Biology subjects

Sacha, P.

Publications and source records attributed to Sacha, P..

5 recordsLinked to original sources

bioRxiv2025

A novel screening assay for insulin receptor

Insulin is a key hormone in glucose homeostasis. Its lack causes severe health complications and has to be compensated by regular administration of insulin. Despite intense long-lasting research, a more stable and efficient substitute has yet to be discovered to alleviate patients issues. Here we report the development of a new assay for screening potential insulin receptor ligands based on the DNA-linked inhibitor antibody assay (DIANA). Our assay meets the need for a fast, sensitive, non-radioactive method as an alternative to the commonly used radioligand receptor binding assay.

biochemistry↗

bioRxiv2024

Discovery and isolation of novel capsaicinoids and their TRPV1-related activity

Chilis contain capsaicin and other structurally related molecules known as capsaicinoids. Capsaicins target protein, the transient receptor potential cation channel subfamily V member 1 (TRPV1), has been linked to many post-activation effects, including changes in metabolism and pain sensation. Capsaicinoids also bind to TRPV1, but current studies often disregard non-capsaicin interactions. To fill in these gaps, we screened 40 different chili varieties derived from four Capsicum species by means of untargeted metabolomics and a rat TRPV1 (rTRPV1) calcium influx activation assay. The resulting capsaicinoid profiles were specific to each variety but only partially corresponded with species delimitations. Based on rTRPV1 activation elicited by crude chili extracts, capsaicinoids act in an additive manner and a capsaicinoid profile can serve as a gauge of this activation. In addition, we isolated eighteen capsaicinoids, including five previously unreported ones, and confirmed their structure by NMR and MS/MS. We then tested rTRPV1 activation by 23 capsaicinoids and three related compounds. This testing revealed that even slight deviations from the structure of capsaicin reduce the ability to activate the target, with a mere single hydroxylation on the acyl tail reducing potency towards rTRPV1 by more than 100-fold. In addition, we tested how rTRPV1 activity changes in the presence of capsaicin together with non-activating capsaicin analogs and weakly activating capsaicinoids and found both classes of molecules to positively modulate the effects of capsaicin. This demonstrates that even such compounds have measurable pharmacological effects, making a case for the use and study of natural chili extracts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/620944v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d56d02org.highwire.dtl.DTLVardef@e7a66dorg.highwire.dtl.DTLVardef@5f409forg.highwire.dtl.DTLVardef@18b88f7_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Smith, J. (2025) https://BioRender.com/a61h668 HighlightsO_LI5 novel capsaicinoids (vanilloids) and 13 other capsaicinoids were isolated from chilis. C_LIO_LIThe slightest deviation in the capsaicin structure results in decreased TRPV1 activity. C_LIO_LIA single hydroxylation can reduce potency to TRPV1 100-fold. C_LIO_LINon-pungent vanilloids modulate capsaicin-TRPV1 activity. C_LI

pharmacology and toxicology↗

bioRxiv2024

Sensitive quantification of fibroblast activation protein and high-throughput screening for inhibition by FDA-approved compounds

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. In order to identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on the library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2,667 compounds within just few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Notably, prodrugs of cephalosporin antibiotics, reverse-transcriptase inhibitors, and one elastase inhibitor were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, underscores the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/600598v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1c19d55org.highwire.dtl.DTLVardef@405b3aorg.highwire.dtl.DTLVardef@1255281org.highwire.dtl.DTLVardef@14bc80e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

bioengineering↗

bioRxiv2019

MCC950/CRID3 potently targets the NACHT domain of wildtype NLRP3 but not disease-associated mutants for inflammasome inhibition

The NLRP3 inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndromes (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight in the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome. Here, we show that the NACHT domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labelling of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance, MCC950/CRID3 failed to inhibit NLRP3- driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1{beta} and IL-18 in LPS-challenged wildtype mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wildtype NLRP3 as the molecular target of MCC950/CRID3, and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wildtype NLRP3, but not CAPS-associated mutants.

immunology↗