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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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Related in: MedlinePlus

Overview of the HCV replicon and host responses showing the observed combination activity. Dose-matrix data were obtained by testing all pairs of serially diluted (two-fold) concentrations for each pair of probes (Supplementary Table S1). Activity values A=−log10(T/U) were calculated at each dosing point by comparing the treated T viability level to untreated values V obtained from ∼20 vehicle-treated wells arranged on each experimental plate (see Materials and methods). Dose matrices were obtained with at least four replicates. Uncertainties on the activity, estimated from the scatter between replicates (see Materials and methods), were typically ∼0.1, averaged across each dosing matrix (Supplementary Table S1). For each pair of chemical probes, the level of interaction observed between the agents is shown as a circle scaled to the synergy score S, relative to a ‘superposed effect' (SPE) model of non-interaction (see Materials and methods). The SPE model interpolates smoothly between single-agent effects that can be either inhibitory or stimulatory. Positive S (solid or red circles) correspond to synergistic interaction when both agents are inhibitory, and to the dominance at high combined concentrations of the inhibitory agent when one of the agents has stimulatory activity (e.g. squalestatin). Negative S corresponded to antagonism between inhibitors or dominance of the stimulatory agent. A synergy score of 1 means that there was a volume of 1 between the observed response and the SPE model surface, integrated over the dose matrix. The scale of these scores depends on the number and density of concentration points tested, and thus is mainly useful for comparisons between combinations, rather than providing an absolute reference level for synergy. Given that the individual activity measurements tended to have standard errors of σa∼0.1, only synergy scores outside of ±3 can be considered significant, when integrated over the 64 combination dosing points. (A) In the replicon assay, combinations targeting sterol pathway enzymes downstream of OSC mostly appear to synergize more toward antiviral activity, and those upstream of OSC mostly appear to show ‘epistasis,' where the effect of modulating an upstream target dominates over those of downstream targets at high combined concentrations. (B) Most combinations in the host toxicity assay produce responses that are close to the SPE expectation, indicating the model represents typical levels of interaction between compounds reasonably well. Where it does occur, strong synergistic viability inhibition (S>3) is associated with compounds (e.g. clomiphene, terconazole and triparanol) that also show strong synergies outside of the sterol pathway (with TOFA and clomiphene, or GGTI-286), and host activity within the pathway obvious mechanism-dependent patterns.
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f3: Overview of the HCV replicon and host responses showing the observed combination activity. Dose-matrix data were obtained by testing all pairs of serially diluted (two-fold) concentrations for each pair of probes (Supplementary Table S1). Activity values A=−log10(T/U) were calculated at each dosing point by comparing the treated T viability level to untreated values V obtained from ∼20 vehicle-treated wells arranged on each experimental plate (see Materials and methods). Dose matrices were obtained with at least four replicates. Uncertainties on the activity, estimated from the scatter between replicates (see Materials and methods), were typically ∼0.1, averaged across each dosing matrix (Supplementary Table S1). For each pair of chemical probes, the level of interaction observed between the agents is shown as a circle scaled to the synergy score S, relative to a ‘superposed effect' (SPE) model of non-interaction (see Materials and methods). The SPE model interpolates smoothly between single-agent effects that can be either inhibitory or stimulatory. Positive S (solid or red circles) correspond to synergistic interaction when both agents are inhibitory, and to the dominance at high combined concentrations of the inhibitory agent when one of the agents has stimulatory activity (e.g. squalestatin). Negative S corresponded to antagonism between inhibitors or dominance of the stimulatory agent. A synergy score of 1 means that there was a volume of 1 between the observed response and the SPE model surface, integrated over the dose matrix. The scale of these scores depends on the number and density of concentration points tested, and thus is mainly useful for comparisons between combinations, rather than providing an absolute reference level for synergy. Given that the individual activity measurements tended to have standard errors of σa∼0.1, only synergy scores outside of ±3 can be considered significant, when integrated over the 64 combination dosing points. (A) In the replicon assay, combinations targeting sterol pathway enzymes downstream of OSC mostly appear to synergize more toward antiviral activity, and those upstream of OSC mostly appear to show ‘epistasis,' where the effect of modulating an upstream target dominates over those of downstream targets at high combined concentrations. (B) Most combinations in the host toxicity assay produce responses that are close to the SPE expectation, indicating the model represents typical levels of interaction between compounds reasonably well. Where it does occur, strong synergistic viability inhibition (S>3) is associated with compounds (e.g. clomiphene, terconazole and triparanol) that also show strong synergies outside of the sterol pathway (with TOFA and clomiphene, or GGTI-286), and host activity within the pathway obvious mechanism-dependent patterns.

Mentions: After determining single-agent activities for all the chemical agents tested in this study, we designed dose-matrix experiments with concentrations centered around each drug's IC50 when possible. To evaluate the synergistic activity of compounds in combination, we compared the observed activity across each drug combination inhibition response surface to a superposition of effect (SPE) model that is derived from the single-agent activity curves (see Materials and methods). Unlike the standard reference models for combination effects (Greco et al, 1995) the SPE model smoothly interpolates between the activities of both inhibitory and stimulatory single agents, highlighting as synergies or antagonisms antiviral responses that fall above or below the SPE expectation. An overview of the replicon responses and the corresponding host combination activity is presented in Figure 3. The dose-matrix response surfaces for all the compound crosses performed in the HCV replicon assay and Huh-7 host cells are shown in Supplementary Figures S2 and S3.


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)

Overview of the HCV replicon and host responses showing the observed combination activity. Dose-matrix data were obtained by testing all pairs of serially diluted (two-fold) concentrations for each pair of probes (Supplementary Table S1). Activity values A=−log10(T/U) were calculated at each dosing point by comparing the treated T viability level to untreated values V obtained from ∼20 vehicle-treated wells arranged on each experimental plate (see Materials and methods). Dose matrices were obtained with at least four replicates. Uncertainties on the activity, estimated from the scatter between replicates (see Materials and methods), were typically ∼0.1, averaged across each dosing matrix (Supplementary Table S1). For each pair of chemical probes, the level of interaction observed between the agents is shown as a circle scaled to the synergy score S, relative to a ‘superposed effect' (SPE) model of non-interaction (see Materials and methods). The SPE model interpolates smoothly between single-agent effects that can be either inhibitory or stimulatory. Positive S (solid or red circles) correspond to synergistic interaction when both agents are inhibitory, and to the dominance at high combined concentrations of the inhibitory agent when one of the agents has stimulatory activity (e.g. squalestatin). Negative S corresponded to antagonism between inhibitors or dominance of the stimulatory agent. A synergy score of 1 means that there was a volume of 1 between the observed response and the SPE model surface, integrated over the dose matrix. The scale of these scores depends on the number and density of concentration points tested, and thus is mainly useful for comparisons between combinations, rather than providing an absolute reference level for synergy. Given that the individual activity measurements tended to have standard errors of σa∼0.1, only synergy scores outside of ±3 can be considered significant, when integrated over the 64 combination dosing points. (A) In the replicon assay, combinations targeting sterol pathway enzymes downstream of OSC mostly appear to synergize more toward antiviral activity, and those upstream of OSC mostly appear to show ‘epistasis,' where the effect of modulating an upstream target dominates over those of downstream targets at high combined concentrations. (B) Most combinations in the host toxicity assay produce responses that are close to the SPE expectation, indicating the model represents typical levels of interaction between compounds reasonably well. Where it does occur, strong synergistic viability inhibition (S>3) is associated with compounds (e.g. clomiphene, terconazole and triparanol) that also show strong synergies outside of the sterol pathway (with TOFA and clomiphene, or GGTI-286), and host activity within the pathway obvious mechanism-dependent patterns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2913396&req=5

f3: Overview of the HCV replicon and host responses showing the observed combination activity. Dose-matrix data were obtained by testing all pairs of serially diluted (two-fold) concentrations for each pair of probes (Supplementary Table S1). Activity values A=−log10(T/U) were calculated at each dosing point by comparing the treated T viability level to untreated values V obtained from ∼20 vehicle-treated wells arranged on each experimental plate (see Materials and methods). Dose matrices were obtained with at least four replicates. Uncertainties on the activity, estimated from the scatter between replicates (see Materials and methods), were typically ∼0.1, averaged across each dosing matrix (Supplementary Table S1). For each pair of chemical probes, the level of interaction observed between the agents is shown as a circle scaled to the synergy score S, relative to a ‘superposed effect' (SPE) model of non-interaction (see Materials and methods). The SPE model interpolates smoothly between single-agent effects that can be either inhibitory or stimulatory. Positive S (solid or red circles) correspond to synergistic interaction when both agents are inhibitory, and to the dominance at high combined concentrations of the inhibitory agent when one of the agents has stimulatory activity (e.g. squalestatin). Negative S corresponded to antagonism between inhibitors or dominance of the stimulatory agent. A synergy score of 1 means that there was a volume of 1 between the observed response and the SPE model surface, integrated over the dose matrix. The scale of these scores depends on the number and density of concentration points tested, and thus is mainly useful for comparisons between combinations, rather than providing an absolute reference level for synergy. Given that the individual activity measurements tended to have standard errors of σa∼0.1, only synergy scores outside of ±3 can be considered significant, when integrated over the 64 combination dosing points. (A) In the replicon assay, combinations targeting sterol pathway enzymes downstream of OSC mostly appear to synergize more toward antiviral activity, and those upstream of OSC mostly appear to show ‘epistasis,' where the effect of modulating an upstream target dominates over those of downstream targets at high combined concentrations. (B) Most combinations in the host toxicity assay produce responses that are close to the SPE expectation, indicating the model represents typical levels of interaction between compounds reasonably well. Where it does occur, strong synergistic viability inhibition (S>3) is associated with compounds (e.g. clomiphene, terconazole and triparanol) that also show strong synergies outside of the sterol pathway (with TOFA and clomiphene, or GGTI-286), and host activity within the pathway obvious mechanism-dependent patterns.
Mentions: After determining single-agent activities for all the chemical agents tested in this study, we designed dose-matrix experiments with concentrations centered around each drug's IC50 when possible. To evaluate the synergistic activity of compounds in combination, we compared the observed activity across each drug combination inhibition response surface to a superposition of effect (SPE) model that is derived from the single-agent activity curves (see Materials and methods). Unlike the standard reference models for combination effects (Greco et al, 1995) the SPE model smoothly interpolates between the activities of both inhibitory and stimulatory single agents, highlighting as synergies or antagonisms antiviral responses that fall above or below the SPE expectation. An overview of the replicon responses and the corresponding host combination activity is presented in Figure 3. The dose-matrix response surfaces for all the compound crosses performed in the HCV replicon assay and Huh-7 host cells are shown in Supplementary Figures S2 and S3.

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