In a study published in the journal Nature communications, researchers looked at ReFRAME (short for Reuses, Focused Rescue and Accelerated Medchem), a drug reuse library, for respiratory syncytial virus (RSV) drugs. They identified lonafarnib as a potent inhibitor of the RSV fusion protein and investigated its therapeutic potential against an RSV infection.
Study: Drug repurposing screen identifies lonafarnib as a respiratory syncytial virus fusion protein inhibitor. Image credit: joshimerbin / Shutterstock
Record
RSV causes severe lower respiratory tract infections in young children, immunocompromised individuals, and older adults, with millions of annual hospital admissions and deaths. The recent coronavirus disease 2019 (COVID-19) pandemic and related interventions have resulted in a shift in RSV epidemiology, with transient suppression and resurgence of RSV circulation, raising concerns about increased infections.
Treatment of RSV infection is currently symptomatic. While ribavirin shows in vitro effectiveness, it is not very effective in patients. Palivizumab provides prophylaxis but is expensive, offers only a partial reduction in hospitalization rates, and faces challenges such as the rapid development of resistance. Although nirsevimab was recently approved for the prevention of RSV in neonates, there is still a lack of treatment options.
Various antiviral strategies are being developed against RSV, including immunoglobulins. Reuse libraries containing licensed drugs or compounds in clinical development serve as repositories with the potential for accelerated therapeutic applications. The researchers in the present study screened the ReFRAME library and identified lonafarnib as an RSV fusion protein inhibitor, while demonstrating its therapeutic potential.
About the study
The library (of 12,000 molecules) was screened using a recombinant RSV subtype A strain GFP (short for green fluorescent protein) reporter virus. Cell viability was determined using an MTT (abbreviation for 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide). The primary success criteria were RSV infection ≤ 16% and cell viability ≥ 80%. Fourteen molecules met the primary criteria and an additional 16 molecules were selected. Two farnesyl-S-transferase inhibitors, lonafarnib and tipifarnib, were evaluated and compared for their inhibitory effects on RSV infection. To identify the potential viral target of lonafarnib, passages of RSV reporter virus with increasing doses of lonafarnib were performed. The resulting virus populations were sequenced and analyzed for mutations. The study additionally included orthogonal infection assays, plaque reduction assays, RSV lentovirus pseudotype assays, and RSV F protein-membrane cell fusion assays. Surface plasmon resonance and crystallization experiments were performed to investigate the interaction of lonafarnib with a recombinant subtype A pre-fusion F protein of the RSV.
The therapeutic effects of lonafarnib were evaluated by inoculating A549 cells with HRSV-A-GFP, treating with lonafarnib or ribavirin 24 hours after inoculation, and monitoring viral shedding over time. The effect of the drug in a more natural model of RSV infection and cell entry was investigated using the immortalized human basal cell line BCi-NS1.1, which was further differentiated into pseudostratified ciliated epithelium.
Six mice were treated with oral lonafarnib or vehicle control and infected with an RSV reporter virus. Animal weight was monitored and on day 4, tissues were removed and lung RSV copy number was measured.
![A review and validation process. Hep-2 B cells were infected with rHRSV-A-GFP in the presence of 5 µM compound. 48 h later, infection and cell viability were quantified via GFP and MTT readings. Dashed lines indicate primary success criteria and dots represent means of two technical replicates. C HEp-2 cells were infected with HRSV-A-Luc at an MOI of 0.01 and treated with the indicated concentrations of compound. 24 h later, the supernatant was transferred to new cells for a second round of infection. Luminescence was quantified 24 h after inoculation of both rounds of infection. Cell viability was measured by MTT reading in treated but uninfected cells. Mean ± SD of three independent experiments. Known RSV inhibitors (F protein: presatovir, N protein: RSV604, IMPDH inhibitors (AVN944, mycophenolic acid), HSP90 inhibitors (radiciol, HSP990). 4-Sulfocalix[6]arene hydrate (4SC6AH, unknown target); The source data is provided as a source data file.](https://d2jx2rerrg6sh3.cloudfront.net/images/news/ImageForNews_771592_17076972756075995.jpg)
ONE Verification and validation process. si HEp-2 cells were infected with rHRSV-A-GFP in the presence of 5 μM compound. 48 h later, infection and cell viability were quantified via GFP and MTT readings. Dashed lines indicate primary success criteria and dots represent means of two technical replicates. do HEp-2 cells were infected with HRSV-A-Luc at an MOI of 0.01 and treated with the indicated concentrations of compound. 24 h later, the supernatant was transferred to new cells for a second round of infection. Luminescence was quantified 24 h after inoculation of both rounds of infection. Cell viability was measured by MTT reading in treated but uninfected cells. Mean ± SD of three independent experiments. Known RSV inhibitors (F protein: presatovir, N protein: RSV604, IMPDH inhibitors (AVN944, mycophenolic acid), HSP90 inhibitors (radiciol, HSP990). 4-Sulfocalix[6]arene hydrate (4SC6AH, unknown target); The source data is provided as a source data file.
Results and discussion
Twenty-one molecules, including lonafarnib, demonstrated antiviral activity against RSV. Lonafarnib is approved for Hutchinson-Gilford progeria syndrome and is in phase III clinical trials for hepatitis delta virus infections. Lonafarnib, but not tipifarnib, demonstrated inhibition of RSV infection as evidenced by reduced reporter virus activity, plaque reduction, and suppressed syncytia formation in infected cells. In addition, lonafarnib, not tipifarnib, was found to interact with pre-fusion protein F at a binding site previously observed for other fusion inhibitors.
Lonafarnib-exposed viral populations accumulated two coding mutations (T335I and T400A) within the RSV fusion protein, leading to phenotypic resistance to lonafarnib. Further, lonafarnib was found to inhibit RSV entry into cells by binding the fusion protein and inhibiting membrane fusion. This inhibition was found to be overcome by resistance mutations in the fusion protein.
In vitro, combinations of lonafarnib and ribavirin showed little inhibitory or slight synergistic activity at selected doses. Lonafarnib treatment after inoculation in A549 cells reduced the spread of HRSV GFP by 30% compared to controls. In the BCi-NS1.1 cell culture model, both apical and basolateral lonafarnib prophylactic treatment dose-dependently inhibited RSV infection, resulting in a 10- to 15-fold reduction in viral load. Therapeutic application of basolateral lonafarnib alone also reduced viral load by approximately 50% in a clinical isolate RSV infection.
In vivo, animals treated with lonafarnib showed significantly reduced reporter virus signal in the lung and nose compared to controls. On day 4, a dose-dependent decrease in viral ribonucleic acid was observed in the lungs of treated mice and there was less weight loss compared to controls. However, cellular infiltrates were observed in the lungs of lonafarnib-treated mice.
conclusion
In conclusion, the study identified lonafarnib as a potential therapeutic candidate for RSV treatment, highlighting the utility of drug repurposing studies. The findings demonstrate the promising antiviral activity of lonafarnib in cell culture as well as mouse models of RSV infection. Further research is needed to confirm the findings.
