Exploring off-targets and off-systems for adverse drug reactions via chemical-protein interactome--clozapine-induced agranulocytosis as a case study

Abstract

In the era of personalized medical practice, understanding the genetic basis of patient-specific adverse drug reaction (ADR) is a major challenge. Clozapine provides effective treatments for schizophrenia but its usage is limited because of life-threatening agranulocytosis. A recent high impact study showed the necessity of moving clozapine to a first line drug, thus identifying the biomarkers for drug-induced agranulocytosis has become important. Here we report a methodology termed as antithesis chemical-protein interactome (CPI), which utilizes the docking method to mimic the differences in the drug-protein interactions across a panel of human proteins. Using this method, we identified HSPA1A, a known susceptibility gene for CIA, to be the off-target of clozapine. Furthermore, the mRNA expression of HSPA1A-related genes (off-target associated systems) was also found to be differentially expressed in clozapine treated leukemia cell line. Apart from identifying the CIA causal genes we identified several novel candidate genes which could be responsible for agranulocytosis. Proteins related to reactive oxygen clearance system, such as oxidoreductases and glutathione metabolite enzymes, were significantly enriched in the antithesis CPI. This methodology conducted a multi-dimensional analysis of drugs' perturbation to the biological system, investigating both the off-targets and the associated off-systems to explore the molecular basis of an adverse event or the new uses for old drugs.

Figure 1. Workflow of construction and mining of the binomial antithesis chemical-protein interactome (CPI).

(a) Binding conformations and raw docking scores were derived from the CPI with each column representing the drug molecule and each row representing the protein. (b) The 2DIZ transformation was applied to the CPI comprising 255 drugs and 410 protein pockets. (c) The OLZ and CLZ columns were extracted from the CPI where their Z′ score differences for each protein were measured by A-scores. The p values for each achieved A-score were calculated by simulating a random background. (d) Proteins were ranked according to their p values. In this case, Hsp70 was selected, proteins belonging to the same biological function (anti-apoptosis system or Hsp70's neighbor in HPRD network) were selected and then their expression changes in CLZ treatment were investigated (green bars indicated the rankings of the Hsp70 related genes when ordered by the change after CLZ treatment) and tested for significance by randomly selecting the same probe number in the genome background for permutation.

Figure 2. Structural comparison of clozapine and olanzapine towards HSP70 protein.

(a) The structural difference between CLA and OLZ. (b, c) Binding conformation of CLZ and OLZ towards the Hsp70 ligand binding pocket. The whole molecule of CLZ binds deep into the pocket, leaving the chlorine atom at the surface. However, the major part of the OLZ molecule is not accommodated in the deep pocket due to the steric hindrance of the methyl on the heterocycle of OLZ. The figures were drawn using PyMOL.

Figure 3. Site-moiety map analysis of the Hsp70 pocket.

(a) The van der Waals-interacting anchor site with three essential residues (R272, R342 and G339). (b) Percentages of the functional group among all docked drug molecules. The binding conformation of CLZ (c) and OLZ (d) towards this site. The molecule directions are also indicated in the 2D molecule structures at the top right corner of (c, d). Bottom left of (d) shows the direction of the OLZ as if it wants to interact using the same pattern as CLZ but significant steric hindrance makes insertion into the pocket in this way difficult.

Figure 4. Clozapine disturbance effect towards the Hsp70 systems.

Compared with the genome background, genes related to anti-apoptosis (a) or Hsp70's neighbor in HPRD network (c) were generally up regulated in CLZ treated HL60 cell lines, in terms of higher R′ value. The mean R′ of anti-apoptosis (b) or Hsp70's neighbor in HPRD network (d) related gene system was significantly higher than randomly selected genes in the genome background simulated by permutation test.

Figure 5. Off-targets and their off-systems' perturbation after clozapine treatment.

The off-targets, the genes involved in the PPI-based off-systems and the hub genes are in diamond, circle and hexagon shape, respectively. The PPI information from HPRD contains binary PPI and protein complex, and only the former information is visualized in this figure for brief. Red/green indicates the up-/down-regulation of the gene expression after clozapine treatment. Oxidoreductases and gluthathione metabolism related protein are in yellow and purple edges, respectively. The interaction between HSPA1A and NQO1 was highlighted in red line.