# AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway
## 3 Discussion
In our study, however, Golgi dispersion appeared to be transient and was fully recovered within 24 h. This effect was different from GCA and BFA, as the treatment of any of these two inhibitors rendered Golgi dispersion persistent. This discrepancy suggests that GBF1 may interact with AG1478 differently from GCA/BFA. Of note, both catalytic Sec7 [42] and non-catalytic N-terminal [33, 44] domains of GBF1 were required for polioviral replication. Thus, the specific domain(s) of GBF1 that interacts with AG1478 will warrant further study. Targeting host factors, instead of viral proteins, has been an alternative strategy for antiviral therapy holding the promise of producing broad-spectrum antivirals and at the same time raising the barrier for the emergence of drug resistant mutants. One major concern for targeting host factors is their potential cellular toxicity, as the candidate host factor may also play important roles in host cell homeostasis. In our study, several unique characteristics about the interaction between AG1478 and GBF1 suggest that the future therapy based on this finding may be safe. First, the inhibition of GBF1 by AG1478 only caused transient Golgi dispersion and it was fully recovered within 24 h. In contrast, two other GBF1 inhibitors, GCA and BFA, caused more extended dispersion of Golgi apparatus. This is also consistent with the kinetics of ARF1 inhibition. As a result, AG1478 treatment did not cause significant cellular toxicity compared to GCA or BFA. Second, influenza appeared to hijack a significant portion of GBF1 for its own protein transport in the cytoplasm. Thus, the effect of AG1478 on the Golgi-cytoplasmic trafficking of GBF1 may preferentially impact viral production since the normal cellular function is kept at a much lower level under this pathological condition. It will then be very interesting to find out whether the mode of action of GBF1 in the IAV-related vesicle trafficking is different from the normal. Third, the normal function of GBF1 could be restored by washing out AG1478. This is significant because the GBF1 system appears to be intact after inhibition and the future dosing can be carefully formulated to target certain windows of the infection while minimizing unwanted side effects. Although this strategy holds the promise to overcome drug resistance, we did not test if IAV can produce a drug-resistant strain in the presence AG1478. A BFA-resistant mutant of polio virus was selected previously. However, this resistant phenotype was cell-type-specific (i.e., BFA-resistant phenotype is restrained in the cells that were used for selection, but not in other cell types), and the replication of the mutant was slow and prone to cellular innate defense such as the IFN system. Thus, even though mutants bypassing host-factor-targeting drugs can emerge, they appear to be less adaptive and have trade-off defects in the evasion of immune surveillance, suggesting that these mutants can be either cleared automatically by the host immunity or be treated by an immune modulator (e.g., CpG etc.) to boost host immune response. In summary, through a serendipitous finding, we discovered a potent anti-influenza drug candidate--AG1478. Its antiviral activity is IFN independent and is mediated by targeting GBF1, a regulator of intracellular trafficking. GBF1 appears to be hijacked by IAV for its own transport via the interaction between GBF1 and M1 as well as its associated vRNP. The treatment of AG1478 disrupted this interaction and potentially impaired vRNP transport leading to markedly decreased IAV production. Further development on this candidate will lead to novel anti-influenza therapy.