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Chemical combinations elucidate pathway interactions and regulation relevant to Hepatitis C replication.

Owens CM, Mawhinney C, Grenier JM, Altmeyer R, Lee MS, Borisy AA, Lehár J, Johansen LM - Mol. Syst. Biol. (2010)

Bottom Line: However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients.There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway.Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.

View Article: PubMed Central - PubMed

Affiliation: CombinatoRx, Incorporated, Cambridge, MA 02142, USA.

ABSTRACT
The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.

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Validation experiments. (A) Evidence of epistasis at the level of HCV protein expression, after 96 h in Huh-luc/neo-ET replicon cells, between upstream-targeting simvastatin and downstream-targeting U18666A. Protein bands were quantified using densitometry and levels of expressed HCV proteins NS3 and NS5A are shown as ratios normalized to GAPDH. (B) Sterol pathway regulation revealed by HMGCR protein expression in HuH-7 cells 16 h after exposure to chemical inhibitors, showing feedback effects for probes targeting the upper end of the pathway. Antibodies specific for HMGCR and GAPDH were used to probe western blots of proteins separated by 10% Bis–Tris SDS/PAGE (see Materials and methods). (C) Confirmation that agents targeting the upper and lower pathway have respectively pro- and antiviral effects at 72 h in quantitative RT–PCR experiments on Huh-luc/neo-ET cells. Averaged expression levels from triplicate experiments were calculated after normalizing replicon copy number to total cellular RNA (see Materials and methods), and error bars show 95% confidence (two standard deviations).
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f5: Validation experiments. (A) Evidence of epistasis at the level of HCV protein expression, after 96 h in Huh-luc/neo-ET replicon cells, between upstream-targeting simvastatin and downstream-targeting U18666A. Protein bands were quantified using densitometry and levels of expressed HCV proteins NS3 and NS5A are shown as ratios normalized to GAPDH. (B) Sterol pathway regulation revealed by HMGCR protein expression in HuH-7 cells 16 h after exposure to chemical inhibitors, showing feedback effects for probes targeting the upper end of the pathway. Antibodies specific for HMGCR and GAPDH were used to probe western blots of proteins separated by 10% Bis–Tris SDS/PAGE (see Materials and methods). (C) Confirmation that agents targeting the upper and lower pathway have respectively pro- and antiviral effects at 72 h in quantitative RT–PCR experiments on Huh-luc/neo-ET cells. Averaged expression levels from triplicate experiments were calculated after normalizing replicon copy number to total cellular RNA (see Materials and methods), and error bars show 95% confidence (two standard deviations).

Mentions: In the HCV replicon assay, however, strong mechanism-dependent patterns emerged, which are highlighted in Figure 4. In many cases, chemical combinations elicited Bateson-type epistatic responses, where one chemical agent's response predominates over the effect of the other chemical agent (Boone et al, 2007). Combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway elicited responses epistatic to the effect of targeting enzymes downstream of SQLE (Figure 4A). This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48-8071. Treatment with squalestatin prevents the SQLS substrate, FPP from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48-8071 treatment. However, less clearly understood are the epistatic effects observed between simvastatin and either squalestatin or U18666A. In these two conflicting examples, simvastatin is observed in one case to antagonize the proviral effect of squalestatin treatment and in another to antagonize the antiviral effect of U18666A. Confirmation of simvastatin's epistasis over U18666A was provided by western blot analysis (Figure 5A). The concentration of each compound was selected for an absence of impact on host proliferation. The results suggest that the observed contradictory effect of simvastatin is not an artifact. It is likely that several simultaneous and competing processes govern the outcome for these combinations, including HMGCR protein inhibition, unspecific cytotoxicity as measured by ATP depletion and HMGCR transcriptional regulation.


Chemical combinations elucidate pathway interactions and regulation relevant to Hepatitis C replication.

Owens CM, Mawhinney C, Grenier JM, Altmeyer R, Lee MS, Borisy AA, Lehár J, Johansen LM - Mol. Syst. Biol. (2010)

Validation experiments. (A) Evidence of epistasis at the level of HCV protein expression, after 96 h in Huh-luc/neo-ET replicon cells, between upstream-targeting simvastatin and downstream-targeting U18666A. Protein bands were quantified using densitometry and levels of expressed HCV proteins NS3 and NS5A are shown as ratios normalized to GAPDH. (B) Sterol pathway regulation revealed by HMGCR protein expression in HuH-7 cells 16 h after exposure to chemical inhibitors, showing feedback effects for probes targeting the upper end of the pathway. Antibodies specific for HMGCR and GAPDH were used to probe western blots of proteins separated by 10% Bis–Tris SDS/PAGE (see Materials and methods). (C) Confirmation that agents targeting the upper and lower pathway have respectively pro- and antiviral effects at 72 h in quantitative RT–PCR experiments on Huh-luc/neo-ET cells. Averaged expression levels from triplicate experiments were calculated after normalizing replicon copy number to total cellular RNA (see Materials and methods), and error bars show 95% confidence (two standard deviations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913396&req=5

f5: Validation experiments. (A) Evidence of epistasis at the level of HCV protein expression, after 96 h in Huh-luc/neo-ET replicon cells, between upstream-targeting simvastatin and downstream-targeting U18666A. Protein bands were quantified using densitometry and levels of expressed HCV proteins NS3 and NS5A are shown as ratios normalized to GAPDH. (B) Sterol pathway regulation revealed by HMGCR protein expression in HuH-7 cells 16 h after exposure to chemical inhibitors, showing feedback effects for probes targeting the upper end of the pathway. Antibodies specific for HMGCR and GAPDH were used to probe western blots of proteins separated by 10% Bis–Tris SDS/PAGE (see Materials and methods). (C) Confirmation that agents targeting the upper and lower pathway have respectively pro- and antiviral effects at 72 h in quantitative RT–PCR experiments on Huh-luc/neo-ET cells. Averaged expression levels from triplicate experiments were calculated after normalizing replicon copy number to total cellular RNA (see Materials and methods), and error bars show 95% confidence (two standard deviations).
Mentions: In the HCV replicon assay, however, strong mechanism-dependent patterns emerged, which are highlighted in Figure 4. In many cases, chemical combinations elicited Bateson-type epistatic responses, where one chemical agent's response predominates over the effect of the other chemical agent (Boone et al, 2007). Combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway elicited responses epistatic to the effect of targeting enzymes downstream of SQLE (Figure 4A). This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48-8071. Treatment with squalestatin prevents the SQLS substrate, FPP from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48-8071 treatment. However, less clearly understood are the epistatic effects observed between simvastatin and either squalestatin or U18666A. In these two conflicting examples, simvastatin is observed in one case to antagonize the proviral effect of squalestatin treatment and in another to antagonize the antiviral effect of U18666A. Confirmation of simvastatin's epistasis over U18666A was provided by western blot analysis (Figure 5A). The concentration of each compound was selected for an absence of impact on host proliferation. The results suggest that the observed contradictory effect of simvastatin is not an artifact. It is likely that several simultaneous and competing processes govern the outcome for these combinations, including HMGCR protein inhibition, unspecific cytotoxicity as measured by ATP depletion and HMGCR transcriptional regulation.

Bottom Line: However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients.There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway.Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.

View Article: PubMed Central - PubMed

Affiliation: CombinatoRx, Incorporated, Cambridge, MA 02142, USA.

ABSTRACT
The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.

Show MeSH
Related in: MedlinePlus