The COPD GWAS gene ADGRG6 instructs function and injury response in human iPSC-derived type II alveolar epithelial cells.

Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.

Deep learning assisted multi-omics integration for survival and drug-response prediction in breast cancer.

BACKGROUND: Survival and drug response are two highly emphasized clinical outcomes in cancer research that directs the prognosis of a cancer patient. Here, we have proposed a late multi omics integrative framework that robustly quantifies survival and drug response for breast cancer patients with a focus on the relative predictive ability of available omics datatypes. Neighborhood component analysis (NCA), a supervised feature selection algorithm selected relevant features from multi-omics datasets retrieved from The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) databases. A Neural network framework, fed with NCA selected features, was used to develop survival and drug response prediction models for breast cancer patients. The drug response framework used regression and unsupervised clustering (K-means) to segregate samples into responders and non-responders based on their predicted IC50 values (Z-score). RESULTS: The survival prediction framework was highly effective in categorizing patients into risk subtypes with an accuracy of 94%. Compared to single-omics and early integration approaches, our drug response prediction models performed significantly better and were able to predict IC50 values (Z-score) with a mean square error (MSE) of 1.154 and an overall regression value of 0.92, showing a linear relationship between predicted and actual IC50 values. CONCLUSION: The proposed omics integration strategy provides an effective way of extracting critical information from diverse omics data types enabling estimation of prognostic indicators. Such integrative models with high predictive power would have a significant impact and utility in precision oncology.

Bioinformatics and Molecular Insights to Anti-Metastasis Activity of Triethylene Glycol Derivatives.

The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1' pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial-mesenchymal transition (EMT) signaling.

Function and structure-based screening of compounds, peptides and proteins to identify drug candidates.

Drug discovery in simple words is all about finding small molecular compounds that possess the potential to interact with specific bio-macromolecules, mainly proteins, thereby bringing a desired effect in the functioning of the target molecules. Virtual screening of large compound libraries using computational approaches has come up as a great alternative to cost and labor-intensive high-throughput screening carried out in laboratories. Virtual high-throughput screening enormously reduces the number of compounds for systematic analysis using biochemical assays before entering the clinical trials. Here, we first give a brief overview of the rationale behind virtual screening, types of virtual screening - structure-based, ligand-based and inverse virtual screening, and challenges that need to be addressed to improve the existing strategies. Subsequently, we describe the methodology adopted for virtual screening of small molecules, peptides and proteins. Finally, we use few case studies to provide a better insight to the application of computer-aided high-throughput screening.

Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea.

BACKGROUND: Brassica juncea var. Varuna is an economically important oilseed crop of family Brassicaceae which is vulnerable to abiotic stresses at specific stages in its life cycle. Till date no attempts have been made to elucidate genome-wide changes in its transcriptome against high temperature or drought stress. To gain global insights into genes, transcription factors and kinases regulated by these stresses and to explore information on coding transcripts that are associated with traits of agronomic importance, we utilized a combinatorial approach of next generation sequencing and de-novo assembly to discover B. juncea transcriptome associated with high temperature and drought stresses. RESULTS: We constructed and sequenced three transcriptome libraries namely Brassica control (BC), Brassica high temperature stress (BHS) and Brassica drought stress (BDS). More than 180 million purity filtered reads were generated which were processed through quality parameters and high quality reads were assembled de-novo using SOAPdenovo assembler. A total of 77750 unique transcripts were identified out of which 69,245 (89%) were annotated with high confidence. We established a subset of 19110 transcripts, which were differentially regulated by either high temperature and/or drought stress. Furthermore, 886 and 2834 transcripts that code for transcription factors and kinases, respectively, were also identified. Many of these were responsive to high temperature, drought or both stresses. Maximum number of up-regulated transcription factors in high temperature and drought stress belonged to heat shock factors (HSFs) and dehydration responsive element-binding (DREB) families, respectively. We also identified 239 metabolic pathways, which were perturbed during high temperature and drought treatments. Analysis of gene ontologies associated with differentially regulated genes forecasted their involvement in diverse biological processes. CONCLUSIONS: Our study provides first comprehensive discovery of B. juncea transcriptome under high temperature and drought stress conditions. Transcriptome resource generated in this study will enhance our understanding on the molecular mechanisms involved in defining the response of B. juncea against two important abiotic stresses. Furthermore this information would benefit designing of efficient crop improvement strategies for tolerance against conditions of high temperature regimes and water scarcity.

Multi-ancestry genome-wide association analyses improve resolution of genes and pathways influencing lung function and chronic obstructive pulmonary disease risk.

Lung-function impairment underlies chronic obstructive pulmonary disease (COPD) and predicts mortality. In the largest multi-ancestry genome-wide association meta-analysis of lung function to date, comprising 580,869 participants, we identified 1,020 independent association signals implicating 559 genes supported by ≥2 criteria from a systematic variant-to-gene mapping framework. These genes were enriched in 29 pathways. Individual variants showed heterogeneity across ancestries, age and smoking groups, and collectively as a genetic risk score showed strong association with COPD across ancestry groups. We undertook phenome-wide association studies for selected associated variants as well as trait and pathway-specific genetic risk scores to infer possible consequences of intervening in pathways underlying lung function. We highlight new putative causal variants, genes, proteins and pathways, including those targeted by existing drugs. These findings bring us closer to understanding the mechanisms underlying lung function and COPD, and should inform functional genomics experiments and potentially future COPD therapies.

Computational Identification of BCR-ABL Oncogenic Signaling as a Candidate Target of Withaferin A and Withanone.

Withaferin-A (Wi-A), a secondary metabolite extracted from Ashwagandha (Withania somnifera), has been shown to possess anticancer activity. However, the molecular mechanism of its action and the signaling pathways have not yet been fully explored. We performed an inverse virtual screening to investigate its binding potential to the catalytic site of protein kinases and identified ABL as a strong candidate. Molecular docking and molecular dynamics simulations were undertaken to investigate the effects on BCR-ABL oncogenic signaling that is constitutively activated yielding uncontrolled proliferation and inhibition of apoptosis in Chronic Myeloid Leukemia (CML). We found that Wi-A and its closely related withanolide, Withanone (Wi-N), interact at both catalytic and allosteric sites of the ABL. The calculated binding energies were higher in the case of Wi-A at catalytic site (-82.19 ± 5.48) and allosteric site (-67.00 ± 4.96) as compared to the clinically used drugs Imatinib (-78.11 ± 5.21) and Asciminib (-54.00 ± 6.45) respectively. Wi-N had a lesser binding energy (-42.11 ± 10.57) compared to Asciminib at the allosteric site. The interaction and conformational changes, subjected to ligand interaction, were found to be similar to the drugs Imatinib and Asciminib. The data suggested that Ashwagandha extracts containing withanolides, Wi-A and Wi-N may serve as natural drugs for the treatment of CML. Inhibition of ABL is suggested as one of the contributing factors of anti-cancer activity of Wi-A and Wi-N, warranting further in vitro and in vivo experiments.

Computational Insights into the Potential of Withaferin-A, Withanone and Caffeic Acid Phenethyl Ester for Treatment of Aberrant-EGFR Driven Lung Cancers.

The anticancer activities of Withaferin-A (Wi-A) and Withanone (Wi-N) from Ashwagandha and Caffeic Acid Phenethyl Ester (CAPE) from honeybee propolis have been well documented. Here, we examined the binding potential of these natural compounds to inhibit the constitutive phosphorylation of epidermal growth factor receptors (EGFRs). Exon 20 insertion mutants of EGFR, which show resistance to various FDA approved drugs and are linked to poor prognosis of lung cancer patients, were the primary focus of this study. Apart from exon 20 insertion mutants, the potential of natural compounds to serve as ATP competitive inhibitors of wildtype protein and other common mutants of EGFR, namely L858R and exon19del, were also examined. The potential of natural compounds was compared to the positive controls such as erlotinib, TAS6417 and poziotinib. Similar to known inhibitors, Wi-A and Wi-N could displace and binds at the ATP orthosteric site of exon19del, L858R and exon20, while CAPE was limited to wildtype EGFR and exon 20 insertion mutants only. Moreover, the binding free energy of the natural drugs against EGFRs was also comparable to the positive controls. This computational study suggests that Wi-A and Wi-N have potential against multiple mutated EGFRs, warranting further in vitro and in vivo experiments.

Potential of Withaferin-A, Withanone and Caffeic Acid Phenethyl ester as ATP-competitive inhibitors of BRAF: A bioinformatics study.

Serine/threonine-protein kinase B-raf (BRAF) plays a significant role in regulating cell division and proliferation through MAPK/ERK pathway. The constitutive expression of wild-type BRAF (BRAFWT) and its mutant forms, especially V600E (BRAFV600E), has been linked to multiple cancers. Various synthetic drugs have been approved and are in clinical trials, but most of them are reported to become ineffective within a short duration. Therefore, combinational therapy involving multiple drugs are often recruited for cancer treatment. However, they lead to toxicity and adverse side effects. In this computational study, we have investigated three natural compounds, namely Withaferin-A (Wi-A), Withanone (Wi-N) and Caffeic Acid Phenethyl ester (CAPE) for anti-BRAFWT and anti-BRAFV600E activity. We found that these compounds could bind stably at ATP-binding site in both BRAFWT and BRAFV600E proteins. In-depth analysis revealed that these compounds maintained the active conformation of wild-type BRAF protein by inducing αC-helix-In, DFG-In, extended activation segment and well-aligned R-spine residues similar to already known drugs Vemurafenib (VEM), BGB283 and Ponatinib. In terms of binding energy, among the natural compounds, CAPE showed better affinity towards both wild-type and V600E mutant proteins than the other two compounds. These data suggested that CAPE, Wi-A and Wi-N have potential to block constitutive autophosphorylation of BRAF and hence warrant in vitro and in vivo experimental validation.

2, 3-Dihydro-3ß-methoxy Withaferin-A Lacks Anti-Metastasis Potency: Bioinformatics and Experimental Evidences.

Withaferin-A is a withanolide, predominantly present in Ashwagandha (Withania somnifera). It has been shown to possess anticancer activity in a variety of human cancer cells in vitro and in vivo. Molecular mechanism of such cytotoxicity has not yet been completely understood. Withaferin-A and Withanone were earlier shown to activate p53 tumor suppressor and oxidative stress pathways in cancer cells. 2,3-dihydro-3ß-methoxy analogue of Withaferin-A (3ßmWi-A) was shown to lack cytotoxicity and well tolerated at higher concentrations. It, on the other hand, protected normal cells against oxidative, chemical and UV stresses through induction of anti-stress and pro-survival signaling. We, in the present study, investigated the effect of Wi-A and 3ßmWi-A on cell migration and metastasis signaling. Whereas Wi-A binds to vimentin and heterogeneous nuclear ribonucleoprotein K (hnRNP-K) with high efficacy and downregulates its effector proteins, MMPs and VEGF, involved in cancer cell metastasis, 3ßmWi-A was ineffective. Consistently, Wi-A, and not 3ßmWi-A, caused reduction in cytoskeleton proteins (Vimentin, N-Cadherin) and active protease (u-PA) that are essential for three key steps of cancer cell metastasis (EMT, increase in cell migration and invasion).

Molecular Insights Into Withaferin-A-Induced Senescence: Bioinformatics and Experimental Evidence to the Role of NFκB and CARF.

Withaferin-A (Wi-A) has been shown to possess anticancer activity. Molecular mechanism(s) of its action has not been fully resolved. We recruited low dose of Wi-A that caused slow growth arrest in cancer cells and was relatively safe for normal cells. Consistently, we detected nuclear translocation of nuclear factor kappa B (NFκB) and activation of p38MAPK selectively in cancer cells. Bioinformatics analyses revealed that Wi-A did not disrupt IKKα/IKKß-Nemo complex that regulates NFκB activity. However, it caused moderate change in the conformation of IKKß-Nemo interacting domain. Experimental data revealed increased level of phosphorylated IκBα in Wi-A-treated cells, suggesting an activation of IKK complex that was supported by nuclear translocation of NFκB. Molecular docking analysis showed that Wi-A did not disrupt; however, decreased the stability of the NFκB-DNA complex. It was supported by downregulation of DNA-binding and transcriptional activities of NFκB. Further analysis revealed that Wi-A caused upregulation of CARF (collaborator of ARF) demonstrating an activation of DNA damage oxidative stress response in both cancer and normal cells. In line with this, upregulation of p21WAF1, p16INK4A, and hypophosphorylated pRB and induction of senescence were observed demonstrating that Wi-A-induced senescence is mediated by multiple pathways in which CARF-mediated DNA damage and oxidative stress play a major role.

Fragment-Based Ligand Designing.

Fragment-based drug design strategies have been used in drug discovery since it was first demonstrated using experimental structural biology techniques such as nuclear magnetic resonance (NMR) and X-ray crystallography. The underlying idea is that existing or new chemical entities with known desirable properties may serve both as tool compounds and as starting points for hit-to-lead expansion. Despite the recent advancements, there remain challenges to overcome, such as assembly of the synthetically feasible structures, development of scoring functions to correlate structure and their activities, and fine tuning of the promising molecules. This chapter first covers the theoretical background needed to understand the concepts and the challenges related to the field of study, followed by the description of important protocols and related software. Case studies are presented to demonstrate practical applications.