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I: Antibody and Compound Drug Discovery for Pan-Coronavirus II: PLD3 and PLD4: A Tale of Two Exonucleases /

Record Type:	Electronic resources : Monograph/item
Title/Author:	I: Antibody and Compound Drug Discovery for Pan-Coronavirus II: PLD3 and PLD4: A Tale of Two Exonucleases // Linghang Steve Peng.
Author:	Peng, Linghang Steve,
Description:	1 electronic resource (232 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 86-03, Section: B.
Contained By:	Dissertations Abstracts International86-03B.
Subject:	Biology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31489076
ISBN:	9798384063728

I: Antibody and Compound Drug Discovery for Pan-Coronavirus II: PLD3 and PLD4: A Tale of Two Exonucleases /
Peng, Linghang Steve,

I: Antibody and Compound Drug Discovery for Pan-Coronavirus II: PLD3 and PLD4: A Tale of Two Exonucleases /Linghang Steve Peng. - 1 electronic resource (232 pages)

Source: Dissertations Abstracts International, Volume: 86-03, Section: B.

Part I: Antibody and Compound Drug Discovery for Pan-coronavirusCOVID-19 is the largest recorded pandemic in human history. The pathogen of COVID-19, SARS-CoV-2, is an enveloped ssRNA virus belonging to the Coronaviridae family. Spike is the major glycoprotein on SARS-CoV-2 responsible for receptor (ACE2) binding and cell entry, and also the target for neutralizing antibodies.In this part of my thesis, we first set up several assays at the beginning of the COVID-19 pandemic, including pseudotyped virus (PSV) assay, proximity-based neutralization (PBN) assay, SARS-CoV-2 live virus neutralization protocols and cell-cell fusion assays. PSV assay uses lenti- or retroviral backbones and coronavirus spike protein to produce replication-incompetent virions that mimic authentic spike-receptor interaction and viral entry process. PBN assay uses split luciferase fused to spike and ACE2 to measure the antibody titer blocking spike-ACE2 interactions in vitro, being a rapid, cell-free alternative of traditional cell-based neutralization assay. These assays have greatly accelerated the research on SARS-CoV-2.Then we applied these assays to specific research projects. Using the PSV assay, we initiated a high throughput compound screening for SARS-CoV-2 viral entry inhibitors in collaboration with Scripps Calibr. Within the ReFRAME library, 18 potent coronavirus-specific entry inhibitors were identified and they exhibited (sub)micromolar IC50 against SARS-CoV-2 PSV and live virus in vitro. The mechanisms of action needed further investigation.Another project is to characterize SARS-CoV-2 broadly neutralizing antibodies (bnAb) in collaboration with NIH. We performed neutralization assays to characterize pan-coronavirus reactive mAbs and identified two conserved epitopes on spike-S2 domain: fusion peptide (FP) and stem helix (SH) region for SARS-CoV-2 bnAbs. Selected bnAbs, including COV44-79 (anti-FP) and COV89-22 (anti-SH), protected animals from SARS-CoV-2 and MERS infection. Sequence analysis of anti-SH bnAbs identified a dominant heavy and light chain V gene combination of VH1-46 and VK3-20, which may instruct future germline-targeting vaccine design for pan-coronaviruses.Part II: PLD3 and PLD4: A Tale of Two ExonucleasesNucleic acid sensing is an important aspect of innate immunity. Endosomal toll-like receptors TLR7/8/9 detect the presence of ssRNA and ssDNA with CpG motifs, respectively and activate pro-inflammatory pathways such as NF-κB. Phospholipase D family members PLD3 and PLD4 are two endolysosomal 5' exonucleases that digest ssDNA and ssRNA to regulate the activation of multiple nucleic acid sensors including TLR7/9 and STING. In Pld3-/-Pld4-/- mouse model, hemophagocytic lymphohistiocytosis (HLH)-like symptoms were observed, and the mice died of severe autoimmunity one month after birth. PLD3 and PLD4 have been genetically associated with neuro-degenerative disorders and autoimmune diseases, respectively. However, the detailed mechanism is unclear.Here, we conducted biochemical experiments to study the enzymology of PLD3 and PLD4. The crystal structures of mouse PLD3 and human PLD4 ectodomain were determined. PLD3 and PLD4 form homodimers, and within each molecule domain A and B form an intra-chain dimer, with a basic catalytic site at the interface. Based on the structure, a two-step Ping-Pong mechanism of catalysis was proposed. In addition, by capturing a pHis intermediate of PLD3, we unexpectedly discovered the 5' phosphatase of PLD3/4. Fluorescence resonance energy transfer (FRET) assay and reporter cell assay were established to measure the enzyme activity of PLD3/4 in vitro. Mutagenesis of key amino acids on PLD3/4 revealed structure-function relationships. We also investigated the biochemical properties of several disease-associated mutants, and found several mutants (hPLD3 I163M and L308P; hPLD4: R235Q and S283L) have reduced enzymatic activity and thermostability.Using the same FRET assay, we carried out a high throughput screening for PLD3 and PLD4 inhibitors. Several compounds with (sub)micromolar IC50 values have been identified, and we confirmed their potency in vitro. Since PLD3 and PLD4 inhibition leads to nucleic acid sensor-driven immune activations, we propose that these inhibitors may have a potential value in cancer immunotherapy and vaccine adjuvant.

English

ISBN: 9798384063728Subjects--Topical Terms:

522710
Biology.
Subjects--Index Terms:

Broadly neutralizing antibodies

I: Antibody and Compound Drug Discovery for Pan-Coronavirus II: PLD3 and PLD4: A Tale of Two Exonucleases /
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520 $a Part I: Antibody and Compound Drug Discovery for Pan-coronavirusCOVID-19 is the largest recorded pandemic in human history. The pathogen of COVID-19, SARS-CoV-2, is an enveloped ssRNA virus belonging to the Coronaviridae family. Spike is the major glycoprotein on SARS-CoV-2 responsible for receptor (ACE2) binding and cell entry, and also the target for neutralizing antibodies.In this part of my thesis, we first set up several assays at the beginning of the COVID-19 pandemic, including pseudotyped virus (PSV) assay, proximity-based neutralization (PBN) assay, SARS-CoV-2 live virus neutralization protocols and cell-cell fusion assays. PSV assay uses lenti- or retroviral backbones and coronavirus spike protein to produce replication-incompetent virions that mimic authentic spike-receptor interaction and viral entry process. PBN assay uses split luciferase fused to spike and ACE2 to measure the antibody titer blocking spike-ACE2 interactions in vitro, being a rapid, cell-free alternative of traditional cell-based neutralization assay. These assays have greatly accelerated the research on SARS-CoV-2.Then we applied these assays to specific research projects. Using the PSV assay, we initiated a high throughput compound screening for SARS-CoV-2 viral entry inhibitors in collaboration with Scripps Calibr. Within the ReFRAME library, 18 potent coronavirus-specific entry inhibitors were identified and they exhibited (sub)micromolar IC50 against SARS-CoV-2 PSV and live virus in vitro. The mechanisms of action needed further investigation.Another project is to characterize SARS-CoV-2 broadly neutralizing antibodies (bnAb) in collaboration with NIH. We performed neutralization assays to characterize pan-coronavirus reactive mAbs and identified two conserved epitopes on spike-S2 domain: fusion peptide (FP) and stem helix (SH) region for SARS-CoV-2 bnAbs. Selected bnAbs, including COV44-79 (anti-FP) and COV89-22 (anti-SH), protected animals from SARS-CoV-2 and MERS infection. Sequence analysis of anti-SH bnAbs identified a dominant heavy and light chain V gene combination of VH1-46 and VK3-20, which may instruct future germline-targeting vaccine design for pan-coronaviruses.Part II: PLD3 and PLD4: A Tale of Two ExonucleasesNucleic acid sensing is an important aspect of innate immunity. Endosomal toll-like receptors TLR7/8/9 detect the presence of ssRNA and ssDNA with CpG motifs, respectively and activate pro-inflammatory pathways such as NF-κB. Phospholipase D family members PLD3 and PLD4 are two endolysosomal 5' exonucleases that digest ssDNA and ssRNA to regulate the activation of multiple nucleic acid sensors including TLR7/9 and STING. In Pld3-/-Pld4-/- mouse model, hemophagocytic lymphohistiocytosis (HLH)-like symptoms were observed, and the mice died of severe autoimmunity one month after birth. PLD3 and PLD4 have been genetically associated with neuro-degenerative disorders and autoimmune diseases, respectively. However, the detailed mechanism is unclear.Here, we conducted biochemical experiments to study the enzymology of PLD3 and PLD4. The crystal structures of mouse PLD3 and human PLD4 ectodomain were determined. PLD3 and PLD4 form homodimers, and within each molecule domain A and B form an intra-chain dimer, with a basic catalytic site at the interface. Based on the structure, a two-step Ping-Pong mechanism of catalysis was proposed. In addition, by capturing a pHis intermediate of PLD3, we unexpectedly discovered the 5' phosphatase of PLD3/4. Fluorescence resonance energy transfer (FRET) assay and reporter cell assay were established to measure the enzyme activity of PLD3/4 in vitro. Mutagenesis of key amino acids on PLD3/4 revealed structure-function relationships. We also investigated the biochemical properties of several disease-associated mutants, and found several mutants (hPLD3 I163M and L308P; hPLD4: R235Q and S283L) have reduced enzymatic activity and thermostability.Using the same FRET assay, we carried out a high throughput screening for PLD3 and PLD4 inhibitors. Several compounds with (sub)micromolar IC50 values have been identified, and we confirmed their potency in vitro. Since PLD3 and PLD4 inhibition leads to nucleic acid sensor-driven immune activations, we propose that these inhibitors may have a potential value in cancer immunotherapy and vaccine adjuvant.
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653 $a PLD4 inhibitors
653 $a SARS-CoV-2
653 $a Responsible for receptor
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