Lycorine, its use to Lycorine, its use to inhibit inhibit flavivirus flavivirus activity through the suppression of activity through the suppression of

viral RNA synthesis.viral RNA synthesis.

An Anti-Flavivirus TherapeuticAn Anti-Flavivirus TherapeuticAvailable for licenseAvailable for license

Presented to you by:Presented to you by:

Health Research, Inc.Health Research, Inc.150 Broadway, Suite 560150 Broadway, Suite 560

Menands, NY 12204Menands, NY 12204www.healthresearch.orgwww.healthresearch.org

Although flaviviruses cause significant human diseases, no antiviral therapy is currently Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. available for clinical treatment of these pathogens.

We have discovered through testing that lycorine exerts its antiviral activity mainly We have discovered through testing that lycorine exerts its antiviral activity mainly through suppression of viral RNA replication. This unique feature of lycorine makes it through suppression of viral RNA replication. This unique feature of lycorine makes it

a a valuablevaluable compound to further explore its potential to inhibit flaviviruses. compound to further explore its potential to inhibit flaviviruses.

Prior inventions demonstrated 30 Prior inventions demonstrated 30 µµM would reduce viral titers of West Nile (WNV). M would reduce viral titers of West Nile (WNV). In our testing, using 1.2 – In our testing, using 1.2 – µµM concentration, lycorine reduced viral titers of West Nile M concentration, lycorine reduced viral titers of West Nile (WNV), dengue and yellow fever viruses by 10(WNV), dengue and yellow fever viruses by 102 2 to 10to 1044 fold. fold.

Modifications of the two hydroxyl groups of lycorine increased the compound’s Modifications of the two hydroxyl groups of lycorine increased the compound’s potency, while again reducing its cytotoxicity. potency, while again reducing its cytotoxicity.

A single-amino acid substitution at Val9Met in West Nile virus 2K peptide between A single-amino acid substitution at Val9Met in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine. NS4A and NS4B confers resistance to lycorine.

Therefore, taken together these results establish lycorine as a potential RNA inhibitor Therefore, taken together these results establish lycorine as a potential RNA inhibitor and its potential for the compound’s therapeutic development for antiviral therapy. and its potential for the compound’s therapeutic development for antiviral therapy.

Currently, no effective antiviral therapy has been approved for the treatment of Currently, no effective antiviral therapy has been approved for the treatment of flavivirus infections. Human vaccines are currently available for Japanese encephalitis flavivirus infections. Human vaccines are currently available for Japanese encephalitis

virus (JEV), yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV).virus (JEV), yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV).

There are three major concerns in the development of a dengue vaccine:There are three major concerns in the development of a dengue vaccine:

There are four serotypes of Dengue i.e. DENV1, DENV2, DENV3 and DENV4. There are four serotypes of Dengue i.e. DENV1, DENV2, DENV3 and DENV4.

Secondary infection is the single greatest risk factor for developing Dengue Secondary infection is the single greatest risk factor for developing Dengue hemorrhagic fever and/or Dengue shock syndrome (DHF/DSS). hemorrhagic fever and/or Dengue shock syndrome (DHF/DSS).

If you are infected with DENV1, you will develop a lifelong immunity to DENV1. If you are infected with DENV1, you will develop a lifelong immunity to DENV1. If your secondary infection is with DENV 2, 3 or 4, you may develop DHF/DSS. If your secondary infection is with DENV 2, 3 or 4, you may develop DHF/DSS.

Therefore, it is necessary to develop of a vaccine to simultaneously immunize against Therefore, it is necessary to develop of a vaccine to simultaneously immunize against ALL four DENV serotypes.ALL four DENV serotypes.

More than 50 million cases of (DENV) were reported annually compared to 200,000 More than 50 million cases of (DENV) were reported annually compared to 200,000 cases of (YFV) and 50,000 cases of (JEV).cases of (YFV) and 50,000 cases of (JEV).

Lycorine suppresses RNA synthesisLycorine suppresses RNA synthesis

Three WNV luceriferase reporting based assays were used to dissect the inhibitory Three WNV luceriferase reporting based assays were used to dissect the inhibitory steps of lycorine. steps of lycorine.

First, a VLP infection based assay allowed identification of viral entry and First, a VLP infection based assay allowed identification of viral entry and replication (Fig 1C). replication (Fig 1C).

Second, use of replicon containing cell lines allowed screening for inhibitors of viral Second, use of replicon containing cell lines allowed screening for inhibitors of viral replication (Fig 4A). replication (Fig 4A).

Third, a transient replicon assay allowed differentiation between inhibitors of Third, a transient replicon assay allowed differentiation between inhibitors of translation and inhibitors of RNA synthesis (Fig 4B). Analysis of the compound in translation and inhibitors of RNA synthesis (Fig 4B). Analysis of the compound in the above systems indicated that whereas lycorine only weakly reduces viral the above systems indicated that whereas lycorine only weakly reduces viral translation (<30% luceriferase activity), it significantly suppresses RNA synthesis translation (<30% luceriferase activity), it significantly suppresses RNA synthesis (>99% luceriferase activity).(>99% luceriferase activity).

The reduction of RNA synthesis could be caused by the compound mediated The reduction of RNA synthesis could be caused by the compound mediated suppression of viral translation. Alternatively, lycorine could suppress both suppression of viral translation. Alternatively, lycorine could suppress both translation and RNA synthesis, leading to the dramatic reduction of RNA synthesis. translation and RNA synthesis, leading to the dramatic reduction of RNA synthesis.

To differentiate between the above two possibilities, a time-of-addition To differentiate between the above two possibilities, a time-of-addition experiment was performed.experiment was performed.

Time of Addition Experiment confers suppression of RNA synthesisTime of Addition Experiment confers suppression of RNA synthesis

The results showed that lycorine gradually declined its anti-WNV activity, when the The results showed that lycorine gradually declined its anti-WNV activity, when the time of addition was varied from 0 to 10 hrs. post infection; the compound completely time of addition was varied from 0 to 10 hrs. post infection; the compound completely lost inhibitory activity when added at >10 hrs. post infection (Fig 4C). (Chambers et al, lost inhibitory activity when added at >10 hrs. post infection (Fig 4C). (Chambers et al, 1990;Puig-Basagoti et al, 2005). If lycorine only inhibited the step of translation, it 1990;Puig-Basagoti et al, 2005). If lycorine only inhibited the step of translation, it would have completely lost its antiviral activity when added at a time point earlier would have completely lost its antiviral activity when added at a time point earlier than 10 hrs. post infection ( i.e. @ 6 hrs. post infection). Therefore, the time-of-than 10 hrs. post infection ( i.e. @ 6 hrs. post infection). Therefore, the time-of-addition results clearly indicate that the compound inhibits a step beyond viral addition results clearly indicate that the compound inhibits a step beyond viral translation. These data, together with the identification of the resistance determinant translation. These data, together with the identification of the resistance determinant in the 2K peptide (Fig 6), led to the conclusion that lycorine suppresses WNV mainly in the 2K peptide (Fig 6), led to the conclusion that lycorine suppresses WNV mainly through suppression of viral RNA synthesis.through suppression of viral RNA synthesis.

Identification of a Single-Amino Acid Mutation in the 2K Peptide as a Identification of a Single-Amino Acid Mutation in the 2K Peptide as a Resistance DeterminantResistance Determinant

Two approaches were taken to identify the potential target(s) of lycorine. The first Two approaches were taken to identify the potential target(s) of lycorine. The first approach was to test the compound in biochemical enzyme assays using recombinant approach was to test the compound in biochemical enzyme assays using recombinant protease (with NS2B), NTPase, RdRp, and MTase proteins. None of the enzyme protease (with NS2B), NTPase, RdRp, and MTase proteins. None of the enzyme activities were suppressed by lycorine. The second approach was to select compound-activities were suppressed by lycorine. The second approach was to select compound-resistant WNV.resistant WNV.

Resistant WNV cell cultures were selected. Engineering of the mutations (recovered Resistant WNV cell cultures were selected. Engineering of the mutations (recovered from the resistant viruses) into an infectious clone (Fig 6) and a replicon (Fig 8A) of from the resistant viruses) into an infectious clone (Fig 6) and a replicon (Fig 8A) of WNV allowed for the mapping of the resistance determinant to a single amino-acid WNV allowed for the mapping of the resistance determinant to a single amino-acid change (Val9Met) in the 2K peptide.change (Val9Met) in the 2K peptide.

Two panels of recombinant viruses were prepared to identify the lycorine-resistant Two panels of recombinant viruses were prepared to identify the lycorine-resistant determinant. determinant.

The first panel of viruses contained the mutations in the E gene. Each of the four The first panel of viruses contained the mutations in the E gene. Each of the four mutations in the E region was individually engineered into an infectious cDNA clone of mutations in the E region was individually engineered into an infectious cDNA clone of WNV. WNV.

Transfection of BHK-21 cells with the genome-length RNA’s resulted in four mutant Transfection of BHK-21 cells with the genome-length RNA’s resulted in four mutant viruses (Fig 6B): C1161U and U1789C (from Selection I) and A1287C and C1418U viruses (Fig 6B): C1161U and U1789C (from Selection I) and A1287C and C1418U (from Selection II). (from Selection II).

The four mutant viruses exhibited different plaque morphologies: the two viruses The four mutant viruses exhibited different plaque morphologies: the two viruses containing the silent mutations (C1161U and A1787C) yielded plaques similar to those containing the silent mutations (C1161U and A1787C) yielded plaques similar to those of the WT virus, whereas the mutant viruses containing amino acid changes in the E of the WT virus, whereas the mutant viruses containing amino acid changes in the E protein (C1418U and U1789C) generated smaller plaques than those of the WT virus. protein (C1418U and U1789C) generated smaller plaques than those of the WT virus.

Resistance assays showed that, after treatment of infected cells (0.1 MOI) with Resistance assays showed that, after treatment of infected cells (0.1 MOI) with lycorine (1.2 lycorine (1.2 µµM lycorine for 42 Hrs.), none of the four mutants yielded viral titers that M lycorine for 42 Hrs.), none of the four mutants yielded viral titers that were significantly higher than the titers of the WT virus (Fig. 6C). The results indicate were significantly higher than the titers of the WT virus (Fig. 6C). The results indicate that the E mutations are not responsible for resistance. Notably, the smaller plaques that the E mutations are not responsible for resistance. Notably, the smaller plaques (Fig 6B) and the lower titers from mock-treated infections for mutant viruses U1789C (Fig 6B) and the lower titers from mock-treated infections for mutant viruses U1789C and C1418U (Fig 6C) suggest that the amino acid changes in the E gene negatively and C1418U (Fig 6C) suggest that the amino acid changes in the E gene negatively affect viral replication.affect viral replication.

The second panel of viruses was prepared to examine the mutation G6817A in the 2K The second panel of viruses was prepared to examine the mutation G6817A in the 2K peptide. The plaque morphology of G6871A virus was similar to that of the WT virus peptide. The plaque morphology of G6871A virus was similar to that of the WT virus (Fig 6B). Remarkably, the G6871A virus showed a resistance level close to those of the (Fig 6B). Remarkably, the G6871A virus showed a resistance level close to those of the P12 viruses from all three selections (Fig 6C). Sequencing of the G6871A mutant virus P12 viruses from all three selections (Fig 6C). Sequencing of the G6871A mutant virus indicated that the engineered mutation was retained without extra changes; indicated that the engineered mutation was retained without extra changes; furthermore, the G6871A mutation was retained after passing the mutant virus in Vero furthermore, the G6871A mutation was retained after passing the mutant virus in Vero cells for five rounds (a total of 10 days). These results demonstrate that the G6871A cells for five rounds (a total of 10 days). These results demonstrate that the G6871A mutation in the 2K peptide is the major determinant for lycorine resistance. Sequence mutation in the 2K peptide is the major determinant for lycorine resistance. Sequence alignment (Fig 6D) indicates that the mutated Val at amino-acid position 9 of the 2K alignment (Fig 6D) indicates that the mutated Val at amino-acid position 9 of the 2K peptide (total of 23 amino acids ) is conserved among the members of the JEV-peptide (total of 23 amino acids ) is conserved among the members of the JEV-serocomplex; and a Val at position 10 is conserved among the members of the DENV-serocomplex; and a Val at position 10 is conserved among the members of the DENV-serocomplex; whereas no Val is found at a similar position in the 2K peptides from serocomplex; whereas no Val is found at a similar position in the 2K peptides from members of the YFV- and TBEV-serocomplexes.members of the YFV- and TBEV-serocomplexes.

Replication Kinetics of WT and 2K-Mutant Viruses in the presence and Replication Kinetics of WT and 2K-Mutant Viruses in the presence and absence of lycorineabsence of lycorine

The G6871A mutation was further characterized by comparing the replication kinetics The G6871A mutation was further characterized by comparing the replication kinetics between the WT and the mutant (MT) viruses in the presence and absence of lycorine between the WT and the mutant (MT) viruses in the presence and absence of lycorine inhibitor. The Vero cells were infected at synchronized times with the WT and the inhibitor. The Vero cells were infected at synchronized times with the WT and the G6871A MT viruses, and treated with or without 1.2 G6871A MT viruses, and treated with or without 1.2 µµM lycorine. As expected, the M lycorine. As expected, the compound inhibited WT virus more dramatically than it did on the MT virus (Fig 7A). compound inhibited WT virus more dramatically than it did on the MT virus (Fig 7A). Western blotting analysis showed that at the 16 hr. post infection, no viral proteins Western blotting analysis showed that at the 16 hr. post infection, no viral proteins could be detected in cells infected with either WT or MT viruses. The expression levels could be detected in cells infected with either WT or MT viruses. The expression levels of viral NS1, NS3, and NS5 increased from 24-36 hr. post infection, but decreased at 48 of viral NS1, NS3, and NS5 increased from 24-36 hr. post infection, but decreased at 48 hr. post infection. The decrease in protein expression at 48 hrs. p.i. was due to hr. post infection. The decrease in protein expression at 48 hrs. p.i. was due to cytopathic effects i.e. cell lysis. At each time point, lycorine treatment suppressed viral cytopathic effects i.e. cell lysis. At each time point, lycorine treatment suppressed viral protein expression. These results confirm the critical role of the G6871A mutation in protein expression. These results confirm the critical role of the G6871A mutation in lycorine resistance.lycorine resistance.

Improvement of the Lycorine compoundImprovement of the Lycorine compound

Studies were done to synthesize seven lycorine analogues for cytotoxicity and antiviral Studies were done to synthesize seven lycorine analogues for cytotoxicity and antiviral activity. Three regions of the parental lycorine compound were modified. One or two activity. Three regions of the parental lycorine compound were modified. One or two hydroxyl groups at the C1 and C2 positions of lycorine were substituted with other hydroxyl groups at the C1 and C2 positions of lycorine were substituted with other chemical groups. All seven analogues that contained these modifications showed that chemical groups. All seven analogues that contained these modifications showed that CC CC 50 50 values higher than the value for parental lycorine. The results clearly indicate values higher than the value for parental lycorine. The results clearly indicate that modifications at the two hydroxyl groups of lycorine could be used to improve the that modifications at the two hydroxyl groups of lycorine could be used to improve the antiviral profile.antiviral profile.

Summary

We have discovered through testing that lycorine exerts its antiviral activity We have discovered through testing that lycorine exerts its antiviral activity mainly through suppression of viral RNA replication.mainly through suppression of viral RNA replication. Modifications of the two Modifications of the two hydroxyl groups of lycorine increased the compound’s potency, while reducing hydroxyl groups of lycorine increased the compound’s potency, while reducing its cytotoxicity. Therefore, this system could be used to engineer targeted its cytotoxicity. Therefore, this system could be used to engineer targeted mutations into West Nile virus, resulting in attenuated virus strains that could mutations into West Nile virus, resulting in attenuated virus strains that could potentially be used for vaccine development.potentially be used for vaccine development.

Inventor BiographyInventor Biography

Pei-Yong Shi, Ph.D., Head of Dengue UnitPei-Yong Shi is heading the dengue unit at Novartis Institute for Tropical Diseases (NITD). He received his Ph.D. in flavivirus replication in 1995 from Georgia State University, USA. After a postdoctoral training at Yale University, he joined Bristol-Myers Squibb as a Principal Scientist from 1998 to 2000. He then moved to the Wadsworth Center, New York State Department of Health. His group at the Wadsworth Center developed the first infectious clone of the epidemic strain of West Nile virus, discovered two cap methylation activities of flavivirus NS5 protein, identified essential RNA elements for flavivirus replication, and established various novel platforms that have been used to screen and study inhibitors of flaviviruses. He is a member of Editorial Boards for both Journal of Virology and Virology. Dr. Shi joined NITD in August 2008.