| 1. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
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In: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
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Research review (peer-reviewed)abstract
- Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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| 3. |
- Prieto-Díaz, Rubén, et al.
(author)
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Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists
- 2023
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 66:1, s. 890-912
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Journal article (peer-reviewed)abstract
- The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.
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| 4. |
- Val, Cristina, et al.
(author)
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Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A(1) Antagonists
- 2022
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 65:3, s. 2091-2106
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Journal article (peer-reviewed)abstract
- We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A(1)AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarily evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R-4 and R-6 of the pyrimidine core but most importantly the prominent role to the unprecedented A(1)AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A(1) and A(2A)ARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.
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