Photopharmacological Applications for Cherenkov Radiation Generated by Clinically Used Radionuclides

Translational photopharmacological applications are limited through irradiation by light showing wavelengths within the bio-optical window. To achieve sufficient tissue penetration, using wavelengths >500 nm is mandatory. Nevertheless, the majority of photopharmacological compounds respond to irradiation with more energetic UV light, which shows only a minor depth of tissue penetration in the µm range. Thus, we became interested in UV light containing Cherenkov radiation (CR) induced as a by-product by clinically employed radionuclides labeling specific tissues. Therefore, CR may be applicable in novel photopharmacological approaches. To provide evidence for the hypothesis, we verified the clinically established radionuclides 68Ga and 90Y but not 18F in clinically used activities to be capable of generating CR in aqueous solutions. We then investigated whether the generated CR was able to photoactivate the caged kinase inhibitor cagedAZD5438 as a photoresponsive model system. Herein, 21% uncaging of the model system cagedAZD5438 occurred by incubation with 90Y, along with a non-specific compound decomposition for 68Ga and partly for 90Y. The findings suggest that the combination of a clinically employed radionuclide with an optimized photoresponsive agent could be beneficial for highly focused photopharmacological therapies

Photoswitchable Azo- and Diazocine-Functionalized Derivatives of the VEGFR-2 Inhibitor Axitinib

In this study, we aimed at the application of the concept of photopharmacology to the approved vascular endothelial growth factor receptor (VEGFR)-2 kinase inhibitor axitinib. In a previous study, we found out that the photoisomerization of axitinib's stilbene-like double bond is unidirectional in aqueous solution due to a competing irreversible [2+2]-cycloaddition. Therefore, we next set out to azologize axitinib by means of incorporating azobenzenes as well as diazocine moieties as photoresponsive elements. Conceptually, diazocines (bridged azobenzenes) show favorable photoswitching properties compared to standard azobenzenes because the thermodynamically stable Z-isomer usually is bioinactive, and back isomerization from the bioactive E-isomer occurs thermally. Here, we report on the development of different sulfur-diazocines and carbon-diazocines attached to the axitinib pharmacophore that allow switching the VEGFR-2 activity reversibly. For the best sulfur-diazocine, we could verify in a VEGFR-2 kinase assay that the Z-isomer is biologically inactive (IC50 >> 10,000 nM), while significant VEGFR-2 inhibition can be observed after irradiation with blue light (405 nm), resulting in an IC50 value of 214 nM. In summary, we could successfully develop reversibly photoswitchable kinase inhibitors that exhibit more than 40-fold differences in biological activities upon irradiation. Moreover, we demonstrate the potential advantage of diazocine photoswitches over standard azobenzenes

Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKI alpha activity and circadian rhythms

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase I alpha (CKI alpha) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKI alpha activity together with the accompanying modulation of cellular circadian rhythms in which CKI alpha is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications

Light-controlled inhibition of BRAFV600E kinase

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues

Prospects and limitations of photopharmacological conepts for kinase inhibitors

Die Photopharmakologie bietet die Möglichkeit, Wirkungen von Arzneistoffen räumlich und zeitlich mit Licht zu steuern. Bisher kann auf verschiedene Konzepte wie Photoschaltung, Photoabspaltung oder die photodynamische Therapie zurückgegriffen werden, um photoresponsive Arzneistoffe zu entwickeln. Kinasen eignen sich sehr gut als Zielprotein in diesem Zusammenhang, da sie eine Schlüsselrolle in der menschlichen Zelle übernehmen und so z.B. die Regulierung von Wachstum und Differenzierung steuern. Eine unkontrollierte Überaktivierung von Kinasen kann zu verschiedenen Krebsarten führen, welche mit Kinaseinhibitoren wiederum therapiert werden können. Die Kombination von Kinaseinhibitoren mit photopharmakologischen Konzepten (vorranging Photoschaltung und Photoabspaltung) könnte die Wirksamkeit der konventionellen Behandlung erhöhen, bei gleichzeitiger Reduzierung systemischer Nebenwirkungen. Diese Arbeit zielt auf eine Weiterentwicklung bestehender Photopharmakologiekonzepte für Kinaseinhibitoren ab, um die mögliche Anwendung in der Klinik voranzutreiben. Außerdem wurden die entwickelten photoschaltbaren Kinaseinhibitoren in einem Kinom-Aktivitäts-Profiling untersucht, um den Einfluss der Wirkstoffe auf das gesamte Kinasenetzwerk zu ermitteln. Die synthetisierten photoresponsiven Inhibitoren wurden photochemisch charakterisiert und anschließend in Form von Kinaseassays, Kinom-Profiling und Zellproliferationsassays biologisch getestet. Im ersten Teil der Arbeit wurden mehrere Photoschalter biologisch evaluiert. Für den zugelassenen Kinaseinhibitor Axitinib konnte eine isomerenabhängige biologische Wirkung (auf VEGFR2, auf HUVEC und im Kinom-Profiling) gezeigt werden. Jedoch ließ sich das Stilben nicht reversibel in wässrigen Medien schalten. Die darauffolgende Funktionalisierung mit Diazocinen vereinte optimierte photochemische Parameter mit einem ausreichenden pharmakologischen Wirkunterschied. Die eingeschränkte Wirksamkeit eines photoschaltbaren BRAFV600E-Inhibitors durch Kinomüberaktiverung konnte durch das Kinom-Profiling erklärt werden. Während ein Kinom-Profiling der Chronoschalter nicht gelang, konnte ein deutlicher Wirkunterschied im Profiling der RET-Stilben-Schalter festgestellt werden. Die im zweiten Teil untersuchten Photoschutzgruppen DMNB und BODIPY für CDK2-Inhibitoren führten unter Bestrahlung entweder zur erwarteten Abspaltung und CDK2-Hemmung (DMNB) oder zu erheblichen photodynamischen Effekten (BODIPY). Die untersuchten Verbindungen leisten einen wichtigen Beitrag für die (Weiter-)Entwicklung photoresponsiver Kinaseinhibitoren. Weiterführende Studien im Tiermaßstab erscheinen so wahrscheinlicher (Photoschalter) oder sollten als nächstes durchgeführt werden (Photoabspaltung). Weiterhin wird eine Ergänzung der biologischen Testung von Kinaseinhibitoren um das Kinom-Profiling als sinnvoll erachtet, da es das Portfolio in der Testung sinnvoll erweitert.Photopharmacology offers a spatiotemporal control of the effects of drugs using light. Up to now, various concepts such as photoswitching, photocleavage or photodynamic therapy can be used to develop photoresponsive drugs. Kinases are very well suited as target proteins for the following applications, as they fulfil a key role in the human cell and therefore control cell functions, e.g., the regulation of growth and differentiation. Uncontrolled overactivation of kinases can lead to various types of cancer, which can be treated with kinase inhibitors. Combining kinase inhibitors with photopharmacological concepts (preferential photoswitching and photocleavage) could increase the efficacy of treatment while reducing systemic side effects. This work aims at the advancement of existing photopharmacological concepts for kinase inhibitors to improve their potential clinical application. In addition, the developed photoswitchable kinase inhibitors have been investigated in a kinome activity profiling to determine the influence of the drugs on the entire kinase network. The synthesized photoresponsive inhibitors were photochemically characterized and subsequently biologically tested using kinase assays, kinome profiling and cell proliferation assays. In the first part of the work, several photoswitches were biologically evaluated. For the approved kinase inhibitor axitinib, an isomer-dependent biological effect (on VEGFR2, on HUVEC and in kinome profiling) could be shown. However, stilbenes could not be reversibly switched in aqueous media. The subsequent functionalization with diazocines combined good photochemical parameters with a sufficient difference in effect. The limited effectiveness of the photoswitchable BRAFV600E inhibitor due to kinome overactivation could be explained by kinome profiling. While kinome profiling of the chronoswitches was not successful, a clear difference in effectiveness in the profiling of the RET stilbene switches was observed. In the second part, the photoremovable protecting groups DMNB and BODIPY for CDK2 inhibitors were investigated. Under irradiation, caged inhibitors led either to the expected cleavage and CDK2 inhibition (DMNB) or to significant photodynamic effects (BODIPY). The investigated compounds contribute significantly to the (further) development of photoresponsive kinase inhibitors. Further studies in animal scale thus seem more likely (photocleavage) or should be carried out next (photoswitching). Furthermore, the addition of kinome profiling to the biological testing of kinase inhibitors is considered useful as it expands the testing portfolio