Kinase inhibitors inhibit FECH by blocking the protoporphyrin binding site

FECH is not a kinase and has no obvious nucleotide binding site. Hence the question arises as to where a small molecule kinase inhibitor binds the protein. First, I studied the PPIX binding site because binding of inhibitors in this area would completely shut down the catalytic activity of the enzyme. In a published crystal structure of FECH, the PPIX site is occupied by three cholate molecules²⁶⁴. The presence of cholate was found to be required in order to keep the purified protein soluble. A Kinobeads competition experiment using cholate and recombinant FECH R115L enzyme Figure 41: FECH activity for Vemurafenib, MK-2461, Linsitinib and Neratinib. a) Chromatograms for heme absorption at 400 nm for K-562 lysates. Differentiated K-562* cells (black line) contain high amounts of hemin, undifferentiated K-562 lysates spiked with 30 µM hemin (orange line) and lysates of HEK 293 (blue line) cells show that hemin can be detected in cells with active heme biosynthesis. b) Increasing Vemurafenib concentrations decrease the amount of heme in K-562 cells. c) 1 µM MK-2461, Linisitinib and Neratinib also decrease amount of heme in K-562 cells. d) Change in K562 cells heme content after 6 days of treatment with 1 µM MK-2461, Neratinib, Linsitinib and Vemurafenib relative to vehicle treated cells. e) Inhibition of recombinant FECH in vitro with increasing concentrations of Vemurafenib.

69 showed a decrease in binding at increasing concentrations of cholate (Figure 42a) indicating that kinobeads bind FECH via its active site.

Given that kinobeads comprise immobilized kinase inhibitors, the aforementioned kinase inhibitors that bind and/or inhibit FECH may act by the same mechanism. To follow up on this hypothesis, PPIX was immobilized onto sepharose beads and a competition pulldown experiment using Vemurafenib as the competitor was performed. FECH binding to the PPIX beads was monitored by Western Blotting and the results clearly show a dose dependent decrease of FECH binding to PPIX beads, demonstrating that Vemurafenib indeed binds to the PPIX site in FECH (Figure 42b). To evaluate the binding mode of further FECH inhibitors identified in this study, pulldowns from K-562 cell lysates were performed using PPIX beads in competition with the respective drugs (5 µM) or PPIX (5 µM) as positive and Dabrafenib (5 µM) as negative control. In line with the Kd values Figure 42: FECH binding of kinase inhibitors. (a) Increasing concentrations of cholate reduce binding of FECH to kinobeads. (b) Increasing Vemurafenib concentrations can compete FECH binding to PPIX immobilized on beads, demonstrating that Vemurafenib binds in the PPIX pocket of FECH. (c) Analogous binding site determination experiments for 18 kinase inhibitors in triplicates. PPIX is included as a positive control and Dabrafenib serves as a negative control. While Vemurafenib, CUDC-101, Cyc-116, Linsitinib, MK-2461, Neratinib, GSK-690693, Cabozantinib, Crenolanib, MK-8033 and GSK-1070916 show decreased binding of FECH to PPIX beads, Gefitinib, Alectinib, Axitinib, Nilotinib, Nintedanib and Momelotinib do not, implying the presence of an alternative binding site for these inhibitors on FECH. Error bars depict the standard error of the mean (SEM).

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obtained from Kinobeads experiments, Vemurafenib, CUDC 101, Cyc-116, Linsitinib, MK-2461, Neratinib, GSK-690693, and Crenolanib reduced FECH binding to the PPIX beads by 50% or more compared to the DMSO control. This data clearly indicate that the PPIX binding site of FECH can be targeted by kinase inhibitors (Figure 42c). However, the binding data were not entirely conclusive for the compounds Cabozantinib, MK-8033, GSK-1070916, Gefitinib, Alectinib, Axitinib, Nilotinib, Nintedanib and Momelotinib raising the possibility that there may be one or several other sites in the FECH structure to which these inhibitors may bind.

Unfortunately, co-crystallization FECH with kinase inhibitors failed since high cholate concentrations are required for protein stabilization and cholate molecules are direct competitors of the kinase inhibitors (see above). Therefore, docking studies were performed in collaboration with Bjoern-Oliver Gohlke (Structural Bioinformatics Group, Charité-Universitätsmedizin, Berlin, Germany)²⁶³ for all FECH inhibitors identified in the study based on the published crystal structure of FECH²⁰⁷ (PDB 3w1w). In this structure, three cholate molecules occupy the active site and one salicylic acid molecule is found in the dimer interface. Interestingly, for Vemurafenib, Neratinib, Linsitinib, MK-2461 and CUDC-101 up to three drug molecules could be docked into the active site akin to the three cholate molecules in the original structure (Figure 43a, b). For other kinase inhibitors, GSK1070916, Gefitinib, Alectinib, Axitinib, Nilotinib, Nintedanib and Momelotinib, no reasonable docking results could be obtained for the PPIX site, suggesting that these molecules may bind to an alternative site. The prime candidate for such a site is the dimerization region because active FECH is a dimer and the crystal structure of the dimer accommodates a salicylic acid molecule.

Figure 43: Docking studies using the crystal structure of FECH. (a) Structure of the FECH dimer with both the PPIX and dimerization sites occupied by Vemurafenib. Three Vemurafenib molecules can be placed into the PPIX binding site. The first molecule docked into the PPIX site is shown in gold, the second molecule when the first is already in place is shown in red and orange depicts the third and final molecule of Vemurafenib in the PPIX binding pocket (b) Detail of the PPIX binding site with three molecules of Vemurafenib stacked inside the pocket. (c) Detail of the dimerization site with Vemurafenib docked at the dimer interface.

71 Hence, additional docking studies for this dimerization site were performed (Figure 43c). Overall, FECH binders showed higher docking scores compared to those kinase inhibitors that showed no binding. Axitinib showed the highest docking score for the dimerization site and did not compete FECH binding in the PPIX pulldown assay indicating that it may indeed bind to the dimerization site.

Alectinib, Gefitinib, GSK-1070916, Nilotinib and Momelotinib also scored higher at the dimerization site than kinase inhibitors that showed no FECH binding raising the possibility that some molecules actually exert their FECH inhibitory effect by disrupting the dimerization site or inducing a conformational change that renders the enzyme inactive²⁰⁷. Docking data also indicate that some inhibitors, including MK-2461 or Cyc-116, appear to fit into both potential binding sites, possibly suggesting a mixed mode of FECH binding and inhibition. However, further biochemical and structural experimentation will be required to substantiate the significance of the presumed second binding site for kinase inhibitors on FECH.