An antiviral targeting strategy based on the inducible interference with cytomegalovirus nuclear egress complex

Kicuntod, Jintawee; Häge, Sigrun; Lösing, Josephine; Kopar, Serli; Muller, Yves A.; Marschall, Manfred

doi:10.1016/j.antiviral.2023.105557

Cited by 3 publications

(9 citation statements)

References 52 publications

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Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

“…The NEC has been validated as a promising target for antiherpesviral strategies via numerous approaches [ 88 , 92 , 93 , 94 , 95 , 96 , 97 , 98 ]. The latest proof-of-concept was established, termed ‘NLS-Hook-GFP’, in order to assess the efficacy of HCMV replication upon an intracellular expression of the pUL53 hook fragment [ 98 ]. A lentiviral gene transfer procedure was utilized to produce a primary fibroblast (HFF) population, in which the intracellular expression of a nuclear localization signal (NLS, amino acids 13–26 of pUL53) fused to the HCMV-specific pUL53 hook fragment (Hook, amino acids 59–87 of pUL53) and the GFP reporter ( Figure 5 A) could be induced by the addition of dox.…”

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

“…Over the period of 2–3 days (d) post-induction, the recombinantly expressed NLS-Hook-GFP could be observed to occur at its typical localization in the nucleoplasm via confocal imaging ( Figure 5 C, images 5, 8, 9, and 12; see the lamin A/C nuclear envelope signal in images 6 and 10 and the DAPI counterstaining of nuclei in images 7 and 11). The specific NEC interaction of this expressed construct was identified by an in vitro assembly assay as previously described [ 71 , 97 , 98 ]. The data revealed that NLS-Hook-GFP specifically interacted with the HCMV groove protein (pUL50; Figure 5 B); however, it did not exhibit an interaction with the HCMV parental hook protein (pUL53).…”

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

“…The quantity of HCMV genome equivalents was diminished in the increasing expression levels of the inducible Hook construct over a period of 9 d post-infection (i.e., three rounds of viral replication). The positive signals of pUL53 and pp28 from the Wb analysis were prominently reduced as compared to non-induced cells [ 98 ]. The replication levels of HCMV strain AD169 under NLS-Hook-GFP expression were decreased compared to a similar tendency when using a second laboratory strain, TB40, and a clinically relevant strain, Toledo ( Figure 5 D).…”

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

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‘Getting Better’—Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

Tillmanns,

Kicuntod,

Lösing

et al. 2024

IJMS

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The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

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Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

Section: The Multifaceted Aspects Of Nec-directed Antiviral Drug Targ...mentioning

confidence: 99%

See 2 more Smart Citations

‘Getting Better’—Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

Tillmanns,

Kicuntod,

Lösing

et al. 2024

IJMS

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“…Concerning novel targeting strategies of anti-HCMV drugs, our group and many other researchers have studied a variety of different options of direct-acting and host-directed antivirals [ 27 , 32 , 33 ]. Very recently, we focused on the NEC as a highly promising target, which is composed of viral core components (core NEC) and, additionally, NEC-associated host and virus proteins (multicomponent NEC) [ 8 , 24 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. A crucial step towards an improved level of discovery of NEC-directed small molecules was our experimental resolution of crystallization-based 3D structures of α-, β- and γ-herpesviral core NECs [ 41 , 42 , 43 ].…”

Section: Introductionmentioning

confidence: 99%

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target

Tillmanns

Häge

Borst

et al. 2023

Cells

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Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50–pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50–pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50–pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.

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Combined Treatment with Host-Directed and Anticytomegaloviral Kinase Inhibitors: Mechanisms, Synergisms and Drug Resistance Barriers

Wild,

Karner,

Eickhoff

et al. 2023

Pharmaceutics

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Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

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An antiviral targeting strategy based on the inducible interference with cytomegalovirus nuclear egress complex

Cited by 3 publications

References 52 publications

‘Getting Better’—Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

‘Getting Better’—Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target

Combined Treatment with Host-Directed and Anticytomegaloviral Kinase Inhibitors: Mechanisms, Synergisms and Drug Resistance Barriers

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