Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches [Corrigendum]

Pathan AAK, Panthi B, Khan Z, et al. OncoTargets and Therapy. 2016;9:2575–2584.  On page 2582, panel A, B, C of Figure 5 had typographical and color issues.Read the original articl

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC), Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion: Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. Keywords: antitumor agent, K-Ras, molecular docking, molecular modeling, virtual screenin

Diospyros assimilis root extract assisted biosynthesised silver nanoparticles and their evaluation of antimicrobial activity

The current research study focuses on biosynthesis of silver nanoparticles (Ag NPs) for the first time from silver acetate employing methanolic root extract of Diospyros assimilis. The UV-Vis absorption spectrum of biologically synthesised nanoparticles displayed a surface plasmon peak at 428nm indicating the formation of Ag NPs. The influence of metal ion concentration, reaction time and amount of root extract in forming Ag NPs by microscopic and spectral analysis was thoroughly investigated. Structural analysis from transmission electron microscopy confirmed the nature of metallic silver as face-centered cubic (FCC) crystalline with an average diameter of 17nm, which correlates with an average crystallite size (19nm) calculated from X-ray diffraction analysis. Further, the work was extended for the preliminary examination of antimicrobial activity of biologically synthesised Ag NPs that displayed promising activity against all the tested pathogenic strains

Polymorphisms in RAD51 and their relation with breast cancer in Saudi females

Sahar Tulbah,1 Huda Alabdulkarim,2 Mohammad Alanazi,3 Narasimha Reddy Parine,3 Jilani Shaik,3 Akbar Ali Khan Pathan,3 Abdullah Al-Amri,3 Wajahatullah Khan,4 Arjumand Warsy1 1Department of Biochemistry, College of Science, King Saud University, Center of Scientific and Medical Colleges, 2Department of Hematology/Oncology, King Fahad Medical City Hospital, Comprehensive Cancer Center, 3Department of Biochemistry, College of Science, King Saud University, 4Basic Sciences Department, College of Science and Health Professions, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Abstract: The present study aimed at investigating the relationship between rs1801320 (G>C), rs1801321 (G>T), and rs2619681 (C>T) RAD51 gene polymorphisms and the risk of breast cancer development in Saudi females. The genotypes were analyzed using TaqMan genotyping assay and polymerase chain reaction-restriction fragment length polymorphism. The genotype and allele frequencies were computed using chi-square or Fisher’s exact test (two-tailed) by SPSS 21 software. The results showed that rs1801321G>T GG genotype and G allele frequency were strongly (P<0.0001) related to an elevated risk of breast cancer, while the mutant T allele appeared to provide protection against breast cancer development as observed from the significantly lower (P<0.0001) frequencies of the TT and GT genotypes in cancer patients compared to the healthy controls. The variant rs1801320G>C showed no significant differences in the frequencies of the genotypes and alleles in the patients and the control groups. The CC genotype and C allele frequency of rs2619681 (C>T) variant were significantly (P=0.012) higher in cancer patients, whereas the T allele showed a protective effect against cancer development. The frequencies of the three single-nucleotide polymorphisms did not differ in cancer patients with different tumor grades and human epidermal growth factor receptor 2 status (+ or -). However, the genotype frequency of rs1801320 (135G>C) differed in the patients with estrogen receptor (ER)+ and ER-, where CC genotype showed a significantly higher prevalence in the females with ER- who were suffering from breast cancer. In addition, the frequency of C allele of rs2619681 (C>T) was also significantly higher in the breast cancer patients who were ER+ and progesterone receptor (PR)+ compared to those with ER- and PR-. In the Saudi females, rs1801320 did not show an association with risk of breast cancer. Taken together, the results suggest that RAD51 rs1801321 polymorphism may be involved in the etiology of breast cancer in the Saudi females; however, further studies are necessary to confirm this relation. Keywords: RAD51, breast cancer, Saudi Arabia, single nucleotide polymorphis

Effect of smoking on the genetic makeup of toll-like receptors 2 and 6

Muhammad Kohailan,1 Mohammad Alanazi,1 Mahmoud Rouabhia,2 Abdullah Alamri,1 Narasimha Reddy Parine,1 Abdullah Alhadheq,1 Santhosh Basavarajappa,3 Abdul Aziz Abdullah Al-Kheraif,3 Abdelhabib Semlali1 1Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Département de Stomatologie, Faculté de Médecine Dentaire, Groupe de Recherche en Écologie Buccale, Université Laval, Québec City, QC, Canada; 3Dental Biomaterial Research Chair, Department of Dental Health, College of Applied Medical Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia Background: Cigarette smoking is a major risk factor for lung cancer, asthma, and oral cancer, and is central to the altered innate immune responsiveness to infection. Many hypotheses have provided evidence that cigarette smoking induces more genetic changes in genes involved in the development of many cigarette-related diseases. This alteration may be from single-nucleotide polymorphisms (SNPs) in innate immunity genes, especially the toll-like receptors (TLRs).Objective: In this study, the genotype frequencies of TLR2 and TLR6 in smoking and nonsmoking population were examined.Methods: Saliva samples were collected from 177 smokers and 126 nonsmokers. The SNPs used were rs3804100 (1350 T/C, Ser450Ser) and rs3804099 (597 T/C, Asn199Asn) for TLR2 and rs3796508 (979 G/A, Val327Met) and rs5743810 (745 T/C, Ser249Pro) for TLR6.Results: Results showed that TLR2 rs3804100 has a significant effect in short-term smokers (OR =2.63; P=0.04), and this effect is not observed in long-term smokers (>5 years of smoking). Therefore, this early mutation may be repaired by the DNA repair system. For TLR2 rs3804099, the variation in genotype frequencies between the smokers and control patients was due to a late mutation, and its protective role appears only in long-term smokers (OR =0.40, P=0.018). In TLR6 rs5743810, the TT genotype is significantly higher in smokers than in nonsmokers (OR =6.90). The effect of this SNP is observed in long-term smokers, regardless of the smoking regime per day.Conclusion: TLR2 (rs3804100 and rs3804099) and TLR6 (rs5743810) can be used as a potential index in the diagnosis and prevention of more diseases caused by smoking. Keywords: polymorphism, toll-like receptor, genotyping, smoking, TLR2, TLR

Toll-like receptor-4 as a predictor of clinical outcomes of estrogen receptor-negative breast cancer in Saudi women

Abdelhabib Semlali,1 Maroua Jalouli,2 Narasimha Reddy Parine,1 Abdullah Al Amri,1 Maha Arafah,3 Abdulrahman Al Naeem,4 Sanaa Abdullah Ajaj,5 Mahmoud Rouabhia,6 Mohammad Saud Alanazi1 1Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Centre de recherche du CHU de Québec, L’Hôtel-Dieu de Québec, Department of Molecular Biology, Medical Biochemistry and Pathology, Université Laval, Quebec, QC, Canada; 3College of Medicine, King Saud University, 4Department of Women’s Imaging, King Fahad Medical City, 5Department of Family Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 6Groupe de Recherche en Écologie Buccale, Department of Stomatology, Faculty of Dentistry, Université Laval, Quebec, QC, Canada Abstract: The aim of this study was to investigate the association of the common polymorphisms of Toll-like receptor 4 (TLR-4) with breast cancer development in the Saudi Arabian population. Four TLR-4 polymorphisms (rs2770150, rs10759931, rs10759932, and rs4986790) were studied using 127 breast cancer patients and 117 controls. Relative expression of TLR-4 protein in the breast tumor and the matched normal breast tissues was determined in a large cohort of 70 clinical breast samples in a tissue micro-array format by immunohistochemistry using a specific anti-TLR-4 antibody. Our results demonstrated an increase in TLR-4 expression in estrogen receptor (ER)-, postmenopausal breast cancer patients compared to normal. We also demonstrated that the G allele of single-nucleotide polymorphism rs10759931 was found to be significantly higher in frequency among patients (36.3%) compared to the control group (26.7%), suggesting that this polymorphism is strongly associated with the development of breast cancer in this ethnic population. In addition, the TLR-4 polymorphism rs2770150 was shown to be highly correlated with breast cancer in patients over 48 years of age. The TLR-4 polymorphism rs4986790 was also found to be associated with this malignancy in the ER- patient groups. Our results suggested firstly that the variation in TLR-4 gene expression may influence breast cancer development and secondly a closely linked association between TLR-4 gene polymorphism and ER status. Our study provides support for a better understanding of the implication of TLR-4 polymorphism in breast tumorigenesis and for its eventual use as a cancer biomarker. Keywords: Toll-like receptor 4, single-nucleotide polymorphisms, breast cancer, Saudi population, innate immunity, estrogen recepto

No genetic relationship between TLR2 rs4696480, rs3804100, and rs3804099 gene polymorphisms and female breast cancer in Saudi populations

Abdelhabib Semlali,1 Mikhlid Almutairi,2 Narasimha Reddy Parine,1 Abdullah Al Amri,1 Jilani P Shaik,1 Abdulrahman Al Naeem,3 Sana Abdulla Ajaj,4 Mahmoud Rouabhia,5 Mohammad Saud Alanazi1 1Department of Biochemistry, 2Zoology Department, College of Science, King Saud University, 3Department of Women’s Imaging, King Fahad Medical City, 4Family Medicine Department, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; 5Groupe de Recherche en Écologie Buccale, Département de Stomatologie, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada Abstract: Breast cancer (BC) is the most common cause of cancer-related deaths among women in the Kingdom of Saudi Arabia. An association between the dysregulation of innate immunity, primarily the deregulation of Toll-like receptors (TLRs), and BC development was described a long time ago. Several studies have reported that BC risk factors appear to be related to the interaction between certain genes and exposure to various environmental factors. Here, we investigated the potential correlation of three TLR2 single-nucleotide polymorphisms (SNPs; rs3804100, rs4696480, and rs3804099) with the development of BC in female patients from Saudi Arabia. We collected 126 blood samples from women with BC and 146 blood samples from healthy women without any clinical signs of BC. The genotypic frequencies of TLR2 polymorphisms were assayed. Our results showed that the genotypic and allelic frequencies of TLR2 did not differ significantly between BC patients and healthy controls. However, the distributions of rs3804100 (1350 T/C) genotypes in BC groups were 1%, 19%, and 80% for CC, CT, and TT, respectively. In the control group, the rs3804100 (1350 T/C) genotype distributions were 3%, 18%, and 79% for CC, CT, and TT, respectively. The SNP rs3804100 homozygous “TT” genotype was not associated with the risk of developing BC in the BC patients compared with controls (odds ratio [OR], 4.5; confidence interval [CI], 0.49–41.02; P=0.145). The TLR2 rs4696480 AA genotype was observed in 23% of BC patients compared to 18% of control individuals, the AT genotype was seen in 40% of BC patients and 46% of control individuals, and the TT genotype was observed in 37% of BC patients and 36% of normal controls. Our results did not show any difference in genotypic frequency between BC patients and normal controls for the TLR2 rs3804099 SNP; however, the (C) phenotypic frequency was 49% in BC patients and 53% in controls. The (T) phenotypic frequency was 51% and 47% in BC patients and normal patients, respectively. These findings indicate that there is no association between the TLR2 polymorphisms tested and BC susceptibility in the female population from the Kingdom of Saudi Arabia. We suggest using other TLR2 SNPs to investigate the possible relationship between innate immunity deregulation by disruption of TLR2 and potential BC development. Keywords: breast cancer, genotyping, polymorphism, Toll-like receptor

CYP19A1 gene polymorphism and colorectal cancer etiology in Saudi population: case–control study

Fatimah Basil Al-Mukaynizi,1 Mohammed Alanazi,1 Sooad Al-Daihan,1 Narasimha Reddy Parine,1 Majid Almadi,2 Abdulrahman Aljebreen,2 Nahla Azzam,2 Othman Alharbi,2 Maha Arafah,3 Arjumand Warsy4 1Department of Biochemistry, College of Science, 2Department of Internal Medicine, 3Department of Pathology, College of Medicine, 4Central Laboratory, Female Center for Scientific & Medical Colleges, King Saud University, Riyadh, Saudi Arabia Background: Considerable interest is directed toward the enzyme aromatase (CYP19A1) and the development of cancer, due to CYP19A1’s role in estrogen biosynthesis. Several cancers display excessive intra-tumor accumulation of estrogens, and aromatase inhibitors are used for treatment. The CYP19A1 gene exhibits polymorphism and mutations that can alter its expression or aromatase activity and influence estrogen production. We designed this study to investigate the link between CYP19A1 polymorphism and susceptibility to colorectal cancer (CRC) development in Saudis. Patients and methods: Blood samples from 100 CRC patients and 100 healthy controls were drawn for DNA extractions. Three polymorphic sites, rs4774585, rs936308, and rs4775936, were genotyped using Taqman genotyping by real-time polymerase chain reaction. Allelic and genotype frequencies were calculated and compared in the two groups. Results: All single nucleotide polymorphisms (SNPs) were polymorphic in Saudis, and comparison of allele frequencies showed several differences when compared to other populations. None of the SNPs were associated with the risk of CRC development in Saudis (P>0.05). Some gender and location (colon or rectal) differences were observed. Discussion: The results of this study highlighted the genetic heterogeneity existing between populations in the prevalence of different SNPs and their relation to disease state. It showed that, although rs4774585, rs936308, and rs4775936 are involved in CRC development in several populations, their role is not significant in the etiology of CRC in Saudis; however, some SNPs do increase susceptibility or resistance to CRC development as judged from the odds ratio. Further large-scale studies are warranted to clarify the role of the CYP19A1 development in CRC. Keywords: aromatase, CYP19A gene, polymorphism, SNPs, colorectal cancer, rs4774585, rs936308, rs477593

Potential role of Toll-like receptor 2 expression and polymorphisms in colon cancer susceptibility in the Saudi Arabian population

Abdelhabib Semlali,1,2 Narasimha Reddy Parine,2 Nouf S Al-Numair,3,4 Mikhlid Almutairi,5 Yousef M Hawsawi,3 Abdullah Al Amri,2 Abdulrahman M Aljebreen,6,7 Maha Arafah,6 Majid A Almadi,6,7 Nahla Ali Azzam,6,7 Othman Alharbi,6,7 Mohammad Saud Alanazi2 1Groupe de Recherche en Écologie Buccale, Département de stomatologie, Faculté de Médecine Dentaire, Université Laval, Quebec City, QC, Canada; 2Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 3Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia; 4College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi Arabia; 5Zoology Department, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 6College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; 7Division of Gastroenterology, King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia Background: Inflammation is a fundamental factor that contributes to the development and progression of several types of cancer including colon cancer. Toll-like receptors (TLRs) and their signaling pathways have been reported to be associated with chronic inflammation and thereby induced cancer. Our aim was to investigate the expression and polymorphisms of TLR2 and their association with colon cancer. Methods: Real-time PCR and immunohistochemistry were used to investigate TLR2 gene expression and to evaluate the potential risk of predisposition to colon cancer caused by three tagging single-nucleotide polymorphisms (SNPs) on TLR2, including rs3804100, rs4696480, and rs3804099. TaqMan assay was conducted on samples from 115 patients with colon cancer and 102 age- and sex-matched normal individuals. Results: We found that, TLR2 was highly expressed in epithelial colon cancer cells and both TLR2 mRNA and protein levels, and significantly decreased in tumor tissues compared to normal tissues. Two of three TLR2 SNPs increased the risk of colon cancer. However, TLR2 rs3804099 increased the risk of colon cancer development by more than 3.8- and 5-fold in female patients and patients aged less than 57 years, respectively. The T allele of TLR2 rs3804100 showed a significant association with patients less than 57 years. In silico analysis of the TLR2 nucleotide substitution in SNP rs3804100 and rs3804099 determined that 67% and 70% probability of these single nucleotide variants alter splicing phenotypes, rs3804100 more specifically result on activating an additional splice site. Genotype and allele frequencies of rs4696480 were similar between the overall study populations. Thus, TLR2 rs4696480 appear to be not involved in colon cancer in our study population. Conclusions: There was a significant link between innate immunity deregulation through disruption of the TLRs and potential development of colon cancer. These SNPs can be used as screening markers for predicting colon cancer risk earlier in life to implement necessary prevention. Keywords: colon cancer, gene expression, genotyping, polymorphism, Toll-like receptors, innate immunit

Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients

Abdelhabib Semlali,1 Narasimha Reddy Parine,1 Abdullah Al Amri,1 Arezki Azzi,2 Maha Arafah,3 Muhammad Kohailan,1 Jilani P Shaik,1 Majid Abdulrahman Almadi,4,5 Abdulrahman M Aljebreen,3,4 Othman Alharbi,3,4 Nahla Ali Azzam,3,4 Mahmoud Rouabhia,6 Mohammad Saud Alanazi1 1Genome Research, Department of Biochemistry, College of Sciences, King Saud University, 2College of Medicine, Al Imam Muhammad Ibn Saud Islamic University, 3College of Medicine, King Saud University, 4Division of Gastroenterology, King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi Arabia; 5Division of Gastroenterology, McGill University Health Center, Montreal General Hospital, Montreal, 6Groupe de Recherche en Écologie Buccale, Département de Stomatologie, Faculté de Médecine Dentaire, Université Laval, Québec City, QC, Canada Objective: The authors aimed to explore the relationship between the expression/polymorphisms of TLR-9 and susceptibility to colon cancer development in the Saudi Arabian population. Methods: In total, blood samples from 115 patients with colon cancer and 102 participants without colon cancer were analyzed in this study. Three single-nucleotide polymorphisms (SNPs) were selected from the TLR-9 gene, including two sites within the TLR-9 gene’s promoter region (rs352144 and rs187084) and one site in a TLR-9 intron region (rs5743839). Odds ratios (ORs) and 95% confidence intervals (CIs) were computed from logistic regression models after adjusting for age, gender, and tumor localization. To investigate the differential expression of TLR-9 in colon cancer, TLR-9 expression was evaluated using quantitative real-time reverse transcription polymerase chain reaction on 40 matched normal and colon tissues. Results: The authors found that TLR-9 expression was decreased in colon cancer tissues as compared with that in normal tissues. Moreover, significant associations between the TLR-9 rs187084 SNP and colon cancer risk were observed in female patients only. In rs187084, the T allele had a significantly lower frequency (2.8 times) in female cancer patients than in controls (0.27 vs 0.41). The TLR-9 rs352139 and rs352144 SNPs were significantly associated with colon cancer development when the tumor was located in the rectal area. Conclusion: The findings support the hypothesis that TLR-9 has an anticancer role in colon cancer development. Furthermore, genetic variation may influence colon cancer development, and SNPs in TLR-9 could serve as biomarkers for decision making in the treatment of females with rectal cancer. Keywords: Innate, immunity, TLR polymorphisms, rs187084, rs352139, rs35214