Discovery of pH-Selective Marine and Plant Natural Product Inhibitors of Cathepsin B Revealed by Screening at Acidic and Neutral pH Conditions
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ABSTRACT:
SUBMITTER: Phan V
PROVIDER: S-EPMC9330179 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.
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Project description:Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors.
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Project description:PurposeTo test the effects of acidic vs. neutral pH glycyrrhizin (GLY) on the unwounded and wounded normal mouse cornea and after infection with Pseudomonas aeruginosa isolates KEI 1025 and multidrug-resistant MDR9.MethodsAcidic or neutral GLY vs. phosphate-buffered saline (PBS) was topically applied to normal or wounded corneas of C57BL/6 mice. In unwounded corneas, goblet cells and corneal nerves were stained and quantitated. After wounding, corneas were fluorescein stained and photographed using a slit lamp. Mice also were infected with KEI 1025 or MDR9 and the protective effects of GLY pH evaluated comparatively.ResultsIn the unwounded cornea, application of acidic or neutral GLY vs. PBS reduced the number of bulbar conjunctival goblet cells but did not alter corneal nerve density. Similar application of GLY to scarified corneas delayed wound closure. After KEI 1025 infection, none of the GLY vs. PBS-treated corneas perforated; GLY treatment also decreased plate count (neutral pH more effective) and reduced MPO and several cytokines. Similarly, for MDR9, GLY at either pH was protective and also enhanced the effects of moxifloxacin to which MDR9 is resistant.ConclusionAcidic or neutral pH GLY decreased goblet cell number but had no effect on nerve density. After corneal wounding, GLY at either pH (1) delayed wound closure and, (2) after infection, decreased keratitis when used alone or in combination with moxifloxacin. Neutral pH did not alter the therapeutic effect of GLY and would be preferred if used clinically.
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Project description:Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆Tm = -1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.
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Project description:Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.
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Project description:Marine natural products (MNPs) are recognized for their structural complexity, diversity, and novelty. The vast majority of MNPs are pharmacologically relevant through their ability to modulate macromolecular targets underlying human diseases. Angiogenesis is a fundamental process in cancer progression and metastasis. Targeting angiogenesis through selective modulation of linked protein kinases is a valid strategy to discover novel effective tumor growth and metastasis inhibitors. An in-house marine natural products mini-library, which comprises diverse MNP entities, was submitted to the Lilly's Open Innovation Drug Discovery platform. Accepted structures were subjected to in vitro screening to discover mechanistically novel angiogenesis inhibitors. Active hits were subjected to additional angiogenesis-targeted kinase profiling. Some natural and semisynthetic MNPs, including multiple members of the macrolide latrunculins, the macrocyclic oxaquinolizidine alkaloid araguspongine C, and the sesquiterpene quinone puupehenone, showed promising results in primary and secondary angiogenesis screening modules. These hits inhibited vascular endothelial growth factor (VEGF)-mediated endothelial tube-like formation, with minimal cytotoxicity at relevant doses. Secondary kinase profiling identified six target protein kinases, all involved in angiogenesis signaling pathways. Molecular modeling and docking experiments aided the understanding of molecular binding interactions, identification of pharmacophoric epitopes, and deriving structure-activity relationships of active hits. Marine natural products are prolific resources for the discovery of chemically and mechanistically unique selective antiangiogenic scaffolds.
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Project description:The Ca2+ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca2+ transporters. The only published NMR chemical shift assignments for Ca2+-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca2+-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).
| S-EPMC9510097 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2022-12-02 | GSE213441 | GEO
Project description:Synthetic melanin nanoparticles have value in metal chelation, photoprotection, and biocompatibility. Applications of these materials have been reported in optics, biomedicine, and electronics. However, precise size control has remained relatively difficult-especially for materials below 1000 nm. In this paper we describe the synthesis of ultrasmall synthetic nanoparticles with size of 9.4-31.4 nm in weakly acidic and neutral conditions via UV-irradiation. Size control of these particles was possible by varying the pH from 6.4-10.0. We then used UV-vis, FTIR, and nuclear magnetic resonance to investigate the mechanism of UV-induced polymerization. The data show that reactive oxygen species from UV irradiation oxidizes intermediates of the reaction and accelerates the formation of these synthetic melanin structures.
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