See what discoveries Cayman scientists presented at scientific conferences in 2023.

Cayman scientists regularly present discoveries from our ongoing research and development activities with the larger research community at scientific conferences. Explore the methods and tools we used to make our discoveries and see how they can be used to support your research in our 2023 scientific poster recap.

Topics covered include:

Lipidomics & Lipid Biochemistry

Characterization of Extracellular Vesicle Lipids in Pre-Diabetic Mice

Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from cells by a variety of mechanisms. These particles are known to contain a variety of cargo, such as lipids, proteins, nucleic acids, and other metabolites that are believed to be responsible for their cell signaling activity.

In this study, we have performed untargeted lipidomics analysis on isolated plasma EVs from healthy and diet-induced obese, prediabetic C57BL6/J mice to study potential differences in their lipid profiles.

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Lipidomics Analysis Reveals Changes in the Regulation of Lipid Metabolism in a Surgical Bone Defect Model

Advances in liquid chromatography and mass spectrometry instrumentation and in specialized software enable identification and relative quantitation of a wide variety of lipid species, providing powerful tools for the investigation of lipid profile changes over time.

The present study illustrates those advances by identifying large numbers of lipid molecular species in a variety of tissues collected at different times post-surgery from a femoral defect model in rats treated with a prostaglandin receptor agonist.

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A Streamlined Synthetic Approach to SPMs

Researchers have synthesized over 20 specialized pro-resolving lipid mediators (SPMs) from just a few commercially available starting materials. With these building blocks, a major portion of any SPMs and their derivatives can be synthesized.

In this poster, five of these integral pieces and their useful synthetic qualities are outlined, with special attention given to the synthesis of resolvin D1 (RvD1).

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Immunopeptidome Profiling

CT26 Neoantigen Presentation and Immunogenicity

Vaccination against tumor antigens is one method being explored to generate immune responses against tumors and may be particularly effective in conjunction with therapies currently in the clinic, such as checkpoint inhibition. Identification of antigens for use in a vaccine is the pinch point of this method.

Using immunopeptidome profiling, we have narrowed a collection of published potential neoantigens to those presented by MHC class I in the CT26 mouse colorectal cancer cell line. These peptides were synthesized and used to immunize BALB/c mice, and immunogenicity of the peptide mix was evaluated by ELISpot to assess IFN-γ responses in immunized mice. These experiments support a complete workflow for the selection of potential cancer neoantigens that experimentally exhibit both MHC presentation and immunogenicity.

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Immunopeptidome Profiling of Xenograft Glioma Samples

Characterization of the peptides presented by MHC molecules (pMHC) on cancer cells and their immunogenic potential is key for generating anti-cancer immune responses. Experimental identification of pMHC is performed using MHC immunoprecipitation and mass spectrometry sequencing of eluted peptides.

In the current study, we characterized the immunopeptidome of two human glioma lines, U87 and DIPG, which were grown in nude mice. As one potential complication of cells grown in mice may be contribution of mouse pMHC to the overall peptide list, we tested specific depletion of mouse MHC prior to immunoprecipitation of human MHC complexes.

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Hit Identification & Drug Discovery 

Discovery of Novel Heterocycle Inhibitors: Hit to Lead Compound in <10 Months

Cayman Chemical identified a certain lipid-based protein target of interest implicated in various disease states including cancer and inflammation. In less than ten months, we developed and deployed our primary and secondary biochemical and biophysical screening assays and carried out in silico screening of multiple commercial compound libraries to identify and confirm various virtual hits. We furthermore executed an iterative structure-activity relationship (SAR) program utilizing computer-aided drug design (CADD) and synthetic and medicinal chemistry to produce over 100 novel compounds targeting the lipid-based biological target.

During this exceptionally successful program, we deployed, in addition to our activity-based primary screen, multiple biophysical methods, including the use of our thermal shift assay (TSA), surface plasmon resonance (SPR), and X-ray crystallography techniques.

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Fragment-Based Drug Discovery (FBDD) Approach for TNF-α

Attempts to design small molecule TNF-α inhibitors have not yet led to approved products. In this study, we followed a fragment-based screening approach to provide insight into designing small molecule inhibitors directly targeting TNF-α.

In collaboration with Key Organics Limited, we conducted a fragment-based screening study against TNF-α using our Integrated Medicinal/Computational Chemistry Platform. A surface plasmon resonance (SPR) "clean screen" of BIONET Premium Fragment Library was run to identify and remove fragments that bind non-specifically to the Biacore CM7 Sensor Chip. After this initial clean-up step, "binding level screens" were carried out for the remaining fragments to identify binders against the target protein and exclude fragments with atypical binding behavior.

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Immunomodulation of Primary Human T Cell Activation Using High-throughput Screen of FDA-approved Small Molecule Drugs

The incorporation of immune function assays into structure-activity relationship (SAR) studies can provide valuable guidance for drug development. In this high-throughput screen (HTS), we focus on the health and function of primary human T cells through utilization of an AlphaLISA IL-2 assay for IL-2 secretion and CellTiter-Glo® assay for cell survival and proliferation.

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KMN-159 Induces Osteogenesis In Novel 3D Model Of Osseointegration

Prostaglandin E₂ (PGE₂) is known to cause bone formation with activation of the EP₄ receptor being primarily responsible for its anabolic actions. Current bone anabolic therapies are typically protein-based biological therapies that require special formulations, lack stability, and are costly.

Here, we show that KMN-159, a small molecule EP₄ receptor agonist, stimulates osteogenesis in a novel in vitro 3D model of osseointegration. We found that KMN-159 increased the expression of osteoblastic differentiation factors and promoted osteoblast function and bone remodeling in this model. KMN-159 elevated the Opg:Rankl ratio, indicating a shift towards bone formation.

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Biophysical Characterization 

Biochemical and Biophysical Characterization of Human Secretory Phospholipases

Secretory phospholipase A₂ (sPLA₂) catalyzes the hydrolysis of sn-2 acyl bond of membrane-bound phospholipids yielding a free fatty acid and a lysophospholipid. The sPLA₂ family contains 10 catalytically active (IB, IIA, IID, IIE, IIF, III, V, X, and XIIA) and one inactive isoform (XIIB) in mammals. This family of enzymes are disulfide-rich, low molecular weight, Ca²⁺-requiring lipolytic enzymes with a His-Asp catalytic dyad. Individual mammalian sPLA₂s have distinct enzymatic properties and display distinct tissue expression patterns, suggesting that each enzyme acts on a distinct phospholipid membrane.

Here we describe the comparative activity of sPLA₂-IIA, -IID, -IIE, -V, and -X in the presence and absence of inhibitors.

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Biophysical Characterization of MR1 (K43A)/β2M Complex

MR1 (MHC-related protein 1) is an antigen-presenting molecule that presents small molecule ligands to mucosal-associated invariant T cells (MAIT cells). MR1 associates with β2-microglobulin (β2M) and can bind with microbial and non-microbial metabolites, which causes the MR1 molecule to translocate from the endoplasmic reticulum to the cell surface.

Here, we describe the expression and two-step purification of the MR1 (K43A)/β2M stable complex in which size exclusion chromatography (SEC) was able to separate aggregated protein from the stable heterodimer complex.

Assay Development

LC-MS/MS Validation of a Novel 3'3'-cGAMP Monoclonal ELISA

3'3'-cGAMP is a bacterial second messenger produced from ATP and GTP by specific dinucleotide cyclases. It binds to riboswitches to regulate motility, biofilm formation, virulence, and colonization through gene transcription. Current detection methods utilize mass spectrometry, which can be both costly and timely. In addition to that, the isolation and purification of CDNs can be complicated and tedious. Rapid and accurate detection methods are critical to enable researchers to study and/or identify the relevant biological pathways.

This poster shows LC-MS/MS validation of a novel 3'3'-cGAMP monoclonal enzyme-linked immunosorbent assay (ELISA). The data provided will demonstrate the ELISA is sensitive, specific, and in good correlation with LC-MS/MS.

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The Roles of ENPP1 Activity and 2'3'-cGAMP Degradation in Ovarian Cancer Cell Lines

Recent studies have shown that ENPP1 may play a role in cancer cell proliferation, migration, and invasion in ovarian and other types of cancer.

In this study, we investigated whether ENPP1 activity and its hydrolysis of 2'3'-cAMP correlate with ENPP1 expression levels in ovarian cancer cell lines. We also tested whether published ENPP1 inhibitors can inhibit ENPP1 activity in cell-free assays and cell culture model, and if inhibition of ENPP1 affects the viabilities of ovarian cancer cell lines.

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Antibody Production 

Development and Testing of IHC-Specific Antibodies at Cayman Chemical

Immunohistochemistry (IHC) is a preferred application in clinical and academic research that allows for the specific detection of a target of interest using antibodies. Researchers can determine organ and cell type information in normal and disease state tissues, provided that antibodies are available. However, tools and reagents necessary for IHC testing can be costly and/or not accessible to some labs.

Cayman Chemical has extensive experience with antigen and antibody development and has now optimized tissue sectioning and IHC testing on formalin-fixed, paraffin-embedded (FFPE) tissue blocks.

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