Two hearts beat as one: the debate over RAS dimers continues.

A recent report by Yun et al. describes the detection of RAS dimers using intact mass spectrometry and investigates the role that membrane lipids, nucleotide state, and binding partners have in their formation.

An Overview on Prevalence and Detection Approaches of BRAF V600E Mutation in Anaplastic Thyroid Carcinoma: A Systematic Review and Meta-Analysis.

Background: BRAF V600E mutation is proved critical in the progression and invasion of thyroid cancer, and as a prognostic biomarker. As anaplastic thyroid cancer (ATC) is a rare and aggressive form of thyroid cancer, this study was conducted to provide a view on prevalence of BRAF V600E as well as the best molecular diagnostic method in ATC patients. Methods: A comprehensive literature search was performed from their inception to Oct 2022 in PubMed, Scopus, Google Scholar, and Web of Science (WoS). The data of the prevalence of ATC were extracted. Moreover, the diagnostic feature of the available diagnostic tools was extracted to measure the sensitivity and specificity. To pool the prevalence data, we used meta-proportion analysis and diagnostic meta-analysis was conducted to determine the specificity and sensitivity of the immunohistochemistry method in detecting BRAF V600E mutation among patients with ATC. Results: Overall, 34 studies were included in this meta-analysis. The incidence of BRAF V600E was shown 33% in the 978 patients. The sensitivity and specificity of IHC in detecting BRAF V600E were detected 78.9% (95%CI: 60.1-97.2), and 69.7% (95%CI: 41.2-98.1), respectively. Conclusion: IHC had an acceptable prognostic profile for detecting BRAF V600E in ATC patients. The diagnosis of BRAF mutation is critical in clinical trials and may be helpful for choosing proper-targeted therapy strategies in ATC patients.

An overview of RAF kinases and their inhibitors (2019-2023).

Protein kinases (PKs) including RAF, perform a principal role in regulating countless cellular events such as cell growth, differentiation, and angiogenesis. Overexpression and mutation of RAF kinases are significant contributors to the development and spread of cancer. Therefore, RAF kinase inhibitors show promising outcomes as anti-cancer small molecules by suppressing the expression of RAF protein, blocking RAS/RAF interaction, or inhibiting RAF enzymes. Currently, there are insufficient reports about approving drugs with minimal degree of toxicity. Therefore, it is an urgent need to develop new RAF kinase inhibitors correlated with increased anticancer activity and lower cytotoxicity. This review outlines reported RAF kinase inhibitors for cancer treatment in patents and literature from 2019 to 2023. It highlights the available inhibitors by shedding light on their chemical structures, biochemical profiles, and current status. Additionally, we highlighted the hinge region-binding moiety of the reported compounds by showing the hydrogen bond patterns of representative inhibitors with the hinge region for each class. In recent years, RAF kinase inhibitors have gained considerable attention in cancer research and drug development due to their potential to be studied under clinical trials and their demonstration of various degrees of efficacy and safety profiles across different cancer types. However, addressing challenges related to drug resistance and safety represents a major avenue for the optimization and enhancement of RAF kinase inhibitors. Strategies to overcome such obstacles were discussed such as developing novel pan-RAF inhibitors, RAF dimer inhibitors, and combination treatments.

NF2-Related Schwannomatosis (NF2): Molecular Insights and Therapeutic Avenues.

NF2-related schwannomatosis (NF2) is a genetic syndrome characterized by the growth of benign tumors in the nervous system, particularly bilateral vestibular schwannomas, meningiomas, and ependymomas. This review consolidates the current knowledge on NF2 syndrome, emphasizing the molecular pathology associated with the mutations in the gene of the same name, the NF2 gene, and the subsequent dysfunction of its product, the Merlin protein. Merlin, a tumor suppressor, integrates multiple signaling pathways that regulate cell contact, proliferation, and motility, thereby influencing tumor growth. The loss of Merlin disrupts these pathways, leading to tumorigenesis. We discuss the roles of another two proteins potentially associated with NF2 deficiency as well as Merlin: Yes-associated protein 1 (YAP), which may promote tumor growth, and Raf kinase inhibitory protein (RKIP), which appears to suppress tumor development. Additionally, this review discusses the efficacy of various treatments, such as molecular therapies that target specific pathways or inhibit neomorphic protein-protein interaction caused by NF2 deficiency. This overview not only expands on the fundamental understanding of NF2 pathophysiology but also explores the potential of novel therapeutic targets that affect the clinical approach to NF2 syndrome.

Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Subcellular dynamics of ethylene signaling drive plant plasticity to growth and stress: Spatiotemporal control of ethylene signaling in Arabidopsis.

Volatile compounds, such as nitric oxide and ethylene gas, play a vital role as signaling molecules in organisms. Ethylene is a plant hormone that regulates a wide range of plant growth, development, and responses to stress and is perceived by a family of ethylene receptors that localize in the endoplasmic reticulum. Constitutive Triple Response 1 (CTR1), a Raf-like protein kinase and a key negative regulator for ethylene responses, tethers to the ethylene receptors, but undergoes nuclear translocation upon activation of ethylene signaling. This ER-to-nucleus trafficking transforms CTR1 into a positive regulator for ethylene responses, significantly enhancing stress resilience to drought and salinity. The nuclear trafficking of CTR1 demonstrates that the spatiotemporal control of ethylene signaling is essential for stress adaptation. Understanding the mechanisms governing the spatiotemporal control of ethylene signaling elements is crucial for unraveling the system-level regulatory mechanisms that collectively fine-tune ethylene responses to optimize plant growth, development, and stress adaptation.

Intracellular Ellagic Acid Derived from Goat Urine DMSO Fraction (GUDF) Predicted as an Inhibitor of c-Raf Kinase.

BACKGROUND: Dietary chemicals and their gut-metabolized products are explored for their anti-proliferative and pro-cell death effects. Dietary and metabolized chemicals are different from ruminants such as goats over humans. METHODS: Loss of cell viability and induction of death due to goat urine DMSO fraction (GUDF) derived chemicals were assessed by routine in vitro assays upon MCF-7 breast cancer cells. Intracellular metabolite profiling of MCF-7 cells treated with goat urine DMSO fraction (GUDF) was performed using an in-house designed vertical tube gel electrophoresis (VTGE) assisted methodology, followed by LC-HRMS. Next, identified intracellular dietary chemicals such as ellagic acid were evaluated for their inhibitory effects against transducers of the c-Raf signaling pathway employing molecular docking and molecular dynamics (MD) simulation. RESULTS: GUDF treatment upon MCF-7 cells displayed significant loss of cell viability and induction of cell death. A set of dietary and metabolized chemicals in the intracellular compartment of MCF-7 cells, such as ellagic acid, 2-hydroxymyristic acid, artelinic acid, 10-amino-decanoic acid, nervonic acid, 2,4-dimethyl-2-eicosenoic acid, 2,3,4'- Trihydroxy,4-Methoxybenzophenone and 9-amino-nonanoic acid were identified. Among intracellular dietary chemicals, ellagic acid displayed a strong inhibitory affinity (-8.7 kcal/mol) against c-Raf kinase. The inhibitory potential of ellagic acid was found to be significantly comparable with a known c-Raf kinase inhibitor sorafenib with overlapping inhibitory site residues (ARG450, GLU425, TRP423, VA403). CONCLUSION: Intracellular dietary-derived chemicals such as ellagic acid are suggested for the induction of cell death in MCF-7 cells. Ellagic acid is predicted as an inhibitor of c-Raf kinase and could be explored as an anti-cancer drug.

RAF-like protein kinases mediate a deeply conserved, rapid auxin response.

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

Unveiling the domain-specific and RAS isoform-specific details of BRAF kinase regulation.

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various types of cancer and mutant RAS, the upstream regulator of BRAF, is a driver of up to one-third of all cancers. BRAF interacts with RAS and undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer, which propagates downstream signaling. Because of BRAF's complex regulation mechanism, the exact order and magnitude of its activation steps have yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation through pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activation of BRAF in a RAS isoform-specific manner. Moreover, we quantified the binding affinities between BRAF N-terminal and kinase domains (KD) to reveal their individual roles in autoinhibition. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the N-terminal domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insight into the activation mechanism of BRAF kinase to guide the development of new therapeutic strategies for cancer treatment.

A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization.

Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Tyrosine kinase inhibitors, such as imatinib, have been used as first-line therapy for the treatment of GISTs. Although these drugs have achieved considerable efficacy in some patients, reports of resistance and recurrence have emerged. Extracellular signal-regulated kinase 1/2 (ERK1/2) protein, as a member of the mitogen-activated protein kinase (MAPK) family, is a core molecule of this signaling pathway. Nowadays, research reports on the important clinical and prognostic value of phosphorylated-ERK (P-ERK) and phosphorylated-MAPK/ERK kinase (P-MEK) proteins closely related to raf kinase inhibitor protein (RKIP) have gradually emerged in digestive tract tumors such as gastric cancer, colon cancer, and pancreatic cancer. However, literature on the expression of these downstream proteins combined with RKIP in GIST is scarce. This study will focus on this aspect and search for answers to the problem. AIM: To detect the expression of RKIP, P-ERK, and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease. Try to establish a new prognosis evaluation model using RKIP and P-ERK in combination with analysis and its prognosis evaluation efficacy. METHODS: The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information. These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020. Immunohistochemical method was used to detect the expression of RKIP, P-ERK, and P-MEK proteins in GIST tissue samples from these patients. Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data. A Nomogram was used to represent the new prognostic evaluation model. The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors, based on two risk classification systems [the new risk grade model vs the modified National Institutes of Health (NIH) 2008 risk classification system]. Receiver operating characteristic (ROC) curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems. RESULTS: In GIST tissues, RKIP protein showed positive expression in the cytoplasm and cell membrane, appearing as brownish-yellow or brown granules. The expression of RKIP was related to GIST tumor size, NIH grade, and mucosal invasion. P-ERK protein exhibited heterogeneous distribution in GIST cells, mainly in the cytoplasm, with occasional presence in the nucleus, and appeared as brownish-yellow granules, and the expression of P-ERK protein was associated with GIST tumor size, mitotic count, mucosal invasion, and NIH grade. Meanwhile, RKIP protein expression was negatively correlated with P-ERK expression. The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis. However, RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance. Furthermore, we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model. ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system. CONCLUSION: Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size, NIH 2008 staging, and tumor invasion, and P-ERK expression was also related to mitotic count. The expression of the two proteins had a certain negative correlation. The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients. The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.

BRAF V600E Mutation of Non-Small Cell Lung Cancer in Korean Patients.

Background and Objectives: BRAF mutational status in resected non-small cell lung cancer (NSCLC) in the Korean population is poorly understood. We explored BRAF (particularly BRAF V600E) mutational status among Korean patients with NSCLC. Materials and Methods: This study included 378 patients with resected primary NSCLC who were enrolled from January 2015 to December 2017. The authors obtained formalin-fixed paraffin-embedded (FFPE) tissue blocks and performed peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR) for detecting BRAF V600, real-time PCR for detecting BRAF V600E, and immunohistochemical analyses using the mutation-specific Ventana VE1 monoclonal antibody. For positive cases in any methods mentioned above, direct Sanger sequencing was additionally performed. Results: The PNA-clamping method revealed the BRAF V600 mutation in 5 (1.3%) of the 378 patients. Among these five patients, real-time PCR, direct Sanger sequencing detected BRAF V600E mutations in three (0.8%) patients. Thus, two cases showed differences in their PNA-clamping and the others. Direct Sanger sequencing of PNA-clamping PCR product was performed for two cases showing negative results on direct Sanger sequencing; both contained BRAF mutations other than V600E. All patients harboring BRAF mutations had adenocarcinomas, and all patients with V600E mutation exhibited minor micropapillary components. Conclusions: Despite the low incidence of the BRAF mutation among Korean patients with NSCLC, lung adenocarcinoma patients with micropapillary components should be prioritized in terms of BRAF mutation testing. Immunohistochemical staining using Ventana VE1 antibody may serve as a screening examination for BRAF V600E.

Design, synthesis and characterisation of a novel type II B-RAF paradox breaker inhibitor.

The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.

Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib.

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

Discovery of New Quinolone-Based Diarylamides as Potent B-RAFV600E/C-RAF Kinase Inhibitors Endowed with Promising In Vitro Anticancer Activity.

The emergence of cancer resistance to targeted therapy represents a significant challenge in cancer treatment. Therefore, identifying new anticancer candidates, particularly those addressing oncogenic mutants, is an urgent medical demand. A campaign of structural modifications has been conducted to further optimize our previously reported 2-anilinoquinoline-diarylamides conjugate VII as a B-RAFV600E/C-RAF inhibitor. Considering the incorporation of a methylene bridge between the terminal phenyl and cyclic diamine, focused quinoline-based arylamides have been tailored, synthesized, and biologically evaluated. Among them, the 5/6-hydroxyquinolines 17b and 18a stood out as the most potent members, with IC50 values of 0.128 µM, 0.114 µM against B-RAFV600E, and 0.0653 µM, 0.0676 µM against C-RAF. Most importantly, 17b elicited remarkable inhibitory potency against the clinically resistant B-RAFV600K mutant with an IC50 value of 0.0616 µM. The putative binding mode of 17b and 18a were studied by molecular docking and molecular dynamics (MD). Moreover, the antiproliferative activity of all target compounds has been examined over a panel of NCI-60 human cancer cell lines. In agreement with cell-free assays, the designed compounds exerted superior anticancer impact over the lead quinoline VII against all cell lines at a 10 µM dose. Notably, both 17b and 18b showed highly potent antiproliferative activity against melanoma cell lines with growth percent under -90% (SK-MEL-29, SK-MEL-5, and UACC-62) at a single dose, while 17b maintained potency with GI50 values of 1.60-1.89 µM against melanoma cell lines. Taken together, 17b, a promising B-RAFV600E/V600K and C-RAF kinase inhibitor, may serve as a valuable candidate in the arsenal of anticancer chemotherapeutics.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression.

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

Computational assessment of chemicals from Morinda citrifolia as potential inhibitors of B-Raf kinase in hepatocellular carcinoma treatment.

Hepatocellular carcinoma (HCC) is a tumour pathology that lacks specific treatment and is predominantly resistant to chemotherapy. The inhibitory activity of Morinda citrifolia, an evergreen tree commonly called Noni, against various carcinomas especially HCC is widely acclaimed. This study was to assess the phytochemical constituents of the plant for inhibitory activity against B-Raf kinase (3C4C) in order to design drugs for HCC treatment. Molecular docking, pharmacophore modelling, induced-fit docking, molecular dynamics (MD) simulations and ADMET predictions were the computational techniques employed in this study to detect potential inhibitors of B-Raf kinase from 135 compounds of Morinda citrifolia. Soranjidiol, Thiamine, Lucidin, 2-Methyl-1,3,5-Trihydroxyanthraquinone and Rubiadin were the five top-scoring compounds ranging from -8.39 to -8.22 kcal/mol, however, the standard ligand, PLX4720, scored -11.26 kcal/mol. The five compounds, like PLX4720 demonstrated hydrogen bond interactions with active site amino acid residues such as GLN 530, CYS 532 and ASP 594. The main energy contributor to the interactions between the compounds and B-Raf kinase were pi-stacking, hydrogen bond, van der Waals and covalent energy. Better docking scores obtained in the induced-fit docking further validates the inhibitory potential of the Soranjidiol against the flexible protein. In MD simulations, Soranjidiol revealed good stability in the active site of the protein since significant conformational changes were not evident. These five compounds, unlike the standard compound, demonstrated adequate druglike properties and good safety profiles. Therefore, further studies should be undertaken so as to develop them into drugs against HCC.Communicated by Ramaswamy H. Sarma.

Recent Trends in Rationally Designed Molecules as Kinase Inhibitors.

Protein kinases modulate the structure and function of proteins by adding phosphate groups to threonine, tyrosine, and serine residues. The phosphorylation process mediated by the kinases regulates several physiological processes, while their overexpression results in the development of chronic diseases, including cancer. Targeting of receptor tyrosine kinase pathways results in the inhibition of angiogenesis and cell proliferation that validates kinases as a key target in the management of aggressive cancers. As such, the identification of protein kinase inhibitors revolutionized the contemporary anticancer therapy by inducing a paradigm shift in the management of disease pathogenesis. Contemporary drug design programs focus on a broad range of kinase targets for the development of novel pharmacophores to manage the overexpression of kinases and their pathophysiology in cancer pathogenesis. In this review, we present the emerging trends in the development of rationally designed molecular inhibitors of kinases over the last five years (2016-2021) and their incipient role in the development of impending anticancer pharmaceuticals.

Vicagrel is hydrolyzed by Raf kinase inhibitor protein in human intestine.

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04 ± 1.99 µM, 434.60 ± 12.46 nM/min/mg protein, and 21.69 ± 0.28 ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24 ± 0.04 mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.