Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors

Abstract

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

Proteome-wide Profiling of Clinical PARP Inhibitors Reveals Compound-Specific Secondary Targets

“…Whereas CIT was assigned a high SAINT score and displayed a high fold change, its large size of 230 kDa and the relatively few peptides identified for this protein (Table S1) suggest that CIT is likely not a strong niraparib interactor. The mitochondrial enzyme FECH was recently found to bind to many small molecule kinase inhibitors (Klaeger et al, 2016; Savitski et al, 2014) and was observed in our data with niraparib, but, similarly to CIT, showed only low spectral counts (Table S1). The most prominent unique niraparib candidate identified was DCK, which is responsible for the monophosphorylation of deoxycytidine, deoxyadenosine, and deoxyguanosine in the rate-limiting step of the nucleotide salvage pathway.…”

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

“…The increasing power and adoption of ABPP is revealing the potential for off-target pharmacology with current drugs and chemical tools. For example, a recent report highlights a chemical proteomics approach that revealed the enzyme ferrochelatase (FECH) as a surprisingly common off-target of kinase inhibitors; notably, 29 of the 226 clinical kinase inhibitors tested, including approved drugs vemurafenib and neratinib, bind to the protoporphyrin pocket of FECH at low- or sub-micromolar concentrations (Klaeger et al., 2016); this is known as “pharmacophore crossing” (see Box 1). …”

Chemoproteomics‐Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors

“…Cell lines were kindly provided by the NCI‐Frederick Cancer DCTD Tumor/Cell line repository, were not found in the database of commonly misidentified cell lines (ICLAC) and were tested negatively for mycoplasm contamination. Cell lysis and Bradford assay were performed as described previously . Kinase affinity matrices were prepared in house as published elsewhere …”

“…Cell lysis and Bradford assay were performed as described previously . Kinase affinity matrices were prepared in house as published elsewhere …”

“…We systematically employed chemical proteomics alongside the medicinal chemistry campaign to profile the potency and selectivity of each and every synthesized molecule in lysates of cancer cells. We hypothesized that this new methodology, which can be described as “chemoproteomics‐aided drug discovery”, could facilitate lead selection and lead optimization by establishing, in a timely manner, the affinity of the molecules for around 250 native kinases and many other ATP binding proteins as well as potentially toxic off‐targets . This synchronous approach indeed provided valuable guidance during the drug‐discovery process and protein crystallography added detailed molecular insight into drug–protein interactions and structure–affinity relationships at the atomic level.…”