Search Results (29)

Small (monomeric) GTP-binding proteins (smgs; Cdc42 and Rac1) play requisite roles in islet beta cell function, including glucose-stimulated insulin secretion. In addition, emerging evidence suggests that sustained (constitutive) activation of smgs (e.g., Rac1) culminates in the genesis of islet beta cell dysfunction under the duress of chronic hyperglycemia. It is noteworthy that functions (i.e., activation–deactivation) of smgs in many cells, including the islet beta cell, have been shown to be under the regulatory control of at least three factors, namely the guanine nucleotide exchange factors (GEFs), the GTPase-activating proteins (GAPs), and the GDP-dissociation inhibitors (GDIs). The overall objective of this review is to highlight our current understanding of the regulatory roles of the RhoGDIβ-Rac1-CARD9 signalome in the pathology of beta cell dysfunction under chronic hyperglycemic stress. For brevity, this review is structured by an overview of smgs and their regulatory proteins/factors in the beta cell, followed by a discussion of potential roles of the RhoGDIβ-Rac1-CARD9 axis in the onset of cellular dysfunction under the duress of metabolic stress. Overall conclusions, potential knowledge gaps, and opportunities for future research in this field of islet biology are highlighted in the last section. Full article

Background: KRAS-G12D mutations drive 20–50% of pancreatic/biliary cancers yet remain challenging to target due to GTP-pocket conservation and high cellular GTP levels. While allosteric inhibitors targeting the SWII pocket (e.g., MRTX1133) show promise, limited chemical diversity and paradoxical cellular/enzymatic activity relationships necessitate the exploration of novel scaffolds. This study aims to develop KRAS-G12D inhibitors and PROTACs to offer a selection of new chemical entities through systematic structure–activity optimization and evaluate their therapeutic potential through PROTAC derivatization. Methods: Eleven compounds featuring heterocyclic cores (pyrimidine/pyrido[4,3-d]pyrimidine/5,6,7,8-tetrahydroprodo[3,4-d]pyrimidine) were designed via structure-based drug design. Antiproliferative activity against KRAS-G12D (Panc1), KRAS-G13D (HCT116) and wild-type (A549) cells was assessed using the CCK-8 assay. KRAS-G12D enzymatic inhibition was measured using a GTPase activity assay. Molecular docking simulations (Sybyl 2.0; PDB:7RPZ) elucidated binding modes. Two PROTACs were synthesized from lead compounds by conjugating E3 ligase linkers. All the novel inhibitors and PROTACs were characterized by means of NMR or HRMS. Results: Compound 10c demonstrated selective anti-proliferation in Panc1 cells (IC₅₀ = 1.40 μM) with 4.9-fold greater selectivity over wild-type cells, despite weak enzymatic inhibition (IC₅₀ > 10 μM). Docking revealed critical hydrogen bonds between its protonated 3,8-diazabicyclo[3.2.1]octane moiety and Asp12/Gly60. The enzymatic inhibitor 10k showed potent KRAS-G12D inhibition (IC₅₀ = 0.009 μM) through homopiperazine-mediated interactions with Glu92/His95. Derived PROTACs 26a/b exhibited reduced potency (IC₅₀ = 3–5 μM vs. parental 10k: 2.22 μM), potentially due to impaired membrane permeability. Conclusions: Eleven novel KRAS-G12D inhibitors with a seven-membered ring pharmacophore were synthesized. Compound 10c showed strong anti-proliferative activity, while 10k exhibited potent enzymatic inhibition. Two PROTACs were designed but showed no clear advantage over 10k. This study provides valuable insights for KRAS-targeted drug development. Full article

In the Nicotiana tabacum flower development study, we identified SCI1 (Stigma/style Cell-cycle Inhibitor 1), a regulator of cell proliferation. SCI1 interacts with NtCDKG;2 (N. tabacum Cyclin-Dependent Kinase G;2), a homolog of human CDK11, which is responsible for RanGTP-dependent microtubule stabilization, regulating spindle assembly rate. In a Y2H screening of a cDNA library using NtCDKG;2 as bait, a RanBP1 (Ran-Binding Protein 1) was revealed as its interaction partner. RanBP1 is an essential regulatory protein of the RanGTPase system, contributing to the formation of the Ran gradient, which modulates different important cellular processes. RanBP1 is crucial in the nuclear import/export machinery during interphase and spindle checkpoint formation during cell division. These processes are well studied in animals, but very little is known about them in plants. We confirmed NtCDKG;2 and NtRanBP1 interaction by pairwise Y2H and characterized the localization of both proteins during plant cell division. We demonstrated the presence of NtRanBP1 in the cytoplasm during interphase and its nuclear arrest at mitosis onset. Meanwhile, we showed that NtCDKG;2 is localized in the mitotic spindle during cell division, indicating an analogous function to the human CDK11. We propose that the phosphorylation of the nuclear export signal at RanBP1 by NtCDKG;2 may be responsible for the reported nuclear arrest. Full article

Despite significant advancements in the field of oncology, cancers still pose one of the greatest challenges of modern healthcare. Given the cytoskeleton’s pivotal role in regulating mechanisms critical to cancer development, further studies of the cytoskeletal elements could yield new practical applications. Septins represent a group of relatively well-conserved GTP-binding proteins that constitute the fourth component of the cytoskeleton. Septin 9 (SEPT9) has been linked to a diverse spectrum of malignancies and appears to be the most notable septin member in that category. SEPT9 constitutes a biomarker of colorectal cancer (CRC) and has been positively correlated with a high clinical stage in breast cancer, cervical cancer, and head and neck squamous cell carcinoma. SEPT9_i1 represents the most extensively studied isoform of SEPT9, which substantially contributes to carcinogenesis, metastasis, and treatment resistance. Nevertheless, the mechanistic basis of SEPT9_i1 oncogenicity remains to be fully elucidated. In this review, we highlight SEPT9’s and SEPT9_i1’s structures and interactions with Hypoxia Inducible Factor α (HIF-1 α) and C-Jun N-Terminal Kinase (JNK), as well as discuss SEPT9_i1’s contribution to aneuploidy, cell invasiveness, and taxane resistance—key phenomena in the progression of malignancies. Finally, we emphasize forchlorfenuron and other septin inhibitors as potential chemotherapeutics and migrastatics. Full article

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme’s dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2’s conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters k_inact and K_I. Their impact on the enzyme’s ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity. Full article

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase. Full article

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway. Full article

The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed. Full article

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes viral encephalitis leading to neural damage, is a major threat in most Asian countries. The RNA-dependent RNA polymerase (RdRp) present in the viral genome is the key component for genome replication, making it an attractive target for antiviral drug development. In this study, the natural products from Echinacea angustifolia were retrieved for structure-based virtual screening against JEV–RdRp. The top six compounds (Echinacoside, Echinacin, Rutin, Cynaroside, Quercetagetin 7-glucoside, and Kaempferol-3-glucoside) were obtained based on the highest negative docking score, ADMET (absorption, distribution, metabolism, excretion, and toxicity), and molecular interaction. The computational analysis of these selected compounds against the co-crystallized ligands, i.e., ATP and GTP, were performed. Further, 100 ns molecular dynamic simulation and post-free binding energy calculation of all the selected compounds complexed with JEV–RdRP were performed to check the stability of the complexes. The obtained results showed considerable stability and intermolecular interaction with native ligand-binding site residues of JEV–RdRp. Hence, selected natural compounds are admissible inhibitors of JEV–RdRp protein and can be considered for future antiviral drug development studies. Full article

Bladder pathologies, very common in the aged population, have a considerable negative impact on quality of life. Novel targets are needed to design drugs and combinations to treat diseases such as overactive bladder and bladder cancers. A promising new target is the ubiquitous Rho GTPase Rac1, frequently dysregulated and overexpressed in bladder pathologies. We have analyzed the roles of Rac1 in different bladder pathologies, including bacterial infections, diabetes-induced bladder dysfunctions and bladder cancers. The contribution of the Rac1 protein to tumorigenesis, tumor progression, epithelial-mesenchymal transition of bladder cancer cells and their metastasis has been analyzed. Small molecules selectively targeting Rac1 have been discovered or designed, and two of them—NSC23766 and EHT 1864—have revealed activities against bladder cancer. Their mode of interaction with Rac1, at the GTP binding site or the guanine nucleotide exchange factors (GEF) interaction site, is discussed. Our analysis underlines the possibility of targeting Rac1 with small molecules with the objective to combat bladder dysfunctions and to reduce lower urinary tract symptoms. Finally, the interest of a Rac1 inhibitor to treat advanced chemoresistance prostate cancer, while reducing the risk of associated bladder dysfunction, is discussed. There is hope for a better management of bladder pathologies via Rac1-targeted approaches. Full article

Mangrove secondary metabolites have many unique biological activities. We identified lead compounds among them that might target KRAS^G12C. KRAS is considered to be closely related to various cancers. A variety of novel small molecules that directly target KRAS are being developed, including covalent allosteric inhibitors for KRAS^G12C mutant, protein–protein interaction inhibitors that bind in the switch I/II pocket or the A59 site, and GTP-competitive inhibitors targeting the nucleotide-binding site. To identify a candidate pool of mangrove secondary metabolic natural products, we tested various machine learning algorithms and selected random forest as a model for predicting the targeting activity of compounds. Lead compounds were then subjected to virtual screening and covalent docking, integrated absorption, distribution, metabolism and excretion (ADME) testing, and structure-based pharmacophore model validation to select the most suitable compounds. Finally, we performed molecular dynamics simulations to verify the binding mode of the lead compound to KRAS^G12C. The lazypredict function package was initially used, and the Accuracy score and F1 score of the random forest algorithm exceeded 60%, which can be considered to carry a strong ability to distinguish the data. Four marine natural products were obtained through machine learning identification and covalent docking screening. Compound 44 and compound 14 were selected for further validation after ADME and toxicity studies, and pharmacophore analysis indicated that they had a favorable pharmacodynamic profile. Comparison with the positive control showed that they stabilized switch I and switch II, and like MRTX849, retained a novel binding mechanism at the molecular level. Molecular dynamics analysis showed that they maintained a stable conformation with the target protein, so compound 44 and compound 14 may be effective inhibitors of the G12C mutant. These findings reveal that the mangrove-derived secondary metabolite compound 44 and compound 14 might be potential therapeutic agents for KRAS^G12C. Full article

Ewing sarcoma (EwS) is an aggressive primary bone cancer in children and young adults characterized by oncogenic fusions between genes encoding FET-RNA-binding proteins and ETS transcription factors, the most frequent fusion being EWSR1-FLI1. We show that EGR2, an Ewing-susceptibility gene and an essential direct target of EWSR1-FLI1, directly regulates the transcription of genes encoding key enzymes of the mevalonate (MVA) pathway. Consequently, Ewing sarcoma is one of the tumors that expresses the highest levels of mevalonate pathway genes. Moreover, genome-wide screens indicate that MVA pathway genes constitute major dependencies of Ewing cells. Accordingly, the statin inhibitors of HMG-CoA-reductase, a rate-limiting enzyme of the MVA pathway, demonstrate cytotoxicity in EwS. Statins induce increased ROS and lipid peroxidation levels, as well as decreased membrane localization of prenylated proteins, such as small GTP proteins. These metabolic effects lead to an alteration in the dynamics of S-phase progression and to apoptosis. Statin-induced effects can be rescued by downstream products of the MVA pathway. Finally, we further show that statins impair tumor growth in different Ewing PDX models. Altogether, the data show that statins, which are off-patent, well-tolerated, and inexpensive compounds, should be strongly considered in the therapeutic arsenal against this deadly childhood disease. Full article

The Ras-Raf-MEK-ERK signaling pathway, the first well-established MAPK pathway, plays essential roles in cell proliferation, survival, differentiation and development. It is activated in over 40% of human cancers owing to mutations of Ras, membrane receptor tyrosine kinases and other oncogenes. The Raf family consists of three isoforms, A-Raf, B-Raf and C-Raf. Since the first discovery of a truncated mutant of C-Raf as a transforming oncogene carried by a murine retrovirus, forty years of extensive studies have provided a wealth of information on the mechanisms underlying the activation, regulation and biological functions of the Raf family. However, the mechanisms by which activation of A-Raf and C-Raf is accomplished are still not completely understood. In contrast, B-Raf can be easily activated by binding of Ras-GTP, followed by cis-autophosphorylation of the activation loop, which accounts for the fact that this isoform is frequently mutated in many cancers, especially melanoma. The identification of oncogenic B-Raf mutations has led to accelerated drug development that targets Raf signaling in cancer. However, the effort has not proved as effective as anticipated, inasmuch as the mechanism of Raf activation involves multiple steps, factors and phosphorylation of different sites, as well as complex interactions between Raf isoforms. In this review, we will focus on the physiological complexity of the regulation of Raf kinases and their connection to the ERK phosphorylation cascade and then discuss the role of Raf in tumorigenesis and the clinical application of Raf inhibitors in the treatment of cancer. Full article

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility. Full article

Around 20% of all malignancies harbour activating mutations in RAS isoforms. Despite this, there is a deficiency of RAS-targeting agents licensed for therapeutic use. The picomolar affinity of RAS for GTP, and the lack of suitable pockets for high-affinity small-molecule binding, precluded effective therapies despite decades of research. Recently, characterisation of the biochemical properties of KRAS-G12C along with discovery of its ‘switch-II pocket’ have allowed development of effective mutant-specific inhibitors. Currently seven KRAS-G12C inhibitors are in clinical trials and sotorasib has become the first one to be granted FDA approval. Here, we discuss historical efforts to target RAS directly and approaches to target RAS effector signalling, including combinations that overcome limitations of single-agent targeting. We also review pre-clinical and clinical evidence for the efficacy of KRAS-G12C inhibitor monotherapy followed by an illustration of combination therapies designed to overcome primary resistance and extend durability of response. Finally, we briefly discuss novel approaches to targeting non-G12C mutant isoforms. Full article