A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

Schaar, Hilde M. van der; Leyssen, Pieter; Thibaut, Hendrik Jan; Palma, Armando De; Linden, Lonneke van der; Lanke, Kjerstin; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; MacLeod, Angus M.; Mitchell, Dale R.; Palmer, Norman D.; Poël, Hervé Van de; Andrews, Martin; Neyts, Johan; Kuppeveld, Frank J. M. van

doi:10.1128/aac.01175-13

Cited by 105 publications

(123 citation statements)

References 58 publications

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“…We found that the viral RNAs from both plaques contained one nucleotide substitution, G5318A, resulting in an amino acid change from alanine to threonine in the 3A sequence. The same mutation was reported previously to confer resistance to a diverse class of so-called enviroxime-like compounds, believed to target a PI4KIII␤-dependent step in the viral replication cycle (10,(43)(44)(45)(46). We confirmed that this mutation was responsible for the resistance to E7(2) by genetically engineering the corresponding change into the poliovirus genome (not shown).…”

Section: E7(2) Affects Binding Of Cellular and Viral Factors To Membrsupporting

confidence: 72%

“…The mutation was the same as the previously described one conferring resistance to so-called enviroxime-like compounds; thus, E7 (2) can be added to this broad class of seemingly unrelated molecules. They are believed to target either PI4KIII␤ (major enviroximelike compounds) (10,45) or oxysterol binding protein (OSBP) (minor enviroxime-like compounds) (14). The amount of PI4KIII␤ associated with the replication membranes in the presence of E7(2) was not affected; however, this inhibitor induced a decrease in recruitment of GBF1 to membranes.…”

Section: Discussionmentioning

confidence: 95%

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New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

Ford-Siltz

Viktorova

Zhang

et al. 2014

J Virol

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Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase III␤ (PI4KIII␤)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIII␤ remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C-and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. IMPORTANCEDiverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

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Section: E7(2) Affects Binding Of Cellular and Viral Factors To Membrsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 95%

New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

Ford-Siltz

Viktorova

Zhang

et al. 2014

J Virol

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“…Herein we describe an experimental approach to use HDX‐MS to define dynamic regions within a protein complex, in this case the type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11, and use this information to generate optimized constructs for X‐ray crystallography. PI4KIIIβ is a lipid kinase that plays a key role in mediating membrane trafficking,21 and is also an emerging drug target for both antiviral22, 23, 24, 25 and antimalarial therapeutics 26. The combined HDX‐MS and X‐ray crystallographic analysis reveals novel aspects of the PI4KIIIβ‐Rab11 complex, specifically conformational changes induced in the switch regions of activated Rab11, as well as novel molecular details of PI4K inhibitor interactions.…”

Section: Introductionmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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“…In this model, virusinduced pancreatic tissue damage is the most prominent symptom and, hence, it served as the basis to study viral fitness. Employing this model, we compared the virulence and pathogenicity of wt CVB3 and CVB3 3A-H 57 Y, either in the absence or presence of compound 2 (a recently published PI4KIII␤ inhibitor of enterovirus replication both in vitro and in vivo [7]). Mice (n ϭ 5 per group) were infected intraperitoneally with equivalent amounts (5 ϫ 10 5 TCID 50 /ml) of either wt virus or CVB3 3A-H 57 Y, which was previously determined to result in a peak of pancreatic symptoms at day 3 postinfection (data not shown).…”

mentioning

confidence: 99%

“…In recent years, several studies identified the essential membrane-modifying host cell factor phosphatidylinositol-4-kinase III␤ (PI4KIII␤) as the target of broad-spectrum and highly potent inhibitors of enterovirus replication (1)(2)(3)(4)(5)(6)(7)(8). However, following lengthy selection processes, coxsackievirus B3 (CVB3) mutants that are resistant to these inhibitors emerged and were shown to harbor single point mutations in the nonstructural protein 3A (4,7,8). The H 57 Y mutation (3A-H 57 Y) was identified most frequently and provided the strongest resistance against PI4KIII␤ inhibitors, as well as to depletion of this factor.…”

mentioning

confidence: 99%

Fitness and Virulence of a Coxsackievirus Mutant That Can Circumnavigate the Need for Phosphatidylinositol 4-Kinase Class III Beta

Thibaut

Schaar

Lanke

et al. 2014

J Virol

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e Coxsackieviruses require phosphatidylinositol-4-kinase III␤ (PI4KIII␤) for replication but can bypass this need by an H 57 Y mutation in protein 3A (3A-H 57 Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as virulent as wild-type virus, even when mice were treated with a PI4KIII␤ inhibitor. Our data suggest that upon emergence, the 3A-H 57 Y mutant has the fitness to establish a resistant population with a virulence similar to that of wild-type virus.

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A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

Cited by 105 publications

References 58 publications

New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

Fitness and Virulence of a Coxsackievirus Mutant That Can Circumnavigate the Need for Phosphatidylinositol 4-Kinase Class III Beta

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