Kinase Activity Assay Research Articles

Synthesis and biological evaluation of a new class of azole urea compounds as Akt inhibitors with promising anticancer activity in pancreatic cancer models

The PI3K/Akt pathway is crucial in numerous cellular functions such as cell growth, survival proliferation and movement in both normal and cancer cells. It plays also a key role in epithelial-mesenchymal transitions and angiogenesis during the tumorigenesis processes. Since many transformative events in cancer are driven by increased PI3K/Akt pathway signaling, Akt is considered a valuable target for developing new therapies against various tumor types, including pancreatic cancer. This is because the PI3K/AKT/mTOR pathway is a key downstream effector of RAS, and RAS activation is the most prominent genetic alteration in pancreatic cancer. Herein we report the synthesis and the biological evaluation of a new series of azole urea compounds that exhibited promising antiproliferative and antimigratory activities against pancreatic cancer cells through an Akt inhibition mechanism. These effects were demonstrated using a variety of assays, including Sulforhodamine B, cell-cycle, wound-healing, and kinase activity, apotposis and ELISA assays. Additionally, the anticancer properties of the most active compound in the series were confirmed in the 3D spheroid model of PATU-T cells.

STEM-18. A HIGH THROUGHPUT NEUROSPHERE BASED COLONY FORMATION ASSAY TO VALIDATE DRUG AND RADIATION EFFECTIVENESS IN PATIENT DERIVED XENOLINES

Abstract Glioblastoma (GBM) is a primary malignant brain tumor developed from normal astrocytes. GBM patients’ median survival is less than 2 years despite safe surgical resection, fractionated conventional radiation, and temozolomide (TMZ) therapy. Few currently used drugs are successful in GBM although there are many FDA approved blood-brain barrier (BBB) penetrant drugs, for other uses that have not been screened in GBM. We utilized a panel of 734 of these BBB penetrant drugs in different GBM patient derived xenolines (PDX) along with acquired radio-resistant derivatives (-RT) including X1441, JX14P, JX14P-RT, JX39P, JX39P-RT and normal human astrocytes (NHA) as control and screened for viability with Cell Titer-Glo. We identified multiple candidate drugs that preferentially killed GBM PDX cells at 10 µM relative to NHA control. As we used stem cell media (serum free) in our drug screening study, it indicates that the potent drugs are also effective in killing cancer stem cells or brain tumor initiating cells. These drugs had targets ranging from PDGFR, DNA/RNA synthesis, as well as other kinase inhibitors. We developed a high throughput (HT), neurosphere based colony formation assay (CFA) in 96-well plate to screen multiple drugs and doses in combination with radiation. For visualizing the colonies, we use a Cytation 5 imager at 4X resolution and count the colonies based on parameters including object size, width, intensity and others. We plan on validating our drug screen and HT-CFA assay with high throughput PamChip kinase activity assays and with in vivo orthotopic tumor PDX models.

9-Methylfascaplysin Prevents Neuroinflammation and Synaptic Damage via Cell-Specific Inhibition of Kinases inAPP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a leading neurodegenerative disorder without effective treatments. The nonlinear dynamic nature of AD pathophysiology suggested that multiple pharmacological actions of anti-AD drugs should be elucidated. 9-Methylfascaplysin (9-MF) was previously designed and synthesized as a novel anti-AD candidate. In this study, 9-MF at low concentrations significantly prevented cognitive impairments with similar efficacy as donepezil in APP/PS1 transgenic mice. In addition, 9-MF potently reduced β-amyloid (Aβ)-associated neuroinflammation and tau-associated synaptic damage invivo. 9-MF-regulated microglia-specific differentially phosphorylated proteins (DPPs) were mainly enriched in neuroinflammation, while 9-MF-regulated neuron-specific DPPs were enriched in synaptic regulation, as revealed by a quantitative phosphoproteomic approach. A phosphoproteome-kinome algorithm further identified that rho-associated coiled-coil kinase 2 (ROCK2) and glycogen synthase kinase 3β (GSK3β) ranked high in 9-MF-downregulated kinase perturbations. 9-MF possessed high affinities for ROCK2 and GSK3β, which was confirmed by invitro kinase activity assay. The protective effects of 9-MF were abolished by ROCK2 knockdown in Aβ-treated BV2 microglial cells, and by GSK3β knockdown in glyceraldehyde-treated SH-SY5Y neuronal cells, respectively. All these results supported that 9-MF produced anti-AD effects via cell-specific inhibition of ROCK2 and GSK3β in microglia and neurons, respectively.

Enhancing Kinase Activity Detection with a Programmable Lanthanide Metal-Organic Framework via ATP-to-ADP Conversion.

Precise modulation of host-guest interactions between programmable Ln-MOFs (lanthanide metal-organic frameworks) and phosphate analytes holds immense promise for enabling novel functionalities in biosensing. However, the intricate relationship between these functionalities and structures remains largely elusive. Understanding this correlation is crucial for advancing the rational design of fluorescent biosensor technology. Presently, there exists a large research gap concerning the utilization of Ln-MOFsto monitor the conversion of ATP to ADP, which poses a limitation for kinase detection. In this work, we delve into the potential of Ln-MOFs to amplify the fluorescence response during the kinase-mediated ATP-to-ADP conversion. Six Eu-MOFs were synthesized and Eu-TPTC ([1,1':4',1″]-terphenyl-3,3'',5,5''-tetracarboxylic acid) was selected as a ratiometric fluorescent probe, which is most suitable for high-precision detection of creatine kinase activity through the differential response from ATP to ADP. The molecular -level mechanism was confirmed by density functional theory. Furthermore, a simple paper chip-based platform was constructed to realize the fast (20 min) and sensitive (limit of detection is 0.34 U/L) creatine kinase activity detection in biological samples. Ln-MOF-phosphate interactions offer promising avenues for kinase activity assays and hold the potential for precise customization of analytical chemistry.

Comparative CNS Pharmacology of the Bruton's Tyrosine Kinase (BTK) Inhibitor Tolebrutinib Versus Other BTK Inhibitor Candidates for Treating Multiple Sclerosis.

Tolebrutinib is a covalent BTK inhibitor designed and selected for potency and CNS exposure to optimize impact on BTK-dependent signaling in CNS-resident cells. We applied a translational approach to evaluate three BTK inhibitors in Phase 3 clinical development in MS with respect to their relative potency to block BTK-dependent signaling and exposure in the CNS METHODS: We used in vitro kinase and cellular activation assays, alongside pharmacokinetic sampling of cerebrospinal fluid (CSF) in the non-human primate cynomolgus to estimate the ability of these candidates (evobrutinib, fenebrutinib, and tolebrutinib) to block BTK-dependent signaling inside the CNS. In vitro kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib, while fenebrutinib, a classical reversible antagonist with a Ki value of 4.7 nM and slow off-rate (1.54 x 10-5 s-1), also had an association rate 1760-fold slower (0.00245 μM-1 * s-1). Estimates of cellular potency were largely consistent with the in vitro kinase assays, with an estimated IC50 of 0.7 nM for tolebrutinib against 33.5 nM for evobrutinib and 2.9 nM for fenebrutinib. We then observed that evobrutinib, fenebrutinib, and tolebrutinib achieved similar levels of exposure in non-human primate CSF after oral doses of 10 mg/kg. However, tolebrutinib CSF exposure (4.8 ng/mL) (kp,uu CSF=0.40) exceeded the IC90 (the estimated concentration inhibiting 90% of kinase activity) value, while evobrutinib (3.2 ng/mL) (kp,uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp,uu CSF=0.15) failed to reach the estimated IC90 values. Tolebrutinib was the only candidate of the three that attained relevant CSF exposure in non-human primates.

1.8-cineole prevents platelet activation and aggregation by activating the cAMP pathway via the adenosine A2A receptor

AimsDysregulated platelet aggregation is a fatal condition in many bacterial- and virus-induced diseases. However, classical antithrombotics cannot completely prevent immunothrombosis, due to the unaddressed mechanisms towards inflammation. Thus, targeting platelet hyperactivation together with inflammation might provide new treatment options in diseases, characterized by immunothrombosis, such as COVID-19 and sepsis. The aim of this study was to investigate the antiaggregatory effect and mode of action of 1.8-cineole, a monoterpene derived from the essential oil of eucalyptus leaves, known for its anti-inflammatory proprieties. Main methodsPlatelet activity was monitored by measuring the expression and release of platelet activation markers, i.e., P-selectin, CD63 and CCL5, as well as platelet aggregation, upon treatment with 1.8-cineole and stimulation with several classical stimuli and bacteria. A kinase activity assay was used to elucidate the mode of action, followed by a detailed analysis of the involvement of the adenylyl-cyclase (AC)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway by Western blot and ELISA. Key findings1.8-cineole prevented the expression and release of platelet activation markers, as well as platelet aggregation, upon induction of aggregation with classical stimuli and immunological agonists. Mechanistically, 1.8- cineole influences the activation of the AC-cAMP-PKA pathway, leading to higher cAMP levels and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Finally, blocking the adenosine A2A receptor reversed the antithrombotic effect of 1.8-cineole. SignificanceGiven the recognized anti-inflammatory attributes of 1.8-cineole, coupled with our findings, 1.8-cineole might emerge as a promising candidate for treating conditions marked by platelet activation and abnormal inflammation.

PKM2 promotes glioma progression by mediating CTNNB1 expression

ABSTRACT Background Glioma is a common intracranial tumor, exhibiting a high degree of aggressiveness and invasiveness. Pyruvate kinase M2 (PKM2) is overexpressed in glioma tissues. However, the biological role of PKM2 in glioma is unclear. Methods The qRT-PCR, CCK-8, Transwell, flow cytometry detection, western blot assays, ELISA assay, and pyruvate kinase activity assays were performed in glioma cells transfected with PKM2 shRNA to explore the function of PKM2 in glioma progression. Then, STRING website was used to predict the proteins that interacted with PKM2, and Co-IP assay was conducted to further validate their interaction. Subsequently, the above experiments were performed again to find the effect of catenin beta 1 (CTNNB1) overexpression on PKM2-deficient glioma cells. The transplanted tumor models were also established to further validate our findings. Results PKM2 was up-regulated in glioma cells and tissues. After inhibiting PKM2, the proliferation, migration, glycolysis, and EMT of glioma cells were significantly decreased, and the proportion of apoptosis was increased. The prediction results of STRING website showed that CTNNB1 and PKM2 had the highest interaction score. The correlation between CTNNB1 and PKM2 was further confirmed by Co-IP test. PKM2 knockdown suppressed glioma cell proliferation, migration, glycolysis, and EMT, while CTNNB1 overexpression rescued these inhibitory effects. Correspondingly, PKM2 knockdown inhibited glioma growth in vivo. Conclusion In summary, these findings indicated that PKM2 promotes glioma progression by mediating CTNNB1 expression, providing a possible molecular marker for the clinical management of gliomas.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

Sex-specific cardiovascular remodeling leads to a divergent sex-dependent development of heart failure in aged hypertensive rats

IntroductionThe prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level.MethodsWe studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE).ResultsTG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes.ConclusionHere we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt

Ethnopharmacological relevanceLicorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. Aim of the studyThis study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. Materials and methodsSW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. ResultsWighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a “closed” conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. ConclusionWighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Association mechanism between Arabidopsis immune coreceptor BAK1 and Pseudomonas syringae effector HopF2

Brassinosteroid activated kinase 1 (BAK1) is a cell-surface coreceptor which plays multiple roles in innate immunity of plants. HopF2 is an effector secreted by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 into Arabidopsis and suppresses host immune system through interaction with BAK1 as well as its downstream kinase MKK5. The association mechanism of HopF2 to BAK1 remains unclear, which prohibits our understanding and subsequent interfering of their interaction for pathogen management. Herein, we found the kinase domain of BAK1 (BAK1-KD) is sufficient for HopF2 association. With a combination of hydrogen/deuterium exchange mass spectrometry and mutational assays, we found a region of BAK1-KD N-lobe and a region of HopF2 head subdomain are critical for intermolecular interaction, which is also supported by unbiased protein-protein docking with ClusPro and kinase activity assay. Collectively, this research presents the interaction mechanism between Arabidopsis BAK1 and P. syringae HopF2, which could pave the way for bactericide development that blocking the functioning of HopF2 toward BAK1.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Abstract 644: PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines

Abstract Ovarian cancer is the most lethal malignant disease of female. Treatment options for ovarian cancer are mostly the use of chemotherapeutics, surgery, or radiotherapy. However, due to resistance to drugs, relapse occurs and thus 5 years survival rate is only 47%. The tumor suppressor protein p53 works as a transcription factor to transcribe several proteins such as p21, p16 and PUMA that lead to halt cell cycle and also induce apoptosis when there is a damage in DNA. Mutation in several regions of p53 (mutp53) is quite common in cancers and can lead to the accumulation of p53 with loss of functions or sometimes gain of oncogenic functions. Thus there is a search for drugs that can degrade the mut53 and can restore the functions of normal p53. In this research, it was observed that the treatment of ovarian cancer cell lines (ES-2, HEY and OVCAR3) with atypical protein kinase C-iota (aPKCɩ) specific inhibitors ICA-1S can cause the degradation of mutp53 (53.6%, N=6 trials). Treatment with ICA-1S also lowered the level of phosphorylated p53 (ser15, 87%, N=3 trials). However, the level of MDM2 protein (murine double minute 2), a known p53 E3 ligase was not significantly (98%, N=2 trials) affected by ICA-1S in ES-2 cell line with mut p53 as well as p21 was also not affected. However level of PUMA was decreased both in ES-2 and HEY cell lines. Immunoprecipitation (IP) with aPKCɩ specific beads showed the aPKCɩ is associated with p53, though reverse IP with p53 beads was not able to show the association. Application of proteasome inhibitor MG-132 at a single dose of 0.5µM could not completely inhibit the degradation of mut53 and WTp53 in ICA-1S treated ES-2 cell line and HEY cell line, respectively, indicating the drug may be affecting the transcription of p53 as well. It is therefore postulated that aPKCɩ can phosphorylate p53, stabilizing it and may be affecting the transcription of p53 as well in those cell lines. Further research (kinase activity assay, immunoprecipitation, immunofluorescence) is going on to reveal the mode of this degradation. Citation Format: Mahfuza Marzan, Nuzhat Nowshin Oishee, Abigail Olatunji, Mildred Acevedo-Duncan. PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 644.

Abstract 4669: Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations

Abstract Background: Differences in profiles of acquired mutations of resistance to BTKis might have implications for treatment sequencing. We evaluated preclinical activity of BTKis (ibrutinib [Ibr], acalabrutinib [Acala], zanubrutinib [Zanu], and pirtobrutinib [Pirto]) against multiple clinically relevant BTK mutations and report on the clinical impact of BTK C481S in patients (pts) treated with Ibr. Methods: Inhibition of kinase activity by BTKis was evaluated in a cell-free kinase activity assay (Nanosyn). Cell-killing activity on CRISPR-generated BTK mutant knock-in TMD8 cells was assessed after 3 days of BTKi treatment. Time from first detection of C481S mutation to progressive disease (PD) was evaluated in pts treated with Ibr with previously untreated (n=247) or relapsed/refractory (R/R; n=172) CLL using pooled data from 5 clinical trials. Results: In vitro kinase activity was highest for Ibr compared with Acala or Zanu against C481S (Ibr IC50: 26.3 nM; Zanu: 84.8 nM; Acala: 338 nM) and T474I (Ibr: 1.2 nM; Zanu: 4.4 nM; Acala: 43.6 nM). Ibr maintained cell-killing activity against all tested BTK mutants, whereas limited activity was seen with Acala against C481S and A428D; with Zanu against C481S, L528W, and A428D; and with Pirto against T474I, L528W, V416L, and A428D (Table). In pts treated with Ibr, C481S was detected prior to clinical PD in 3 previously untreated pts and in 26 pts with R/R CLL; median (range) time from first detection of C481S to PD in these pts was 17.0 mo (11.2-34.3) and 10.0 mo (2.6-35.7), respectively. Conclusion: This study demonstrates activity of Ibr against all evaluated BTK variants, which are associated with resistance to covalent and noncovalent BTKis. Clinical data shows that pts who develop C481S mutations continue to derive benefit from Ibr for several months before PD. Together, these data suggest a role for Ibr in the treatment of pts with a broad spectrum of BTK variants. Table. Cell-killing activity of BTKis against wild type and mutant BTK EC50 (nM) BTK Wild type C481S T474I L528W V416L A428D Ibr 0.6 414.0 1.9 181.4 0.5 205.3 Acala 3.9 2973.8 43.3 3.5 106.3 3000.0 Zanu 0.7 2322.0 11.7 3000.0 0.6 1726.0 Pirto 8.2 12.6 2727.5 3000.0 1898.0 3000.0 Citation Format: Matthew S. Davids, Inhye E. Ahn, Jacob J. Riehm, Melih Acar, Vincent Girardi, Sima Patel, Tarikere Gururaja, Jennifer Woyach, Lloyd T. Lam, Edith Szafer-Glusman. Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4669.

Abstract 4704: Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers

Abstract The RAF protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the RAS GTPases and as the initiating kinases in the ERK cascade. In human cancer, RAF activity is frequently dysregulated due to mutations in the RAF family members (ARAF, BRAF, and CRAF) or to alterations in upstream RAF regulators, including RAS and receptor tyrosine kinases. The first and second generations of RAF inhibitors have yielded dramatic responses in malignant melanomas containing BRAF mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In addition, cancers with hyperactive RAS exhibit intrinsic resistance to these drugs. In particular, issues related to the dimerization of the RAF kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. We have established biochemical HotSpotTM kinase assay, NanoBRETTM and NanoBITTM cell assay platforms for High Through Screening of kinase inhibitors. Here, we demonstrate that the 3rd generation of pan-RAF inhibitors LY3009120, LXH254, and Belvarafenib inhibit ARAF, BRAF, CRAF, BRAF(V600E), and CRAF(R391W) kinase activity in biochemical HotSpotTM assay. Our NanoBRETTM target engagement and NanoBITTM cellular assay data show that LY3009120, LXH254, and Belvarafenib bind to KRAS(G12C) primed BRAF and CRAF, and block BRAF and CRAF dimerization. Furthermore, our results show these inhibitors can block the downstream ERK phosphorylation in cellular HTRF assay and induce caspase-3/7 activation in Western blot assay in the triple negative breast cancer MDA-MB-231 cells. Taken together, our results indicate the biochemical HotSpotTM kinase activity assay, and NanoBRETTM target engagement and NanoBITTM cellular assays can serve as great platforms to facilitate RAF drug discovery against human cancers. Citation Format: Jianghong Wu, Yong Wan, Shuguang Liang, Peter Gallagher, Li Liang, Christian Loch, Haiching Ma. Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4704.

Abstract 648: Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays

Abstract Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use. However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. We compared the potency of a selection of WEE1 inhibitors when different substrates were used. While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly. Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.

Structure-based investigation of pyruvate dehydrogenase kinase-3 inhibitory potential of thymoquinone, targeting lung cancer therapy

Protein kinases are an attractive therapeutic target for cardiovascular, cancer and neurodegenerative diseases. Cancer cells demand energy generation through aerobic glycolysis, surpassing “oxidative phosphorylation” (OXPHOS) in mitochondria. The pyruvate dehydrogenase kinases (PDKs) have many regulatory roles in energy generation balance by controlling the pyruvate dehydrogenase complex. Overexpression of PDKs is associated with the overall survival of cancer. PDK3, an isoform of PDK is highly expressed in various cancer types, is targeted for inhibition in this study. PDK3 has been shown to binds strongly with a natural compound, thymoquinone (TQ), which is known to exhibit anti-cancer potential. Detailed interaction between the PDK3 and TQ was carried out using spectroscopic and docking methods. The overall changes in the protein's structures after TQ binding were estimated by UV–Vis spectroscopy, circular dichroism and fluorescence binding studies. The kinase activity assay was also carried out to see the kinase inhibitory potential of TQ. The enzyme inhibition assay suggested an excellent inhibitory potential of TQ towards PDK3 (IC50 = 5.49 μM). We observed that TQ forms a stable complex with PDK3 without altering its structure and can be a potent PDK3 inhibitor which may be implicated in cancer therapy after desired clinical validation.

Melatonin Inhibits AGS Cell Proliferation by Binding to the ATP Binding Site of CDK2 Under Hyperglycemic Conditions.

Cancer cells utilize glucose as their primary energy source. The aggressive nature of cancer cells is therefore enhanced in hyperglycemic conditions. This study has been adopted to investigate the therapeutic potential of melatonin against such aggressive proliferation of AGS cells-a human gastric cancer cell line, under hyperglycemic conditions. AGS cells were incubated with high glucose-containing media, and the effects of melatonin have been evaluated, therein. Cell proliferation, ROS generation, flow-cytometric analysis for cell cycle and apoptosis, wound healing, immunoblotting, zymography, reverse zymography assays, in-silico analysis, and kinase activity assays were performed to evaluate the effects of melatonin. We observed that melatonin inhibited the hyperglycemia-induced cell proliferation in a dose-dependent manner. It further altered the expression and activity of MMP-9 and TIMP-1. Moreover, melatonin inhibited AGS cell proliferation by arresting AGS cells in the G0/G1 phase after binding in the ATP binding site of CDK-2, thereby inhibiting its kinase activity. In association, a significant decrease in the expression of cyclin D1, cyclin E, CDK-4, and CDK-2 were observed. In conclusion, these findings suggest that melatonin has anti-gastric cancer potential. Melatonin could therefore be included in future drug designs for gastric cancer-hyperglycemia co-morbidity treatment.

Plasmonic nanoparticle-assisted single-molecule dynamic binding for protein kinase activity digital counting.

In this work, we design a new approach for digital counting-based protein kinase activity assay by using plasmonic nanoparticle-assisted single-molecule dynamic binding. This method breaks the concentration-dependent limitation in single-molecule detection and displays good selectivity and sensitivity with a detection limit as low as 0.0001 U mL-1 for the protein kinase A (PKA) assay, which would find broad applications in drug screening and medical investigations.

An electrochemical biosensor for T4 polynucleotide kinase activity assay based on host-guest recognition between phosphate pillar[5]arene@MWCNTs and thionine.

T4 polynucleotide kinase helps with DNA recombination and repair. In this study, an electrochemical biosensor was developed for a T4 polynucleotide kinase activity assay and inhibitor screening based on phosphate pillar[5]arene and multi-walled carbon nanotube nanocomposites. The water-soluble pillar[5]arene was employed as the host to complex thionine guest molecules. The substrate DNA with a 5'-hydroxyl group initially self-assembled on the gold electrode surface through chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 polynucleotide kinase. Titanium dioxide nanoparticles served as a bridge to link phosphorylated DNA and phosphate pillar[5]arene and multi-walled carbon nanotube composite due to strong phosphate-Ti4+-phosphate chemistry. Through supramolecular host-guest recognition, thionine molecules were able to penetrate the pillar[5]arene cavity, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 polynucleotide kinase concentration in the range of 10-5 to 15 U mL-1 with a detection limit of 5 × 10-6 U mL-1. It was also effective in measuring HeLa cell lysate-related T4 polynucleotide kinase activity and inhibitor screening. The proposed method offers a unique sensing platform for kinase activity measurement, holding great potential in nucleotide kinase-target drug development, clinical diagnostics, and inhibitor screening.