Identification of pseudobaptigenin as a novel polyphenol-based multi-target antagonist of different hormone receptors for breast cancer therapeutics.

Breast cancer (BC) is one of the most prevalent cancers in the world and is one of the major reasons for the death of women worldwide. BC is majorly categorized based on the presence or absence of three cell receptors ER, PR and HER2. The latest treatment for BC involves interfering with the production and action of hormones such as estrogen and progesterone. These hormones bind with receptors such as ER and PR and enhance the growth and proliferation of the BC cells. Although the available are effective, the increasing resistance and side effects related to hormonal imbalance are significant and hence there is a need for designing. On the other hand, plant-derivative products have gained a lot of popularity for their promising anti-cancerous activities. Polyphenols are one such group of plant derivatives that have proven to be useful against cancer. In the present study, an in-silico approach was used to search for a polyphenol that can inhibit ER. In this work, a total of 750 polyphenols were taken into consideration. This number was narrowed down to 55, based on their ADMET properties. These 55 polyphenols were then docked to the receptors, ER, PR and HER2. The molecular docking was followed by Molecular Dynamics (MD) simulations. Based on molecular docking and MD simulation results it was concluded that Pseudobaptigenin has the potential to be an inhibitor of ER, PR and HER2.Communicated by Ramaswamy H. Sarma.

Comparative structural and dynamics study of free and gRNA-bound FnCas9 and SpCas9 proteins.

The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.

Comprehensive Assessment of Indian Variations in the Druggable Kinome Landscape Highlights Distinct Insights at the Sequence, Structure and Pharmacogenomic Stratum.

India confines more than 17% of the world's population and has a diverse genetic makeup with several clinically relevant rare mutations belonging to many sub-group which are undervalued in global sequencing datasets like the 1000 Genome data (1KG) containing limited samples for Indian ethnicity. Such databases are critical for the pharmaceutical and drug development industry where diversity plays a crucial role in identifying genetic disposition towards adverse drug reactions. A qualitative and comparative sequence and structural study utilizing variant information present in the recently published, largest curated Indian genome database (IndiGen) and the 1000 Genome data was performed for variants belonging to the kinase coding genes, the second most targeted group of drug targets. The sequence-level analysis identified similarities and differences among different populations based on the nsSNVs and amino acid exchange frequencies whereas a comparative structural analysis of IndiGen variants was performed with pathogenic variants reported in UniProtKB Humsavar data. The influence of these variations on structural features of the protein, such as structural stability, solvent accessibility, hydrophobicity, and the hydrogen-bond network was investigated. In-silico screening of the known drugs to these Indian variation-containing proteins reveals critical differences imparted in the strength of binding due to the variations present in the Indian population. In conclusion, this study constitutes a comprehensive investigation into the understanding of common variations present in the second largest population in the world and investigating its implications in the sequence, structural and pharmacogenomic landscape. The preliminary investigation reported in this paper, supporting the screening and detection of ADRs specific to the Indian population could aid in the development of techniques for pre-clinical and post-market screening of drug-related adverse events in the Indian population.

Molecular signatures of long-term hepatocellular carcinoma risk in nonalcoholic fatty liver disease.

Prediction of hepatocellular carcinoma (HCC) risk is an urgent unmet need in patients with nonalcoholic fatty liver disease (NAFLD). In cohorts of 409 patients with NAFLD from multiple global regions, we defined and validated hepatic transcriptome and serum secretome signatures predictive of long-term HCC risk in patients with NAFLD. A 133-gene signature, prognostic liver signature (PLS)-NAFLD, predicted incident HCC over up to 15 years of longitudinal observation. High-risk PLS-NAFLD was associated with IDO1+ dendritic cells and dysfunctional CD8+ T cells in fibrotic portal tracts along with impaired metabolic regulators. PLS-NAFLD was validated in independent cohorts of patients with NAFLD who were HCC naïve (HCC incidence rates at 15 years were 22.7 and 0% in high- and low-risk patients, respectively) or HCC experienced (de novo HCC recurrence rates at 5 years were 71.8 and 42.9% in high- and low-risk patients, respectively). PLS-NAFLD was bioinformatically translated into a four-protein secretome signature, PLSec-NAFLD, which was validated in an independent cohort of HCC-naïve patients with NAFLD and cirrhosis (HCC incidence rates at 15 years were 37.6 and 0% in high- and low-risk patients, respectively). Combination of PLSec-NAFLD with our previously defined etiology-agnostic PLSec-AFP yielded improved HCC risk stratification. PLS-NAFLD was modified by bariatric surgery, lipophilic statin, and IDO1 inhibitor, suggesting that the signature can be used for drug discovery and as a surrogate end point in HCC chemoprevention clinical trials. Collectively, PLS/PLSec-NAFLD may enable NAFLD-specific HCC risk prediction and facilitate clinical translation of NAFLD-directed HCC chemoprevention.

Decrypting the mechanistic basis of CRISPR/Cas9 protein.

CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies.

BACKGROUND: Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach. METHODS: We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map ( https://clue.io/ ). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA­dependent RNA­polymerase (RdRp) and 3C­like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. RESULTS: Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Molecular Signature Predictive of Long-Term Liver Fibrosis Progression to Inform Antifibrotic Drug Development.

BACKGROUND & AIMS: There is a major unmet need to assess the prognostic impact of antifibrotics in clinical trials because of the slow rate of liver fibrosis progression. We aimed to develop a surrogate biomarker to predict future fibrosis progression. METHODS: A fibrosis progression signature (FPS) was defined to predict fibrosis progression within 5 years in patients with hepatitis C virus and nonalcoholic fatty liver disease (NAFLD) with no to minimal fibrosis at baseline (n = 421) and was validated in an independent NAFLD cohort (n = 78). The FPS was used to assess response to 13 candidate antifibrotics in organotypic ex vivo cultures of clinical fibrotic liver tissues (n = 78) and cenicriviroc in patients with nonalcoholic steatohepatitis enrolled in a clinical trial (n = 19, NCT02217475). A serum protein-based surrogate FPS was developed and tested in a cohort of compensated cirrhosis patients (n = 122). RESULTS: A 20-gene FPS was defined and validated in an independent NAFLD cohort (adjusted odds ratio, 10.93; area under the receiver operating characteristic curve, 0.86). Among computationally inferred fibrosis-driving FPS genes, BCL2 was confirmed as a potential pharmacologic target using clinical liver tissues. Systematic ex vivo evaluation of 13 candidate antifibrotics identified rational combination therapies based on epigallocatechin gallate, which were validated for enhanced antifibrotic effect in ex vivo culture of clinical liver tissues. In patients with nonalcoholic steatohepatitis treated with cenicriviroc, FPS modulation was associated with 1-year fibrosis improvement accompanied by suppression of the E2F pathway. Induction of the PPARα pathway was absent in patients without fibrosis improvement, suggesting a benefit of combining PPARα agonism to improve the antifibrotic efficacy of cenicriviroc. A 7-protein serum protein-based surrogate FPS was associated with the development of decompensation in cirrhosis patients. CONCLUSION: The FPS predicts long-term fibrosis progression in an etiology-agnostic manner, which can inform antifibrotic drug development.

A blood-based prognostic liver secretome signature and long-term hepatocellular carcinoma risk in advanced liver fibrosis.

BACKGROUND: Accurate non-invasive prediction of long-term hepatocellular carcinoma (HCC) risk in advanced liver fibrosis is urgently needed for cost-effective HCC screening; however, this currently remains an unmet need. METHODS: A serum-protein-based prognostic liver secretome signature (PLSec) was bioinformatically derived from previously validated hepatic transcriptome signatures and optimized in 79 patients with advanced liver fibrosis. We independently validated PLSec for HCC risk in 331 cirrhosis patients with mixed etiologies (validation set 1 [V1]) and thereafter developed a score with clinical prognostic variables. The score was then validated in two independent cohorts: validation set 2 (V2): 164 patients with advanced liver fibrosis due to hepatitis C virus (HCV) infection cured after direct-acting antiviral therapy; validation set 3 (V3): 146 patients with advanced liver fibrosis with successfully-treated HCC and cured HCV infection. FINDINGS: An 8-protein blood-based PLSec recapitulated transcriptome-based hepatic HCC risk status. In V1, PLSec was significantly associated with incident HCC risk (adjusted hazard ratio [aHR], 2.35; 95% confidence interval [CI], 1.30-4.23). A composite score with serum alpha-fetoprotein (PLSec-AFP) was defined in V1, and validated in V2 (adjusted odds ratio, 3.80 [95%CI, 1.66-8.66]) and V3 (aHR, 3.08 [95%CI, 1.78-5.31]; c-index, 0.74). PLSec-AFP outperformed AFP alone (Brier score, 0.165 vs. 0.186 in V2; 0.196 vs. 0.206 in V3, respectively). CONCLUSIONS: The blood-based PLSec-AFP can accurately stratify patients with advanced liver fibrosis for long-term HCC risk and thereby guide risk-based tailored HCC screening.

Determination of the glucuronide metabolite of ON 013100, a benzylstyrylsulfone antineoplastic drug, in colon cancer cells using LC/MS/MS.

ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6 Da) was found in the drug-resistant cells upon a 1h incubation with ON 013100 (20 µg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84 µg/ml at 0 h through 0.21 µg/ml at 6h to below detection limit of 8.0 ng/ml at 9 h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41 µg/ml (0 h) to 0.06 µg/ml (9 h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88 µg/ml to 0.94 µg/ml.

Mechanism of action of ferrocene derivatives on the catalytic activity of topoisomerase IIalpha and beta--distinct mode of action of two derivatives.

Topoisomerase II is found to be present in two isoforms alpha and beta, and both the isoforms are regulated in cancerous tissue. Development of isoform-specific topoisomerase II poisons has been of great interest for cancer-specific drug targeting. In the present investigation using quantitative structure-activity analysis of ferrocene derivatives, we show that two derivatives of ferrocene, azalactone ferrocene and thiomorpholide amido methyl ferrocene, can preferentially inhibit topoisomerase IIbeta activity. Thiomorpholide amido methyl ferrocene shows higher inhibition of catalytic activity (IC(50) = 50 microM) against topoisomerase IIbeta compared to azalactone ferrocene (IC(50) = 100 microM). The analysis of protein DNA intermediates formed in the presence of these two compounds suggests that azalactone ferrocene readily induces formation of cleavable complex in a dose-dependent manner, in comparison with thiomorpholide amido methyl ferrocene. Both the compounds show significant inhibition of DNA-dependent ATPase activity of enzyme. These results suggest that azalactone ferrocene inhibits DNA passage activity of enzyme leading to the formation of cleavable complex, while thiomorpholide amido methyl ferrocene competes with ATP binding resulting in the inhibition of catalytic activity of enzyme. In summary, thiomorpholide amido methyl ferrocene and azalactone ferrocene show distinctly different mechanisms in inhibition of catalytic activity of topoisomerase IIbeta.