Project PXD014346

Summary

Title

Small Molecule Degraders of the Hepatitis C Virus Protease Reduce Susceptibility to Resistance Mutations

Description

Targeted protein degradation (TPD) has emerged as a promising new drug development paradigm. We leveraged this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a ‘reversible-covalent’ inhibitor that binds to the hepatitis C virus (HCV) protease active site was conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. We developed an optimized degrader, DGY-08-097, that potently inhibits HCV in a cellular infection model and demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.

Sample Processing Protocol

Stable cells expressing the HCV NS3-eGFP protein fusion and the mCherry reporter were seeded at a density of 5 x 106 cells/well in a T75 flask. Expression of the fusion protein was induced for 24 hours using 1 g/mL tetracycline (MilliporeSigma T3383). Cells were treated in biological triplicates for 4 hours with DMSO, DGY-08-097 (1 mM) or DGY-08-097-BUMP (1 mM). The cells were lifted with Versene (Thermo Fisher Scientific 15040-066), washed with PBS twice, and pellets were snap-frozen at -80°C until processed. Lysis buffer (8 M urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, supplemented with protease and phosphatase inhibitors (Roche)) was added to the cell pellets, which were then homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 – 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample was reduced and alkylated as previously described{An, 2017 #49}. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37°C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 10-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. The sample channels were combined at a 1:1:1:1:1:1:1:1:1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid, and acidified (pH 2−3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (Phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions, and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on an EasySpray ES803 75 μm inner diameter microcapillary column (Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6–27% acetonitrile in 1.0% formic acid with a flow rate of 350 nL/min. Each analysis used a MS3-based TMT method as described previously{McAlister, 2014 #51}. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data-dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 55%, AGC target set to 1.5 x 105 and a maximum injection time of 150 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 1.5 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Data Processing Protocol

Proteome Discoverer 2.2 (Thermo Fisher Scientific) was used for .RAW file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot human database (September 2016) containing the HCV NS3 protein sequence with both the forward and reverse sequences. Database search criteria are as follows: tryptic with two missed cleavages, a precursor mass tolerance of 20 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of cysteine (57.02146 Da), static TMT labelling of lysine residues and N-termini of peptides (229.16293 Da), and variable oxidation of methionine (15.99491 Da). TMT reporter ion intensities were measured using a 0.003 Da window around the theoretical m/z for each reporter ion in the MS3 scan. Peptide spectral matches with poor quality MS3 spectra were excluded from quantitation (summed signal-to-noise across 10 channels < 200 and precursor isolation specificity < 0.5), and resulting data was filtered to only include proteins that had a minimum of 2 unique peptides identified. Reporter ion intensities were normalised and scaled using in-house scripts in the R framework{, 2017 #52}. Statistical analysis was carried out using the limma package within the R framework{Ritchie, 2015 #50}.

Contact

Eric Fischer, Dana-Farber Cancer Institute
Eric Fischer, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA., Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA ( lab head )

Submission Date

24/06/2019

Publication Date

03/07/2019

Publication

    de Wispelaere M, Du G, Donovan KA, Zhang T, Eleuteri NA, Yuan JC, Kalabathula J, Nowak RP, Fischer ES, Gray NS, Yang PL. Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations. Nat Commun. 2019 10(1):3468 PubMed: 31371704