Carbamazepine-Mediated Adverse Drug Reactions: CBZ-10,11-epoxide but Not Carbamazepine Induces the Alteration of Peptides Presented by HLA-B∗15:02

Abstract

Among patients treated with the anticonvulsive and psychotropic drug carbamazepine (CBZ), approximately 10% develop severe and life-threatening adverse drug reactions. These immunological conditions are resolved upon withdrawal of the medicament, suggesting that the drug does not manifest in the body in long term. The HLA allele B∗15:02 has been described to be a genomic biomarker for CBZ-mediated immune reactions. It is not well understood if the immune reactions are triggered by the original drug or by its metabolite carbamazepine-10,11-epoxide (EPX) and how the interaction between the drug and the distinct HLA molecule occurs. Genetically engineered human B-lymphoblastoid cells expressing soluble HLA-B∗15:02 molecules were treated with the drug or its metabolite. Functional pHLA complexes were purified; peptides were eluted and sequenced. Applying mass spectrometric analysis, CBZ and EPX were monitored by analyzing the heavy chain and peptide fractions separately for the presence of the drug. This method enabled the detection of the drug in a biological situation post-pHLA assembly. Both drugs were bound to the HLA-B∗15:02 heavy chain; however, solely EPX altered the peptide-binding motif of B∗15:02-restricted peptides. This observation could be explained through structural insight; EPX binds to the peptide-binding region and alters the biochemical features of the F pocket and thus the peptide motif. Understanding the nature of immunogenic interactions between CBZ and EPX with the HLA immune complex will guide towards effective and safe medications.

Figure 1

Characteristics of peptides presented by HLA-B∗15:02. (a) Length distribution of low and high binding peptides presented by HLA-B∗15:02. (b) Length distribution of peptides presented by HLA-B∗15:02 without treatment (blue), after incubation with CBZ (red) and EPX (green). (c) Preferred AAs of low binding peptides presented by HLA-B∗15:02. (d) Preferred AAs of high binding peptides presented by HLA-B∗15:02. All AAs with a frequency higher than 15% (dashed line) are given for position p1-p3, p5, and pΩ. AAs are distinguished into polar positive (blue), polar neutral (green), nonpolar aliphatic (grey), and nonpolar aromatic (purple) AAs.

Figure 2

CBZ and EPX bind to the HLA heavy chain and not to the peptides. sB∗15:02 molecules were produced in the HLA class I-negative LCL721.221 cell line without treatment and in the presence of CBZ or EPX and purified via affinity chromatography. The amount of the drugs was measured mass spectrometrically (CBZ shown as ★, EPX shown as ◆). The threshold for drug detections is represented by a dashed line. The trimeric complexes (a) were analyzed as well as retentate and peptide fractions after separation of low (b) and high binding peptides (c). The amount of drug measured in the HLA heavy-chain fraction is displayed in black; the amount measured in the peptide fraction is shown in grey.

Figure 3

Alteration of peptides bound to B∗15:02/CBZ or B∗15:02/EPX complexes. The alterations of AA frequency after treatment with CBZ (red) and EPX (green) are depicted at p1 (a), p2 (b), p3 (c), and pΩ (d). The graphs for CBZ and EPX were adjusted to the data of AA frequencies obtained without drug treatment (black). The data have been obtained from triplicate experiments.

Figure 4

Deamidation at p4 is altered after treatment with EPX for B∗15:02. (a) Given is the amount of modified peptides presented by B∗15:02 w/o treatment (blue), after treatment with CBZ (red) and EPX (green). (b) Frequency of deamidation of modified peptides derived from B∗15:02.

Figure 5

Modelling of peptides bound to empty and EPX bound HLA∗B15:02. The homology model of HLA-B∗15:02 (grey) is based on the structure of HLA-B∗15:01 (1XR8). The peptide SQATPHSSY (blue) could be solely detected without treatment, whereas following treatment with EPX (green), the peptide VSQqKLQAEAQ (orange) was detected. (a) Top view on the PBR of the pHLA complex in the absence and presence of EPX. (b) Side view on the PBR, the alpha helix of the alpha 2 domain is not shown. (c) Surface model of peptides and EPX binding to the F pocket (colored brownish). The AAs interacting with the peptide or the drug via hydrogen bridge bonds are depicted. (d) The oxygen atom in the carboxamide moiety of EPX interacts with two AAs of the F pocket.