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High-throughout screening assay for identification of several high-risk HPV inhibitors

Icosagen has developed a specific 3D cell-based assay to identify human papillomavirus (HPV) DNA replication inhibitors in high-throughput format. In a proof of concept (PoC) study, 5 high-risk HPV specific inhibitors were identified that influence Tdp1 dependent DNA repair.

There are number of systems for conducting high-throughput screening (HTS) of available chemical libraries is a widely used technique to identify new inhibitors of various diseases. Most of the work has been done in human primary epithelial keratinocytes. However, using these cells is relatively time consuming and expensive, especially for high-throughput screening.

In this assay, a U2OS-based model system with dual-luciferase system was used to measure cell growth and toxicity of the compounds and HPV genome replication. U2OS cells which are derived from moderately differentiated osteosarcoma, have an adherent eptihelial morphology and carry wild-type p53 and pRB genes that have proven to be useful for studying various aspects of HPV life cycle.

Introduction

Several types of human papillomaviruses (HPVs) may induce transformation of infected cells. Two main genera of HPVs are classified as mucosotropic α and cutaneous β HPVs, both including high-risk cancer-associated species. Regardless of being extensively studied for decades, treatment for HPV-related infections/diseases remain challenging. Three vaccines against clinically relevant HPV types have been developed with their efficacy approved. However, various social, economic and religious concerns limit their effect. Moreover, as the vaccines are not effective against established infection, it needs to administered at a very early age. Therefore no effective and specific cure against an ongoing HPV infection has not yet been developed.

The absence of antiviral drugs has driven investigations into the details of the molecular mechanisms of the HPV life cycle, which is tightly linked to epithelial differentation. Viral helicase E1 and transcription factor E2 are phosporoteins, which play a pivotal role in the viral life cycle. For effective HPV genome replication, two viral proteins E1 and E2 form a complex. This protein-protein interaction has been an attractive target for developing anti-HPV drugs.

In addition to targeting HPV proteins, the viral genome itself could be target as well. Several sequence-specific DNA binding compounds have been developed, that bind to AT-rich regions near the E1 and E2 binding sites in the HPV replication origin and effictively reduce the stably maintained episomal viral genomes.

There are number of systems for conducting high-throughput screening (HTS) of available chemical libraries that is a widely used technique to identify new inhibitors of various diseases. Most of the work has been done in human primary epithelial keratinocytes. However, using these cells is relatively time consuming and expensive, especially for high-throughput screening.

Cell-based assay for high-throughput-screen

1. Develop assay system for HT-screen
In Icosagen's U2OS cell-based assay, the dual-luciferase system is used to measure simultaneously:
  • Cell growth (reflecting the cytotoxicity of the compounds)
  • HPV genome replication intensity
U2OS cells, which are derived from moderately differentiated osteosarcoma, have an adherent epithelial morphology and they carry wild-type p53 and pRB genes that are useful for studying various aspects of HPV life cycle.
 
This proprietary system enables to study all three replication stages of HPVs. The viral replication mechanism itself as well as the replication intermediates of HPV genome that are similar to keratinocyte cell-lines. The gene expression of HPVs in U2OS cells is almost identical to the keratinocytes, making it suitable for identification of new anti-HPV drugs.
 
2. Generation of HPV marker genomes
We generated HPV 18, HPV 16 and HPV 5 marker genomes containing reporter genes for rapid and easy quantification of viral copy number.
 
3. Pilot study — screening of NCI Diversity Set IV Library

Icosagen model system was used to screen NCI Diversity Set IV library, which consists of different classes of biologically active compounds. 5 compounds were identified that inhibited HPV18 initial amplification in low-micro molar range. None of the identified compounds inhibit E1 and E2 dependent URR replication. Besides initial amplification four out of five compounds successfully inhibited stable maintenance phase of the viral replication. In addition three compounds inhibited vegetative amplification, which takes place in highly differentiated upper epithelia.

These inhibitors or their analogues are therefore capable of eliminating different stages of HPV infection. The identified compounds do not inhibit E1 and E2 dependent URR replication, indicating that different cellular proteins could be used to facilitate HPV genome replication and/or that the replication mechanism of viral genome differs from the URR plasmid replication.

Results

  • We developed universal assay system for HT-screening of HPV inhibitor
 
  • We identified 5 high risk HPV specific inhibitors with IC50 in high nanomolar to low micromolar range
 
  • We identified Tdp1 and PARP1 as suitable drug targets

Regardless of the three existing vaccines,
there is need for antivirals against HPV infection because
vaccines are only preventive and other types of therapies
have proven to be unsuccesful.

Patents

1.   Title: Method and kit for identifying compounds capable of inhibiting human papillomavirus replication
      International Application No. PCT/EE2010/000010
 
2.   Title: Assay system to identify HPV replication inhibitors in HT-screen
      International Application No.PCT/EP20/057898

This assay is available for licensing

For more information, please contact sales@icosagen.com

Scientific publication

1 Toots M, et al. (2017) Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay. PLoS Pathog 13(2): e1006168.

Read the paper here

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