Small-Molecule Probes Targeting the Viral PPxY-Host Nedd4 Interface Block Egress of a Broad Range of RNA Viruses

Abstract

Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress. IMPORTANCEThere is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.

Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

“…Overall, the inhibition of M protein function by both AP3B1-derived polypeptides and overexpressed full-length AP3B1 supports the general concept that binding interfaces between host factors and buddingrelevant viral proteins can be targeted for disruption as an effective antiviral approach (47,57). Indeed, small-molecule budding inhibitors have recently been identified that target the PTAP-Tsg101 interface in the case of HIV-1 (58,59) or the PPxY-Nedd4 interface in the cases of Ebola virus, Marburg virus, Lassa virus, and rabies virus (60). Similar approaches that target the AP3B1-M binding interface may prove useful in the identification of new therapeutics for paramyxovirus infections.…”

A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus

“…As with our other tuberculosis-focused research 32, 33 , and computational approaches to repositioning compounds 34 we embrace the essentiality for computational predictions to be interrogated through rigorous experimental studies. For example at least two in silico docking studies screened commercially available compounds 14, 15 . We propose that docking FDA approved drugs could also be a viable first step to identifying potential compounds that could be used.…”

“…Several studies have identified non-FDA approved drugs including an in silico docking approach to identify molecules targeting the viral Nedd4-PPxY interface 14 . These molecules were similar to the FDA benzimidazole and aminoquinoline 8, 9 compounds that were active against EBOV.…”

Host Cell Factors Involved in Filovirus Infection

“…It has been demonstrated that several small molecules that interfere with the late domain-mediated interactions between VP40 and host factors efficiently inhibit replication of a broad range of RNA viruses, including filoviruses in vitro [93,94]. Importantly, one of these molecules protected mice from lethal ebolavirus challenge [93].…”