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

Abstract

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.

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

“…Based on the previously described core NEC deletion mutant ΔUL50, in the context of pUL50-complementing cells, the system was extended, allowing for investigations of both core NEC proteins in parallel. This sophisticated recombinant HCMV ΔUL50-ΣUL53 was designed for the individual and conditional expression of pUL50 and/or pUL53 in the context of HCMV replication [ 88 ]. Based on the BAC HCMV AD169 ΔUL50 [ 89 ], the ORF of pUL50 was deleted but pUL50 expression can be complemented by doxycycline (dox)-inducible pUL50 overexpressing cells.…”

“…This limitation can be overcome by normalizing investigated antiviral effects to their solvent control, allowing comparisons between the different infectious settings. Taken together, HCMV ΔUL50-ΣUL53 represents a very valuable tool for the functional characterization of this heterodimer in general as well as of core NEC affecting small molecules and peptides [ 88 ].…”

“…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 ].…”

“…During the course of validating herpesviral NECs as suitable and effective targets of a novel type of antiviral drug discovery, several reports described methodological approaches, relevant mechanistic insight, and NEC targeting concepts to prepare antiviral research and compound screenings [ 71 , 88 , 92 , 93 , 95 , 96 , 97 , 102 , 103 , 104 , 105 ]. The approach to target a viral core NEC structure is challenging in the way that, first of all, it demands the identification of specific PPI inhibitors instead of conventional enzymatic inhibitors.…”

“…This stands in contrast to the massively increased molecular understanding of NEC targeting requirements, as achieved through the description of high-resolution 3D structures and additional information on NEC biochemistry [ 65 , 66 , 68 , 69 , 72 , 75 ]. Current approaches have recently been reported based on the utilization of covalently target-binding small molecules (see Section 2.4 [ 88 , 95 ]).…”