Sequential Blockage as a Theoretical Basis for Drug Synergism

Black, Martin L.

doi:10.1021/jm00338a014

Cited by 24 publications

(9 citation statements)

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“…Moreover, the combination of sunitinib and erlotinib had a synergistic effect on HCV entry. Synergy between antimicrobial drugs often results from a sequential blocking mechanism, implying a block of two steps in a common pathway that is critical for viral replication (100,116). The observed synergy thus suggests that, in the context of HCV entry, some of the AAK1-and GAK-phosphorylated targets may be distinct.…”

Section: Discussionmentioning

confidence: 99%

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

Neveu

Ziv-Av

Barouch-Bentov

et al. 2015

J Virol

121

155

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Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively. IMPORTANCEUnderstanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1-and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways. H epatitis C virus (HCV) is a major global health problem, estimated to infect 170 million people worldwide (1, 2). HCV persistence results in severe liver disease, including cirrhosis, liver failure, and hepatocellular carcinoma (reviewed in reference...

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Section: Discussionmentioning

confidence: 99%

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

Neveu

Ziv-Av

Barouch-Bentov

et al. 2015

J Virol

121

155

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“…Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3), which catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, has been regarded as one of the crucial targets for bacterial and protozoal chemotherapy. The enzyme was shown to be the molecular site of action of a number of antifolates (2,8,32).…”

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confidence: 99%

“…Despite the wide application of these drug combinations, the mechanism of potentiation has not been well elucidated. It has generally been accepted that sequential blocking of different enzymes in the same metabolic pathway can lead to synergism (7,18,33). However, an observation that sulfonamides can be moderately potent inhibitors of bacterial dihydrofolate reductase from Escherichia coli led to an alternative hypothesis based on simultaneous inhibition of dihydrofolate reductase by both pyrimethamine and sulfonamides as a mechanism of potentiation (31).…”

mentioning

confidence: 99%

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

Sirawaraporn¹,

Yuthavong²

1986

Antimicrob Agents Chemother

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Dihydrofolate reductase was partially purified from a pyrimethamine-sensitive Plasmodium chabaudi clone and a pyrimethamine-resistant clone derived from it and used in a study of the inhibitory effect of pyrimethamine and sulfadoxine, both alone and in combination. Kinetic analysis of the inhibitory effect of sulfadoxine against the enzyme from pyrimethamine-sensitive and -resistant parasites revealed that the drug inhibited the former enzyme competitively, with an inhibition constant (K1.) of 0.7 + 0.4 mM, but inhibited the latter enzyme noncompetitively, with Ki, and K1i of 8.9 ± 1.2 and 4.1 ± 1.2 mM, respectively. Previous studies also showed competitive inhibition by pyrimethamine on the former enzyme and noncompetitive inhibition on the latter enzyme, with some 200-fold-lower affinity. Sulfadoxine and pyrimethamine exhibited a mutually potentiating effect on the enzyme activity, as revealed by the concave isoboles and the fractional inhibitions of less than unity. A potentiating effect was observed for the enzymes from both sources and was not dependent on the degree of the purification of the enzyme. Our results can be explained by assuming simultaneous binding of two inhibitors on the enzyme.

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“…An early theoretical treatment of synergy between two drugs (BLACK 1963) made the assumption that enzyme was in excess. This work has since been considered to contain mathematical errors (JACKSON 1991); current models of synergism for folate inhibitors (JACKSON 1991) predict synergy based on a circular model of substrate flow and are relatively parameter independent (GRINDEY et al 1975).…”

Section: A 'Log[ Le 392mentioning

confidence: 99%

A correlation with type of sulfonamide resistance gene in Escherichia coli and synergy between trimethoprim and sulfamethoxazole

Valentine

Hawk

Christian

et al. 1992

J. Basic Microbiol.

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Plasmids carrying type I or II sulfonamide-resistance (Sur) genes were evaluated for their effect on synergy between trimethoprim (Tmp) and sulfamethoxyzole (Smx) in E. coli. Strain J53 containing each of three plasmids (R1, pSa, and R388) with the type I Sur gene displayed a synergistic response to Tmp/Smx; strain LE392 containing a plasmid (RSF1010) with the type II Sur gene displayed no synergy. The difference in synergy between type I and type II Sur genes might be explained by the difference in amount of resistant enzyme produced.

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Sequential Blockage as a Theoretical Basis for Drug Synergism

Cited by 24 publications

References 0 publications

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

A correlation with type of sulfonamide resistance gene in Escherichia coli and synergy between trimethoprim and sulfamethoxazole

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