Km and Ki Dataset for Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

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Last modified date
  • July 8, 2022
Creator
  • Swanstrom, Ron
    • ORCID: 0000-0001-7777-0773
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
  • Spielvogel, Ean
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
Abstract
  • Ki values were determined against all UMASS inhibitors (Table 1). Brackets above the bars represent significant p-values between the two groups using the unpaired t-test. Data were pooled in different ways for the following analyses: A) The Ki values against the inhibitors with the larger R1-2 moiety (n=34) were more potent against resistant proteases compared to the inhibitors with the smaller R1-1 moiety (n=35) B) Ki values for the inhibitors with the R2-2 group (n=10) showed a trend toward being more potent against the highly mutated proteases compared to the other R2 groups (n=40). C) Ki values for enzymes with the I50V mutation (n=33) showed greater resistance to the inhibitors compared to enzymes with the I84V mutation (n=22). D) The R1-2 moiety provided increased potency to enzymes with the I84V mutation (n=20) but not the I50V mutation (n=20). E) The R2-2 moiety was more potent against the enzymes with I84V mutation (n=10) compared to the enzymes with the I50V mutation (n=10). The unpaired t-test was used to assess differences in Ki values.
Methodology
  • Km Assay. Km values were determined as previously described (Henes et al., 2019; Lockbaum et al., 2019; Matayoshi et al., 1990; Windsor and Raines, 2015). Briefly, a 10-amino acid substrate containing the natural MA/CA cleavage site with an EDANS/DABCYL FRET pair was dissolved in 8% DMSO at 40 nM and 6% DMSO at 30 nM. The 30 nM substrate was 4/5 serially diluted from 30 nM to 6 nM. HIV-1 protease was diluted to 120 nM and, and 5 µl were added to the 96-well plate to obtain a final concentration of 10 nM. Fluorescence was observed using a PerkinElmer Envision plate reader with an excitation at 340 nm and emission at 492 nm, and monitored for 200 counts. A FRET inner filter effect correction was applied as previously described (Liu et al., 1999). Data corrected for the inner filter effect was analyzed with Prism7. Datasets were uploaded to the Carolina Digital Repository. Ki Assay. Enzyme inhibition constants (Ki values) were determined as previously described (Henes et al., 2019; Lockbaum et al., 2019; Matayoshi et al., 1990; Windsor and Raines, 2015). Briefly, in a 96-well plate, inhibitors were serially diluted down from 2000-10,000 nM depending on protease resistance. All samples were incubated with 5 nM protein for 1 hour. A 10-amino acid substrate containing an optimized protease cleavage site (Windsor and Raines, 2015), purchased from Bachem, with an EDANS/DABCYL FRET pair was dissolved in 4% DMSO at 120 µM. Using a PerkinElmer Envision plate reader, 5 µl of the 120 µM substrate were added to the 96-well plate to a final concentration of 10 µM. Fluorescence was observed with an excitation at 340 nm and emission at 492 nm and monitored for 200 counts. Data was analyzed with Prism7. Datasets were uploaded to the Carolina Digital Repository.
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Kind of data
  • Other
Resource type
  • Dataset
Rights statement
  • In Copyright
License
  • CC0 1.0 Universal
Language
  • English

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