Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models.

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In this study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal "plugs" aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictive ability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homo-hexameric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable hetero-hexameric channels did not. From the perspective of the conformational energetics of WT and variant Cx26 hexamers, we propose that unaffected carriers could result from a conformational predominance of the WT and pore-shrinkage-incapable hetero-hexamers, while mice with homozygous variants can only harbor an unstable and dysfunctional N-termini-blocking V37M homo-hexamer. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

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.

Phosphatidylserine Exposed Lipid Bilayer Models for Understanding Cancer Cell Selectivity of Natural Compounds: A Molecular Dynamics Simulation Study.

Development of drugs that are selectively toxic to cancer cells and safe to normal cells is crucial in cancer treatment. Evaluation of membrane permeability is a key metric for successful drug development. In this study, we have used in silico molecular models of lipid bilayers to explore the effect of phosphatidylserine (PS) exposure in cancer cells on membrane permeation of natural compounds Withaferin A (Wi-A), Withanone (Wi-N), Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC). Molecular dynamics simulations were performed to compute permeability coefficients. The results indicated that the exposure of PS in cancer cell membranes facilitated the permeation of Wi-A, Wi-N and CAPE through a cancer cell membrane when compared to a normal cell membrane. In the case of ARC, PS exposure did not have a notable influence on its permeability coefficient. The presented data demonstrated the potential of PS exposure-based models for studying cancer cell selectivity of drugs.

Computational Approaches for Designing Highly Specific and Efficient sgRNAs.

The easily programmable CRISPR/Cas9 system has found applications in biomedical research as well as microbial and crop applications, due to its ability to create site-specific edits. This powerful and flexible system has also been modified to enable inducible gene regulation, epigenome modifications and high-throughput screens. Designing efficient and specific guides for the nuclease is a key step and also a major challenge in effective application. This chapter describes rules for sgRNA design and important features to consider while touching upon bioinformatics advances in predicting efficient guides. Computational tools that suggest improved guides, depending on application, or predict off-targets have also been mentioned and compared.

Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53Y220C mutant: bioinformatics and experimental evidence.

Mutations in the tumor suppressor protein p53 is a prevalent feature in majority of cancers resulting in inactivation of its activities related to control of cell cycle progression and proliferation. p53Y220C is one of the common hotspot mutations that causes decrease in its thermodynamic stability. Some small molecules have been shown to bind to the mutated site and restore its wild type thermodynamics and tumor suppressor function. In this study, we have explored the potential of caffeic acid phenethyl ester (CAPE-a bioactive compound from propolis) to interact with p53Y220C and restore its wild type p53 (p53wt) transcription activation and tumor suppressor activities. We recruited computational methods, viz. molecular docking, molecular dynamics simulations and free energy calculations to study the interaction of CAPE at the mutation crevice and found that it has potential to restore p53wt function of the p53Y220C mutant similar to a previously described restoration molecule PK7242. We provide cell-based experimental evidence to these predictions and suggest CAPE as a potential natural drug for treatment of p53Y220C mutant harboring cancers.

Combination of Withaferin-A and CAPE Provides Superior Anticancer Potency: Bioinformatics and Experimental Evidence to Their Molecular Targets and Mechanism of Action.

We have earlier reported anticancer activity in Withaferin A (Wi-A), a withanolide derived from Ashwagandha (Withania somnifera) and caffeic acid phenethyl ester (CAPE), an active compound from New Zealand honeybee propolis. Whereas Wi-A was cytotoxic to both cancer and normal cells, CAPE has been shown to cause selective death of cancer cells. In the present study, we investigated the efficacy of Wi-A, CAPE, and their combination to ovarian and cervical cancer cells. Both Wi-A and CAPE were seen to activate tumor suppressor protein p53 by downregulation of mortalin and abrogation of its interactions with p53. Downregulation of mortalin translated to compromised mitochondria integrity and function that affected poly ADP-ribose polymerase1 (PARP1); a key regulator of DNA repair and protein-target for Olaparib, drugs clinically used for treatment of breast, ovarian and cervical cancers)-mediated DNA repair yielding growth arrest or apoptosis. Furthermore, we also compared the docking capability of Wi-A and CAPE to PARP1 and found that both of these could bind to the catalytic domain of PARP1, similar to Olaparib. We provide experimental evidences that (i) Wi-A and CAPE cause inactivation of PARP1-mediated DNA repair leading to accumulation of DNA damage and activation of apoptosis signaling by multiple ways, and (ii) a combination of Wi-A and CAPE offers selective toxicity and better potency to cancer cells.

Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence.

Fucoxanthin is commonly found in marine organisms; however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin; hence, it warrants further attention for basic mechanistic studies as well as drug development.

CRISPcut: A novel tool for designing optimal sgRNAs for CRISPR/Cas9 based experiments in human cells.

The ability to direct the CRISPR/Cas9 nuclease to a unique target site within a genome would have broad use in targeted genome engineering. However, CRISPR RNA is reported to bind to other genomic locations that differ from the intended target site by a few nucleotides, demonstrating significant off-target activity. We have developed the CRISPcut tool that screens the off-targets using various parameters and predicts the ideal genomic target for -guide RNAs in human cell lines. sgRNAs for four different types of Cas9 nucleases can be designed with an option for the user to work with different PAM sequences. Direct experimental measurement of genome-wide DNA accessibility is incorporated that effectively restricts the prediction of CRISPR targets to open chromatin. An option to predict target sites for paired CRISPR nickases is also provided. The tool has been validated using a dataset of experimentally used sgRNA and their identified off-targets. URL: http://web.iitd.ac.in/crispcut.

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.

Identifying synthetic lethal targets using CRISPR/Cas9 system.

Synthetic lethality occurs when co-occurrence of two genetic events is unfavorable for the survival of the cell or organism. The conventional approach of high throughput screening of synthetic lethal targets using chemical compounds has been replaced by RNAi technology. CRISPR/Cas9, an RNA guided endonuclease system is the most recent technology for this work. Here, we have discussed the major considerations involved in designing a CRISPR/Cas9 based screening experiment for identification of synthetic lethal targets. It mainly includes CRISPR library to be used, cell types for conducting the experiment, the most appropriate screening strategy and ways of selecting the desired phenotypes from the complete cell population. The complete knockdown of genes can be achieved using CRISPR/Cas9 knockout libraries. For higher quality loss-of-function screens, haploid cells with defective homology-directed DNA repair mechanism could be used. Two widely used screening formats include arrayed and pooled screens followed by negative or positive selection of the cells with desired phenotype. However, pooled screening format with negative selection of cells serves the best. The advantages of using CRISPR/Cas9 system over the other RNAi approaches have also been discussed. Finally, some studies using CRISPR/Cas9 for genome-wide knockout screening in human cells and computational approaches for identification of synthetic lethal interactions have been discussed.