Keemiliste ja geneetiliste meetodite ühitamine proteiinkinaaside fotoluminestsentsanalüüsiks

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Doktoritöös kasutati geneetilisi ja keemilisi meetodeid sensorsüsteemide loomiseks proteiinkinaaside analüüsiks. Proteiinkinaasid katalüüsivad valkude fosforüülimist, see on oluline reaktsioon valkude modifitseerimisel, mille kaudu reguleeritakse valkude aktiivsust rakkudes. Valkude fosforüülimine võimaldab suurendada proteoomi mitmekesisust, seeläbi mõjutatakse rakkude normaalset ja patoloogilist toimimist. Valkude fosforüülimistasakaalude nihkumine PKde häiritud aktiivsuse tõttu on paljude raskete haiguste (vähktõve erinevad vormid, diabeet jne) põhjuseks või märguandeks nende tekkimise kohta. Sellest tuleneval on proteiinkinaasid olnud 21. sajandi olulisteks ravimiarenduse sihtmärkideks. Viimase 15 aasta jooksul on 30 proteiinkinaaside inhibiitorit jõudnud vähiravimitena haiglatesse. Lisaks olulisele rollile ravimiarenduses on proteiinkinaasid tähtsad biomarkerid vähktõve erinevate vormide ning teistele haiguste tuvastamisel. Sellest tuleneb ka suur nõudmine analüüsimeetodite järele, mis võimaldaksid määrata kinaaside kontsentratsiooni ja aktiivsust kliinilistes proovides, näiteks kehavedelikes ja biopsiaproovides. Viimasel aastakümnel on mõistetud, et just kahe uurimisala, sünteetilise keemia ja valkude konstrueerimise geneetiliste meetodite kooskasutamine on vajalik uudsete omadustega molekulide ja nende komplekside konstrueerimiseks. Need struktuurid lisaksid uudseid võimalusi biomeditsiiniliste uuringute läbiviimiseks, ravimiarenduseks ning haiguste tuvastamiseks. Käesolevas töös ühitati keemilised ja geneetilised meetodid selleks, et välja arendada uudsed sensorsüsteemid PKde ja nend inhibiitoride uurimiseks. Ühelt poolt toodeti imetajate rakkudes mitmete proteiinkinaaside (PKAc, CK2α, and PIM-kinaasid) liitvalke fluorestseeruvate valkudega. Teisalt konstrueeriti ARC-inhibiitoritest lähtuvalt uudsete luminestsentsomadustega kõrge afiinsusega sondid kinaasidede jaoks [ARC-Lum(Fluo)-sondid]. Seoses PIM-kinaaside (PIM1, PIM2 ja PIM3) olulise tähtsusega nii ravimarenduse sihtmärkidena kui ka vähktõve biomarkeritena, on abivahendite väljatöötamine nende kinaaside uurimiseks suure praktilise väärtusega. PIM2 liitvalke punase fluorestseeruva valguga (TagRFP) ekspresseeriti imetajate rakkudes kahel erineval kujul (TagRFP-PIM2 ja PIM2-TagRFP). Töös näidati, need erineva märgise paigutusega liitvalgud omavad kompleksis ARC-Lum(Fluo)-sondidega erinevaid luminestsentsomadusi, mis tuleneb erinevast Försteri-tüüpi energiaülekandest vastasmõjus olevate luminofooride vahel. Töös näidati esmakordselt, et kompleksis on võimalik energiaülekanne seleeni-sisaldava aromaatse süsteemi ergastatud tripletest olekust kinaasiga seotud fluorestseeruvale valgule, mille tulemusena fluorestsentsvärvilt kiiratav valgus on küll värvile omase kiirgusspektriga, kuid enam kui 10000 korda pikema elueaga. See tulemus loob eelduse rakusiseste sensorsüsteemide loomiseks raku plasmamembraani läbivate ARC-Lum(Fluo)-sondide ja rakkudes ekspresseeritud proteiinkinaasi ja TagRFP liitvalkude baasil, mis võimaldaks kaardistada proteiinkinaaside aktiivsust elusrakkudes.Phosphorylation reaction is an important post-translational protein modification procedure in cells, which is carried out by protein kinases (PKs). This modification leads to increase in the diversity of the proteome that influences various aspects of normal and pathological physiology. Dysregulation of protein phosphorylation balances, caused by the aberrant activity of PKs is a cause or consequence of several complex diseases such as cancers, inflammatory disorders, cardio-vascular diseases and diabetes. Therefore PKs have become important drug targets in the 21st century. In recent 15 years, 30 small-molecule PK inhibitors have been approved for use in clinical practice. More than 500 PKs, constituting almost 2.5 % proteins coded by the human genome, are carrying out the phosphorylation of various proteins. In addition to being a potential drug targets, PKs also serve as biomarkers for cancers and other diseases, as altered expression level of various PKs is observed in a variety of malignancies. Thus there is high demand for analytical methods that enable determination of expression and activity levels of specific PKs in clinical samples, such as bodily fluids and cancerous tissues. Thus high throughput assays for screening PK inhibitors also form an important component of drug development pipeline. During the last decade, it has been understood that merging of two disciplines, synthetic chemistry and protein engineering, is needed to construct molecules and their complexes with new functionalities that can create novel opportunities for biomedical research, drug development and disease diagnostics. In this thesis combination of chemical and genetic approaches was used for the development of analytical tools for the characterization of PKs and PK inhibitors. PKs (PKAc, CK2α, and PIM kinases) were fused with fluorescent proteins (FPs). On the other hand, ARC-based small-molecule PK inhibitors were developed into protein binding-responsive ARC-Lum probes possessing unique photoluminescent properties. Joint application of PKs fused with fluorescent proteins and ARC-Lum probes enabled the construction of sensor systems that can be used for specific and sensitive determination of PKs in biological samples and as research tools for mapping and monitoring PK activity in living cells

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases

Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements

The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches

A covalent calmodulin inhibitor as a tool to study cellular mechanisms of K-Ras-driven stemness

This article is part of the Research Topic Ras and other GTPases in Cancer: From Basic to Applied Research https://doi.org/10.3389/978-2-88974-081-9Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.Peer reviewe

Influence of recycled basalt-aramid fibres integration on the mechanical and thermal properties of brake friction composites

In the brake friction composites(BFCs), fibres take part in significant attention as reinforcement in governing mechanical and thermal-mechanical properties. The current investigation aims to develop hybrid brake friction composites using recycled basalt- aramid fibre integration and to characterise for its mechanical and thermal properties. The experiments related to thermal (heat swell, loss of ignition and thermal conductivity) and mechanical (tensile, compression, flexural and impact) properties were conducted as per industrial standards. From the experimental investigations, it was concluded that fibre inclusion in the BFCs enhanced the mechanical and thermal properties considerably. Further, with the aid of scanning electron microscope (SEM), fracture interfaces of the tested friction composites were analyzed for various characteristics like pullout, void, fibre-matrix bonding etc

Identification of an H-Ras nanocluster disrupting peptide

AbstractThe Ras-MAPK pathway is critical to regulate cell proliferation and differentiation. Its dysregulation is implicated in the onset and progression of numerous types of cancers. To be active, Ras proteins are membrane anchored and organized into nanoclusters, which realize high-fidelity signal transmission across the plasma membrane. Nanoclusters therefore represent potential drug targets. However, targetable protein components of signalling nanoclusters are poorly established.We previously proposed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering by stabilizing stacked dimers of H-Ras and Raf via a direct interaction of dimeric Gal1 with the Ras binding domain (RBD) in particular of B-Raf. Here, we provide further supportive evidence for this model. We establish that the B-Raf preference emerges from divergent regions of the Raf RBDs that were proposed to interact with Gal1. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B-Raf-RBD. Its 23-mer core fragment is thus sufficient to interfere with Gal1-enhanced H-Ras nanocluster, reduce MAPK-output and cell viability inHRAS-mutant cancer cell lines.Our data therefore suggest that the interface between Gal1 and the RBD of B-Raf can be targeted to disrupt Gal1-enhanced H-Ras nanoclustering. Collectively, our results support that Raf-proteins are integral components of active Ras nanoclusters

Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.</p

PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity

The trafficking chaperone PDE6D (also referred to as PDE delta) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a "chemical spring" into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block sternness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future

PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity

The trafficking chaperone PDE6D (also referred to as PDE?) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a "chemical spring" into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block stemness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents