Clinical implications of FECH inhibition

Biochemical follow-up studies could confirm FECH as a general off-target of kinase inhibitors.

Potential implications of FECH inhibition shall now be discussed in more detail.

The discovery that other kinase inhibitors can also inhibit FECH was unexpected and the fact that more than 10% of all clinically used kinase inhibitors show this off-target effect was even more surprising. This raises the question if FECH inhibition by kinase inhibitors is also of clinical relevance.

Partial FECH deficiency in humans leads to the development of a disease called erythropoietic protoporphyria (EPP)^{265, 266}, which is characterized by cutaneous signs of acute and painful photosensitivity^{267, 268}. Photosensitivity also occurs in at least 50% of all patients receiving Vemurafenib therapy^269-271, whereas photosensitivity is a much lesser issue in patients treated with Dabrafenib²⁷². The fact that Vemurafenib is a FECH inhibitor and Dabrafenib is not, makes FECH inhibition a strong candidate for a molecular mechanism explaining the photosensitive phenotype observed in Vemurafenib patients. It has been shown that decreased FECH activity leads to protoporphyrin accumulation in a number of tissues and cell types including erythrocytes^{265, 273}. Protoporphyrin IX is an endogenous photosensitizer as it absorbs light in a range from 320 to 595 nm and can induce the production of reactive oxygen species (ROS) that, in turn, may contribute to pro-inflammatory processes²⁷⁴. Vemurafenib patients also show elevated PPIX levels after UVA-radiation275, 276,277 further substantiating the link between FECH as an off-target and the clinical phenotype. Further evidence comes from the pharmacology of Vemurafenib. With a dose of 960 mg twice daily and a reported plasma concentration of AUC0-24 of 1741±639 µM x h²⁷⁰, the Vemurafenib concentration in the human body is certainly high enough to inhibit FECH completely and systemically. It is therefore not far-fetched to speculate that FECH inhibition by Vemurafenib is the molecular mechanism that explains the photosensitive phenotype in patients treated with this drug.

Based on this rational and the data presented above, photosensitivity due to FECH inhibition may also be a relevant issue for Cabozantinib for which plasma concentrations in the mid micromolar range have been reported²⁵⁷. Similar considerations may apply to Crenolanib²⁷⁸, Alectinib²⁷⁹, Axitinib²⁸⁰, or Nilotinib²⁸¹ for which photosensitivity has also been described as a clinical side effect.

For other kinase inhibitors such as Neratinib or MK-2461, FECH inhibition may be irrelevant as the clinical doses of these compounds do not reach the levels required for FECH inhibition in-vivo²⁸².

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Cyc-116 is currently the most potent FECH inhibitor (Kdapp= 0.7 µM) but no plasma concentrations have been reported so far and there are also no reports yet on clinical observations from a completed Phase I clinical trial. Notably, the photosensitizing effect of small molecule inhibitors can also be used for photodynamic therapy of cancer. For example, the heme precursor 5-aminolevulinic acid (ALA) is a commonly used prodrug to upregulate PPIX content in cancer cells.

Upon light excitation, PPIX forms reactive oxygen species that kill nearby tumor cells^{283, 284}. It has been shown that not only ALA treatment but also FECH inhibition leads to PPIX accumulation²⁸⁵. Therefore, on a more speculative note, a kinase inhibitor that is also a FECH inhibitor may be useful for photodynamic therapy, in case the kinase inhibitor is otherwise very well tolerated.

Since the photosensitive phenotype characteristic for patients with genetic FECH deficiency resembles that of Vemurafenib treated skin cancer patients, FECH inhibition by this drug is a likely molecular mechanism by which this toxicity occurs. Given that about 13% of all kinase inhibitors are also FECH inhibitors, it would seem prudent to consider including a FECH assay in the pre-clinical development of kinase inhibitor drugs in general.

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4 General Discussion

Molecular targeted therapies including small molecule kinase inhibitors have shown great success over the years. As these inhibitors are mostly directed towards the ATP-binding pocket of a kinase, they are not always only acting on their intended target. With the rising use of kinase screening panels, selectivity and off-targets of inhibitors can nowadays be assessed kinome-wide and in routine manner71, 72, 74, 75, 286, 287. However, the available assays often concentrate on recombinant protein kinases in an artificial environment. Kinobeads, a chemical proteomic technology, can be used to profile protein binding to these inhibitors in endogenous conditions without prior modification of the compound of interest. Using this setup, 242 inhibitors currently in clinical trials were profiled against the proteome of four cell lines. This enabled the identification of 221 kinases and 32 other nucleotide, FAD or heme binders as targets of small molecules.

The presented study is the most thorough, systematic selectivity evaluation of small molecule inhibitors currently evaluated in humans. Other large-scale screens profiled a smaller number of clinical molecules against over 400 recombinant kinases^{72, 74} or focused on large tool compound libraries²⁸⁷. Moreover, these studies mostly profiled one or two concentrations of a compound and determined remaining residual activity. The Kinobeads profiling was performed with eight concentrations of inhibitors, enabling the generation of dose-response curves, as well as EC50 and Kdapp values. This dose dependent evaluation has allowed to rank targets according to their affinity and to better estimate therapeutic windows in several applications. Furthermore, a dose response generates more solid target hypotheses than one single data point alone. As chemical proteomics assays measure mostly binding and generate target hypotheses, recombinant activity assays are needed to confirm these assumptions. Correlation of recombinant activity assays and binding assays is often poor due to screening activity on kinase domain level only²⁸⁸. The screen was performed in native lysate; hence the setup is closer to natural occurring protein abundances, their proteoforms and their influences on specific drug-protein interactions. The native biological settings are better represented than when inhibition is evaluated on proteins in isolation^{77, 90}. As shown for Dabrafenib, activity data confirmed the results of the binding assay. CDK2 binding of Dabrafenib was identified both in the Kinobeads assay and the KinomeScan profiling (Dataset 20131, LINCS²¹¹) and, so far, remains the only case, where binding seems not to be affecting activity.

The reason for this might be that CDK2, when paired with a cyclin to measure activity or performing its function in vivo, is not affected by Dabrafenib anymore.

Kinobeads profiling is, of course, dependent on the provided protein resource and the used immobilized compounds. The dataset is lacking information on kinases, which are not expressed in the used cell lines. As shown for MET- and EGFR-inhibitor profiling, additional cell lines or use of tissue can give further insights into the target space. Despite an already good kinome coverage with the current version of Kinobeads⁹² and a mix of four cell lines, some kinases like ALK, LRRK2, VEGFR or lipid kinases could not be enriched or competed in this screen. Further improvements in the design of such immobilized probes can help to overcome this issue. Mass spectrometric readout in the applied bottom-up approach also cannot determine the mutation status of the underlying protein²⁸⁹, thus, compounds that might specifically interact with mutant versions of one protein⁷³ cannot be distinguished in this setup. Hence, conclusions on inhibited mutants can only be inferred from RNAseq data for the respective cell line or tissue used. These shortcomings of proteomic technologies are compensated by other interesting discoveries not possible with recombinant

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kinases assays. Chemical proteomics and cellular thermal shift assays facilitate the detection of non-kinase off-targets. Using Kinobeads, other ATP-binding proteins can be profiled but also proteins completely lacking a respective ATP-binding site. Previous studies already described NQO2 as an off-target of kinase inhibitors91, 99, 290. With the screen, nine more inhibitors could be identified, binding to NQO2 with nanomolar affinity (30 inhibitors up to 1 µM). The physiological relevance of this common off-target remains unclear. It might provide a therapeutic benefit in some cases as NQO2 inhibition can lead to a reduction of NFκB activation²⁹¹. A more recent such case is Ferrochelatase which has been identified as an off-target of the approved BRAF inhibitor Vemurafenib¹¹⁰ that is used for the treatment of unresectable or metastatic melanoma carrying a BRAF(V600E) mutation²⁹². The present study has shown that a substantial number of clinically used kinase inhibitors (26 compounds) also target the enzyme Ferrochelatase (FECH). Biochemical and structural data revealed that several inhibitors bind to the protoporphyrin site in the enzyme whereas others potentially interact with the protein in the dimerization domain. FECH deficiency leads to erythropoietic protoporphyria, which is accompanied by severe photosensitivity^{267, 268}. Photosensitivity is also a side effect in Vemurafenib therapy^{276, 277}, thus FECH inhibition is likely the underlying molecular reason for this side effect. The photosensitivity phenotype can be prevented by application of sunscreen, the effect of protoporphyrin IX accumulation in lipid tissues, however, is not fully assessed yet²⁶⁵. As about 12% of all drugs in clinical trials target this protein, inclusion of potential FECH binding in the preclinical evaluation of a promising inhibitor candidate, therefore, seems rational to estimate off-target toxicities. Besides these two now well-characterized biomolecules, the screen also revealed further less studied non-kinase proteins to be targets of these compounds. Several kinase inhibitors, like Alisertib and Crizotinib, were potent binders of the Acetyl-CoA dehydrogenases ACAD10 and ACAD11. They might bind inhibitors via their FAD co-factor binding site. The information on ACAD function is sparse, but they presumably play a role in fatty-acid metabolism. ACAD11 has been shown to be involved in oxidative phosphorylation and tumor survival under conditions of glucose starvation. In this case, ACAD inhibition might provide therapeutic opportunities and a beneficial effect when targeted as an off-target during cancer therapy²⁹³.

Still, the majority of the 242 clinical inhibitors targeted mainly kinases. These 221 kinases identified in the drug screen represent more than 40% of the kinome that can be addressed by drugs, which is a considerable improvement to the known target space just a few years ago⁶¹. On the one hand, this highlights a Kinobeads point of view on the druggable kinome²³²; on the other hand, it enables improved characterization of potential off-targets and selectivity of these inhibitors.

The polypharmacology of inhibitors is of great interest during the drug development process. The analysis of inhibitors in clinical trials revealed no specific trend towards either more selective or less selective inhibitors. Benefits of one group over the other are still disputed. Selective inhibitors, so called ‘magic bullets’²⁹⁴, have shown potential, but also might lack efficacy in specific cancers if their target is not important for survival or if signaling can be reconstituted by other proteins. Reasonable selectivity has to be a criterion, when drugs are administered chronically or in other indications than cancer²³⁷. Some unselective inhibitors target a defined subset of kinases. These selectively unselective inhibitors, also referred to as ‘magic shotguns’²⁹⁴, are often more efficacious in clinical settings²⁹⁵. One reason could be that partial inhibition of multiple targets might be more effective than selective and complete inhibition of one target²⁹⁶. However, these multi-kinase inhibitors might lead to more side effects, which need to be considered in rational drug design and monitored in clinical trials. In fact, therapeutic effects in some cancers are dependent on targeting multiple

75 targets at once which could either be achieved by one multi-kinase inhibitor or a combination of several selective molecules^{35, 297}. Here, combinations of a variety of selective drugs seem to offer a more effective and flexible approach than the application of multi-kinase inhibitors. Despite successful results for selected examples like BRAF and MEK inhibitors, many combination therapies suffer from dosing errors and drug-drug interactions²⁹⁸.

Overall, targeted therapies require patient stratification based on the molecular tumor subtype to ensure treatment success with kinase inhibitors. These efforts are now increasing in personalized cancer therapy and medical oncology²⁹⁹. Advances in routine DNA or RNA sequencing of cancer cell lines or patient tissue revealed a huge variety of kinases mutated in tumors^{300, 301}.

Apart from known and well-studied proteins involved in disease, also previously uncharacterized proteins can be potential drug targets. How these kinases interfere with signaling and whether they might be a suitable therapeutic target is still a matter of ongoing research. Even with the rise of genome wide studies, most of protein research is still focused on the same 10% of proteins³⁰². Molecular tools like antibodies or small molecules are needed to study the function and implications of a protein. Each molecule needs to be thoroughly characterized to confirm, that it is suitable to be used as chemical probe against the intended target^{235, 303}. The results of the drug screen may help to identify potential inhibitors for kinases that have so far lacked interest in drug discovery. Furthermore, these structures can serve as scaffold for the generation of new inhibitors with increased selectivity for a kinase, also facilitating thorough evaluation in basic research. The drug screen already identified highly selective molecules (e.g. Lapatinib, Capmatinib or Rabusertib) that might be used as chemical probes for the investigation of their target proteins.

Another problem in targeting cancer with small molecules is tumor resistance. After a few months of treatment, the tumor often adopts to the treatment by mutation of the targeted protein such that the drug cannot bind anymore or by upregulation of an alternative pathway. For example, the BRAF(V600E) inhibitors Vemurafenib and Dabrafenib show very promising results, but after two to twelve months the tumor developed resistance against these inhibitors^{270, 304}. Elevated CRAF levels have been observed in some studies, indicating a circumvention of BRAF signaling³⁰⁵. Combination therapy with inhibitors targeting the downstream signaling node MEK has significantly prolonged survival and postponed tumor resistance²⁵⁰. Other known resistance mechanisms include higher levels of c-MET and EPHA2³⁰⁶ or mutations of the oncogene, like observed for BCR-ABL (e.g. T315I)⁵⁰ and EGFR (e.g. T790M)³⁰⁷ and lead to the administration of another drug. The screen can also identify potential useful combination treatments or alternative drugs to overcome resistance.

The molecular characterization of tumors also identifies certain drug targets involved in various entities. Here, polypharmacology of drugs can have advantage as an already well-known drug can also be of use in other indications. The additional use of Imatinib in gastro intestinal stromal tumors (GIST) due to its additional KIT-inhibition has paved the way for so-called kinase inhibitor repurposing^{43, 44}. Drug repositioning in general can reduce research and economic efforts that arise with the development of a new drug against a new target³⁰⁸. The compounds used in this study are all in clinical evaluation, making them easy candidates for repurposing. They have already been optimized in preclinical studies and have been tested for toxicity or are currently in such a phase I trial. Therefore, another phase 1 safety trials can be skipped and the drug can directly be evaluated for efficacy in a phase II trial. Often, many drugs are applied and evaluated in off-label uses by practicing physicians³⁰⁹.

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The potential of repositioning drugs for personalized therapy is already reflected in clinical trial design. Basket trials are based on molecular subtyping of cancers and patients are then grouped into the trial based on their genetic phenotype rather than their cancer entity^{310, 311}. This was firstly evaluated for Imatinib and patients expressing Imatinib sensitive targets³¹². Furthermore, patients with various cancers driven by a BRAF(V600E) mutation could be successfully treated with Vemurafenib³¹³. Of course, certain entities showed better response than others, but rare diseases with no further treatment option (e.g. Erdheim-Chester) showed promising response. This ‘basket’

design of clinical studies provides a good option for evaluation of molecularly targeted therapies and is rapidly adaptable for novel biomarkers. Several such trials are now active and investigate BRAF or MET inhibitors but also hedgehog inhibitors or PD-1 antagonists.

The resource presented here now provides further drug candidates and molecular rationales for repurposing drugs. This can be achieved by either using the drug against the same drug target in another disease setting or by using the drug because of an off-target effect. The screen provides opportunities for either ways. Ozanne et al. discovered that Dasatinib and Bosutinib inhibit SIK2 and thus induce anti-inflammatory macrophages. This effect could be of advantage in the treatment of chronic inflammatory diseases³¹⁴. The screen of 242 clinical inhibitors revealed 25 inhibitors for SIK2, which can now also be investigated in regard to inflammation. Another rising target of interest is NTRK1 (TrkA). Sequencing revealed a TPM3-NTRK1 fusion in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) patients as tumor driving mutation^{255, 256}. At the beginning of this project, no selective inhibitor was known to target NTRK1. Besides the known TrkA inhibitors Lestaurtinib and Entrectinib, selectivity profiling revealed 18 additional compounds that can bind to NTRK1 with nanomolar affinity. As these inhibitors are all targeting many other kinases as well, it might be more likely that they serve as starting point for medicinal chemistry approaches trying to develop selective NTRK1 inhibitors. Another promising example for drug repositioning is the here presented example of Cabozantinib. FLT3 inhibition was already described for Cabozantinib²⁵⁷ and potent FLT3 inhibition could be confirmed in the Kinobeads pulldown. FLT3 represents a therapeutic target in acute myeloid leukemia (AML)²⁵⁸ and FLT3 internal tandem duplications (ITD) were observed in patients³¹⁵. Treatment of AML is still lacking personalized medicine approaches and selective inhibition of FLT3-ITD might provide one therapeutic option. The convincing in vitro and in vivo data of a joint study conducted based on the results of this screen as well as other studies²⁶⁰ strongly supports a clinical evaluation of Cabozantinib in AML.

In summary, the target landscape of kinase inhibitors currently evaluated in clinical trials was successfully profiled using the Kinobeads technology. This work provides a rich resource of data describing the molecular landscape of clinically evaluated small molecule kinase inhibitors and targets thereof. Examples on how this information can be used were highlighted. Besides the aspects on selectivity, selected inhibitors and targets were further characterized. Noteworthy is the discovery of the non-kinase off-target FECH, whose inhibition most likely is one molecular reason for the observed side effect of photosensitivity. The most exciting aspect of the study is the demonstration that re-purposing kinase drugs is feasible and can be approached in a systematic fashion. In order to facilitate further exploitation of the data, the derived target profiling information will be made publicly available. It can help the community to improve the understanding of the mode-of-action of cancer drugs and aid in accelerating the development of novel therapeutic regimens.

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Abbreviations

ABPP activity based protein profiling ACN acetonitrile

CATDS concentration and target dependent selectivity

CETSA cellular thermal shift assay CEM chain ejection model

CID collision induced dissociation CK1 casein kinase group

CMGC cyclin dependent kinase, MAP kinase, GSK kinase, casein kinase 2 group

CML chronic myeloid leukemia

EC50 effective concentration for half maximal inhibition

iBAQ intensity based absolute quantification

IC50 inhibitory concentration for half maximal inhibition

IEM ion evaporation model ITD internal tandem duplication ITDR isothermal dose response

Kd dissociation constant

Kdapp apparent dissociation constant LC liquid chromatography

Kdapp apparent dissociation constant LC liquid chromatography