonmelanoma skin cancers - basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) - to MCPyV infection. The current study describes the phylogenetic information of MCPyV isolated from Iranian non-MCC (nonmelanoma skin cancers) focusing on tumorigenesis of mutations in large tumor (LT) antigen (LT-Ag) fragment.

Methods: Sixty patients with BCC and 20 patients with SCC were included in this study (48 males and 32 females; average age 65 years). The MCPyV-DNA copy number in positive samples was measured by quantitative real-time PCR. Then, mutational analysis of the MCPyV LT gene was carried out by direct sequencing.

Results: While MCPyV DNA was detected in 6 (10%) of 60 BCC samples, no viral genome was found in SCCs. There was no distinct association of MCPyV positivity with gender, age, or type of tumor (BCC or SCC) (p value >0.05). Quantitative real-time PCR revealed that the median number of viral DNA copies per cell was 0.7 in 6 MCPyV-positive BCC samples. Furthermore, full-length LT-Ag sequencing of positive samples indicated no stop codon or frameshift mutations compared to reference sequences.

Conclusion: Considering the important role of the LT-Ag in the pathogenicity of MCPyV, non-synonymous mutations compared with the reference proteins triggered relevant amino acid substitutions. Overall, the results showed no tumor-associated mutations in the LT-Ag sequence of MCPyVs from positive samples.
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http://dx.doi.org/10.1159/000510254DOI Listing
September 2020

Computational Approach for Rational Design of Fusion Uricase with PAS Sequences.

Int J Mol Cell Med 2020 ;9(1):90-103

Enzyme Technology Laboratory, Department of Biochemistry,Genetic and Metabolism Research Group, Pasteur Institute of Iran,Tehran, Iran.

Tumor lysis syndrome is a life-threatening condition for humans due to the lack of urate oxidase. In this study, several variants of PASylated uricase from the species were analyzed computationally to find the appropriate fusions to solve short half-life and stability concern. The method was performed using Rosetta software to structurally characterize the PAS sequences. The 3D structures of fusions were predicted for fused C- or N-terminally PAS sequences in different length to the uricase. The refinement and energy minimization steps revealed that physicochemical and conformational properties of fusions improved while the structures possessed prolonged PAS sequences. Molecular docking results showed that the highest binding affinity to uric acid belonged to uricase-PAS1-100 by the formation of six hydrogen and four non-hydrogen bonds. Altogether, the results indicated that the PASylation process would be promising upon the production of urate oxidase with improved solubility and stability.
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http://dx.doi.org/10.22088/IJMCM.BUMS.9.1.90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422847PMC
January 2020

Effect of sodium butyrate on HDAC8 mRNA expression in colorectal cancer cell lines and molecular docking study of LHX1 - sodium butyrate interaction.

EXCLI J 2020 23;19:1038-1051. Epub 2020 Jul 23.

Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran.

Colorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of cancer related deaths worldwide. The Histone Deacetylase 8 (HDAC8) gene is a gene with unique features which can be used as a potential target for drug design. The LHX1 transcription factor is an important transcription factor for this gene. The aim of this study was to investigate the effect of sodium butyrate (NaB) as a histone deacetylase inhibitor (HDACi) on the expression of the HDAC8 gene in the colorectal cancer cell line, and the molecular docking of the LHX1 transcription factor with NaB. For this purpose, HCT-116 and HT-29 cell lines were treated with different concentrations of NaB (6.25 mM to 150 mM) at 24, 48 and 72 hours. Subsequently, RNA was extracted from the treated and untreated cells and cDNA was synthesized. Quantitative Real-Time-PCR was done to investigate the mRNA expression of HDAC8. Molecular docking was also performed to investigate the interaction between NaB and LHX1. Based on Real-time-PCR results, the concentration of 150 mM of NaB after 24 hours in HT-29 and HCT-116 cell lines caused a significant reduction in mRNA expression of HDAC8 (P<0.05). After 48 hours of treatment, there was a significant decrease in the mRNA expression of HDAC8 at all concentrations (P<0.05). The docking results showed that LHX1 and NaB interacted best at the lowest energy levels. Our results also showed that NaB bonded strongly to LHX1. In addition, our results demonstrated that NaB bound to the LHX1 transcription factor and inhibited the function of this factor and consequently decreased the transcription from the HDAC8 gene which resulted in cell death. Future studies are needed to assess the likely molecular mechanisms of NaB action on gene expression.
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http://dx.doi.org/10.17179/excli2020-2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415931PMC
July 2020

In silico and in vivo anti-malarial investigation on 1-(heteroaryl)-2-((5-nitroheteroaryl)methylene) hydrazine derivatives.

Malar J 2020 Jun 29;19(1):231. Epub 2020 Jun 29.

Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran.

Background: Resistance of Plasmodium falciparum against common anti-malarial drugs emphasizes the need of alternative and more effective drugs. Synthetic derivatives of 1-(heteroaryl)-2-((5-nitroheteroaryl)methylene) hydrazine have showed in vitro anti-plasmodial activities. The present study aimed to evaluate the molecular binding and anti-plasmodial activity of synthetic compounds in vivo.

Methods: The molecular docking was used to study the binding of compounds to haem and Plasmodium falciparum lactate dehydrogenase (PfLDH). Acute toxicity of the synthetic compounds was evaluated based on the modified up & down method. The anti-plasmodial activity of the compounds was conducted by the two standard tests of Peters' and of Rane, using chloroquine-sensitive Plasmodium berghei in mice. Also, the toxicity to the internal organs of mice was evaluated on the seventh day after the treatment in addition to the histopathology of their liver. Compound 3 that showed high activity in the lowest dose was selected for further pharmacodynamic studies.

Results: According to the docking studies, the active site of PfLDH had at least four common residues, including Ala98, Ile54, Gly29, and Tyr97 to bind the compounds with the affinity, ranging from - 8.0 to - 8.4 kcal/mol. The binding mode of ligands to haem revealed an effective binding affinity, ranging from - 5.1 to - 5.5 kcal/mol. Compound 2 showed the highest  % suppression of parasitaemia (99.09%) at the dose of 125 mg/kg/day in Peters' test. Compound 3, with 79.42% suppression, was the best in Rane's test at the lowest dose (31 mg/kg/day). Compound 3 was confirmed by the pharmacodynamic study to have faster initial parasite elimination in the lowest concentration. The histopathology of the livers of mice did not reveal any focal necrosis of hepatocytes in the studied compounds.

Conclusions: The docking studies verified Pf LDH inhibition and the inhibitory effect on the haemozoin formation for the studied compounds. Accordingly, some compounds may provide new avenues for the development of anti-malarial drugs without liver toxicity, although further studies are required to optimize their anti-plasmodial activity.
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http://dx.doi.org/10.1186/s12936-020-03269-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322848PMC
June 2020

Genome diversity and evolutionary characteristics of clinical isolates of circulating in Iran.

Iran J Microbiol 2020 Feb;12(1):1-10

Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran.

Background And Objectives: The re-emergence of pertussis still is being reported all over the world. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main reasons of infection resurgence. Waning immunity is also an important factor contributing to resurgence of pertussis.

Materials And Methods: The genetic diversity and evolutionary characteristics of circulating Iranian isolates of during February 2015 to October 2018 was investigated by pulsed-field gel electrophoresis (PFGE) and subsequently and alleles were characterized. The next generation genome sequencing was then used to compare the genomics of and of selected isolates from PFGE dendrogram.

Results: PFGE differentiated 62 clinical isolates and vaccine and reference strains into 19 PFGE profiles, indicating the higher level of heterogeneity in the population during 2015-2018. The predominant genotype harbored pertussis toxin promoter allele, and the expansion of isolates, were also observed in our population.

Conclusion: No changes in allelic profile of predominant clone in recent years was observed but antigenic divergence between recently circulating isolates and the vaccine strain has been progressed and significantly was higher than previous studies. The comparative genomic analysis of the and isolates indicate that changes in genome structure including 32 unique SNPs and three unique indels may have contributed to the expansion of the clone. We compared and isolates in pathogenicity-associated genes and found five of them were specific for the isolates. The polymorphisms in pathogenicity-associated genes suggest structural adaptations for these virulence factors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163038PMC
February 2020

Effects of tosyl-l-arginine methyl ester (TAME) on the APC/c subunits: An in silico investigation for inhibiting cell cycle.

J Mol Graph Model 2020 06 5;97:107563. Epub 2020 Feb 5.

Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

The anaphase-promoting complex/cyclosome (APC/c) is requisite for controlling mitosis, which is activated by Cdh1 and Cdc20 activators. Dysregulation of APC/c is observed in many cancers and is known as a targeted drug particularly in cancer drug resistance. It was shown that tosyl-l-arginine methyl ester (TAME), via mimicking isoleucine-arginine (IR) tail of co-activators, inhibits APC/c functions. However, structure details and interaction of TAME with APC/c are poorly defined. In the current study, a well-established set of computational methods was used to identify the best binding pocket in order to inhibit APC activity. Therefore, the interaction of IR tail and Cbox of co-activators, as well as TAME as an inhibitor, as an inhibitor, with APC3 and APC8 subunits of APC/c were analyzed, regarding structure, molecular docking, molecular dynamics, and free binding energy. The results indicated that TAME bound to APC3 with a higher binding affinity (∼-7.3 kcal/mol) than APC8 (∼-5.7 kcal/mol). Also, the binding free energy value obtained for the APC3-TAME was -22.25 ± 1.12 kcal/mol. According to binding free energies, van der Waals energy was the major favorable contributor to the ligand binding. These results offer that TAME had more affinity to interact with the APC3 subunit, at the IR binding pocket than the APC8 subunit at the Cbox binding pocket. In conclusion, IR binding pocket can serve as an appropriate potential target for TAME as an inhibitor of APC/c.
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http://dx.doi.org/10.1016/j.jmgm.2020.107563DOI Listing
June 2020

In silico mutagenesis in recombinant human keratinocyte growth factor: Improvement of stability and activity in addition to decrement immunogenicity.

J Mol Graph Model 2020 06 28;97:107551. Epub 2020 Jan 28.

Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

The recombinant human keratinocyte growth factor (rhKGF) is clinically applied to decrease the incidence and duration of cancer therapeutic agents. Particularly, it is extensively used for oral mucositis after chemotherapy-induced damage of different human cancers. However, the usage of rhKGF in treatment is limited owing to its short half-life, poor stability, immunogenicity, tendency to aggregate, and side effects. Therefore, there is a need to enhance the stability and to reduce immunogenicity of rhKGF for therapeutic applications. In this study, the stability, activity, and immunogenicity of rhKGF were improved using computational methods. The several mutations were generated based on sequence alignment, amino acids physic-chemical properties, and the structure simulation. The 3D structure of rhKGF and proposed mutants were predicted by Modeller v9.15 program, and then were evaluated using PROSESS, PROCHECK, and ProSA web tools. Afterwards, the effect of these mutants on rhKGF structure, stability, activity, and its interaction with fibroblast growth factor receptor2-IIb (FGFR2-IIb) was analyzed through utilizing GROMACS molecular dynamics simulations and docking tools, respectively. Also, binding free energies were calculated by the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. We found that F63Y, R121K, and combine1 (K38R, F63Y, K72E, N105S) mutants lead to reduction of the number of T-cell epitopes. However, all of the selected mutants, except for R121K, could considerably increase stability and affinity of the rhKGF to FGFR2-IIb, in silico. In conclusion, this study, for the first time, offered that the combine1 and F63Y mutants could highly improve the stability and activity of rhKGF and even reduce immunogenicity without having any significant effect on the biological functions of rhKGF.
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http://dx.doi.org/10.1016/j.jmgm.2020.107551DOI Listing
June 2020

Effect of Sodium Butyrate on mRNA Expression as a Transcription Factor of HDAC8 in Human Colorectal Cancer Cell Lines.

Avicenna J Med Biotechnol 2019 Oct-Dec;11(4):317-324

Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran.

Background: LHX1 is an important transcription factor for the gene. The aim of this study was to investigate the effect of Sodium Butyrate (SB), as a histone deacetylase inhibitor, on the expression of gene in colorectal cancer cell lines.

Methods: HT-29 and HCT-116 cell lines were treated with 6.25 to 200 concentrations of SB at 24, 48, and 72 . The cytotoxicity effect on cell viability was evaluated by MTT assay. The 50% Inhibiting Concentration (IC) was determined graphically. Quantitative real-time PCR was performed to investigate the mRNA expression level.

Results: Our study revealed that SB inhibited the proliferation of these cell lines in a concentration and time-dependent manner. The IC values for HT-29 cell line were 65, 18.6, and 9.2 after 24, 48, and 72 of treatment, respectively. The IC values for HCT-116 cell line were 35.5, 9.6, and 10 after 24, 48, and 72 of treatment, respectively. Furthermore, real-time PCR findings demonstrated that the mRNA expression in treated HT-29 cell line significantly increased in comparison with untreated cells (p<0.05). However, in treated HCT-116 cell line, SB led to a significant decrease in the level of mRNA (p<0.05), as compared to untreated cells.

Conclusion: In this study, different effects of SB on mRNA expression level were revealed in two distinct human colorectal cancer cell lines.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925397PMC
January 2020

Graft versus host disease and microchimerism in a patient.

Allergy Asthma Clin Immunol 2019 22;15:47. Epub 2019 Aug 22.

1Molecular Medicine Department, Pasteur Institute of Iran, Pasteur St., 12 Farvardin Ave., Tehran, 1316943551 Iran.

Background: The lymphohematopoietic cells originating from feto-maternal trafficking during pregnancy may cause microchimerism and lead to materno-fetal graft versus host disease (GVHD) in severe combined immunodeficiency (SCID) patients. However, definitive diagnosis between GVHD and Omenn's syndrome is often difficult based on clinical and immunological phenotypes particularly in the patients with hypomorphic mutations.

Case Presentation: A 3-year-old girl with a history of erythroderma and immunodeficiency was studied. The whole exome sequencing method was used to find the disease-causing variants, and T-A cloning and Quantitative Florescence Polymerase Chain Reaction (QF-PCR) methods were utilized to detect the presence of mosaicism or microchimerism. We identified a homozygous missense Janus Kinase 3 mutation (, c.2324G>A, p.R775H) as a new disease-causing variant in the patient, and the presence of microchimerism with maternal origin was proven as an underlying cause of her clinical presentation.

Conclusion: The findings highlighted the importance of appropriate diagnostic approach in GVHD cases with hypomorphic mutations. When analyzing the results of the next generation sequencing, the possibility of microchimerism should be considered based on the context of the disease.
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http://dx.doi.org/10.1186/s13223-019-0361-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704686PMC
August 2019

Whole Exome Sequencing of an X-linked Thrombocytopenia Patient with Normal Sized Platelets.

Avicenna J Med Biotechnol 2019 Jul-Sep;11(3):253-258

Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.

Wiskott-Aldrich Syndrome () is a rare X-linked recessive Primary Immunodeficiency (PID) caused by mutations in gene which encodes a protein known as WASp. WASp plays important roles in cytoskeletal functions that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration. WASp defect particularly causes platelets abnormality which is presented in forms of decrease of Mean Platelet Volume (MPV) and thrombocytopenia in most conditions; nevertheless, some studies reported patients with a normal or large size of platelets in recent years. This phenomenon is unique and the exact mechanism of thrombocytopenia with a normal or large size of platelets is still unknown. In this study, Next Generation Sequencing (NGS) was utilized to discover the causing mutation in gene; furthermore, an attempt was made to evaluate the possibility of other mutations or genes especially WASp interacting proteins and inherited platelet disorder genes in patient clinical symptoms for the purpose of understanding the origin of such unique symptom and to perform further analysis if it is required. Therefore, clinical manifestations and immunologic functions of the patient were checked and Whole Exome Sequencing (WES) was performed to analyze all exonic variations which can be associated with patient phenotypes. Finally, a novel de novo mutation in gene which truncates WASp to half of its normal size was determined as the only cause of clinical manifestation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626504PMC
August 2019

Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches.

Immunol Lett 2019 08 24;212:106-113. Epub 2019 Jun 24.

Biotechnology Research Centre, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Current advances in antibody engineering driving the strongest growth area in biotherapeutic agents development. Affinity improvement that is mainly important for biological activity and clinical efficacy of therapeutic antibodies, has still remained a challenging task. In the human body, during a course of immune response affinity maturation increase antibody activity by several rounds of somatic hypermutation and clonal selection in the germinal center. The final outputs are antibodies representing higher affinity and specificity against a particular antigen. In the realm of biotechnology, exploring of mutations which improve antibody affinity while preserving its specificity and stability is an extremely time-consuming and laborious process. Recent advances in computational algorithms and DNA sequencing technologies help researchers to redesign antibody structure to achieve desired properties such as improved binding affinity. In this review, we briefly described the principle of affinity maturation and different corresponding in vitro techniques. Also, we recapitulated the most recent advancements in the field of antibody affinity maturation including computational approaches and next-generation sequencing (NGS).
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http://dx.doi.org/10.1016/j.imlet.2019.06.009DOI Listing
August 2019

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli.

Comput Biol Chem 2019 Jun 29;80:225-233. Epub 2019 Mar 29.

Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Background: The recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing.

Objective: The aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli.

Methods: Thirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package.

Results And Conclusion: Computational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.
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http://dx.doi.org/10.1016/j.compbiolchem.2019.03.003DOI Listing
June 2019

Identification of new DNA gyrase inhibitors based on bioactive compounds from streptomyces: structure-based virtual screening and molecular dynamics simulations approaches.

J Biomol Struct Dyn 2020 02 27;38(3):791-806. Epub 2019 Mar 27.

Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

DNA gyrase enzyme has vital role in bacterial survival and can be considered as a potential drug target. Owing to the appearance of resistance to gyrase-targeted drugs, especially fluoroquinolone, screening new compounds which bind more efficiently to the mutant binding pocket is essential. Hence, in this work, using Smina Autodock and through structure-based virtual screening of StreptomeDB, several natural products were discovered based on the SimocyclinoneD8 (SD8) binding pocket of GyrA subunit of DNA gyrase. After evaluation of binding affinity, binding modes, critical interactions and physicochemical and pharmaceutical properties, three lead compounds were selected for further analysis. Afterward 60 ns molecular dynamics simulations were performed and binding free energies were calculated by the molecular mechanics/Poisson-Boltzmann surface area method. Also, interaction of the selected lead compounds with the mutated GyrA protein was evaluated. Results indicated that all of the selected compounds could bind to the both wild-type and mutated GyrA with the binding affinities remarkably higher than SimocyclinoneD8. Interestingly, we noticed that the selected compounds comprised angucycline moiety in their structure which could sufficiently interact with GyrA and block the DNA binding pocket of DNA gyrase, . In conclusion, three DNA gyrase inhibitors were identified successfully which were highly capable of impeding DNA gyrase and can be considered as potential drug candidates for treatment of fluoroquinolone-resistant strains.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2019.1588784DOI Listing
February 2020

Two Novel Tri-Aryl Derivatives Attenuate the Invasion-Promoting Effects of Stromal Mesenchymal Stem Cells on Breast Cancer.

Anticancer Agents Med Chem 2019 ;19(8):1002-1011

Physiology and Pharmacology Department, Pasteur Institute of Iran, Tehran, Iran.

Background: The concept of Epithelial-Mesenchymal Transition (EMT) to promote carcinoma progression has been recognized as a venue for research on novel anticancer drugs. Triaryl template-based structures are one of the pivotal structural features found in a number of compounds with a wide variety of biological properties including anti-breast cancer. Among the various factors triggering EMT program, cyclooxygenase-2 (COX-2), NF-κB as well as the transforming growth factor-beta (TGF-β) have been widely investigated.

Objective: Here, we aim to investigate the effect of two novel compounds A and B possessing triaryl structures, which interact with both COX-2 and TGF-β active sites and suppress NF-κB activation, on EMT in a co-culture system with breast cancer and stromal cells.

Methods: MDA-MB-231 and bone-marrow mesenchymal stem (BM-MS) cells were co-cultured in a trans-well plate. Migration, matrigel-based invasion and colony formation in soft agar assays along with Real- time PCR and Western blot analysis were performed to examine the effect of compounds A and B on the invasive properties of MDA-MB-231 cells after 72 hours of co-culturing with BM-MSCs. In addition, TGF-beta interaction was investigated by Localized Surface Plasmon Resonance (LSPR).

Results: BM-MSCs enhanced migration, invasion and anchorage-independent growth of the co-cultured MDAMB- 231 cells. A reduction in E-cadherin level concomitant with an increase in vimentin and N-cadherin levels following the co-culture implied EMT as the underlying process. Compounds A and B inhibited invasion and anchorage-independent growth of breast cancer cells co-cultured with BM-MSCs at 10µM. The observed inhibitory effects along with an increase in E-cadherin and a reduction in vimentin and ZEB2 levels suggest that the anti-invasive properties of compounds A and B might proceed through the blockade of stromal cell-induced EMT, mediated by their interaction with TGF-beta.

Conclusion: These findings introduce compounds A and B as novel promising agents, which prevent EMT in invasive breast cancer cells.
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http://dx.doi.org/10.2174/1871520619666190212123912DOI Listing
March 2020

The transient production of anti-TNF-α antibody Adalimumab and a comparison of its characterization to the biosimilar Cinorra.

Protein Expr Purif 2019 03 22;155:59-65. Epub 2018 Nov 22.

Department of Genetics & Metabolism, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Recombinant antibodies have emerged over the last few decades as the fastest growing class of therapeutic proteins for autoimmune diseases. Post-translation modifications of antibodies produced by human cell lines are highly consistent with those existing in natural human proteins and this is a major advantage of utilizing these cell lines. Cinorra is a biosimilar form of the antibody Adalimumab, which is an antagonist of TNF-α used for the treatment of autoimmune diseases. Adalimumab and Cinorra were produced by stable expression from CHO cells. The aim of this study was to select HEK cells as a host for producing Adalimumab to reveal whether the antibody produced by this human-derived cell line has similar characterization to Cinorra. Adalimumab was transiently produced in HEK-293T cells, characterized and analyzed for its properties. Circular dichroism spectroscopy confirmed a strong structural similarity of the expressed antibody with Cinorra. Likewise its binding activity and kinetic affinity to TNF-α (EC = 416.5 ng/ml, KD = 3.89 E-10 M,) were highly similar to that of Cinorra (EC = 421.2 ng/ml and KD = 3.34 E-10 M,). Additionally there was near identical neutralization of TNF-α-mediated cellular cytotoxicity (IC of the expressed = 4.93 nM; IC of Cinorra = 4.5 nM). Results indicate that Adalimumab produced by HEK-293T cells possesses a similarly efficient function and biological activity to Cinorra. Consequently, human-derived host cells with human post-translational modifications might potentially provide a basis for the development of Adalimumab with pharmaceutical properties for research and therapeutic use.
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http://dx.doi.org/10.1016/j.pep.2018.11.006DOI Listing
March 2019

Structural and dynamic characterization of human Wnt2-Fzd7 complex using computational approaches.

J Mol Model 2018 Sep 6;24(10):274. Epub 2018 Sep 6.

Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran.

Wnt and Frizzled (Fzd) family members play crucial roles in the self-renewal of tumor-initiating cells. Until now, only a few studies have addressed the distinct mechanism of Wnt-Fzd interactions. In this study, we suggest a possible interaction mode of Wnt2 with the Fzd7 cysteine-rich domain (CRD)-both of which are up-regulated in some types of cancer. A combination of homology modeling, molecular docking and molecular dynamics (MD) simulations was carried out to study this ligand-receptor complex in great detail. The results demonstrated the unique dynamic behavior of Wnt2 upon binding to Fzd7. Interestingly, the β-strand content of the C-terminal binding site of Wnt2 was obviously reduced when bound to Fzd7 CRD. Moreover, the N-terminal and C-terminal binding sites of Wnt2 appeared to interact with the C-terminal and N-terminal binding sites of Fzd7, respectively. Calculation of the binding energies uncovered the pivotal role of electrostatic and hydrophobic interactions in the binding of Wnt2 to Fzd7 CRD. In conclusion, this study provides valuable insights into the mechanism of the Wnt2-Fzd7 CRD interaction for application in colorectal cancer prevention programs. Graphical abstract Flowchart representation of different steps used in this study.
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http://dx.doi.org/10.1007/s00894-018-3788-3DOI Listing
September 2018

In silico studying of the whole protein structure and dynamics of Dickkopf family members showed that N-terminal domain of Dickkopf 2 in contrary to other Dickkopfs facilitates its interaction with low density lipoprotein receptor related protein 5/6.

J Biomol Struct Dyn 2019 Jul 17;37(10):2564-2580. Epub 2018 Nov 17.

a Department of Medical Biotechnology, School of Advanced Technologies in Medicine , Tehran University of Medical Sciences , Tehran , Iran.

Wnt (Wingless Int) signaling pathway has been known to be dysregulated in several human cancers, especially colorectal cancer (CRC). The Dickkopf (DKK) family which consists of four secreted proteins in vertebrates (DKK 1, 2, 3, 4) is one of the most critical antagonist families for Wnt signaling pathway. They typically antagonize Wnt/β-catenin signaling by binding and inhibiting Wnt co-receptors, LRP5/6 (low density lipoprotein receptor related protein 5/6). However, except for DKK1 (Dickkopf 1), details about structure and function of the members of this family are poorly defined. In this study, main Dickkopf family members were analyzed structurally, using protein structure prediction tools, molecular dynamics (MD), molecular docking and energy analyses. Three dimensional structure of whole DKKs was predicted and their interaction with LRP6 was investigated in detail. The results indicated that in DKK family members, a considerable diversity, in the case of structure, activity and physicochemical properties was seen. This diversity was more profound in DKK3 (Dickkopf3). Interestingly, the interaction mode of DKK2 (Dickkopf2) with its receptor, LRP6, was shown to be substantially different from other Dickkopf family members while N-terminal region of this ligand was also involved in the binding to the LRP6-P3P4. Moreover, the cysteine-rich domain 2 (CRD2) of DKK1 and DKK3 had a higher binding affinity to LRP6 in comparison with the whole protein structures. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1491891DOI Listing
July 2019

An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

J Mol Model 2017 Jul 15;23(7):202. Epub 2017 Jun 15.

Biotechnology Research Center, Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran.

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg. Such detailed understanding will probably help to design an improved enzyme.
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http://dx.doi.org/10.1007/s00894-017-3374-0DOI Listing
July 2017

Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound.

Adv Biomed Res 2017 7;6:26. Epub 2017 Mar 7.

Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: Anaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20.

Materials And Methods: The structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated.

Results: It was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown.

Conclusion: The findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds.
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http://dx.doi.org/10.4103/2277-9175.201683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359995PMC
March 2017

designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation.

Res Pharm Sci 2017 Feb;12(1):60-66

Thrombosis and Homeostasis Research Center, Imam Khomeni Hospital Complex, Tehran University of Medical Sciences, Tehran, I.R. Iran.

Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results.
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http://dx.doi.org/10.4103/1735-5362.199048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333481PMC
February 2017

Rational design of DKK3 structure-based small peptides as antagonists of Wnt signaling pathway and in silico evaluation of their efficiency.

PLoS One 2017 24;12(2):e0172217. Epub 2017 Feb 24.

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Dysregulated Wnt signaling pathway is highly associated with the pathogenesis of several human cancers. Dickkopf proteins (DKKs) are thought to inhibit Wnt signaling pathway through binding to lipoprotein receptor-related protein (LRP) 5/6. In this study, based on the 3-dimensional (3D) structure of DKK3 Cys-rich domain 2 (CRD2), we have designed and developed several peptide inhibitors of Wnt signaling pathway. Modeller 9.15 package was used to predict 3D structure of CRD2 based on the Homology modeling (HM) protocol. After refinement and minimization with GalaxyRefine and NOMAD-REF servers, the quality of selected models was evaluated utilizing VADAR, SAVES and ProSA servers. Molecular docking studies as well as literature-based information revealed two distinct boxes located at CRD2 which are actively involved in the DKK3-LRP5/6 interaction. A peptide library was constructed conducting the backrub sequence tolerance scanning protocol in Rosetta3.5 according to the DKK3-LRP5/6 binding sites. Seven tolerated peptides were chosen and their binding affinity and stability were improved by some logical amino acid substitutions. Molecular dynamics (MD) simulations of peptide-LRP5/6 complexes were carried out using GROMACS package. After evaluation of binding free energies, stability, electrostatic potential and some physicochemical properties utilizing computational approaches, three peptides (PEP-I1, PEP-I3 and PEP-II2) demonstrated desirable features. However, all seven improved peptides could sufficiently block the Wnt-binding site of LRP6 in silico. In conclusion, we have designed and improved several small peptides based on the LRP6-binding site of CRD2 of DKK3. These peptides are highly capable of binding to LRP6 in silico, and may prevent the formation of active Wnt-LRP6-Fz complex.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172217PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325476PMC
September 2017

In silico enhancement of the stability and activity of keratinocyte growth factor.

J Theor Biol 2017 04 16;418:111-121. Epub 2017 Jan 16.

Molecular Medicine Department, Biotechnology Research Center., Pasteur Institute of Iran, Iran. Electronic address:

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, has been implicated in some biological processes such as cell proliferation, development and differentiation. High mitogenic activity of this protein has made it very suitable for repairing radiation-and chemotherapy-induced damages. Palifermin, which has been developed from human KGF, is clinically applied to reduce the incidence and duration of cancer therapeutic agents. However, the activity of Palifermin is limited during treatment due to its poor stability. In this study, we have improved the stability and activity of recombinant human KGF (Palifermin) using a computational mutagenesis approach. According to the KGF multiple sequence alignment among different species as well as literature-based information, we have generated several mutations using PyMOL program and evaluated their effects on the stability and activity of KGF in silico. In order to preserve the KGF activity, we did not change the predicted functional residues. Prior to mutagenesis, the 3D structure of rhKGF was predicted by Modeller v9.15 program and quantitative evaluation of predicted models were carried out using VADAR and PROSESS servers. The stability and activity of rhKGF mutants were analyzed using GROMACS molecular dynamics (MD) simulations and docking tools, respectively. The results showed that N159S (N105S in rhKGF sequence) and I172V (I118V in rhKGF) substitutions caused an increased stability and affinity of the rhKGF to Fibroblast growth factor receptor 2 (FGFR2). We will evaluate the effects of favorable mutations on the rhKGF stability and activity in vitro.
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http://dx.doi.org/10.1016/j.jtbi.2017.01.009DOI Listing
April 2017

Molecular Characterization of Animal spp. Isolates from Kermanshah, Western Iran.

Iran J Public Health 2016 Oct;45(10):1315-1321

Dept. of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran.

Background: We evaluated the genetic diversity of samples identified morphologically as spp. from sheep, cattle and goat from Kermanshah Province, western Iran using PCR-RFLP method.

Methods: We used PCR-RFLP analysis of ribosomal ITS1 fragment using RsaI restriction enzyme to investigate the genetic characteristics of species obtained from different hosts (16 sheep, 28 cattle, 4 goats). The species of were confirmed by sequencing the 700 bp region of ribosomal ITS1 gene.

Results: In Kermanshah, was present in 96% of the samples, was found only in two cattle sample. No hybrid forms were detected in the present study.

Conclusion: Our results contribute to clarify the dark spots of genotyping in different parts of Iran.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5149495PMC
October 2016

Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.

Mol Immunol 2016 10 17;78:183-192. Epub 2016 Sep 17.

Vrije University Brussel, Research group Cellular & Molecular Immunology, Brussels, Belgium; VIB, Department of Structural Biology, Vrije University Brussel, Belgium.

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×10 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.
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http://dx.doi.org/10.1016/j.molimm.2016.09.012DOI Listing
October 2016