Fig 1.
The workflow of the virtual screening protocol for screening of similar drugs to abacavir.
Fig 2.
Chemical structures for abacavir and the other seven drugs that were predicted to bind in the F-pocket of HLA-B*57:01.
Fig 3.
Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.
Specific peptides with a terminal valine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC50 values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig 4.
Effects of acyclovir (2 mg/mL) on the affinity of C-terminal residues for HLA-B*57:01.
Values are represented as geometric mean with 95% CI of the fold difference between vehicle/acyclovir treatment. The experiment was run 6 times with each run performed in triplicates. Analyzed for statistical significance by column statistics; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001). The most pronounced affinity increases for HLA-B*57:01 in the presence of 2 mg/mL of acyclovir were found for peptides with a cysteine, isoleucine and valine at the C-terminus.
Table 1.
Peptide affinity (nM) for HLA-B*57:01 in the presence of 2 mg/mL acyclovir.
Fig 5.
Effects of abacavir and acyclovir on HLA-B*57:01 binding specificity.
Specific peptides with a terminal isoleucine that showed an increased affinity for HLA-B*57:01 in the presence of abacavir were tested. Values are represented as geometric mean with 95% CI of two independent runs in triplicates, analyzed for statistical significance by Mann-Whitney U test comparing log IC50 values vs. vehicle; p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig 6.
Acyclovir and abacavir alter the binding specificity of HLA-B*57:01.
The peptide KAAKYRVSV was radiolabeled and tested for binding to HLA-B*57:01 in increasing doses of acyclovir and abacavir. Values are represented as geometric mean with 95% CI of four experimental runs in triplicates. Analysed by statistical significance by one-sided Mann Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 7.
PBMC from a healthy HLA-B*57:01 positive donor (Donor 1) where primed with abacavir at day 0, cultured for 14 days and then restimulated 1:10 with (A) HLA-B*57:01 single antigen line (C1R.B57), (B) with O/N abacavir treated C1R.B57 (C1R.B57.ABC) or (C) with O/N acyclovir treated C1R.B57 (C1R.B57.ACY).
Antigen activated cells were detected by ICS for IFN-γ production and CD8+/ IFN-γ T-cells quantitated using flow cytometry. (D) PBMC from two healthy HLA-B*57:01 positive donors were either primed with abacavir (ABC primed), primed with acyclovir (ACY primed) or had no treatment (Control. PBMC were cultured for 14 days and then stimulated 1:10 with treated and untreated single antigen line, C1R.B57, as indicated.