Summary of Study ST003388
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002099. The data can be accessed directly via it's Project DOI: 10.21228/M89C1V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003388 |
| Study Title | RMC-113 (dual-lipid kinase inhibitor, PIKfyve, PIP4K2C) alters the phosphoinositide regioisomer signature by advanced lipidomics analysis |
| Study Type | lipid stduy of phosphoinostide lipids |
| Study Summary | To determine whether RMC-113 treatment impacts phosphoinositide abundance, lipid extracts derived from uninfected and SARS-CoV-2-infected A549-ACE2 cells were subject to lipidomic analysis. Employing Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry (PRMC-MS), we comprehensively profiled all eight PI classes and their acyl chain variants (defined by the carbon number and the saturation level). Seven of the eight phosphoinositide classes (except for PI(3,4)P2) were detected in all tested conditions. Upon SARS-CoV-2 infection, the abundance of multiple PI classes was increased relative to uninfected samples, albeit with some variability across independent experiment. This increase was most pronounced with the abundant acyl chain (38:4), yet a similar trend was observed with other acyl chains. Notably, RMC-113 treatment in infected cells caused a 1.5-2-fold increase in the abundance of PI3P and PI5P—the substrates of PIKfyve and PIP4K2C, respectively6, 36—relative to DMSO controls. No concomitant reduction in the levels of the respective phosphorylated products, PI(3,5)P2 and PI(4,5)P2, was detected in RMC-113- vs. DMSO-treated infected cells, likely due to intact activity of enzymes not targeted by RMC-113, such as PIP4K (1A/1B/1C). Nevertheless, the product-to-substrate ratios of PIKfyve and PIP4K2C were reduced in both uninfected and infected cells upon RMC-113 treatment relative to DMSO |
| Institute | Stanford University |
| Department | medicine |
| Laboratory | Einav lab |
| Last Name | Mishra |
| First Name | Manjari |
| Address | 300 pasteur drive, stanford |
| manjari1@stanford.edu | |
| Phone | 6503849709 |
| Submit Date | 2024-07-13 |
| Num Groups | 4 (uninfected and SARS-CoV-2 infected samples and RMC-113-treated and untreated samples) |
| Study Comments | na |
| Publications | https://www.biorxiv.org/content/10.1101/2024.04.15.589676v1.full |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Chear Study | na |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-08-28 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002099 |
| Project DOI: | doi: 10.21228/M89C1V |
| Project Title: | PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2 |
| Project Type: | Lipidomics of Human lung epithelial cells |
| Project Summary: | In this study, we calculated the peak area abundance of phosphoinositide lipids in SARS-Cov2 infected, uninfected and dual lipid kinase (PIP4K2C and PIKfyve) inhibitor treated (RMC-113,) (PI, PIP, PIP2 and PIP3) Human lung epithelial cells. We discovered PIP4K2C’s (Phosphatidylinositol-5-phosphate 4-kinase, type II, gamma) roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 proteins and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113(dual lipid kinase inhibitor (PIP4K2C and PIKfyve). These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C (Phosphatidylinositol-5-phosphate 4-kinase, type II, gamma) and PIKfyve (a FYVE finger-containing phosphoinositide kinase) as a candidate strategy to combat emerging viruses. |
| Institute: | Stanford School of Medicine |
| Department: | medicine |
| Laboratory: | Einav lab |
| Last Name: | mishra |
| First Name: | manjari |
| Address: | 3332 middlefield road |
| Email: | manjari1@stanford.edu |
| Phone: | 6503849709 |
| Funding Source: | NIH |
| Publications: | https://www.biorxiv.org/content/10.1101/2024.04.15.589676v1.full |
| Contributors: | Marwah Karim, Manjari Mishra, Chieh-Wen Lo, Sirle Saul, Halise Busra Cagirici, Do Hoang Nhu Tran, Aditi Agrawal, Luca Ghita, Amrita Ojha, Michael P. East, Karen Anbro Gammeltoft, Malaya Kumar Sahoo, Gary L. Johnson, Soumita Das, Dirk Jochmans, Courtney A. Cohen, Judith Gottwein, John Dye, Norma Neff, Benjamin A. Pinsky, Tuomo Laitinen, Tatu Pantsar, Antti Poso, Fabio Zanini, Steven De Jonghe, Christopher R M Asquith, Shirit Einav |
Subject:
| Subject ID: | SU003509 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Human lung epithelial cells (A549-ACE2) |
| Cell Passage Number: | 15 |
| Cell Counts: | 1*10^6 cells |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA367933 | DMSO SARS-CoV2 | Human lung epithelial cells A549-ACE2 | Control |
| SA367934 | DMSO uninfected | Human lung epithelial cells A549-ACE2 | Control |
| SA367935 | RMC-113 SARS-CoV2 | Human lung epithelial cells A549-ACE2 | treated |
| SA367936 | RMC-113 uninfected | Human lung epithelial cells A549-ACE2 | treated |
| Showing results 1 to 4 of 4 |
Collection:
| Collection ID: | CO003502 |
| Collection Summary: | In this study, we have used lung epithelial A549-ACE2 cell line for our experiment in both SARS-CoV2 infected and uninfected cells including treatment with our lab-made dual lipid kinase inhibitor RMC-113 |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR003518 |
| Treatment Summary: | The cells are treated with dual lipid kinase inhibitor RMC-113 |
| Treatment Dose: | 5uM |
| Treatment Doseduration: | 24hrs |
| Treatment Vehicle: | methanol |
| Cell Storage: | 37 Celsius 5% co2 |
| Cell Growth Container: | petriplate |
| Cell Growth Rate: | 32hrs doubling time |
| Cell Media: | DMEM |
| Cell Envir Cond: | ambient |
| Cell Harvesting: | p-16 |
| Cell Pct Confluence: | 80% |
Sample Preparation:
| Sampleprep ID: | SP003516 |
| Sampleprep Summary: | Lipid extraction: 10 pmol each of 4 reference standards (deuterated 5 PI, deuterated 62 PI3P, deuterated 62 PI(4,5)P2) was spiked into methanol volume required for total # of samples. 1 sample is spiked with 37:4 and 38:4 reference standards (Table 1) for retention time matching. 1.5 ml of methanol standard mix was added to 200 ul of plasma or cell pellet. 750 of uL Ultrapure water, 750ul of 2M HCL, and 200 uL of 1 M NaCl were added to above mixture. Mixture was vortexed, then 1 mL of chloroform was added. Tubes were mixed for 2 min. After centrifugation at 1200×g for 4 min at RT, lower organic was transferred to a new tube. Prep for Methylation: 1 mL of Methanol/12 M HCL/H20 (12/12/1/1) was added to the lower phase. 900 uL of 120 mM NaCl was added to the mixture and vortexed. After centrifugation at 1200×g for 4 min at RT, lower organic was transferred to a new tube. Methylation reaction: 50 uL of 2 M trimethylsilyl diazomethane was added to each sample tube. After 10 min, the reaction was quenched with 20 uL of glacial acetic acid. Samples were mixed with 700 uL of chloroform/methanol/UltrapureH20 (3/48/47). Tubes were mixed for 1 min. After centrifugation at 1200×g for 4 min at RT, lower organic was transferred to a 1.5 mL vials dried under N2 stream. Samples were redissolved in 200 uL acetonitrile. Samples were placed on shaker for 10 minutes, then transferred to a 200 ul glass vial insert. Samples were loaded on UPLC autosampler and injected at 30 uL for LCMS analysis. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | 4℃ |
| Sample Derivatization: | methylation |
| Sample Spiking: | yes |
Combined analysis:
| Analysis ID | AN005555 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 |
| Column | Phenomenex Lux i-Cellulose-5 (250 x 4.6mm, 3um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | POSITIVE |
| Units | au |
Chromatography:
| Chromatography ID: | CH004219 |
| Chromatography Summary: | Setup Chiral column to MS and create MRM profiles for each lipid class. Run samples and collect data using parameters in table 2 and 3 below. Table 2. UHPLC Parameters Column: Lux 3 µm i-Cellulose-5, LC Column 250 x 4.6 mm, Part No.: 00G-4755-E0 Detector: MS triple quad Sciex 5500 Injection Volume: 30 µL Sample Temperature: 15 °C Column Temperature: 35 °C Flow rate: 0.5 mL/min. Mobile Phase-A: 5 mM ammonium acetate in Methanol Mobile Phase-B: 5 mM ammonium acetate in Acetonitrile Elution Mode: Gradient see below table Time (min.) A% B% 0 0 100 1 0 100 3 30 70 15 30 70 15.01 0 100 20 0 100 |
| Methods Filename: | LIPIDOMICS_protocol_all_parameters_etc.pdf |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Phenomenex Lux i-Cellulose-5 (250 x 4.6mm, 3um) |
| Column Temperature: | 35 |
| Flow Gradient: | Time (min.) A% B%; 0 0 100; 1 0 100; 3 30 70; 15 30 70; 15.01 0 100; 20 0 100 |
| Flow Rate: | 0.5 mL/min |
| Internal Standard: | PI, PIP, PIP2 |
| Solvent A: | 100% methanol; 5 mM ammonium acetate |
| Solvent B: | 100% acetonitrile; 5 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Solvent C: | none |
MS:
| MS ID: | MS005280 |
| Analysis ID: | AN005555 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Calculate areas under curve of each sample with retention times matching the 37:4 and 38:4 standard mix only sample. Divide areas of each sample by their respective deuterated reference standard to account for experimental and analytical variation. Use analyst 3.0 software to both run samples and analyze and process samples |
| Ion Mode: | POSITIVE |
