Identification of new target proteins of a Urotensin-II receptor antagonist using transcriptome-based drug repositioning approach

Drug repositioning research using transcriptome data has recently attracted attention. In this study, we attempted to identify new target proteins of the urotensin-II receptor antagonist, KR-37524 (4-(3-bromo-4-(piperidin-4-yloxy)benzyl)-N-(3-(dimethylamino)phenyl)piperazine-1-carboxamide dihydrochloride), using a transcriptome-based drug repositioning approach. To do this, we obtained KR-37524-induced gene expression profile changes in four cell lines (A375, A549, MCF7, and PC3), and compared them with the approved drug-induced gene expression profile changes available in the LINCS L1000 database to identify approved drugs with similar gene expression profile changes. Here, the similarity between the two gene expression profile changes was calculated using the connectivity score. We then selected proteins that are known targets of the top three approved drugs with the highest connectivity score in each cell line (12 drugs in total) as potential targets of KR-37524. Seven potential target proteins were experimentally confirmed using an in vitro binding assay. Through this analysis, we identified that neurologically regulated serotonin transporter proteins are new target proteins of KR-37524. These results indicate that the transcriptome-based drug repositioning approach can be used to identify new target proteins of a given compound, and we provide a standalone software developed in this study that will serve as a useful tool for drug repositioning.

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. Purpose: Herbal medicines have been a source of various antiviral compounds. An accelerated repurposing potential of antiviral herbs can provide usable drugs and identify druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos pareira L (Cipa). using an integrative approach. Methods: We analyzed 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. 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.

Connectivity Map Analysis Indicates PI3K/Akt/mTOR Inhibitors as Potential Anti-Hypoxia Drugs in Neuroblastoma

Neuroblastoma (NB) is one of the deadliest pediatric cancers, accounting for 15% of deaths in childhood. Hypoxia is a condition of low oxygen tension occurring in solid tumors and has an unfavorable prognostic factor for NB. In the present study, we aimed to identify novel promising drugs for NB treatment. Connectivity Map (CMap), an online resource for drug repurposing, was used to identify connections between hypoxia-modulated genes in NB tumors and compounds. Two sets of 34 and 21 genes up- and down-regulated between hypoxic and normoxic primary NB tumors, respectively, were analyzed with CMap. The analysis reported a significant negative connectivity score across nine cell lines for 19 compounds mainly belonging to the class of PI3K/Akt/mTOR inhibitors. The gene expression profiles of NB cells cultured under hypoxic conditions and treated with the mTORC complex inhibitor PP242, referred to as the Mohlin dataset, was used to validate the CMap findings. A heat map representation of hypoxia-modulated genes in the Mohlin dataset and the gene set enrichment analysis (GSEA) showed an opposite regulation of these genes in the set of NB cells treated with the mTORC inhibitor PP242. In conclusion, our analysis identified inhibitors of the PI3K/Akt/mTOR signaling pathway as novel candidate compounds to treat NB patients with hypoxic tumors and a poor prognosis.

Identification of PIK3CA multigene mutation patterns associated with superior prognosis in stomach cancer

Background PIK3CA is the second most frequently mutated gene in cancers and is extensively studied for its role in promoting cancer cell resistance to chemotherapy or targeted therapy. However, PIK3CA functions have mostly been investigated at a lower-order genetic level, and therapeutic strategies targeting PIK3CA mutations have limited effects. Here, we explore crucial factors interacting with PIK3CA mutations to facilitate a significant marginal survival effect at the higher-order level and identify therapeutic strategies based on these marginal factors. Methods Mutations in stomach adenocarcinoma (STAD), breast adenocarcinoma (BRCA), and colon adenocarcinoma (COAD) samples from The Cancer Genome Atlas (TCGA) database were top-selected and combined for Cox proportional-hazards model analysis to calculate hazard ratios of mutation combinations according to overall survival data and define criteria to acquire mutation combinations with considerable marginal effects. We next analyzed the PIK3CA + HMCN1 + LRP1B mutation combination with marginal effects in STAD patients by Kaplan-Meier, transcriptomic differential, and KEGG integrated pathway enrichment analyses. Lastly, we adopted a connectivity map (CMap) to find potentially useful drugs specifically targeting LRP1B mutation in STAD patients. Results Factors interacting with PIK3CA mutations in a higher-order manner significantly influenced patient cohort survival curves (hazard ratio (HR) = 2.93, p-value = 2.63 × 10− 6). Moreover, PIK3CA mutations interacting with higher-order combination elements distinctly differentiated survival curves, with or without a marginal factor (HR = 0.26, p-value = 6.18 × 10− 8). Approximately 3238 PIK3CA-specific higher-order mutational combinations producing marginal survival effects were obtained. In STAD patients, PIK3CA + HMCN1 mutation yielded a substantial beneficial survival effect by interacting with LRP1B (HR = 3.78 × 10− 8, p-value = 0.0361) and AHNAK2 (HR = 3.86 × 10− 8, p-value = 0.0493) mutations. We next identified 208 differentially expressed genes (DEGs) induced by PIK3CA + HMCN1 compared with LRP1B mutation and mapped them to specific KEGG modules. Finally, small-molecule drugs such as geldanamycin (connectivity score = − 0.4011) and vemurafenib (connectivity score = − 0.4488) were selected as optimal therapeutic agents for targeting the STAD subtype with LRP1B mutation. Conclusions Overall, PIK3CA-induced marginal survival effects need to be analyzed. We established a framework to systematically identify crucial factors responsible for marginal survival effects, analyzed mechanisms underlying marginal effects, and identified related drugs.

Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition

In traditional systems, a single herbal formulation is often used in the treatment of diverse diseases, including some that are newly emergent and prevalent today. We provide here a multi-omics framework to probe the molecular basis of a multicomponent example herb, Cissampelos pareira L. (Cipa) used in the treatment of hormonal disorders and fever in Ayurveda. Cipa treated MCF7 cells exhibit downregulation of signatures of estrogen response. 38 constituent molecules in Cipa potentially bind (∆G< -7.5) with ERα at the same site as estrogen. Cipa transcriptome signatures in the connectivity map exhibit positive scores with protein translation inhibitors and knockdown signatures of genes linked to the antiviral response. This includes the knockdown signature of RPL7, a coactivator of ESRI with a connectivity score > 99.92. This axis was found to be upregulated in the COVID-19 patient transcriptome. The antiviral activity through ESR1 modulation was validated in the DENV-2 infection model. We further observed 98% inhibition of SARs-COV-2 replication in infected Vero cell cultures with the whole extract. A few of its prominent pure constituents e.g pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. This study provides a novel framework for querying the molecular links of multicomponent Ayurveda formulations and explains their use in the treatment of disparate diseases. The novel biological targets identified here can become potential that could be applicable to more than one viral infection, such as the use of Cipa in dengue and COVID-19.

IMPACT OF TIMETABLE SYNCHRONIZATION ON HUB CONNECTIVITY OF EUROPEAN CARRIERS

This paper evaluates the net impact of timetable synchronization on the connectivity of the key European carriers at their main hubs. We measure hub connectivity using a weighted connectivity score (WCS) that takes into account the number and the trip time related quality of flight connections. Based on WCS, we compare hub performance resulting from the existing schedule against a random expectati on calculated from multiple randomized schedule simulations. In each simulated schedule scenario we randomly vary the flight departure and arrival times within the operation hours at a hub and at outbound stations keeping all other flight parameters from the real schedule unchanged.We observe that the timetable synchronization leverages hub connectivity of most analyzed airlines by 40% to 60%. The highest increase of connectivity is achieved by medium-sized carriers that operate peaky wave systems with flights concentrated in many short and non-overlapping banks, as well as by carriers that organize their flights in directional waves. The lowest increase is achieved by airlines that operate at highly congested airports. At most hubs, connections to long-haul flights operated with wide-body aircraft are better synchronized than connections between short-haul flights.

Metabolic network-guided binning of metagenomic sequence fragments

Motivation: Most microbes on Earth have never been grown in a laboratory, and can only be studied through DNA sequences. Environmental DNA sequence samples are complex mixtures of fragments from many different species, often unknown. There is a pressing need for methods that can reliably reconstruct genomes from complex metagenomic samples in order to address questions in ecology, bioremediation, and human health. Results: We present the SOrting by NEtwork Completion (SONEC) approach for assigning reactions to incomplete metabolic networks based on a metabolite connectivity score. We successfully demonstrate proof of concept in a set of 100 genome-scale metabolic network reconstructions, and delineate the variables that impact reaction assignment accuracy. We further demonstrate the integration of SONEC with existing approaches (such as cross-sample scaffold abundance profile clustering) on a set of 94 metagenomic samples from the Human Microbiome Project. We show that not only does SONEC aid in reconstructing species-level genomes, but it also improves functional predictions made with the resulting metabolic networks. Availability and implementation: The datasets and code presented in this work are available at: https://bitbucket.org/mattbiggs/sorting_by_network_completion/. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.

Vorinostat Reverses Relapse-Specific Drug Resistance Gene Expression Signatures In Childhood Acute Lymphoblastic Leukemia (ALL).

Abstract 3630 Introduction: Despite significant improvements in outcome for childhood ALL, prognosis is dismal for the 20–25% of patients who relapse. Poor outcomes of relapsed ALL warrant new therapeutic approaches. We have previously used gene expression profiling of pediatric ALL diagnosis–relapse paired patient samples to identify the underlying biological pathways that mediate drug resistance. We identified a relapse-specific gene signature characterized by upregulation of genes involved in cell cycle regulation, apoptosis and nucleotide biosynthesis. Downregulation of these genes restored drug sensitivity in cell lines and preclinical models. We searched the Connectivity Map (cmap) (http://www:broadinstitute.org/cmap), a database of over 7,000 gene expression profiles from cell lines treated with 1309 compounds, to identify novel agents that could “reverse” the relapse-specific signature and potentially restore chemosensitivity. The histone deacetylase (HDAC) inhibitor, vorinostat, also known as SAHA emerged as a top candidate agent which could potentially endow a chemosensitive gene expression profile. Methods: The current version of cmap (build02) was used to “query” our relapsed leukemia gene expression signature derived from 49 diagnosis-relapse pairs analyzed on Affymetrix U133plus2 microarrays comprising 154 probes (Hogan et al, ASH 2009). For each drug-disease pair comparison, an enrichment score was calculated for the probe sets representing the up or downregulated signature genes separately using a rank-based Kolmogorov Smirnov statistic (Lamb et al., 2006). The scores were then combined into a single connectivity score for each drug-disease combination, ranging from +1 to -1, representing positive and negative connectivity, respectively. To validate our findings, B lineage (Reh and RS4:11) and T lineage (Molt4) ALL cell lines were treated with different concentrations of vorinostat and quantitative real time PCR (qRT-PCR) was performed to determine the relative expression of relevant relapse-specific target genes. To determine whether vorinostat could enhance chemosensitivity when combined with conventional chemotherapeutic agents, cells were seeded in 96-well plate and treated with varying concentrations of vorinostat in combination with prednisone, cytarabine and etoposide, either simultaneously or sequentially. Apoptosis and cell survival were analyzed by flow cytometry (Becton Dickinson FACScalibur). Results were analyzed using the Calcusyn software (Biosoft) for dose effect analyses which calculates a combination index (CI) where CI>1.1=antagonism, 0.9–1.1=additive and <0.90=synergy. Results: Based on connectivity scores, 11 of 12 instances of vorinostat treated cancer cell lines in the cmap database indicated that this agent was capable of reversing the relapsed ALL gene signature most significantly with the connectivity score of -0.659 (p=0). Subsequently, qRT-PCR demonstrated down regulation (50-80%) of the expression of survivin, FOXM1 and TYMS (genes upregulated at relapse), after treatment with 1 uM of vorinostat in all the cell lines tested, while a 2–3 fold increase in the expression of NR3C1, which encodes a glucocorticoid receptor (gene downregulated at relapse), was noted with same concentration of vorinostat. Additive to synergistic effects were observed between vorinostat and all 3 chemotherapy agents when cells were first treated with vorinostat followed by chemotherapy after a 24, 48 or 72 hour time interval. Sequential administration of vorinostat followed by etoposide was more than additive or synergistic, especially when etoposide was administered 48–72 hours after vorinostat (CI 0.4–0.9). Similar results were observed with prednisone (CI 0.8–1.1) and cytarabine (CI 0.5–0.9), with enhanced synergy in sequential schedules; whereas concomitant administration of vorinostat and chemotherapy resulted in less than additive or antagonistic effect (CI 1.1–1.3). Conclusion: Gene expression based chemical genomics identifies the HDAC inhibitor, vorinostat, as a potential agent which can reverse the relapsed ALL signature and hence reverse drug resistance. Evaluation in ALL cell lines shows that vorinostat is additive to synergistic with chemotherapy, when administered sequentially. Vorinostat applied before conventional chemotherapy may be a promising approach to the treatment of relapsed ALL. Disclosures: No relevant conflicts of interest to declare.

Evaluating quantitative measures of grammatical complexity in spontaneous speech samples

ABSTRACTThe validity of MLU and a measure of syntactic complexity were tested against LARSP on spontaneous speech samples from 87 children, ranging in age from 1;6 to 4;9. Change in some LARSP clausal measures was found across MLU stages up to MLU 4.5. For the measure of syntactic complexity, no such ceiling was found for the clausal connectivity score in LARSP or for average clausal complexity in LARSP. Neither MLU nor the measure of syntactic complexity indexed LARSP phrasal complexity. It is concluded that MLU is a valid measure of clausal complexity up to 4·5 and that our measure of syntactic complexity is more valid at more advanced stages.