A novel homozygous nonsense mutation in NECTIN4 gene in a Pakistani family with ectodermal dysplasia syndactyly syndrome 1.

BACKGROUND: Ectodermal dysplasia syndactyly syndrome 1 (EDSS1) is a rare hereditary disorder characterized by defects in teeth, hair, and nails in association with a fusion of the digits. Genetically, the disease phenotypes are caused by homozygous and compound heterozygous variants in NECTIN4 gene. OBJECTIVE: The main objective of the study was to identify the pathogenic sequence variant(s) for family screening and identification of carriers. METHODS: In the present study, the authors have investigated a large consanguineous family of Pakistani origin segregating autosomal recessive EDSS1. All the coding exons of the NECTIN4 gene were directly sequenced using gene-specific primers. RESULTS: The affected individuals presented the classical EDSS1 clinical features including sparse hair, hypoplastic nails with thick flat discolored nail plates, peg-shaped, conical, and widely spaced teeth with enamel hypoplasia, proximal cutaneous syndactyly of fingers and toes. Sequence analysis of the coding region of the NECTIN4 identified a novel nonsense variant [c.163C>T; p.(Arg55*)] in exon-2 of the gene. Computational analysis of protein structure revealed that the variant induced premature termination at Arg55 located in Ig-like V-loop region leading to loss of Ig-C2 type domains and transmembrane region, and most likely Nectin-4 function will be lost. STUDY LIMITATION: Gene expression studies are absent that would have strengthened the findings of computational analysis. CONCLUSION: The present study expanded the phenotypic and mutation spectrum of the NECTIN4 gene. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families.

A Novel Homozygous Nonsense Variant in the DYM Underlies Dyggve-Melchior-Clausen Syndrome in Large Consanguineous Family.

(1) Background: Dyggve-Melchior-Clausen Syndrome is a skeletal dysplasia caused by a defect in the DYM gene (OMIM number 607461). Pathogenic variants in the gene have been reported to cause Dyggve-Melchior-Clausen (DMC; OMIM 223800) dysplasia and Smith-McCort (SMC; OMIM 607326) dysplasia. (2) Methods: In the present study, large consanguineous families with five affected individuals with osteochondrodysplasia phenotypes were recruited. The family members were analyzed by polymerase chain reaction for homozygosity mapping using highly polymorphic microsatellite markers. Subsequent to linkage analysis, the coding exons and exon intron border of the DYM gene were amplified. The amplified products were then sent for Sanger sequencing. The structural effect of the pathogenic variant was analyzed by different bioinformatics tools. (3) Results: Homozygosity mapping revealed a 9 Mb homozygous region on chromosome 18q21.1 harboring DYM shared by all available affected individuals. Sanger sequencing of the coding exons and exon intron borders of the DYM gene revealed a novel homozygous nonsense variant [DYM (NM_017653.6):c.1205T>A, p.(Leu402Ter)] in affected individuals. All the available unaffected individuals were either heterozygous or wild type for the identified variant. The identified mutation results in loss of protein stability and weekend interactions with other proteins making them pathogenic (4) Conclusions: This is the second nonsense mutation reported in a Pakistani population causing DMC. The study presented would be helpful in prenatal screening, genetic counseling, and carrier testing of other members in the Pakistani community.

A novel homozygous nonsense mutation in NECTIN4 gene in a Pakistani family with ectodermal dysplasia syndactyly syndrome 1

Abstract Background Ectodermal dysplasia syndactyly syndrome 1 (EDSS1) is a rare hereditary disorder characterized by defects in teeth, hair, and nails in association with a fusion of the digits. Genetically, the disease phenotypes are caused by homozygous and compound heterozygous variants in NECTIN4 gene. Objective The main objective of the study was to identify the pathogenic sequence variant(s) for family screening and identification of carriers. Methods In the present study, the authors have investigated a large consanguineous family of Pakistani origin segregating autosomal recessive EDSS1. All the coding exons of the NECTIN4 gene were directly sequenced using gene-specific primers. Results The affected individuals presented the classical EDSS1 clinical features including sparse hair, hypoplastic nails with thick flat discolored nail plates, peg-shaped, conical, and widely spaced teeth with enamel hypoplasia, proximal cutaneous syndactyly of fingers and toes. Sequence analysis of the coding region of the NECTIN4 identified a novel nonsense variant [c.163C>T; p.(Arg55*)] in exon-2 of the gene. Computational analysis of protein structure revealed that the variant induced premature termination at Arg55 located in Ig-like V-loop region leading to loss of Ig-C2 type domains and transmembrane region, and most likely Nectin-4 function will be lost. Study limitation Gene expression studies are absent that would have strengthened the findings of computational analysis. Conclusion The present study expanded the phenotypic and mutation spectrum of the NECTIN4 gene. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families.

ZOMEC via the p-Akt/Nrf2 Pathway Restored PTZ-Induced Oxidative Stress-Mediated Memory Dysfunction in Mouse Model.

A new mechanistic approach to overcome the neurodegenerative disorders caused by oxidative stress in Alzheimer's disease (AD) is highly stressed in this article. Thus, a newly formulated drug (zinc ortho-methyl carbonodithioate (ZOMEC)) was investigated for five weeks on seven-week-old BALB/c male mice. ZOMEC 30 mg/kg was postadministered intraperitoneally during the third week of pentylenetetrazole (PTZ) injection. The brain homogenates of the mice were evaluated for their antioxidant potential for ZOMEC. The results including catalase (CAT), glutathione S transferase (GST), and lipid peroxidation (LPO) demonstrated that ZOMEC significantly reverted the oxidative stress stimulated by PTZ in the mouse brain. ZOMEC upregulated p-Akt/Nrf-2 pathways (also supported by molecular docking methods) to revoke PTZ-induced apoptotic protein markers. ZOMEC reversed PTZ-induced neuronal synapse deficits, improved oxidative stress-aided memory impairment, and inhibited the amyloidogenic pathway in mouse brains. The results suggested the potential of ZOMEC as a new, safe, and neurotherapeutic agent to cure neurodegenerative disorders by decreasing AD-like neuropathology in the animal PTZ model.

A novel homozygous variant in the GLI1 underlies postaxial polydactyly in a large consanguineous family with intra familial variable phenotypes.

Polydactyly is a human inherited disorder caused by to anomalies in the genes involved in autopod development. The disorder segregates in both autosomal recessive and autosomal dominant form. Up till now, eleven genes causing non-syndromic polydactyly, have been identified. This includes ZNF141, GLI3, ZRS in LMBR1, MIPOL1, PITX1, IQCE, GLI1, FMA92A1, KIAA0825, STKLD1, and DACH1. In the present study, we have investigated a large consanguineous family of Pakistani origin segregating polydactyly in autosomal recessive pattern. Clinical examination of affected individuals revealed a non-syndromic form of the disorder. Genetic study based on homozygosity mapping and Sanger sequencing using DNA of the normal and affected individuals found a novel homozygous missense sequence variant [NM_005269.3: c.1133C > T, p.(Ser378Leu)] in the GLI1 located on human chromosome 12q13.3. In silico analysis of the identified variant showed a significant change in the secondary structure of the mutant protein that affects its function. Findings of the present study expand the mutation spectrum of the GLI1. In addition, the study will help in prevention of the disorder through carrier testing and bringing awareness among families affected with polydactyly.

Sulindac acetohydrazide derivative attenuates against cisplatin induced organ damage by modulation of antioxidant and inflammatory signaling pathways.

This study aimed to explore the mechanisms of action of a sulindac acetohydrazide derivative, N'-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide, against anticancer drug cisplatin induced organ damage. Using a rodent model, various markers of organ function and signaling pathways were examined and validated by molecular docking studies. The study involves five groups of animals: control, DMSO, CDDP, CDDP + DMFM, and DMFM. Biochemical enzyme activity, histopathology, tissue antioxidant, and oxidative stress markers were examined. RT-PCR and western blot analyses were conducted for the expression of inducible cyclooxygenase enzyme (COX-2), nuclear factor kappa beta (NF-κB), p65, IL-1, TNF-α, and inducible nitric oxide synthase (iNOS). Flow cytometry analysis of CD4 + TNF-α, CD4 + COX-2, and CD4 + STAT-3 cells in whole blood was performed. Structural and dynamic behavior of DMFM upon binding with receptor molecule molecular docking and dynamic simulations were performed using bioinformatics tools and software. Treatment with DMFM reversed cisplatin-induced malondialdehyde (MDA) and nitric oxide (NO) induction, whereas the activity of glutathione peroxidase (GPx), and superoxide dismutase (SOD) in the kidney, heart, liver, and brain tissues were increased. DMFM administration normalized plasma levels of biochemical enzymes. We observed a marked decline in CD4 + STAT3, TNF-α, and COX2 cell populations in whole blood after treatment with DMFM. DMFM downregulated the expression factors related to inflammation at the mRNA and protein levels, i.e., IL-1, TNF-α, iNOS, NF-κB, STAT-3, and COX-2. Dynamic simulations and in silico docking data supports the experimental findings. Our experimental and in silico results illustrated that DMFM may affect protective action against cisplatin-induced brain, heart, liver, and kidney damage via reduction of inflammation and ROS.

Molecular docking, pharmacokinetic studies, and in vivo pharmacological study of indole derivative 2-(5-methoxy-2-methyl-1H-indole-3-yl)-N'-[(E)-(3-nitrophenyl) methylidene] acetohydrazide as a promising chemoprotective agent against cisplatin induced organ damage.

Cisplatin is an efficient anticancer drug against various types of cancers however, its usage involves side effects. We investigated the mechanisms of action of indole derivative, 2-(5-methoxy-2-methyl-1H-indol-3-yl)-N'-[(E)-(3-nitrophenyl) methylidene] acetohydrazide (MMINA) against anticancer drug (cisplatin) induced organ damage using a rodent model. MMINA treatment reversed Cisplatin-induced NO and malondialdehyde (MDA) augmentation while boosted the activity of glutathione peroxidase (GPx), and superoxide dismutase (SOD). The animals were divided into five groups (n = 7). Group1: Control (Normal) group, Group 2: DMSO group, Group 3: cisplatin group, Group 4: cisplatin + MMINA group, Group 5: MMINA group. MMINA treatment normalized plasma levels of biochemical enzymes. We observed a significant decrease in CD4+COX-2, STAT3, and TNF-α cell population in whole blood after MMINA dosage. MMINA downregulated the expression of various signal transduction pathways regulating the genes involved in inflammation i.e. NF-κB, STAT-3, IL-1, COX-2, iNOS, and TNF-α. The protein expression of these regulatory factors was also downregulated in the liver, kidney, heart, and brain. In silico docking and dynamic simulations data were in agreement with the experimental findings. The physiochemical properties of MMINA predicted it as a good drug-like molecule and its mechanism of action is predictably through inhibition of ROS and inflammation.

Hg(II) sensing, catalytic, antioxidant, antimicrobial, and anticancer potential of Garcinia mangostana and α-mangostin mediated silver nanoparticles.

This study reports synthesis of Garcinia mangostana fruit pericarp (unwanted waste material) and α-mangostin mediated silver nanoparticles (AgNPs). These AgNPs were efficiently produced using 1:10 (extract and salt) ratio under stirring and heating, which was confirmed by surface plasmon resonance (SPR) band in UV-Visible spectroscopic analysis, and size of 73-91 nm determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The synthesized AgNPs were used for Hg(II) detection in tap water, where the limits of detection and quantification were 2.6 µM and 8.9 µM, respectively. Furthermore, the subject AgNPs showed promising catalytic activity in the reduction of dyes and food colours including Congo red (CR), methylene blue (MB), malachite green (MG), methyl orange (MO), para-nitrophenol (PNP), rhodamine B (RdB), zarda yellow (ZY), deep green (DG), and bright red (BR). The synthesized AgNPs were also evaluated for their antioxidant, antimicrobial, and anticancer properties, where α-mangostin and its nanoparticles (Mang-AgNPs) exhibited promising IC50 values of 14.1 and 13.5 µg/mL, respectively against DU-145 cell line validated by in silico molecular docking study. This study is the first report highlighting the application of AgNPs of G. mangostana fruit pericarp extracts, and α-mangostin in Hg(II) detection, dyes degradation, and anticancer potential against DU-145. Finding of this study suggested the suitability of AgNPs as promising solid biosensor in Hg(II) metal detection, dyes reduction, and target in anticancer drug development.

Elucidation of PLK1 Linked Biomarkers in Oesophageal Cancer Cell Lines: A Step Towards Novel Signaling Pathways by p53 and PLK1-Linked Functions Crosstalk.

BACKGROUND: Oesophgeal adenocarcinoma (OAC) is the most frequent cause of cancer death. POLO-like kinase 1 (PLK1) is overexpressed in broad spectrum of tumors and has prognostic value in many cancers including esophageal cancer, suggesting its potential as a therapeutic target. p53, the guardian of genome is the most important tumor suppressors that represses the promoter of PLK1, whereas tumor cells with inactive p53 are arrested in mitosis due to DNA damage. PLK1 expression has been linked to the elevated p53 expression and has been shown to act as a biomarker that predicts poor prognosis in OAC. OBJECTIVES: The aim of the present study was identification of PLK1 associated phosphorylation targets in p53 mutant and p53 normal cells to explore the downstream signaling evets. METHODS: Here we develop a proof-of-concept phospho-proteomics approach to identify possible biomarkers that can be used to identify mutant p53 or wild-type p53 pathways. We treated PLK1 asynchronously followed by mass spectrometry data analysis. Protein networking and motif analysis tools were used to identify the significant clusters and potential biomarkers. RESULTS: We investigated approximately 1300 potential PLK1-dependent phosphopeptides by LCMS/ MS. In total, 2216 and 1155 high confidence phosphosites were identified in CP-A (p53+) and OE33 (p53-) cell lines owing to PLK1 inhibition. Further clustering and motif assessment uncovered many significant biomarkers with known and novel link to PLK1. CONCLUSION: Taken together, our study suggests that PLK1 may serve as a potential therapeutic target in human OAC. The data highlight the efficacy and specificity of small molecule PLK1 kinase inhibitors to identify novel signaling pathways in vivo.

Homozygous variants of EDAR underlying hypohidrotic ectodermal dysplasia in three consanguineous families.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a congenital anomaly characterized by hypohydrosis, hypotrichosis and hypodontia. Mutations in at least four genes (EDAR, EDARADD, WNT10A, TRAF6) have been reported to cause both autosomal recessive and autosomal dominant forms of HED. Mutations in two other genes (EDA and IKBKG) have been reported to cause X-linked HED. OBJECTIVES: To clinically characterize three consanguineous families (A-C) segregating with autosomal recessive HED and identify possible disease-causing variants of EDAR and EDARADD genes. MATERIALS AND METHODS: The genes, EDAR and EDARADD, were sequenced in Family A and C, and exome sequencing was performed in Family B. Additionally, in Family A and C, the effect of the identified variants was examined by analysis of EDAR mRNA, extracted from hair follicles from both affected and unaffected members. RESULTS: Sequence analysis revealed three possible disease-causing EDAR variants including a novel splice acceptor site variant (IVS3-1G > A) in Family A and two previously reported mutations (p.[Ala26Val], p.[Arg25*]) in the two other families. Previously, the nonsense variant p.(Arg25*) was reported only in the heterozygous state. Analysis of the RNA, extracted from hair follicles, revealed skipping of a downstream exon in EDAR and complete degradation of EDAR mRNA in affected members in family A and C, respectively. Computational modelling validated the pathogenic effect of the two variants identified in Family B and C. CONCLUSION: The three variants reported here expand the spectrum of EDAR mutations associated with HED which may further facilitate genetic counselling of families segregating with similar disorders in the Pakistani population.

Sequence Variants in the WNT10B and TP63 Genes Underlying Isolated Split-Hand/Split-Foot Malformation.

Aims: Split-hand/split-foot malformation (SHFM) is a developmental and congenital limb malformation characterized by variable degrees of medial clefting or absence of one or more digits in hands and/or feet. The aim of this study was to identify the underlying cause of three consanguineous Pakistani families showing various types of SHFM-related features. Materials and Methods: Standard molecular methods, including whole-genome sequencing (WGS), whole-exome sequencing (WES), microsatellite markers-based genotyping, and Sanger sequencing were performed to search for the likely causative variants. Results: In family A, WES revealed a novel homozygous missense variant [c.338G>A, p.(Gly113Asp)] in the WNT10B gene. In family B, microsatellite-based genotyping followed by Sanger sequencing revealed a novel homozygous 13 base pairs deletion [c.884-896delTCCAGCCCCGTCT, p.(Phe295Cysfs*87)] in the same gene. In family C, WGS divulged a previously reported heterozygous missense variant [c.956G>A, p.(Arg319His)] in the TP63 gene. Conclusions: Mapping and sequencing genes and variants for severe skeletal disorders, such as SHRM, will facilitate establishing specific genotype-phenotype correlations and providing genetic counseling for the families suffering from such conditions.

Identification and Computational Analysis of Novel TYR and SLC45A2 Gene Mutations in Pakistani Families With Identical Non-syndromic Oculocutaneous Albinism.

Non-syndromic oculocutaneous albinism (nsOCA) is an inherited disorder of melanin biosynthesis with autosomal recessive mode of inheritance, presenting either hypopigmented or depigmented skin, hair, and eyes. It is genetically heterogeneous with seven loci (OCA1-OCA7) reported to date. In the present study, we have reported three consanguineous families (A, B, C) presenting identical nsOCA phenotypes. Sanger sequencing revealed a novel [NM_000372.5: c.826 T > C, p.(Cys276Arg)] and a recurrent variant [NM_000372.5: c.832C > T, p.(Arg278∗)] in tyrosinase (TYR) in families A and B, respectively. Microsatellite marker-based homozygosity mapping linked family C to OCA4. Sequence analysis identified a novel insertion variant (NM_016180.5: c.1331_1332insA) in the SLC45A2. Further, in silico mutagenesis and dynamic simulation approaches revealed that a novel Cys276Arg variant abolished the cysteine bridge and might contribute toward decreased stability of the TYR protein. Our study expands the mutation spectrum of the TYR and SLC45A2 genes and emphasizes that molecular investigations are essential for accurate disease diagnosis.

Screening and computational analysis of colorectal associated non-synonymous polymorphism in CTNNB1 gene in Pakistani population.

BACKGROUND: Colorectal cancer (CRC) is categorized by alteration of vital pathways such as ß-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc. In the present study an attempt was made to screen CTNNB1 gene in colorectal cancer samples from Pakistani population and investigated the association of CTNNB1 gene mutations in the development of colorectal cancer. METHODS: 200 colorectal tumors approximately of male and female patients with sporadic or familial colorectal tumors and normal tissues were included. DNA was extracted and amplified through polymerase chain reaction (PCR) and subjected to exome sequence analysis. Immunohistochemistry was done to study protein expression. Molecular dynamic (MD) simulations of CTNNB1WT and mutant S33F and T41A were performed to evaluate the stability, folding, conformational changes and dynamic behaviors of CTNNB1 protein. RESULTS: Sequence analysis revealed two activating mutations (S33F and T41A) in exon 3 of CTNNB1 gene involving the transition of C.T and A.G at amino acid position 33 and 41 respectively (p.C33T and p.A41G). Immuno-histochemical staining showed the accumulation of ß-catenin protein both in cytoplasm as well as in the nuclei of cancer cells when compared with normal tissue. Further molecular modeling, docking and simulation approaches revealed significant conformational changes in the N-terminus region of normal to mutant CTNNB1 gene critical for binding with Glycogen synthase kinase 3-B (GSK3) and transducin containing protein1 (TrCp1). CONCLUSION: Present study on Pakistani population revealed an association of two non-synonymous polymorphisms in the CTNNB1 gene with colorectal cancer. These genetic variants led to the accumulation of the CTNNB1, a hallmark of tumor development. Also, analysis of structure to function alterations in CTNNB1 gene is crucial in understanding downstream biological events.

Inhibitory Mechanism of An Anticancer Drug, Bexarotene Against Amyloid ß Peptide Aggregation: Repurposing Via Neuroinformatics Approach.

BACKGROUND: Aggregation of Amyloid ß (Aß) peptide is a crucial feature of Alzheimer disease (AD) pathogenesis. In fact, Aß peptides are misfolded and aggregated to frame Amyloid fibrils, which is considered as one of the major contributing events in the onset of AD. All these observations have prompted the researchers to design therapeutic molecules with robust anti-Aß aggregation potential. Interestingly, in the last few decades, drug repurposing has turned into a fruitful and savvy approach for the treatment of several diseases. Bexarotene is an anticancer drug that has been under consideration for its ability to suppress Aß-peptide aggregation. However, the exact mechanistic aspect of suppression of Aß-peptide accumulation has not yet been completely revealed. METHODS: In the present study, we have attempted to decipher the mechanistic aspects of the anti-aggregation potential of bexarotene by using the computational biology approach. RESULTS: We have observed the effect of 'Aß-bexarotene' interaction on the aggregation ability of the Aß-peptide and decoded the involvement of receptor for advanced glycation end products (RAGE) and beta-secretase (BACE-1). A deep structural analysis of Aß upon binding with bexarotene revealed critical binding sites and structural twists involved in Aß aggregation. It is evident from the present that bexarotene could significantly restrain the process of primary nucleation of Aß. In addition, bexarotene showed a strong interaction with RAGE and BACE-1, suggesting them as plausible targets for the neuro-therapeutic action of bexarotene. CONCLUSION: Hence, we could safely suggest that bexarotene is a potent drug candidate that could reduce Aß- peptide aggregation by applying different mechanistic pathways. These results might boost the portfolio of pharmaceutical companies looking for the development of new chemical entities against AD.

Screening and Identification of PLK1-Polo Box Binding Peptides by High-Throughput Sequencing of Phage-Selected Libraries.

BACKGROUND: Human proteome contains a plethora of short linear peptide motifs that is crucial for signaling and other cellular processes. These motifs are difficult to identify due to lack of systematic approach for their detection. OBJECTIVES: Here we demonstrate the use of peptide phage display in combination with high throughput next generation sequencing to identify enriched peptide sequences through biopanning process against polo box domain (PBD) of mitotic polo like kinase 1 (Plk1). METHODS: Purified recombinant Plk1 and two unrelated controls namely B-lymphocyte antigen (CD20) and fluorescent protein (mCherry) were subjected to peptide phage display analysis. Bacterially-propagated phage DNA was amplified by PCR using triplet bar coded primers to tag the pool from each amplicon. RESULTS: Proteomic peptide phage display along with next generation sequencing and Bioinformatics analysis demonstrated several known and putative novel interactions which were potentially related to Plk1-PBD. With our strategy, we were able to identify and characterize several Plk1-PBD binding peptides, as well as define more precisely, consensus sequences. CONCLUSION: We believe that this information could provide valuable tools for exploring novel interaction involved in Plk1 signaling as well as to choose peptides for Plk1 specific drug development.

An Integrative "Omics" Approach, for Identification of Bona Fides PLK1 Associated Biomarker in Esophageal Adenocarcinoma.

BACKGROUND: The rapid expansion of genome-wide profiling techniques offers the opportunity to utilize various types of information collected in the study of human health and disease. Overexpression of Polo like kinase 1 (PLK1) is associated with esophageal adenocarcinoma (OAC), however biological functions and molecular targets of PLK1 in OAC are still unknown. OBJECTIVES: Here we performed integrative analysis of two "omics" data sources to reveal high-level interactions of PLK1 associated with OAC. METHODS: Initially, quantitative gene expression (RPKM) was measured from transcriptomics data set of four OAC patients. In parallel, alteration in phosphorylation levels was evaluated in the proteomics data set (mass spectrometry) in OAC cell line (PLK1 inhibited). Next, two "omics" data sets were integrated and through comprehensive analysis possible true PLK1 targets that may serve as OAC biomarkers were assembled. RESULTS: Through experimental validation, small ubiquitin-related modifier 1 (SUMO1) and heat shock protein beta-1 (HSPB1) were identified as novel phosphorylation targets of PLK1. Consequently in vivo, in situ and in silico experiments clearly demonstrated the interaction of PLK1 with putative novel targets (SUMO1 and HSPB1). CONCLUSION: Identification of a PLK1 dependent biosignature in OAC with high confidence in two omics levels proven the robustness and efficacy of our integrative approach.

In Silico Structure Modeling and Molecular Docking Analysis of Phosphoribosyl Pyrophosphate Amidotransferase (PPAT) with Antifolate Inhibitors.

BACKGROUND: Cancer remains one of the most serious disease worldwide. Robust metabolism is the hallmark of cancer. PPAT (phosphoribosyl pyrophosphate amidotransferase) catalyzes the first committed step of de novo purine biosynthesis. Hence PPAT, the key regulatory spot in De novo purine nucleotide biosynthesis, is an attractive and credible drug target for leukemia and other cancer therapeutics. OBJECTIVE: In the present study, detailed computational analysis has been performed for PPAT protein, the key enzyme in de novo purine biosynthesis which is inhibited by many folate derivatives, hence we aimed to investigate and gauge the inhibitory effect of antifolate derivatives; lomexterol (LTX) methotrexate (LTX), and pipretixin (PTX) with human PPAT to effectively capture and inhibit De novo purine biosynthesis pathway. METHODS: The sequence to structure computational approaches followed by molecular docking experiments was performed to gain insight into the inhibitory mode, binding orientation and binding affinities of selected antifolate derivatives against important structural features of PPAT. RESULTS: Results indicated a strong affinity of antifolate inhibitors for the conserved active site of PPAT molecule encompassing a number of hydrophobic, hydrogen bonding, Vander Waals and electrostatic interactions. CONCLUSION: Conclusively, the strong physical interaction of selected antifolate inhibitors with human PPAT suggests the selective inhibition of De novo purine biosynthesis pathway by antifolate derivatives towards cancer therapeutics.

In silico identification of putative bifunctional Plk1 inhibitors by integrative virtual screening and structural dynamics approach.

Polo like kinase (Plk1) is a master regulator of cell cycle and considered as next generation antimitotic target in human. As Plk1 predominantly expresses in the dividing cells with a much higher expression in cancerous cells, it serves as a discriminative target for cancer therapeutics. Here we implied a novel and promising integrative strategy to identify "bifunctional" Plk1 inhibitors that compete simultaneously with ATP and substrate for their binding sites. We integrated structure-based virtual screening (SBVS) and molecular dynamics simulations with emphasis on unique structural properties of Plk1. Through screening of 20,000 compounds, nearly ~2000 hits were enriched and subjected to SBVS against ATP and substrate binding sites of Plk1. Subsequently, on the basis of their binding abilities to Plk1 kinase and polo box domains, filtration of candidate hits resulted in the isolation of 26 compounds. By exclusion of close analogs or isomers, 10 unique compounds were selected for detailed study. A representative compound was subjected to molecular dynamics simulation assay to have deep structural insights and to gauge critical structural crunch for inhibitor binding against kinase and polo box domains. Our integrative approach may complement high-throughput screening and identify bifunctional Plk1 inhibitors that may contribute in selective targeting of Plk1 to elicit desired biological process.

Inhibition of Janus kinases by tyrosine phosphorylation inhibitor, Tyrphostin AG-490.

Janus kinases (JAKs) belong to a crucial family of tyrosine kinases, implicated in the patho-physiology of multiple cancer types, and serve as striking therapeutic targets. To date, many potent, either ATP-competitive (PTK domain) or non-ATP-competitive JAK inhibitors have been identified. Among them, Tyrphostin AG-490 (2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide) is a well-known ATP-competitive inhibitor. However, its mode of action, details of interacting residues, and induced conformational changes in JAK-specific binding sites remain elusive. Here, through comparative structure analysis, molecular docking, and molecular dynamics simulation assays, we explored comparative binding patterns of AG-490 against JAK1, JAK2, and JAK3. Our results entail noteworthy observations about the binding affinity of AG-490 by illustrating distinctive amino acid residues lying at the conserved ATP-binding domains of JAK family members. By subsequent assessment of their structural homology and conserved structural folds, we highlight intriguing prospects to design more specific and potent inhibitors for selective targeting of JAK family members. Our comparative study provides a platform for the rational design of precise and potent inhibitor for selective targeting of JAK family members.

Disruption of murine Tcte3-3 induces tissue specific apoptosis via co-expression of Anxa5 and Pebp1.

Programmed cell death or apoptosis plays a vital physiological role in the development and homeostasis. Any discrepancy in apoptosis may trigger testicular and neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. Tcte3 (T-complex testis expressed 3) is an accessory component of axonemal and cytoplasmic dynein which expresses predominantly in meiotic and postmeiotic germ cells. It plays an essential role during spermatogenesis; however, to explore its diverse and complex functioning in male germ cell apoptosis, requires further prosecution. Here, 2D-gel electrophoresis, mass spectrometry and qRT-PCR analyses were performed to elucidate the differential expression of genes, in both wild-type and homozygous Tcte3-3 mice. We observed an increased expression of Tcte3 in homozygotes as compared to wild-type testes. Perpetually, an increased expression of Anxa5 and Pebp1, while a lower expression of Rsph1 was detected in Tcte3-3-/- mice. We propose that over-expression of Pebp1 and Anxa5 in Tcte3-3-/- testes might be due to increased apoptosis. To evaluate this possibility, testes specific microarray data set extracted from NCBI gene ontology omnibus (GEO) was used to cluster the possible co-expression partners of Tcte3. Further functional coherence of compiled candidate genes was monitored computationally by studying the common TFBS overlapped at the regulatory regions. Differential expression of Tcte3-3 and its involvement in apoptosis may provide a basis for the investigation of transcriptional specificities of other Tcte3 paralogs (Tcte3-1 and Tcte3-2). A complete understanding of controlling factors which have implications in regulating tissue-specific Tcte3 expression would provide additional insights into the gene control events. The collective knowledge may prove useful for the development of novel therapeutic regimen and would open new avenues in defining selective roles of Tcte3 in germ cell development.