identifying-pd-1pd-l1-inhibitors-with-surface-plasmon-resonance

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating ...

Inhibitors targeting kynurenine-3-monooxygenase (KMO), an enzyme in the neurotoxic kynurenine pathway (KP), are potential therapeutics for KP metabolites-mediated neuroinflammatory and neurodegenerative disorders. Although phytochemicals from Cannabis (C. sativa L.) have been reported to show modulating effects on enzymes involved in the KP metabolism, the inhibitory effects of C. sativa compounds, including phytocannabinoids and non-phytocannabinoids (i.e., cannflavin A; CFA), on KMO remain unknown. Herein, CFA (purified from hemp aerial material at a gram-scale) and a series of phytocannabinoids were evaluated for their anti-KMO activity. CFA showed the most active inhibitory effect on KMO, which was comparable to the positive control Ro 61-8048 (IC50 = 29.4 vs. 5.1 μM, respectively). Furthermore, a molecular docking study depicted the molecular interactions between CFA and the KMO protein and a biophysical binding assay with surface plasmon resonance (SPR) technique revealed that CFA bound to the protein with a binding affinity of 4.1×10−5 M. A competitive SPR binding analysis suggested that CFA and Ro 61-8048 bind to the KMO protein in a competitive manner. Our findings show that C. sativa derived phytochemicals, including CFA, are potential KMO inhibitors, which provides insight into the development of therapeutics targeting the KP and its related pathological conditions.

Gram-Scale Preparation of Cannflavin A from Hemp ( L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two cholinergic enzymes catalyzing the reaction of cleaving acetylcholine into acetate and choline at the neuromuscular junction. Abnormal hyperactivity of AChE and BChE can lead to cholinergic deficiency, which is associated with several neurological disorders including cognitive decline and memory impairments. Preclinical studies support that some cannabinoids including cannabidiol (CBD) and tetrahydrocannabinol (THC) may exert pharmacological effects on the cholinergic system, but it remains unclear whether cannabinoids can inhibit AChE and BChE activities. Herein, we aimed to evaluate the inhibitory effects of a panel of cannabinoids including CBD, Δ8-THC, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabicitran (CBT), cannabidivarin (CBDV), cannabichromene (CBC), and cannabinol (CBN) on AChE and BChE activities.

Inhibitory Effects of Cannabinoids on Acetylcholinesterase and Butyrylcholinesterase Enzyme Activities.

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (M), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 M, their anti-M activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-M activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the M protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active M inhibitors (IC = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10-4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids' alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids' inhibitory effects on SARS-CoV-2 M.

Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity.

Our group has previously reported on the phytochemical composition and biological activities of a phenolic-enriched maple syrup extract (MSX), which showed promising anti-inflammatory effects in several disease models including diabetes and Alzheimer's disease. However, the efficacious doses of MSX and its molecular targets involved in the anti-inflammatory effects are not fully elucidated. Herein, the efficacy of MSX in a peritonitis mouse model was evaluated in a dose-finding study and the underlying mechanisms were explored using data-independent acquisition (DIA) proteomics assay. MSX (at 15, 30 and 60 mg kg) alleviated lipopolysaccharide-induced peritonitis by reducing the levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in the serum and major organs of the mice. Furthermore, DIA proteomics analyses identified a panel of proteins that were significantly altered (both up- and down-regulated) in the peritonitis group, which were counteracted by the MSX treatments. MSX treatment also modulated several inflammatory upstream regulators including interferon gamma and TNF. Ingenuity pathway analysis suggested that MSX may modulate several signaling pathways in the processes of initiation of cytokine storm, activation of liver regeneration, and suppression of hepatocyte apoptosis. Together, these proteomic and  findings indicate that MSX could regulate inflammation signaling pathways and modulate inflammatory markers and proteins, providing critical insight to its therapeutic potential.

Uncovering the anti-inflammatory mechanisms of phenolic-enriched maple syrup extract in lipopolysaccharide-induced peritonitis in mice: insights from data-independent acquisition proteomics analysis.

Blockade of the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis is a promising strategy for cancer immunotherapy. Although antibody-based PD-1/PD-L1 inhibitors have shown remarkable results in clinical cancer studies, their inherent limitations underscore the significance of developing novel PD-1/PD-L1 inhibitors. Small molecule inhibitors have several advantages over antibody-based inhibitors, including favorable tumor penetration and oral bioavailability, fewer side effects, easier administration, preferred biological half-life, and lower cost. However, small molecule inhibitors that directly target the PD-1/PD-L1 interaction are still in the early development stage, partially due to the lack of reliable biophysical assays. Herein, we present a novel PD-1/PD-L1 blockade assay using a surface plasmon resonance (SPR)-based technique. This blockade assay immobilizes human PD-1 on a sensor chip, which interacts with PD-L1 inhibitors or negative PD-L1 binders with human PD-L1 protein at a range of molecular ratios. The binding kinetics of PD-L1 to PD-1 and the blockade rates of small molecules were determined. Compared to other techniques such as PD-1/PD-L1 pair enzyme-linked immunosorbent assay (ELISA) and AlphaLISA immunoassays, our SPR-based method offers real-time and label-free detection with advantages including shorter experimental runs and smaller sample quantity requirements. Key features A SPR protocol screens compounds for their capacity to block the PD-1/PD-L1 interaction. Validation of PD-1/PD-L1 interaction, followed by assessing blockade effects with known inhibitors BMS-1166 and BMS-202, and a negative control NO-Losartan A. Analysis of percentage blockade of PD-1/PD-L1 of the samples to obtain the IC. Broad applications in the discovery of small molecule-based PD-1/PD-L1 inhibitors for cancer immunotherapy. Graphical overview.

Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor.

P2X purinoceptor 4 (P2X4) is an ATP-gated ion channel receptor with diverse neurophysiological functions, and P2X4 modulators hold promise as potential therapeutics for neuropathic pain, neuroinflammation, and neurodegenerative diseases. While several cannabinoids have been reported as modulators of purinoreceptors, their specific purinoreceptor-binding characteristics remain elusive. In this study, we established a comprehensive workflow that included a binding screening platform and a novel surface plasmon resonance (SPR) competitive assay, complemented by computational docking, to identify potential P2X4 binders among a panel of twenty-eight cannabinoids. Through SPR, we determined the binding affinities of cannabinoids ( values ranging from 3.4 × 10 M to 1 × 10 M), along with two known P2X4 antagonists, BX430 ( = 4.5 × 10 M) and 5-BDBD ( = 7.8 × 10 M). The competitive SPR assay validated that BX430 and 5-BDBD acted as non-competitive binders with P2X4. In the following competitive assays, two cannabinoids including cannabidiol (CBD) and cannabivarin (CBV) were identified as competitive P2X4-binders with 5-BDBD, while the remaining cannabinoids exhibited non-competitive binding with either BX430 or 5-BDBD. Our molecular docking experiments further supported these findings, demonstrating that both CBD and CBV shared identical binding sites with residues in the 5-BDBD binding pocket on P2X4. In conclusion, this study provides valuable insights into the P2X4-binding affinity of cannabinoids through SPR and sheds light on the interactions between cannabinoids (CBD and CBV) and P2X4.

Investigating cannabinoids as P2X purinoreceptor 4 ligands by using surface plasmon resonance and computational docking.

Precursors of advanced glycation endproducts, namely, reactive carbonyl species (RCSs), are aging biomarkers that contribute to cell death. However, the impact of RCSs on ferroptosis-an iron-dependent form of cell death-in skin cells remains unknown. Herein, we constructed a cellular model (with human keratinocyte; HaCaT cells) to evaluate the cytotoxicity of the combinations of RCSs (including glyoxal; GO and methyglyoxal; MGO) and erastin (a ferroptosis inducer) using bioassays (measuring cellular lipid peroxidation and iron content) and proteomics with sequential window acquisition of all theoretical mass spectra. Additionally, a data-independent acquisition approach was used to characterize RCSs' and erastin's molecular network including genes, canonical pathways, and upstream regulators. Using this model, we evaluated the cytoprotective effects of two dietary flavonoids including cannflavins A and B against RCSs and erastin-induced cytotoxicity in HaCaT cells. Cannflavins A and B (at 0.625 to 20 µM) inhibited ferroptosis by restoring the cell viability (by 56.6-78.6% and 63.8-81.1%) and suppressing cellular lipid peroxidation (by 42.3-70.2% and 28.8-63.6%), respectively. They also alleviated GO + erastin- or MGO + erastin-induced cytotoxicity by 62.2-67.6% and 56.1-69.3%, and 35.6-54.5% and 33.8-62.0%, respectively. Mechanistic studies supported that the cytoprotective effects of cannflavins A and B are associated with their antioxidant activities including free radical scavenging capacity and an inhibitory effect on glycation. This is the first study showing that cannflavins A and B protect human keratinocytes from RCSs + erastin-induced cytotoxicity, which supports their potential applications as dietary interventions for aging-related skin conditions.

Cannflavins A and B with Anti-Ferroptosis, Anti-Glycation, and Antioxidant Activities Protect Human Keratinocytes in a Cell Death Model with Erastin and Reactive Carbonyl Species.

Our laboratory has established a comprehensive program to investigate the phytochemical composition and nutritional/medicinal properties of phenolic-enriched maple syrup extract (MSX). Previous studies support MSX's therapeutic potential in diverse disease models, primarily through its anti-inflammatory effects. We recently demonstrated MSX's ability to regulate inflammatory signaling pathways and modulate inflammatory markers and proteins in a lipopolysaccharide (LPS)-induced peritonitis mouse model. However, MSX's immunoregulatory properties remain unknown. Herein, we investigated MSX's immunoregulatory properties for the first time using an integrated approach, combining data-dependent acquisition (DDA) and data-independent acquisition (DIA) strategies in a proteomic analysis of spleen tissue collected from the aforementioned peritonitis mouse model. Additionally, we conducted immune cell activation assays using macrophages and T lymphocytes. The DIA analysis unveiled a distinctive expression pattern involving three proteins-Krt83, Thoc2, and Vps16-which were present in both the control and MSX-treated groups but absent in the LPS-induced model group. Furthermore, proteins Ppih and Dpp9 exhibited significant reductions in the MSX-treated group. Ingenuity pathway analysis indicated that MSX may modulate several critical signaling pathways, exerting a suppressive effect on immune responses in various cell types involved in both innate and adaptive immunity. Our  cell assays supported findings from the proteomics, revealing that MSX significantly reduced the levels of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in LPS-stimulated human macrophage cells, as well as the levels of IL-2 in anti-CD3/anti-CD28-induced Jurkat T cells. Taken together, our investigations provide evidence that MSX exerts immune regulatory effects that impact both innate and adaptive immunity, which adds to the data supporting MSX's development as a functional food.

Exploring immunoregulatory properties of a phenolic-enriched maple syrup extract through integrated proteomics and  assays.

Erratum: Correction Notice: Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor.

Skin cells are susceptible to oxidative stress and various types of cell death, including an iron-dependent form known as ferroptosis. Cannabidiol (CBD) can protect skin cells against oxidative stress, but whether this is attributed to the inhibition of ferroptosis is unknown. Herein, we evaluated the anti-ferroptotic effect of CBD in human keratinocytes using biochemical assays (radical scavenging and iron chelating) and cell-based models (for lipid peroxidation and intracellular iron). CBD's anti-ferroptotic effect was further characterized by proteomic analysis. This study identifies anti-ferroptosis as a mechanism of CBD's skin protective effects.

Anti-Ferroptotic Effect of Cannabidiol in Human Skin Keratinocytes Characterized by Data-Independent Acquisition-Based Proteomics.

Guavinoside B (GUB) is a characteristic constituent from guava with strong antioxidant activity; however, its low water solubility limits its utilization. Herein, we investigated the interaction between GUB and zein, a prolamin with self-assembling property, using multiple spectroscopic methods and fabricated GUB-zein-NaCas nanoparticles (GUB-Z-N NPs) via the antisolvent coprecipitation approach. GUB caused fluorescence quenching to zein via the static quenching mechanism. Fourier-transform infrared spectroscopy and computational analysis revealed that GUB bound to zein via van der Waals interaction, hydrogen bond, and hydrophobic forces. The GUB-Z-N NPs were in the nanometric size range (< 200 nm) and exhibited promising encapsulation efficiency and redispersibility after freeze-drying. These particles remained stable for up to 31 days at 4 °C and great resistance to salt and pH variation, and displayed superior antioxidant activity to native GUB. The current study highlights the potential of zein-based nanoparticles as delivery vehicles for GUB in the food industry.

Caseinate-coated zein nanoparticles as potential delivery vehicles for guavinoside B from guava: Molecular interactions and encapsulation properties.

Bottom-up proteomics utilizes sample preparation techniques to enzymatically digest proteins, thereby generating identifiable and quantifiable peptides. Proteomics integrates with other omics methodologies, such as genomics and transcriptomics, to elucidate biomarkers associated with diseases and responses to drug or biologics treatment. The methodologies employed for preparing proteomic samples for mass spectrometry analysis exhibit variability across several factors, including the composition of lysis buffer detergents, homogenization techniques, protein extraction and precipitation methodologies, alkylation strategies, and the selection of digestion enzymes. The general workflow for bottom-up proteomics consists of sample preparation, mass spectrometric data acquisition (LC-MS/MS analysis), and subsequent downstream data analysis including protein quantification and differential expression analysis. Sample preparation poses a persistent challenge due to issues such as low reproducibility and inherent procedure complexities. Herein, we have developed a validated chloroform/methanol sample preparation protocol to obtain reproducible peptide mixtures from both rodent tissue and human cell line samples for bottom-up proteomics analysis. The protocol we established may facilitate the standardization of bottom-up proteomics workflows, thereby enhancing the acquisition of reliable biologically and/or clinically relevant proteomic data. Key features • Tissue/cell pellet sample preparation for bottom-up proteomics. • Chloroform/methanol protein extraction from murine tissue samples. • In-solution trypsin digestion proteomics workflow.

Article	Year
Gram-Scale Preparation of Cannflavin A from Hemp ( L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase. Biology\| PubMed ID: 36290320	2022
Inhibitory Effects of Cannabinoids on Acetylcholinesterase and Butyrylcholinesterase Enzyme Activities. Medical cannabis and cannabinoids\| PubMed ID: 35702400	2022
Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity. Molecules (Basel, Switzerland)\| PubMed ID: 36144858	2022
Uncovering the anti-inflammatory mechanisms of phenolic-enriched maple syrup extract in lipopolysaccharide-induced peritonitis in mice: insights from data-independent acquisition proteomics ... Food & function\| PubMed ID: 37403713	2023
Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor. Bio-protocol\| PubMed ID: 37575393	2023
Investigating cannabinoids as P2X purinoreceptor 4 ligands by using surface plasmon resonance and computational docking. Heliyon\| PubMed ID: 37920520	2023
Cannflavins A and B with Anti-Ferroptosis, Anti-Glycation, and Antioxidant Activities Protect Human Keratinocytes in a Cell Death Model with Erastin and Reactive Carbonyl Species. Nutrients\| PubMed ID: 37960218	2023
Exploring immunoregulatory properties of a phenolic-enriched maple syrup extract through integrated proteomics and assays. Food & function\| PubMed ID: 38019191	2024
Erratum: Correction Notice: Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor. Bio-protocol\| PubMed ID: 38152682	2023
Anti-Ferroptotic Effect of Cannabidiol in Human Skin Keratinocytes Characterized by Data-Independent Acquisition-Based Proteomics. Journal of natural products\| PubMed ID: 38373879	2024
Caseinate-coated zein nanoparticles as potential delivery vehicles for guavinoside B from guava: Molecular interactions and encapsulation properties. Food chemistry\| PubMed ID: 38901076	2024
Chloroform/Methanol Protein Extraction and In-solution Trypsin Digestion Protocol for Bottom-up Proteomics Analysis. Bio-protocol\| PubMed ID: 39210950	2024

Tess Puopolo

Biography

JoVE Journal Publications

Other Publications