A Novel 5-Chloro-N-phenyl-1H-indole-2-carboxamide Derivative as Brain-Type Glycogen Phosphorylase Inhibitor: Potential Therapeutic Effect on Cerebral Ischemia

Abstract

Brain-type glycogen phosphorylase inhibitors are potential new drugs for treating ischemic brain injury. In our previous study, we reported compound 1 as a novel brain-type glycogen phosphorylase inhibitor with cardioprotective properties. We also found that compound 1 has high blood–brain barrier permeability through the ADMET prediction website. In this study, we deeply analyzed the protective effect of compound 1 on hypoxic-ischemic brain injury, finding that compound 1 could alleviate the hypoxia/reoxygenation (H/R) injury of astrocytes by improving cell viability and reducing LDH leakage rate, intracellular glucose content, and post-ischemic ROS level. At the same time, compound 1 could reduce the level of ATP in brain cells after ischemia, improve cellular energy metabolism, downregulate the degree of extracellular acidification, and improve metabolic acidosis. It could also increase the level of mitochondrial aerobic energy metabolism during brain cell reperfusion, reduce anaerobic glycolysis, and inhibit apoptosis and the expression of apoptosis-related proteins. The above results indicated that compound 1 is involved in the regulation of glucose metabolism, can control cell apoptosis, and has protective and potential therapeutic effects on cerebral ischemia-reperfusion injury, which provides a new reference and possibility for the development of novel drugs for the treatment of ischemic brain injury.

A Novel 5-Chloro-N-phenyl-1H-indole-2-carboxamide Derivative as Brain-Type Glycogen Phosphorylase Inhibitor: Validation of Target PYGB

“…After the successful isolation of mouse astrocytes [ 12 ], we established a H/R injury model and implemented it using an adeno-associated virus (AAV2) to further explore whether compound 1 could act against H/R injury in mouse astrocytes by targeting PYGB. Relevant studies have shown that the degree of H/R injury in mouse astrocytes could be reflected by cell viability, LDH leakage rate, intracellular glucose, and ROS level [ 14 , 15 , 16 , 17 ].…”

“…In addition, ATP is an important energy molecule in cells, and the intracellular ATP content is used to evaluate the level of cellular energy metabolism [ 21 , 22 , 23 , 24 ]. Our previous report showed that compound 1 could dramatically reduce cellular ATP and remarkably improve energy metabolism in mouse astrocytes [ 12 ]. In this study, we wanted to determine ATP content further after PYGB knockdown to validate whether compound 1 worked by targeting PYGB.…”

“…Thus, the cellular oxygen consumption rate (OCR) is commonly used to estimate the function of mitochondria [ 27 , 28 , 29 ]. Our previous report indicated that compound 1 could significantly improve the mitochondrial aerobic energy metabolism levels in astrocytes [ 12 ]. Therefore, in order to investigate the effect of compound 1 on the level of cellular mitochondrial aerobic energy metabolism after PYGB knockdown, we examined the cellular oxygen consumption rate with Seahorse XF.…”

“…Therefore, inhibiting their expression could alleviate apoptosis. Moreover, our previous study indicated that compound 1 can inhibit the expression of apoptosis-related proteins Bax and caspase-3, thus alleviating apoptosis [ 12 , 13 ]. In this study, we further applied Western Blot to determine the expression of apoptosis-related proteins caspase-3 and Bax to verify whether the expression of apoptosis-related proteins was inhibited by compound 1 after PYGB knockdown.…”

“…We previously revealed that compound 1 ( Figure 1 ) is a novel glycogen phosphorylase inhibitor involved in glucose metabolism by inhibiting PYGB. Meanwhile, we also found that compound 1 has better inhibitory activity against PYGB (IC 50 of 90.27 nM) and a potential therapeutic effect on brain ischemia [ 12 , 13 ]. In the present study, we initially applied a gene-silencing strategy to downregulate PYGB proteins and investigate the potential protective effect against astrocyte H/R injury with compound 1 by targeting PYGB.…”