Immunolocalization of a PIGR-like Protein in Tetrahymena thermophila

Netrins are pleiotropic signaling molecules which guide axonal development and help regulate processes such as angiogenesis. Netrins can act as chemorepellents for developing axons, and our previous work has shown that several netrins, including netrin-1, netrin-3, and netrin-4, are chemorepellents in Tetrahymena thermophila. In vertebrates, netrin-1 signals through several receptors, including those in the UNC-5 family. UNC-5 family proteins often signal through the src family of tyrosine kinases. We have previously characterized UNC-5 and src-like proteins in Tetrahymena, by immunolocalization and Western blotting. Sequencing of our src-like proteins gave a number of homologous sequences, including the sequence for polymeric immunoglobulin-like receptor (PIGR). With all of these findings in mind, we hypothesized that Tetrahymena might possess a receptor similar to PIGR, which would localize either to the plasma membrane or cilia of Tetrahymena

Biochemical Evidence for Netrin-Signaling Homologues in Tetrahymena thermophila

Netrins are pleiotropic guidance proteins that are involved in developmental signaling of branched structures within vertebrates. However, like many developmental pathways, dysregulation of the netrin pathway has been implicated in cancer progression and metastasis. Since Tetrahymena respond to guidance proteins, showing chemoattractant and chemorepellent behavior, we hypothesized that we could use these organisms as a model system for cancer signaling. We have previously found that netrin-1-peptided, netrin-3-peptide, and recombinant netrin-4 are all chemorepellents in this organism. Since netrin-1-peptide signals through a tyrosine kinase in Tetrahymena, we hypothesized that Tetrahymena might possess tyrosine kinases as well as a receptor homologous to UNC-5, a netrin receptor which relays signals via tyrosine kinases in vertebrates. Using immunoprecipitation with a polyclonal anti-UNC-5-B antibody, we purified a 250 kD protein from Tetrahymena whole cell extract. Similarly, we immunoprecipitated several proteins, including a 60 kD protein and a 75 kD protein using a polyclonal anti-src-antibody. Our purified samples were sent out for identification by mass spectroscopy. Mass spectroscopy indicated that we have purified a number of novel peptides not currently found in the Tetrahymena Genome Database. Our data indicate that the proteome database in this organism is incomplete, and that there are additional proteins waiting to be discovered in this organism

Optimization rules for SARS-CoV-2 M\u3csup\u3epro\u3c/sup\u3e antivirals: Ensemble docking and exploration of the coronavirus protease active site

© 2020 by the authors. Coronaviruses are viral infections that have a significant ability to impact human health. Coronaviruses have produced two pandemics and one epidemic in the last two decades. The current pandemic has created a worldwide catastrophe threatening the lives of over 15 million as of July 2020. Current research efforts have been focused on producing a vaccine or repurposing current drug compounds to develop a therapeutic. There is, however, a need to study the active site preferences of relevant targets, such as the SARS-CoV-2 main protease (SARS-CoV-2 Mpro), to determine ways to optimize these drug compounds. The ensemble docking and characterization work described in this article demonstrates the multifaceted features of the SARS-CoV-2 Mpro active site, molecular guidelines to improving binding affinity, and ultimately the optimization of drug candidates. A total of 220 compounds were docked into both the 5R7Z and 6LU7 SARS-CoV-2 Mpro crystal structures. Several key preferences for strong binding to the four subsites (S1, S1\u27, S2, and S4) were identified, such as accessing hydrogen binding hotspots, hydrophobic patches, and utilization of primarily aliphatic instead of aromatic substituents. After optimization efforts using the design guidelines developed from the molecular docking studies, the average docking score of the parent compounds was improved by 6.59 -log10(Kd) in binding affinity which represents an increase of greater than six orders of magnitude. Using the optimization guidelines, the SARS-CoV-2 Mpro inhibitor cinanserin was optimized resulting in an increase in binding affinity of 4.59 -log10(Kd) and increased protease inhibitor bioactivity. The results of molecular dynamic (MD) simulation of cinanserin-optimized compounds CM02, CM06, and CM07 revealed that CM02 and CM06 fit well into the active site of SARS-CoV-2 Mpro [Protein Data Bank (PDB) accession number 6LU7] and formed strong and stable interactions with the key residues, Ser-144, His-163, and Glu-166. The enhanced binding affinity produced demonstrates the utility of the design guidelines described. The work described herein will assist scientists in developing potent COVID-19 antivirals

Optimization Rules for SARS-CoV-2 Mpro Antivirals: Ensemble Docking and Exploration of the Coronavirus Protease Active Site

Coronaviruses are viral infections that have a significant ability to impact human health. Coronaviruses have produced two pandemics and one epidemic in the last two decades. The current pandemic has created a worldwide catastrophe threatening the lives of over 15 million as of July 2020. Current research efforts have been focused on producing a vaccine or repurposing current drug compounds to develop a therapeutic. There is, however, a need to study the active site preferences of relevant targets, such as the SARS-CoV-2 main protease (SARS-CoV-2 Mpro), to determine ways to optimize these drug compounds. The ensemble docking and characterization work described in this article demonstrates the multifaceted features of the SARS-CoV-2 Mpro active site, molecular guidelines to improving binding affinity, and ultimately the optimization of drug candidates. A total of 220 compounds were docked into both the 5R7Z and 6LU7 SARS-CoV-2 Mpro crystal structures. Several key preferences for strong binding to the four subsites (S1, S1′, S2, and S4) were identified, such as accessing hydrogen binding hotspots, hydrophobic patches, and utilization of primarily aliphatic instead of aromatic substituents. After optimization efforts using the design guidelines developed from the molecular docking studies, the average docking score of the parent compounds was improved by 6.59 −log10(Kd) in binding affinity which represents an increase of greater than six orders of magnitude. Using the optimization guidelines, the SARS-CoV-2 Mpro inhibitor cinanserin was optimized resulting in an increase in binding affinity of 4.59 −log10(Kd) and increased protease inhibitor bioactivity. The results of molecular dynamic (MD) simulation of cinanserin-optimized compounds CM02, CM06, and CM07 revealed that CM02 and CM06 fit well into the active site of SARS-CoV-2 Mpro [Protein Data Bank (PDB) accession number 6LU7] and formed strong and stable interactions with the key residues, Ser-144, His-163, and Glu-166. The enhanced binding affinity produced demonstrates the utility of the design guidelines described. The work described herein will assist scientists in developing potent COVID-19 antivirals

Need for Regulatory Change to Incorporate Beyond A1C Glycemic Metrics

status: publishe