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Updated: May 26, 2025

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication
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A robust fluorogenic substrate for chikungunya virus protease (nsP2) activity.

Sparsh Makhaik1,2, Wioletta Rut3, Shruti Choudhary4

  • 1Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA.

Protein Science : a Publication of the Protein Society
|February 21, 2025
PubMed
Summary
This summary is machine-generated.

A new fluorogenic substrate, acc-CHIK15-dnp, enhances high-throughput screening for chikungunya virus protease (CHIKVP) inhibitors. This tool offers a 30-fold improved signal-to-noise ratio for drug development against chikungunya virus (CHIKV).

Keywords:
FRET protease reporteralphavirus proteaseamino carbamoyl coumarin FRET donorcysteine proteasedinitrophenyl quencherfluorescent substrate design principlesfluorogenic substratepolyprotein junction mimicproteasevirus

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Area of Science:

  • Virology
  • Biochemistry
  • Drug Discovery

Background:

  • Chikungunya virus (CHIKV) is an emerging pathogen causing fever, arthralgia, and myalgia, with no specific antiviral treatments available.
  • The chikungunya virus protease (CHIKVP) is essential for viral replication and a key target for antiviral drug development.
  • Existing methods for screening CHIKVP inhibitors are limited, particularly those reflecting critical polyprotein cleavage sites.

Purpose of the Study:

  • To develop a robust fluorogenic substrate for efficient screening of CHIKVP inhibitors.
  • To investigate the substrate length requirements for CHIKVP recognition.
  • To establish a superior tool for high-throughput screening (HTS) of small molecules targeting CHIKV.

Main Methods:

  • Design and synthesis of a novel fluorogenic substrate, acc-CHIK15-dnp, utilizing a 15-mer peptide from the CHIKV nsP3/4 junction with an ACC-Lys(dnp) donor-quencher pair.
  • Comparison of the signal-to-noise ratio of acc-CHIK15-dnp with the previously reported edab8 substrate.
  • Testing the substrate's specificity against CHIKVP and other alphavirus proteases.

Main Results:

  • The new substrate, acc-CHIK15-dnp, demonstrated a 30-fold improvement in signal-to-noise ratio compared to the edab8 substrate.
  • acc-CHIK15-dnp was specifically recognized by CHIKVP, not by other alphavirus proteases, highlighting the importance of P-side residues for cleavage.
  • The improved substrate enabled significantly enhanced small molecule HTS for CHIKV inhibitors.

Conclusions:

  • The acc-CHIK15-dnp substrate is a highly effective and specific tool for CHIKVP inhibition screening.
  • This advancement facilitates improved drug discovery efforts for chikungunya virus infections.
  • The findings suggest that P-side residues play a critical role in CHIKVP substrate recognition.