Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden.
Cyclic peptoids are potential candidates for diverse biological activities. However, applications of cyclic peptoids are limited by the synthetic difficulties, conformational flexibility of large cyclic peptoids, and lack of secondary amide in the backbone. Herein, an elegant methodology for the synthesis of small and medium‐size cyclic hybrid peptoids is developed. αN‐Alkyl and αN‐acyl substituents in N‐(2‐aminoethyl)glycine monomers enforce intra‐ and intermolecular cyclization to form stable six‐ and 12‐membered cyclic products, respectively. NMR studies show inter‐ and intramolecular hydrogen bonding in six‐ and 12‐membered cyclic peptoids, respectively. Screening of a cyclic peptoid library resulted in the identification of a potential candidate that enhanced autophagic degradation of cargo in a live cell model. Such upregulation of autophagy using small molecules is a promising approach for elimination of intracellular pathogens and neurodegenerative protein aggregates.
Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.
BackgroundMacroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions.ObjectiveIn search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast S. cerevisiae that could measure both selective and general autophagy in real time.MethodsFirefly and Renilla luciferase reporter genes were cloned under POT-1 promoter. Using fatty acid medium the promoter was induced and the luciferase cargo was allowed to build up. The cells were then transferred to starvation conditions to stimulate autophagy and the degradation of luciferase markers was followed with time.Results and conclusionThe assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.
Growing amount of evidence in the last two decades highlight that macroautophagy (generally referred to as autophagy) is not only indispensable for survival in yeast but also equally important to maintain cellular quality control in higher eukaryotes as well. Importantly, dysfunctional autophagy has been explicitly shown to be involved in various physiological and pathological conditions such as cell death, cancer, neurodegenerative, and other diseases. Therefore, modulation and regulation of the autophagy pathway has emerged as an alternative strategy for the treatment of various disease conditions in the recent years. Several studies have shown genetic or pharmacological modulation of autophagy to be effective in treating cancer, clearing intracellular aggregates and pathogens. Understanding and controlling the autophagic flux, either through a genetic or pharmacological approach is therefore a highly promising approach and of great scientific interest as spatiotemporal and cell-tissue-organ level autophagy regulation is not clearly understood. Indeed, chemical biology approaches that identify small molecule effectors of autophagy have thus a dual benefit: the modulators act as tools to study and understand the process of autophagy, and may also have therapeutic potential. In this review, we discuss different strategies that have appeared to screen and identify potent small molecule modulators of autophagy.
Voltage dependent anion channels (VDACs) family of proteins are the most abundant proteins in the outer mitochondrial membrane (OMM). There are three isoforms of VDAC, that are structurally and functionally conserved as far as their ion transport function is concerned. However, there are isoform-specific functions that support distinct pathways. For VDAC2 isoform, an established isoform-specific function is the targeting of the pro-apoptotic Bak protein to the OMM, which stabilizes Bak, leading to its elevated cellular levels. On the surface of OMM, activation and oligomerization of Bak and Bax, another pro-apoptotic protein leads to OMM perforation and release of pro-apoptotic proteins from intermembrane space to the cytoplasm. This is an irreversible step that stimulates the downstream events of apoptotic pathway. Hepatocellular carcinoma (HCC) ranks third in cancer mortality and in the advanced stages is associated with poor prognosis. We observed that unexpectedly, HCC cells are vastly more sensitive to tBid induced apoptosis compared to normal hepatocytes. tBid is a BH3-only pro-apoptotic Bcl-2 family protein, the effect of which is primarily mediated by Bak. We have shown that, in the absence of VDAC2, the Bid-Bak apoptosis pathway is suppressed. VDAC2 protein expression is the lowest in liver and lung among different tissues. We also determined the protein abundances in the HCC lines and normal hepatocytes and found elevated VDAC2 and Bak levels in HCC. Our findings are in line with multiple datasets that show an elevation of VDAC2/Bak expression in human HCC suggesting a potential clinical relevance. We found that combination of an inhibitor of the anti-apoptotic proteins (S63845, a selective Mcl-1 inhibitor), which normally suppresses Bak activation, with an activator of the tBid pathway (TRAIL) enhanced the death of hepatocarcinoma cells with little effect on normal hepatocytes. This treatment strategy was also effective at reducing tumor growth in vivo, but only in tumors expressing VDAC2. Thus, we postulate that increased VDAC2 expression in HCC leads to elevated Bak recruitment to the OMM, which in turn sensitizes HCC to tBid. Therefore, this differential expression provides an excellent opportunity for selective elimination of HCC cells. Citation Format: Piyush Mishra. Moonlighting function of mitochondrial protein VDAC2 provides a potent pharmacological strategy against selective killing of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3707.
Apoptosis, a form of programmed cell death, is an evolutionarily conserved and genetically regulated phenomenon that is pivotal in maintaining tissue homeostasis and is an important player in regulating carcinogenesis. Mitochondria along with its resident proteins are the central player in regulating apoptosis. In recent years, VDAC2, an Outer Mitochondrial Membrane (OMM) protein has been shown to be the key recruitment factor for Bak, a pro-apoptotic protein to the OMM and, therefore is crucial for tBid-induced OMM permeabilization. We have previously found that unexpectedly, hepatocellular carcinoma (HCC) cells are vastly more sensitive to tBid induced apoptosis compared to normal hepatocytes and this difference is owed to high abundance of VDAC2-Bak in HCC. These findings are in line with multiple datasets posted in Oncomine, and protein atlas websites and may have clinical relevance. Combination of S63845, a selective Mcl-1 inhibitor, with TRAIL was effective at reducing tumor growth in vivo, but only in tumors expressing VDAC2. We hypothesized that the liver metastases of some extrahepatic primary tumors might also show enhanced sensitivity to VDAC2-Bak mediated apoptosis as compared to the normal liver tissue. Testing this hypothesis is important for uveal melanoma (UM) that primarily metastasizes in the liver, and these metastases lack an effective treatment. Interestingly, on testing several UM cell lines and patient samples we observed that VDAC2-Bak are upregulated in UM liver metastasis as well as compared to normal liver tissue. As expected, high VDAC2 expressing cell lines were also more sensitive to tBid induced OMM permeabilization. We propose that these differences can be exploited to design an effective therapy for metastasized UM. Citation Format: Piyush Mishra. Heterogeneity of VDAC2-Bak/Bax mediated mitochondrial apoptosis can be exploited to develop effective and selective treatment against metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2541.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.