No abstract

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein generating antigenic determinants for T-cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant antigens to functional immune responses is lacking. The aim of this study was to develop an integrated approach using both animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship between adduct formation, cell death, co-stimulatory signalling and stimulation of a T-cell response. Formation of SMX-derived adducts in antigen presenting cells was dose- and time-dependent, detectable at non-toxic concentrations and dependent on drug metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell co-stimulatory molecule expression and cytokine secretion. Antigen presenting cells cultured with SMX for 16h, the time needed for drug metabolism, stimulated T-cells from sensitized mice and lymphocytes and T-cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T-cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T-cells were stimulated with adducts derived from SMX metabolism in antigen presenting cells, not the parent drug. This study shows that antigen presenting cells metabolize SMX; subsequent protein binding generates a functional T-cell antigen. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.