Novel Endocytosis Inhibitors Block HIV-1 Tat Protein Entry into Neural Cells

Academic Background

HIV-1 (human immunodeficiency virus type 1) infection not only leads to immune system exhaustion but is also closely associated with HIV-associated neurocognitive disorders (HAND). Although combined antiretroviral therapy (cART) has significantly improved the survival rate of people living with HIV, the virus continues to persist in infected individuals and causes neurocognitive impairment in approximately one-third of patients. The neurotoxicity of HIV-1 is primarily related to its viral protein Tat (transactivation of transcription protein). The Tat protein can cross the blood-brain barrier, enter neural cells, induce oxidative stress, DNA damage, and mitochondrial dysfunction, ultimately leading to neuronal death.

Endocytosis is one of the main pathways for viruses to enter cells, and the HIV-1 Tat protein also enters cells via endocytosis. Therefore, blocking the endocytic process of the Tat protein may be an effective strategy to prevent its neurotoxicity. However, existing endocytosis inhibitors have non-specific effects and side effects, which limit their clinical application. To address this issue, this study aims to develop novel endocytosis inhibitors, evaluate their ability to block Tat protein endocytosis, and explore their potential applications in neuroprotection.

Source of the Paper

This paper was collaboratively completed by research teams from multiple institutions in Poland, France, and Ukraine. The main authors include Olga Klaudia Szewczyk-Roszczenko, Piotr Roszczenko, Anna Shmakova, and others, who are respectively from the Medical University of Białystok, Institut Gustave Roussy, Danylo Halytsky Lviv National Medical University, and other institutions. The paper was first published on December 24, 2024, in the journal American Journal of Physiology-Cell Physiology, with the DOI: 10.1152/ajpcell.00723.2024.

Research Process

1. Design and Synthesis of Endocytosis Inhibitors

The researchers designed and synthesized two novel endocytosis inhibitors, LES-6631 and LES-6633. These two compounds are based on a rhodanine scaffold and were synthesized through a Knoevenagel condensation reaction. To improve their water solubility, the researchers further converted them into methanesulfonate forms. During the synthesis process, nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) techniques were used to characterize the compounds.

2. Cell Culture and Differentiation

The study used the human neuroblastoma cell line SH-SY5Y as a model cell. To simulate the differentiated state of neurons, the researchers cultured SH-SY5Y cells in a medium containing retinoic acid and B-27 supplements for 10 days. The differentiation status of the cells was assessed using Ki-67 antibody and phalloidin staining.

3. Cytotoxicity Assessment

To evaluate the cytotoxicity of LES-6631 and LES-6633, the researchers conducted MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays in various cell lines, including breast cancer cells MDA-MB-231, MCF-7, and neuroblastoma SH-SY5Y. The results showed that these two compounds exhibited low cytotoxicity at tested concentrations, with IC50 values higher than 30 μM.

4. Endocytosis Inhibition Experiment

The researchers evaluated the inhibitory effects of LES-6631 and LES-6633 on endocytosis using fluorescently labeled dextran and Tat-Cy5 complexes. After pretreatment for 1 hour, fluorescently labeled Tat-Cy5 or dextran was added to the cells, followed by fixation and microscopic observation. The results showed that LES-6631 and LES-6633 could significantly inhibit endocytosis at low concentrations, outperforming the classical endocytosis inhibitor chlorpromazine.

5. Oxidative Stress and DNA Damage Assessment

To investigate the protective effects of LES-6631 and LES-6633 against oxidative stress and DNA damage induced by the Tat protein, the researchers conducted reactive oxygen species (ROS) detection and alkaline comet assays. The results showed that pretreatment with these two compounds significantly reduced oxidative stress and DNA damage induced by the Tat protein.

6. Glutathione (GSH) and Nitric Oxide (NO) Level Detection

The researchers also evaluated the effects of LES-6631 and LES-6633 on intracellular glutathione (GSH) and nitric oxide (NO) levels. The results showed that these two compounds significantly increased intracellular GSH levels and reduced NO production, indicating their significant effects in alleviating oxidative stress and nitrosative stress induced by the Tat protein.

Main Results

1. Endocytosis Inhibition Effect: LES-6631 and LES-6633 can significantly inhibit the endocytosis of Tat-Cy5 and dextran at low concentrations, outperforming chlorpromazine.
2. Oxidative Stress Protection: These two compounds can significantly reduce oxidative stress induced by the Tat protein, restoring intracellular ROS levels to normal.
3. DNA Damage Protection: Through comet assays, the researchers found that LES-6631 and LES-6633 significantly reduced DNA breaks induced by the Tat protein.
4. GSH and NO Level Regulation: These two compounds significantly increase intracellular GSH levels and reduce NO production, indicating their significant effects in alleviating oxidative stress and nitrosative stress.

Conclusion and Significance

This study successfully developed two novel endocytosis inhibitors, LES-6631 and LES-6633, which effectively block the endocytosis of the HIV-1 Tat protein at low concentrations and significantly alleviate oxidative stress, DNA damage, and nitrosative stress induced by the Tat protein. These compounds exhibit low cytotoxicity and show significant effects in neuroprotection, indicating their potential application value in treating HIV-related neurodegenerative diseases.

Research Highlights

1. Novel Endocytosis Inhibitors: LES-6631 and LES-6633 are novel compounds based on a rhodanine scaffold with highly efficient endocytosis inhibition effects.
2. Multifaceted Protective Effects: These two compounds not only block the endocytosis of the Tat protein but also alleviate oxidative stress, DNA damage, and nitrosative stress, exhibiting multiple protective effects.
3. Low Cytotoxicity: LES-6631 and LES-6633 exhibit low cytotoxicity in various cell lines, indicating good safety.
4. Potential Clinical Applications: The significant effects of these compounds in neuroprotection provide a theoretical basis for their application in the treatment of HIV-related neurodegenerative diseases.

Other Valuable Information

The experimental data of this study are available upon reasonable request, and relevant supplementary materials can be accessed via DOI 10.5281/zenodo.14287922. The research was funded by institutions such as the Medical University of Białystok and the National Research Foundation of Ukraine.