Evaluating the efficacy of harmol in treating herpes simplex virus-induced keratitis

By Vijay Kumar MalesuReviewed by Lily Ramsey, LLMMay 29 2024

In a recent study published in the Virology Journal, a group of researchers evaluated the antiviral efficacy and therapeutic potential of harmol in treating herpes simplex virus type 1 (HSV-1) induced keratitis, including drug-resistant strains, and its ability to enhance the effects of acyclovir (ACV).

Study: Harmol used for the treatment of herpes simplex virus induced keratitis. Image Credit: STEKLO/Shutterstock.com

Background 

Herpes simplex keratitis (HSK) is a common eye infection characterized by corneal damage, leading to opacity and visual impairment. The incidence of HSK is 10-30 per 100,000 in developed countries and higher in developing regions.

Untreated HSK can cause blindness and potentially fatal encephalitis. It is primarily associated with HSV-1 infection. ACV is the standard treatment, but rising drug resistance, especially in immunocompromised patients, necessitates new therapies.

Harmol, a β-carboline alkaloid found in several medicinal plants, has shown antiviral properties. Further research is needed to fully understand harmol's mechanisms of action, optimize its therapeutic potential, and evaluate its long-term safety and efficacy in diverse patient populations.

About the study 

Vero cells from the American Type Culture Collection (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). HSV-1 F strain and ACV-resistant HSV-1/153 were provided at Jingling Hospital, China, and initially isolated at the Guangzhou Institutes of Biomedicine and Health.

These strains were propagated in Vero cells, with supernatants collected, centrifuged to remove debris, and stored at -80°C. Viral titers were determined using the tissue culture infective dose (TCID₅₀) assay.

Harmol, isolated from Peganum harmala Linn and sourced from MedChemExpress, was dissolved in Dimethyl Sulphoxide (DMSO). ACV was obtained from the National Institutes for Food and Drug Control, China.

A natural product library with 502 compounds was screened for anti-HSV-1 activity at ten µM. Vero cells were treated with harmol or ACV, and cell viability was assessed using the Cell Counting Kit 8 (CCK-8) assay.

The 50% cytotoxic concentration (CC₅₀) and effective concentration (EC₅₀ ) were calculated, showing harmol significantly inhibited HSV-1 and HSV-1/153 infections.

In a mouse HSK model, Bagg Albino Laboratory-bred/c (BALB/c) mice were infected with HSV-1 or HSV-1/153 and treated with harmol or ACV.

The antiviral efficacy was evaluated through corneal staining, blepharitis scoring, Real-time optical coherence tomography (RTvue OCT) examination, and in vivo confocal microscopy. Statistical analysis confirmed the significant antiviral activity of harmol, with P < 0.05.

Study results 

Harmol significantly inhibits HSV-1 replication. Screening natural compounds using the Cytopathic Effect (CPE) inhibition assay in Vero cells identified 22 compounds with over 75% viral inhibition. Harmol, exhibiting over 90% inhibition, was selected for further study. Its CC₅₀ was 242.54 µM, with a non-CC₅₀ of 12.5 µM used for in vitro studies. Harmol and ACV (1 µM) both reduced HSV-1-related CPEs.

Harmol's anti-HSV-1 effect was confirmed by measuring Glycoprotein D of Herpes Simplex Virus Type 1 (gD-1) protein expression. Both ACV (1 µM) and harmol (0-50 µM) reduced HSV-1 gD-1 protein levels.

Harmol’s EC₅₀ was 9.34 µM for HSV-1 F and 5.84 µM for HSV-1/153, with Selectivity Index (SI) values of 26.0 and 44.5, respectively. Harmol effectively reduced the replication and progeny production of both HSV-1 strains.

Combining harmol with ACV showed enhanced anti-HSV-1 effects. Harmol and ACV inhibited HSV-1 F gD-1 messenger Ribonucleic Acid (mRNA) expression and progeny virus production more effectively than either treatment alone.

Increasing doses of harmol (0, 3.12, 6.25, 12.5 µM) further reduced gD protein expression, with combination treatment showing superior anti-HSV-1 F effects. Similar outcomes were observed in HSV-1/153-infected cells, indicating harmol enhances ACV’s anti-HSV-1 effect, including on ACV-resistant strains.

A time of addition experiment was conducted to determine the stage at which harmol exerts its antiviral effects during HSV-1 infection. Vero cells infected with HSV-1 were treated with harmol (1 µM) at various times post-infection.

Quantitative polymerase chain reaction (qPCR) assays showed that pretreatment with harmol significantly inhibited HSV-1 replication, with diminished efficacy observed with prolonged addition times. 

Topical application of harmol was tested for safety on mouse corneas. Mice treated with 5 µL harmol (0.01 mg/kg, approximately 100 µM) on days 1, 3, and 5 showed no adverse effects on body weight or corneal transparency compared to Phosphate Buffered Saline (PBS) treatment.

Corneal fluorescein staining confirmed no epithelial damage at five days. Thus, harmol at 0.01 mg/kg showed no toxicity to mouse corneas.

In a mouse HSK model, harmol eyedrop treatment alleviated HSK severity. Compared to untreated HSK mice, harmol-treated mice exhibited reduced corneal opacity, blepharitis scores, and early corneal injury.

Harmol also mitigated HSV-1 F-induced body weight loss. In vivo, confocal microscopy (IVCM) and RTvue OCT confirmed that harmol-treated mice retained ocular structural integrity. 

Harmol’s effectiveness against ACV-resistant HSV-1/153 was also demonstrated. Harmol, but not ACV, significantly reduced blepharitis scores and early corneal lesions and prevented weight loss in HSV-1/153-infected mice. Collectively, harmol effectively combats ACV-resistant HSV infections in vivo.

Conclusions 

To summarize, HSK development is linked to HSV-1 infection, causing vision loss globally. Drug resistance due to overuse presents a major public health challenge, necessitating new treatments.

Harmol inhibits HSV-1 F and HSV-1/153 replication in corneal tissues and alleviates early viral-induced lesions. It shows antiviral activity against RNA viruses and enhances ACV's effects on HSV-1, indicating a different mechanism of action.

Harmol may activate autophagy and affect Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) and AMP- Activated Protein Kinasepathways (AMPK), which are crucial in combating HSV.