Influenza A virus infection is one of the most common respiratory tract diseases in humans. It is the cause of typical seasonal flu as well as major human pandemics worldwide. According to the World Health Organization (WHO) in October 2023, around 3 to 5 million severe influenza cases were recorded, leading to 290,000 to 650,000 deaths across the globe. Influenza Medications Market size was valued at USD 942.6 million in 2023 and is anticipated to witness 2.9% CAGR from 2024-2032, driven by the rising prevalence of influenza.
Currently, the primary drug used clinically for influenza virus infection is Tamiflu (oseltamivir), which targets the viral neuraminidase (NA). Monotherapy against RNA viruses is usually inefficient and thus, combination therapy against the influenza virus is an urgent, unmet need.

TECHNOLOGY
We discovered several highly potent small-molecule inhibitors of the virus. The best of these inhibitors are new chemical entities that manifest ED50 values between ~10 ɳM to ~1 μM in cell-based assays, depending on the viral strain. Using escape mutants strategy, we found that our molecules target known binding pockets in one of the two major surface proteins of the virus- the hemagglutinin (HA) protein. HA facilitates endosome-mediated virus entry into the host cell. Being crucial to viral infection, HA is a well-established drug target, e.g., it is the target of the broad-spectrum antiviral drug arbidol (umifenovir), used clinically in Russia and China. Our measurements, employing an HA-mediated red blood cell hemolysis assay and the biophysical method differential scanning fluorimetry (DSF), further provided evidence that HA is the viral target of our novel inhibitors, suggesting that the inhibitors interfere with HA’s conformational change to its fusogenic form.
Using our novel chemiluminescence-based neuraminidase probe (CLNA) that targets viral neuraminidase activity, we demonstrated that our inhibitors are ~1,000-fold more potent at inhibiting viral replication than amantadine (a previously used anti-flu drug, phased out due to the emergence of escape mutants) against Influenza A Puerto Rico 08/34 (H1N1) in virus-infected cells. Following further optimization, we expect our next generation inhibitors to be improved in potency up to ~10,000-fold relative to amantadine, and around the same order of magnitude as Tamiflu (Oseltamivir).
A preliminary in vivo drug trial with mice was conducted with two of our inhibitors and showed significantly attenuated weight loss and clinical symptoms of lethal influenza disease relative to untreated mice, superior to treatment with the previously used drug amantadine. Using the CLNA probe in infected mice for the first time, we showed that this viral sensor is sensitive and robust, enabling the visualization of viral infection, quantification of virus and progression of the disease within the mice’s bodies through a broad dynamic range, potentially adding important yet missing capabilities to the physician’s toolbox.

Figure 1. (Left) Hemagglutinin trimer in its fusion-inactive form, showing subunits HA1 in light teal, HA2 in grey and the fusion peptides in green, as well as the predicted binding site of our novel inhibitor/s. We propose that the inhibitor depicted in the box in magenta locks the protein in a pre-fusion conformation, preventing the “jack-knife” motion required for exposing of the fusion peptide and subsequent viral fusion with the host cell (Right).

POTENTIAL APPLICATION
A new influenza treatment that inhibits viral replication by targeting the viral HA protein. Applicable both as a monotherapy and in a cocktail with inhibitors of other viral targets (combination therapy).

STAGE OF DEVELOPMENT
Using both ligand-based and structure-based quantitative structure-activity relationship (SAR) we have discovered >20 hits so far, including 18 novel chemical entities. Further drug efficacy experiments in mice and pharmacokinetic studies are underway.

NEXT STEPS
• Thorough mice experiments: optimization of the protocol and the drug application regimen and testing more drug leads in influenza-infected mice.
• Thorough pharmacokinetic studies. Both in vitro and in vivo (in mice).
• Cycles of further optimization based on the SAR computations and feedback from cell-based assays, biophysical assays, mice experiments, and pharmacokinetics.
• Further investigation of the precise mechanism of action: to generate escape mutants with more of our inhibitors, to determine the structure of HA in complex with one or more of the drug leads.

PATENTS
A provisional patent was filed.