Rho Family Research Articles

Associating Preoperative MRI Features and Gene Expression Signatures of Early-stage Hepatocellular Carcinoma Patients using Machine Learning.

Background and AimsThe relationship between quantitative magnetic resonance imaging (MRI) imaging features and gene-expression signatures associated with the recurrence of hepatocellular carcinoma (HCC) is not well studied.MethodsIn this study, we generated multivariable regression models to explore the correlation between the preoperative MRI features and Golgi membrane protein 1 (GOLM1), SET domain containing 7 (SETD7), and Rho family GTPase 1 (RND1) gene expression levels in a cohort study including 92 early-stage HCC patients. A total of 307 imaging features of tumor texture and shape were computed from T2-weighted MRI. The key MRI features were identified by performing a multi-step feature selection procedure including the correlation analysis and the application of RELIEFF algorithm. Afterward, regression models were generated using kernel-based support vector machines with 5-fold cross-validation.ResultsThe features computed from higher specificity MRI better described GOLM1 and RND1 gene-expression levels, while imaging features computed from lower specificity MRI data were more descriptive for the SETD7 gene. The GOLM1 regression model generated with three features demonstrated a moderate positive correlation (p<0.001), and the RND1 model developed with five variables was positively associated (p<0.001) with gene expression levels. Moreover, RND1 regression model integrating four features was moderately correlated with expressed RND1 levels (p<0.001).ConclusionsThe results demonstrated that MRI radiomics features could help quantify GOLM1, SETD7, and RND1 expression levels noninvasively and predict the recurrence risk for early-stage HCC patients.

SmgGDS: An Emerging Master Regulator of Prenylation and Trafficking by Small GTPases in the Ras and Rho Families

Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.

Emerging Roles of Small GTPases in Islet β-Cell Function.

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet β-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the β-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in β-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic β-cells, which contribute to the dysregulation of metabolism and the development of T2D.

A putative structural mechanism underlying the antithetic effect of homologous RND1 and RhoD GTPases in mammalian plexin regulation.

Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations, we showed that RND1 reinforces the plexin dimerization interface, whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase-binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.

Lysophosphatidic Acid Regulates Rho Family of GTPases in Lungs.

The bio-active lipid, lysophosphatidic acid (LPA) interacts with various lysophosphatidic acid receptors (LPARs) to affect a variety of cellular functions, including proliferation, differentiation, survival, migration, morphogenesis and others. The Rho family of small GTPases, is well-known downstream signaling pathways activated by LPA. Among the Rho GTPases, RhoA, Rac1, and Cdc42 are best characterized and LPA-induced activation of the GTPases RhoA, Rac1, and Cdc42 influences a wide range of cellular processes and functions such as cell differentiation, contractile movements, cellular migration, or infiltration. In this review, we will briefly discuss the interplay between LPA and each of these three Rho family proteins, summarizing the main interactions between them. Our discussion will focus mainly on their interplay within lung endothelial and epithelial cells, drawing attention to how these interactions may contribute to pro-inflammatory processes.

Repression of RhoJ expression promotes TGF-β-mediated EMT in human non-small-cell lung cancer A549cells

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC.In the present study, we demonstrated that RhoJ knockdown accelerated TGF-βmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-β–Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC.

Progress in the therapeutic inhibition of Cdc42 signalling.

Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42–GEF, Cdc42–GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.

The Multiple Functions of Rho GTPases in Fission Yeasts.

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus, has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine–nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

MRTF: Basic Biology and Role in Kidney Disease.

A lesser known but crucially important downstream effect of Rho family GTPases is the regulation of gene expression. This major role is mediated via the cytoskeleton, the organization of which dictates the nucleocytoplasmic shuttling of a set of transcription factors. Central among these is myocardin-related transcription factor (MRTF), which upon actin polymerization translocates to the nucleus and binds to its cognate partner, serum response factor (SRF). The MRTF/SRF complex then drives a large cohort of genes involved in cytoskeleton remodeling, contractility, extracellular matrix organization and many other processes. Accordingly, MRTF, activated by a variety of mechanical and chemical stimuli, affects a plethora of functions with physiological and pathological relevance. These include cell motility, development, metabolism and thus metastasis formation, inflammatory responses and—predominantly-organ fibrosis. The aim of this review is twofold: to provide an up-to-date summary about the basic biology and regulation of this versatile transcriptional coactivator; and to highlight its principal involvement in the pathobiology of kidney disease. Acting through both direct transcriptional and epigenetic mechanisms, MRTF plays a key (yet not fully appreciated) role in the induction of a profibrotic epithelial phenotype (PEP) as well as in fibroblast-myofibroblast transition, prime pathomechanisms in chronic kidney disease and renal fibrosis.

The to and fro of Rho

The functions of Ras members are largely governed by the structural dynamics of the nucleotide-binding switch regions. In this issue of Structure, Lin etal. (2021) take a close look at the dynamic equilibrium of RhoA, the founding member of the Rho family of Ras GTPases.

Essential role of smooth muscle Rac1 in severe asthma associated-airway remodelling

Severe asthma is a chronic lung disease characterized by inflammation, airway hyperresponsiveness (AHR) and airway remodelling with hyperplasia of airway smooth muscle cells (aSMC). Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 controls bronchoconstriction. The objective of this study was to assess the role of Rac1 in severe asthma associated airway remodelling. We have studied the level of Rac1 activation by immunofluorescence in bronchial biopsies from severe asthmatic patients and control patients. In order to identify the role of Rac1 in aSMC remodelling, mice with specific deletion of Rac1 in SMC were sensitized to house dust mite to mimic the characteristics of severe allergic asthma in humans. Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from severe asthmatic patients compared to control subjects. The basal proliferation rate of aSMC from severe asthmatics subjects is increased compared to control subjects. We have also observed that bFGF and PDGF-BB, growth factors described to be implicated in airway remodelling, stimulate the proliferation of aSMC from severe asthmatic subjects by 33% and 37% respectively. Moreover, the inhibition of Rac1 by EHT1864 decreased this proliferation by more than 50%. By a biochemical approach we have demonstrated that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the STAT3 signalling pathway. In vivo, we have demonstrated that specific deletion of Rac1 in SMC prevents aSMC hyperplasia and hypertrophy. This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation and airway remodelling. Inhibition of Rac1 dependant signalling pathway may represent an interesting target for the treatment of severe asthma.

Hsa_circ_0008234 inhibits the progression of lung adenocarcinoma by sponging miR-574-5p

circRNAs are a novel type of noncoding RNA (ncRNA) that have been identified as an important regulator of gene expression and play a part in the progression of various diseases. However, the function of circ_0008234 in lung adenocarcinoma (LUAC) remains unknown. Through the GEO (Gene Expression Omnibus) database, circ_0008234 was first found to be downregulated in LUAC tissues. It could inhibit cell growth and accelerate apoptosis in vitro and in vivo. In terms of its possible mechanism, circ_0008234 mainly was present in the cytoplasm and competed with miR-574-5p to regulate RND3 (Rho family GTPase 3). Our results revealed that circ_0008234 inhibited the progression of LUAC through a competing endogenous RNA (ceRNA)-based mechanism and provided potential biomarkers and therapeutic targets for LUAC treatment.

Cdc42 has important roles in postnatal angiogenesis and vasculature formation

Cdc42, a Rho family low molecular weight G protein, has important roles in various cell functions, including cytoskeletal rearrangement, cell adhesion and cell proliferation and differentiation. To investigate the involvement of Cdc42 in the activities of vascular endothelial cells, we generated Cdc42 conditional knockout mice in which Cdc42 was time -specifically deficient in vascular endothelial cells (Cdc42 ​fl/fl; VE-Cad CreERT: Cdc42 cKO). When the Cdc42 gene was deleted after birth, Cdc42 cKO mice were smaller than the control mice, and died between postnatal day 8 (P8) and P10. Necropsy findings confirmed that these mice had various pathological aberrances in the vessels of most organs, such as blood flow congestion and blood cell invasion. Electron microscopic observations also revealed that capillary endothelial cells were detached from the basement membrane as well as phagocytosis of dead endothelial cells induced by macrophages. Moreover, vascular sprouting from aortic rings induced by VEGF-A was diminished in samples from the Cdc42 cKO mice because of an endothelial cell proliferation defect. These results suggest that Cdc42 in vascular endothelial cells has important roles in blood vessel formation after birth.

Pharmacological Modulators of Small GTPases of Rho Family in Neurodegenerative Diseases.

Classical Rho GTPases, including RhoA, Rac1, and Cdc42, are members of the Ras small GTPase superfamily and play essential roles in a variety of cellular functions. Rho GTPase signaling can be turned on and off by specific GEFs and GAPs, respectively. These features empower Rho GTPases and their upstream and downstream modulators as targets for scientific research and therapeutic intervention. Specifically, significant therapeutic potential exists for targeting Rho GTPases in neurodegenerative diseases due to their widespread cellular activity and alterations in neural tissues. This study will explore the roles of Rho GTPases in neurodegenerative diseases with focus on the applications of pharmacological modulators in recent discoveries. There have been exciting developments of small molecules, nonsteroidal anti-inflammatory drugs (NSAIDs), and natural products and toxins for each classical Rho GTPase category. A brief overview of each category followed by examples in their applications will be provided. The literature on their roles in various diseases [e.g., Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD), and Multiple sclerosis (MS)] highlights the unique and broad implications targeting Rho GTPases for potential therapeutic intervention. Clearly, there is increasing knowledge of therapeutic promise from the discovery of pharmacological modulators of Rho GTPases for managing and treating these conditions. The progress is also accompanied by the recognition of complex Rho GTPase modulation where targeting its signaling can improve some aspects of pathogenesis while exacerbating others in the same disease model. Future directions should emphasize the importance of elucidating how different Rho GTPases work in concert and how they produce such widespread yet different cellular responses during neurodegenerative disease progression.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

RLIP76: A Structural and Functional Triumvirate.

Simple SummaryThe RLIP76 protein is present at high levels in multiple cancers, compared with levels in normal cells. In cancer cells it is thought to be important for removal of chemotherapeutics, while in normal cells it has been implicated in endocytosis, stress response and mitochondrial fission. Although RLIP76 is a potential target for ovary, breast, lung, colon, prostate and kidney cancers, only the middle third of the protein has been structurally characterized. Understanding the full structure of RLIP76 will help us to better understand the signalling pathways in which it is involved and by extension its role in cancer.RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications.

The C-type lectin CD93 controls endothelial cell migration via activation of the Rho family of small GTPases

Endothelial cell migration is essential to angiogenesis, enabling the outgrowth of new blood vessels both in physiological and pathological contexts. Migration requires the activation of several signaling pathways, the elucidation of which expands the opportunity to develop new drugs to be used in antiangiogenic therapy. In the proliferating endothelium, the interaction between the transmembrane glycoprotein CD93 and the extracellular matrix activates signaling pathways that regulate cell adhesion, migration, and vascular maturation. Here we identify a pathway, comprising CD93, the adaptor proteins Cbl and Crk, and the small GTPases Rac1, Cdc42, and RhoA, which we propose acts as a regulator of cytoskeletal movements responsible for endothelial cell migration. In this framework, phosphorylation of Cbl on tyrosine 774 leads to the interaction with Crk, which acts as a downstream integrator in the CD93-mediated signaling regulating cell polarity and migration. Moreover, confocal microscopy analyses of GTPase biosensors show that CD93 drives coordinated activation of Rho-proteins at the cell edge of migratory endothelial cells. In conclusion, together with the demonstration of the key contribution of CD93 to the migratory process in living cells, these findings suggest that the signaling triggered by CD93 converges to the activation and modulation of the Rho GTPase signaling pathways regulating cell dynamics.

Subcellular Colocalization of Rho GTPase TCL with Rab Family GTPases

TCL (RhoJ) belongs to the Rho family of GTPases and contributes to tumor-associated angiogenesis. Understanding its basic cell biology is essential for establishing its role in this process. Experiments in several labs have shown plasma membrane and vesicular localizations of TCL, but it is unknown where the GTPase may function in vesicle trafficking. To better understand this aspect of TCL cellular biology, a YFP-tagged version TCL will be separately co-expressed in HeLa cells with twelve different mCherry-tagged versions of Rab GTPases. Images will be captured using an Olympus Fluoview confocal microscope and colocalization assessed both qualitatively and quantitatively using CellSens software. Since many of the Rab GTPases have established vesicular localizations, we hypothesize that analyzing the colocalization of tagged Rabs and TCL will help establish specific vesicular localization patterns of TCL.

Suppressing Lung Cancer Cell Migration and Invasion through Disruption of Rho GTPase Function by Diclofenac and Docosahexaenoic Acid

Background Low survival rates of lung cancer patients and high recurrence rate in non-small cell lung cancer (NSCLC) patients have been attributed to metastasis. The aberrant signaling of GTPases, such as the Rho family of GTPases (Cdc42, Rac1, and RhoA) have been implicated in lung cancer metastasis. Several studies indicate that cyclooxygenases (COX) inhibitors and omega-3 polyunsaturated fatty acids (PUFAs) may help prevent cell migration and invasion, which are critical steps in cancer cell metastasis. We previously reported the inhibitory effect of co-treatment with docosahexaenoic acid (DHA) and diclofenac on the expression of hyperactive small GTPases. This study aimed to investigate the potential anti-metastatic effects of such co-treatment on NSCLC cancer cells as well as to investigate the related mechanisms. Methods We conducted western blotting to detect Rho GTPases (RhoA, Rac1, and CDC42) in A549, NCI-H1299, and NCI-H1975 cells after exposure to DHA and diclofenac. We also conducted immunostaining analyses of F-actin organization and Rho GTPases in A549 cells. We further examined the effect of co-treatment on NSCLC cell migration and invasion. Expression of marker proteins in EMT and MMP-2/9 were detected by western blot analysis. Results Co-treatment with DHA and diclofenac disrupted actin filament assembly as well as inhibited cell migration and invasion. Exposure of A549 cells to DHA (5 μM) and diclofenac (25 μM) resulted in suppression of both the distance of migration by 58.1 ± 2.8% as well as the number of cells that migrated into the wounded area by 62.2 ± 1.8%. In addition, the co-treatment resulted in altered expression of marker proteins involved in EMT by increasing expression of epithelial marker, E-cadherin, while downregulating the expressions of mesenchymal markers, N-cadherin, Fibronectin and Vimentin, ZEB1, and β-catenin. Conclusions Our findings indicate that co-treatment with diclofenac and DHA suppressed NSCLC cell motility and invasion, and is associated with disruption of the actin cytoskeleton and inhibition of the Rho GTPase activity. Our findings suggest that combination of diclofenac and DHA could potentially prevent or control NSCLC metastasis.

Characterization and development of new anti‐toxins targeting Clostridium difficile

Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. One strategy for targeting C. difficile infection is to development treatments that target bacterial virulence. Anti-virulence strategies to treat C. difficile infection are attractive options to antibiotic therapy because the human microbiota is spared and damage to host tissue is minimized. The Tcd toxins, TcdA and TcdB, are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glucosylating and inactivating Rho family GTPases using UDP-glucose as a glucosyl donor. Glucosylation of Rho GTPases leads to cell rounding and eventually cell death. We used kinetic isotope effects (KIEs) and transition state theory to solve the transition state structure of the glycosyltransferase domain of TcdB (TcdB-GTD). This permits the design of transition state analogues which are potent enzyme inhibitors. KIEs compare the chemical steps of enzymatic reaction rates with isotopically labeled substrates and unlabeled substrates and provide critical information on the catalytic mechanism, bond lengths and geometry at the transition state. KIE analysis on the glycohydrolase reaction of TcdB-GTD supported the formation of a late, dissociative glucocation-like transition state where positive charge develops on the anomeric carbon. Glucocationic transition states are best mimicked by cationic nitrogen groups at or near the anomeric carbon. Iminosugars contain a nitrogen atom in the sugar ring in place of oxygen and mimic glucocationic transition states. We tested iminosugars for inhibition of TcdB-GTD and TcdA-GTD. Isofagomine (Ki TcdB-GTD = 1.4 µM and Ki TcdA-GTD = 0.24 µM) and noeuromycin (Ki TcdB-GTD = 10.6 µM and Ki TcdA-GTD = 4.7 µM) were the most potent iminosugars identified. Isofagomine and noeuromycin were shown to exhibit an uncompetitive mechanism of inhibition suggesting that both iminosugars act by binding to the enzyme-UDP complex. Isothermal titration calorimetry experiments with TcdB-GTD supported this finding where isofagomine and noeuromycin could only bind to TcdB-GTD in the presence of UDP with Kd = 0.133 µM and Kd = 0.4 µM respectively. In addition, the X-ray crystal structure of TcdB-GTD in complex with either Isofagomine or noeuromycin with UDP revealed the formation of an ion-pair interaction between the cationic nitrogen atom of the iminosugar and the β-phosphate of UDP. Finally, we confirmed that isofagomine and noeuromycin could prevent Tcd toxin-induced cytotoxicity of IMR90 cells. Firstly, both iminosugars were able to prevent Tcd toxin-induced cell rounding as visualized by light microscopy. Secondly, Western blot analysis of Tcd toxin treated IMR90 cell lysates revealed that both isofagomine and noeuromycin could prevent glucosylation of Rac1 by Tcd toxin. Isofagomine and noeuromycin are effective in preventing Tcd toxin action. Transition state analogues against TcdA and TcdB have the potential to be used as treatments for C. difficile infections.