Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay

Abstract

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors. IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

The HSV-1 ubiquitin ligase ICP0: Modifying the cellular proteome to promote infection

“…Importantly, mutation of ICP0-UBE2 interaction residues inactivate ICP0 function ( Grant et al, 2012 ; Lium and Silverstein, 1997 ; Vanni et al, 2012 ), highlighting the importance of these host interactions in the biological lifecycle of HSV-1. As such, ICP0 represents an attractive drug target for the development of antiviral HSV-1 therapeutics ( Grant et al, 2012 ; Deschamps et al, 2019 ; Boutell and Davido, 2015 ). It remains to be determined if ICP0 can interact with other UBE2 enzymes that may facilitate the differential ubiquitination of substrates or formation of alternative chain types, for example N-terminal methionine-linked (Met1-linked) or K63-linked poly-ubiquitin chains ( Griffiths et al, 2013 ; Metzger et al, 2014 ).…”

The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection

“…3B, the ΔICP4 virus expressed high levels of ICP0 in order to complement the absence of ICP4, whereas in ΔICP27 virus-infected cells, ICP0 expression was reduced. These data prompted us to investigate whether ICP0 has a role in the down-modulation of p62/SQSTM1 because ICP0 is an E3 ubiquitin ligase that degrades hostile host factors (46,47). For this, we exposed HEL cells to two different doses (5 and 10 PFU/cell) of either HSV-1(F), an ICP0-null virus (ΔICP0), or an ICP0 E3 ubiquitin ligase (RING finger [RF]) mutant (with the C116A and C156A mutations) (47).…”

“Non-Essential” Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review

“…Other components of ND10s include the Sp100, Daxx, Mre 11, ATM, ATRX, p53, and others. ICP0 disrupts the ND10s by causing degradation of the different isoforms of PML, Sp100, and potentially of other proteins ( Figure 1 A) [ 34 , 59 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 ] Notably, several components of the ND10 bodies are interferon inducible genes, which underscores the synergy between gene silencing mechanisms and innate immunity in suppressing HSV-1 gene expression. ICP0-null viruses or E3 ubiquitin ligase mutants have viral DNA entrapped in PML-NBs at low MOI and display reduced transactivation activity and ability to block antiviral responses [ 34 , 64 , 78 , 79 , 80 , 81 , 82 , 83 ].…”

“…ICP0 E3 ligase-deficient viruses are hypersensitive to interferon, replicate poorly, and fail to reactivate efficiently from neuronal latency [ 25 , 55 , 67 , 68 , 69 , 70 , 71 ]. Based, on these observations, Dr. Kalamvoki’s group recently developed a high-throughput assay to screen for ICP0-E3 ubiquitin ligases inhibitors [ 72 ]. This assay is proximity based and takes advantage of the fact that ICP0 is autoubiquitinated and degraded during infection and that this ICP0 autoubiquitination can occur in vitro using the purified protein encoded by the exon II of ICP0 (contains the RF domain), UbcH5a, and Ub [ 60 , 73 , 74 ].…”

“…This assay is proximity based and takes advantage of the fact that ICP0 is autoubiquitinated and degraded during infection and that this ICP0 autoubiquitination can occur in vitro using the purified protein encoded by the exon II of ICP0 (contains the RF domain), UbcH5a, and Ub [ 60 , 73 , 74 ]. Screening a small compound library, Dr. Kalamvoki’s group identified potential scaffolds that can interfere with the ICP0 E3 ubiquitin ligase activity [ 72 ].…”