Curated compound collections from the University of Kansas High Throughput Screening laboratory which include Life Chemicals (15,040), ChemBridge (43,736), ChemDiv (56,232), Selleck Bioactives (1649), TimTec (5000), and FDA Repurposed Library (2,286) were used. Compound transfers from source (80 nL of 10 mM stocks) to assay plates were performed using an Echo 550 acoustic liquid handler (Labcyte, Sunnyvale, CA). For most libraries, a target final concentration of 10 μM of compound (in a final volume of 80 μL for the multiplexed reporter gene-based assay described below) was achieved; the FDA Repurposed Library compounds were plated to obtain final concentrations of 2.5 μM. Assay plates were hermetically sealed and stored at -80°C until used. AmpB and nystatin were purchased from Sigma-Aldrich (St. Louis, MO). Synthetic MPLA, lipoteichoic acid (LTA) from S. aureus, PAM2CSK4, Poly(I:C), ultrapure LPS from E. coli K12, flagellin from S. typhimurium, ODN-2006 (Vaccigrade), C12-iE-DAP, and Murabutide were procured from InvivoGen (San Diego, CA). The structures of small molecule PRR agonists synthesized by us are shown in S1 Fig . Responses to a variety of TLR and NLR agonists were first examined using THP1-Blue™ NF-κB reporter cells (InvivoGen, San Diego, CA, Fig 1 ).
Murabutide
Manufactured by InvivoGen
Sourced in Spain, United States
Murabutide is a synthetic molecule that is structurally similar to the bacterial cell wall component muramyl dipeptide. It is commonly used as a research tool in immunology and microbiology studies.
Automatically generated - may contain errors
Lab products found in correlation
C12 ie dap, by InvivoGen (4 mentions)
Poly 1 c, by InvivoGen (4 mentions)
Odn2006, by InvivoGen (3 mentions)
Pam3csk4, by InvivoGen (3 mentions)
M tridap, by InvivoGen (3 mentions)
Pam2csk4, by InvivoGen (2 mentions)
L18 mdp, by InvivoGen (2 mentions)
Tri dap, by InvivoGen (2 mentions)
M trilys, by InvivoGen (2 mentions)
Ie dap, by InvivoGen (2 mentions)
Imiquimod, by InvivoGen (2 mentions)
Nystatin, by Merck Group (1 mentions)
Ultrapure lps from e coli k12, by InvivoGen (1 mentions)
Echo 550 acoustic, by Beckman Coulter (1 mentions)
Flagellin from s typhimurium, by InvivoGen (1 mentions)
Thp1 blue nf κb reporter cells, by InvivoGen (1 mentions)
Lipoteichoic acid lta from s aureus, by InvivoGen (1 mentions)
Milli q integral water purification system, by Merck Group (1 mentions)
Endofit ovalbumin, by InvivoGen (1 mentions)
Ova257 264, by InvivoGen (1 mentions)
9 protocols using murabutide
1
Screening Diverse Compound Libraries for PRR Agonists
2
Murabutide-Adjuvanted OVA Vaccine Preparation
3
Diverse TLR Agonist Library Evaluation
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PAM2CSK4, PAM3CSK4, lipoteichoic acid (LTA), lipopolysaccharide (LPS) from E. coli 055:B4, MPLA, C12-iE-DAP, Murabutide, Poly (I:C) (high molecular weight), flagellin, CL307, ODN2216, ODN2006, and ODN2395 were purchased from InvivoGen (San Diego, CA). The TLR agonists DBS-2-217C,46 C4, IMDQ, Meta-amine,21 (link) EY-2-40,47 (link) XG-1-236,28 (link) MB-564, MB-569,37 (link) MB-152,34 (link) and KHP-3-12648 were synthesized using the routes previously described by us. C27449 was graciously provided by Dynavax Technologies (Berkeley, CA).
4
Endotoxin-Induced Acute Inflammation
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Male mice (8–10 weeks old) were injected intraperitoneally with a non-lethal dose of highly purified LPS (10 mg/kg of highly purified E. coli 0111:B4 purchased from Invivogen) 24 h before a secondary challenge with murabutide at 10 mg/kg (Invivogen). Alternatively, mice were challenged with a lethal dose of highly purified LPS as a model of acute endotoxin septic shock (54 mg/kg of highly purified E. coli 0111:B4 purchased from Invivogen). Mice were monitored twice daily over a 6-day period. The morphology of recruited cells within the peritoneum of mice was determined by cytological examination after centrifugation on glass microscope slides (Cytospin; Shandon), fixation and staining according to the manufacturer's recommendations (Diff; Dade Behring Inc.). For cytokine measurements, serum was taken at 90, 180, 360, and 540 min after secondary MDP challenge.
5
Small-Molecule Reagent Preparation
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All reagents and solvents were procured from Thermo Fisher or Sigma‐Aldrich and employed without further purification. Small‐molecules (Table S3 , Supporting Information), namely 2‐Fucosyllactose, 2‐NP, Baicalein, Eganelisib, Entinostat, KIN‐1408, LCL161, MSA‐2, Picroside, Pidotimod, R848, Ruxolitinib, SR‐717, and Tilorone, were purchased from MedChemExpress; CRX527, MPLA, M‐TriDAP, and Murabutide were obtained from InvivoGen, while RBN2397 was acquired from AmBeed. The compounds were dissolved in dimethyl sulfoxide (DMSO) as appropriate and were utilized without further processing. MilliQ water was sourced from the Waters filtration system.
6
Comprehensive Immune Receptor Agonist Library
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The following compounds were purchased from Invivogen (Ibian Technologies, Zaragoza, Spain): NOD1 and NOD2 agonist kit (#tlrl-nodkit2), containing C12-iE-DAP, iE-DAP, murmyl-dypeptide (MDP), L18-MDP, M-TriDAP, M-TriLYS, murabutide, PGN-ECndi and TriDAP, were purchased from Invivogen (Ibian Technologies, Zaragoza, Spain); Human TLR3/7/8/9 Agonist Kit (#TLRL-KIT3HW3), containing Poly(I:C) (HMW), Poly(I:C) (LMW), Poly(A:U), Imiquimod, R848, CL075, ssRNA40/LyoVec, ssRNA41/LyoVec, ODN2006, ODN2006control, ODN2216, ODN2216control, ODN2395 and ODN2395control; CU-CPT9a, ODN2088, MRT67307, BX795, BAY 11-7082, Pepinh-MYD, Pepinh-MYD Control, Pepinh-TRIF, Pepinh-TRIF Control, L-Kynurenine, 3p-hpRNA, G3-YSD, G3-YSD Control, H-151, G-140, Indirubin, VACV-70/LyoVec, VACV-70c control, HSV-60, Control for CDS Ligands HSV-60/LyoVec, Poly(dA:dT) LyoVec and Poly(dG:dC) LyoVec. NOD inhibitors NOD-IN-1 and ML130 were purchased from Selleckchem (Munich, Germany). Poly(I:C), lipopolysaccharide (LPS, #L6529) and PMA (#P1585) were obtained from Sigma-Merck, Burlington, MA, USA. All compounds were reconstituted in dimethyl sulfoxide (DMSO) or water according to manufacturer’s instructions and stored at −20 °C until use.
7
Polymer Synthesis and Microparticle Production
8
Activation of Pattern Recognition Receptors
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Poly(I:C) high molecular weight, CpG oligodeoxynucleotide (ODN) type C (M362), Resiquimod, Pam3CSK4, Monophosphoryl Lipid A (MPLA), Curdlan (Beta-1,3-glucan from Alcaligenes faecalis), and Murabutide were obtained from Invivogen (San Diego, CA, USA). These mediators were selected for their ability to activate different cell-surface and intracellular PRRs (Table 1 ). Their final concentrations were based on the ability to induce TNF-α production in human monocytes (Resiquimod, Pam3CSK4, MPLA Curdlan, and Murabutide) (Supplementary Figure S1 ) or the manufacturer’s data sheet (Poly(I:C) and CpG ODN). Recombinant human M-CSF and recombinant human RANKL were purchased from Peprotech (London, UK).
9
Innate Immune Receptor Agonist Library
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TLR ligands Pam3CSK4, heat-killed L. monocytogenes, poly I:C (high molecular weight), poly I:C (low molecular weight), lipopolysaccharide (Escherichia coli), flaggellin from Salmonella typhimurium, FSL-1, ssRNA40, and CpG DNA (ODN1826) were purchased from InvivoGen (Mouse TLR1-9 agonist kit). NOD1/2 ligands C12-iE-DAP, iE-DAP, L18-MDP, MDP, M-Tri DAP, M-Tri LYS, Murabutide, PGN-ECndi ultra-pure, PGN-Sandi ultra-pure, and Tri-DAP were purchased from InvivoGen (NOD agonist kit, InvivoGen). E. coli ssDNA and dsDNA, 5 0 ppp-dsRNA, poly (dA:dT), Curdlan, heat-killed E. coli, and heat-killed Candida albicans were also purchased from InvivoGen. Yoda1 was purchased from Sigma (SML1558) and used to stimulate cells at a concentration of 5 mM. The Wnt signaling activator LiCl was purchased from Nacalai. The calpain inhibitor PD 150606, and the Akt inhibitor AZD 5363 were purchased from Sigma and Cayman Chemical, respectively.
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