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Significance The pharmaceutical targeting of mutated BRAF has shown promising clinical outcomes in patients with melanoma. However, more than 300 reported BRAF patient mutations and the occurrence of a kinase-drug resistance mechanism hamper patient-oriented therapies. We have developed an extendable and cell-based kinase conformation reporter platform (KinCon) to predict and compare the effect of kinase drugs. We systematically evaluated drug efficacies by assessing KinCon reporter dynamics in response to different drugs and patient mutations. Our findings suggest that FDA-approved melanoma inhibitors may have the potential to block non-V600E–mutated BRAF activities also in non–small-cell lung cancers (NSCLC). We assume that widespread analyses of KinCon:drug interactions may assist in the future in identifying patient mutation–specific and thus more effective kinase inhibitors., Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non–small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein–protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Live-cell recordings of protein conformations and interactions yield novel mechanistic insights into physiologic and pathologic protein functions. We have developed an adaptable and genetically encoded bioluminescence-based biosensor platform to precisely measure signaling dynamics of full-length RAS GTPases and RAF kinases upon physiologic pathway activation, post-translational modifications, mutagenesis, and drug binding. We have recorded the impact of both patient mutations and bioactive small-molecule binding on kinase conformations and binary interactions of GTP-loaded H, N, KRAS with kinases such as RAF. Systematic profiling of αC-helix-OUT BRAF inhibitors (BRAFi) vemurafenib, dabrafenib, encorafenib, and PLX8394 using the BRAF reporter reporter platform (displaying 10 different patient mutations) revealed differences in drug specificities and efficacies. Unexpectedly, the αC-helix-OUT BRAFi engagement with the catalytic pocket of mutated BRAF stabilized an inactive and intermediate full-length kinase conformation, which provoked the enhancement of binary RAS:RAF interactions. We showed that the interference with RAS interactions and nanoclustering has the potential to antagonize the sequential formation of drug-induced tetrameric RAS:RAF complexes. Recently, we have bioengineered a reporter toolbox for direct in vivo recordings of RAS activity conformations. First, we demonstrated that the wild-type RAS reporter—expressed at levels far below endogenous RAS proteins—responds to physiologic pathway activation. Second, we showed that hot-spot RAS mutations display a similar effect on the intramolecular dynamics. The systematic implementation, combination, and advancement of the GTPase and kinase reporter platform have the potential to be used to identify novel means for regulating or perturbing RAS-RAF-ERK signaling in vivo. Citation Format: Ruth Röck, Johanna Mayrhofer, Omar Torres-Quesada, Florian Enzler, Jakob Troppmair, Eduard Stefan. Surveillance of RAS-RAF dynamics in vivo: Tracking activity conformations and drug-induced interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B45.

Patient mutation–specific BRAF inhibitors alter full-length kinase conformations and promote interactions with GTP-loaded RAS., Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation–containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

BACKGROUND Emergency departments may be a useful information source to describe the demographics and clinical characteristics of patients with acute cocaine-related medical problems. We therefore conducted a retrospective analysis of 165 acute, laboratory-confirmed cocaine intoxications admitted to an urban emergency department in Switzerland between January 2007 and March 2011. RESULTS A total of 165 patients with a mean age of 32 years were included. Most patients were male (73%) and unemployed (65%). Only a minority (16%) had abused cocaine alone while 84% of the patients had used at least one additional substance, most commonly ethanol (41%), opioids (38%), or cannabis (36%) as confirmed by their detection in blood samples. The most frequently reported symptoms were chest pain (21%), palpitations (19%), anxiety (36%) and restlessness (36%). Psychiatric symptoms were present in 64%. Hypertension and tachycardia were observed in 53% and 44% of the patients, respectively. Severe poisonings only occurred in patients with multiple substance intoxication (15%). Severe intoxications were non-significantly more frequent with injected drug use compared to nasal, oral, or inhalational drug use. Severe complications included acute myocardial infarction (2 cases), stroke (one case), and seizures (3 cases). Most patients (75%) were discharged home within 24 h after admission. A psychiatric evaluation in the ED was performed in 24% of the patients and 19% were referred to a psychiatric clinic. CONCLUSIONS Patients with acute cocaine intoxication often used cocaine together with ethanol and opioids and presented with sympathomimetic toxicity and/or psychiatric disorders. Severe acute toxicity was more frequent with multiple substance use. Toxicity was typically short-lasting but psychiatric evaluation and referral was often needed.