TRIM56 protects against nonalcoholic fatty liver disease by promoting the degradation of fatty acid synthase
Abstract
Nonalcoholic fatty liver disease (NAFLD) encompasses a disease continuum from simple steatosis to nonalcoholic
steatohepatitis (NASH). However, there are currently no approved pharmacotherapies for NAFLD, although several drugs
are in advanced stages of clinical development. Because of the complex pathophysiology and heterogeneity of NAFLD,
the identification of potential therapeutic targets is clinically important. Here, we demonstrated that tripartite motif
56 (TRIM56) protein abundance was markedly downregulated in the livers of individuals with NAFLD and of mice fed
a high-fat diet. Hepatocyte-specific ablation of TRIM56 exacerbated the progression of NAFLD, while hepatic TRIM56
overexpression suppressed it. Integrative analyses of interactome and transcriptome profiling revealed a pivotal role of
TRIM56 in lipid metabolism and identified the lipogenesis factor fatty acid synthase (FASN) as a direct binding partner
of TRIM56. TRIM56 directly interacted with FASN and triggered its K48-linked ubiquitination–dependent degradation.
Finally, using artificial intelligence–based virtual screening, we discovered an orally bioavailable small-molecule inhibitor
of FASN (named FASstatin) that potentiates TRIM56-mediated FASN ubiquitination. Therapeutic administration of
FASstatin improved NAFLD and NASH pathologies in mice with an optimal safety, tolerability, and pharmacokinetics
profile. Our findings provide proof of concept that targeting the TRIM56/FASN axis in hepatocytes may offer potential
therapeutic avenues to treat NAFLD.
... TRIM proteins form one of the largest subfamilies of RING (Really Interesting New Gene)-containing ubiquitin ligases. They belong to the highly conserved RING family of E3 ubiquitin ligases, also known as RBCC proteins-a large family of over 100 members found in all multicellular mammals, playing a pivotal role in ubiquitination 8 .Tripartite Motif-Containing Protein 56 (TRIM56), a member of the TRIM family, has been shown to play crucial roles in antiviral defense 9 and protection against nonalcoholic fatty liver disease 10 , however, its impact on pan-cancer prognosis and immune infiltration has not yet been reported. In this study, we found that TRIM56 was differentially expressed in most tumors. ...
TRIM56 plays a role in tumor development through the ubiquitination of several key substrate molecules. However, its relationship with tumor prognosis and immune infiltration remains unclear. The expression and localization of TRIM56 were analyzed from TCGA_GTEx, TCGA and HPA database. The effects of TRIM56 on the proliferation and migration of lung cancer cells A549 were evaluated by CCK-8 and wound healing assays. Correlations between TRIM56 expression and survival in patients were analyzed using the Kaplan-Meier Plotter and a nomogram model. Additionally, the relationship between TRIM56 and immune cell infiltration in tumors was explored via TIMER 2.0. Functional interactions and associated proteins of TRIM56 were examined using GEPIA 2.0 and the STING database. The signaling pathways influenced by TRIM56 were identified through GO and KEGG analyses. TRIM56 expression showed significant variation across 11 different tumor types when compared to normal tissues, with some tumors displaying high expression and others showing the opposite. TRIM56 inhibited the proliferation and migration of A549 cells. High TRIM56 expression was associated with shorter overall survival (OS) in patients with COAD, GBM, and LGG, but with longer OS in BLCA, KIRC, MESO, and SKCM. In BLCA and KIRC, high TRIM56 expression was closely linked to B cells, macrophages, and CD4(+) and CD8(+) T cell infiltration, contributing to a favorable prognosis. TRIM56 appears to affect tumor development through transcriptional regulatory complexes, transcriptional co-regulatory factor activity, and immune-related pathways.TRIM56 may play a critical role in tumor immunity and influence tumor prognosis. It holds potential as both a target for immunotherapy and a prognostic marker.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-97856-w.
... Given that liver tissue lesions can lead to lipid metabolism disorders and abnormal blood lipids [22], we measured the levels of blood lipids (TC, TG, LDL-c, and HDL-c) and liver function indicators (AST and ALT) in mice [1]. Compared with the PBS group, the levels of TC and TG in CS-1M and CS-2M mice showed a gradually increasing trend, which was more pronounced in the CS-2M group. ...
Background: This study aims to investigate the impact of coal dust (silicon dioxide) exposure on dyslipidemia and its underlying mechanisms, with a focus on the association between coal dust exposure and hepatic metabolic disorders. Methods: Clinical data were collected from 5433 coal mine workers to compare the incidence of dyslipidemia between the dust-exposed group and the non-exposed group. A mouse model of silicon dioxide exposure was established to observe hepatic fat accumulation and pathological changes. Liver tissue sequencing was performed to screen for key differential genes. In vitro cell experiments were utilized to identify the molecular mechanisms underlying hepatocyte metabolic abnormalities induced by silicon dioxide exposure. Results: Clinical data revealed that 69.2% of miners in the dust-exposed group developed dyslipidemia, which was higher than the 30.7% in the non-exposed group. Animal data showed that silicon dioxide exposure led to hepatic fat deposition and pathological damage, with the degree of injury positively correlated with exposure time. Liver sequencing identified a significant upregulation of the FMO3 (flavin monooxygenase 3) gene in mouse liver tissue following silicon dioxide exposure, accompanied by enhanced inflammatory responses. Mechanistic studies demonstrated that silicon dioxide activates Kupffer cells to secrete IL-6 (interleukin-6), which induces high expression of FMO3 in hepatocytes through the PKC/YY1 signaling pathway, thereby disrupting lipid metabolism. Conclusions: Silicon dioxide exposure can promote the upregulation of FMO3 expression in hepatocytes by activating Kupffer cells to release IL-6 via the PKC/YY1 pathway, ultimately leading to lipid metabolic disorders and dyslipidemia
... Another study utilized a high-content imaging analysis system to identify the E3 ubiquitin ligase TRIM56 as a suppressor of lipid deposition. The study experimentally demonstrated that TRIM56 promotes the degradation of FASN via the proteasomal pathway by catalyzing K48-linked ubiquitination, thereby suppressing FASN protein expression and downstream lipid synthesis to delay the progression of NAFLD (121). HMG-CoA reductase degradation protein 1 (Hrd1) functions as both an E3 ubiquitin ligase and ERAD complex subunit. ...
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases and is closely associated with metabolic abnormalities. The causes of NAFLD are exceedingly complicated, and it is known that a variety of signaling pathways, endoplasmic reticulum stress, and mitochondrial dysfunction play a role in the pathogenesis of NAFLD. Recent studies have shown that ubiquitination and deubiquitination are involved in the regulation of the NAFLD pathophysiology. Protein ubiquitination is a dynamic and diverse post-translational alteration that affects various cellular biological processes. Numerous disorders, including NAFLD, exhibit imbalances in ubiquitination and deubiquitination. To highlight the significance of this post-translational modification in the pathogenesis of NAFLD and to aid in the development of new therapeutic approaches for the disease, we will discuss the role of enzymes involved in the processes of ubiquitination and deubiquitination, specifically E3 ubiquitin ligases and deubiquitinating enzymes that are important in the regulation of NAFLD.
... The fold change relative to the respective controls was determined using the 2^( -ΔΔCt ) method. Primers were designed using the PrimerQuest software and subsequently synthesised by Sangon Biotech (Shanghai, China) [30]. The sequences of primers used are provided in Table S1. ...
Rationale: Atherosclerotic cardiovascular disease (ASCVD) represents the predominant cause of mortality and morbidity globally. Given the established role of hypercholesterolemia as a significant risk factor for ASCVD, the discovery of new lipid-lowering medications is of paramount importance. ATP citrate lyase (ACLY) is a crucial enzyme in cellular metabolism, providing acetyl-CoA as the building block for the biosynthesis of fatty acids and cholesterol. Consequently, it has emerged as a promising drug target for innovative treatments of lipid metabolic disorders.
Methods: Virtual screening of a natural product library was performed to identify small-molecule ACLY inhibitors, leading to the discovery of isoginkgetin (ISOGK). The lipid-lowering and anti-atherosclerotic effects of ISOGK were validated in hypercholesterolemic diet-induced animal models (mice and hamsters). The inhibitory effects of ISOGK on ACLY enzymatic activity were measured using commercial assay kits. The direct interaction between ISOGK and ACLY was confirmed by surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA). Liver-specific ACLY knockdown mice were generated using GalNAc-conjugated siRNA (GalNAc-siAcly).
Results: ISOGK directly bind to ACLY and inhibit its enzymatic activity in vitro and in vivo. By inhibiting ACLY, ISOGK treatment thus alleviates hypercholesterolemia and atherosclerosis in mice and hamsters. However, ISOGK fails to attenuate lipid accumulation and the expression of lipid-metabolism related genes in Acly knockout or depleted hepatocytes. In vivo, the lipid-lowering and anti-atherosclerotic effects of ISOGK were reversed by hepatic knockdown of Acly via treatment with GalNAc-siAcly in mice.
Conclusions: Taken together, the present study identifies ISOGK as an effective and naturally-occurring small-molecule inhibitor of ACLY that limits hypercholesterolemia and atherosclerosis. ISOGK thus serves as a promising drug lead in cardiovascular therapeutics.
... For example, E3 ubiquitin ligase (TRIM56) slows NAFLD progression by ubiquitinating FASN, a key protein in fatty acid synthesis. TRIM56 is downregulated in human NAFLD liver tissue and dietinduced mouse NAFLD liver tissue, while its gene level remains unchanged, and changes in TRIM56 protein expression occur only in liver cells [87]. These findings shed light on the potential role of lactate modification in the development of NASH, possibly by regulating the transcriptions and protein translations of specific genes involved in the progression of the disease. ...
Metabolic diseases, including cardiovascular diseases, type 2 diabetes mellitus (T2DM), osteoporosis, and non-alcoholic fatty liver disease (NAFLD), constitute a major global health burden associated with chronic morbidity and mortality. Lactate, once considered as a metabolic byproduct, has emerged as a key regulator of cellular reprogramming through lactylation, a novel post-translational modification (PTM) that dynamically couples metabolic flux to chromatin remodeling. Lactylation exerts dual regulatory roles as a signaling molecule via GPR81/GPR4-mediated pathways and as a substrate for the covalent modification of histones and metabolic enzymes. Pathologically, chronic hyperlactatemia suppresses mitochondrial biogenesis, driving metabolic cardiomyopathy through the epigenetic silencing of oxidative metabolism genes. Conversely, exercise-induced lactate surges transiently enhance insulin sensitivity via AMPK/PGC-1α/GLUT4 signaling, resolve inflammation through GPR81-mediated M2 macrophage polarization, and restore mitochondrial function via lactylation-dependent pathways. This review delineates lactylation as a spatiotemporal rheostat: chronic dysregulation perpetuates metabolic disorders, whereas acute exercise-mediated lactylation remodels transcriptional networks to restore metabolic homeostasis. Future research should integrate multiomics to clarify lactylation’s spatiotemporal dynamics, tissue-specific thresholds, metabolism–immunity interactions, and metabolic–epigenetic crosstalk for the precision management of metabolic diseases.
Unlabelled:
The principal limitations of the terms nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favour of a change in nomenclature and/or definition.
Methods:
A modified Delphi process was led by three large pan-national liver associations. Consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria.
Results:
A total of 236 panellists from 56 countries participated in four online surveys and two hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83% and 78%, respectively. 74% of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms 'non-alcoholic' and 'fatty' were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease (SLD) was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). There was consensus to change the definition to include the presence of at least one of five cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic SLD. A new category, outside pure MASLD, termed MetALD was selected to describe those with MASLD who consume greater amounts of alcohol per week (140 to 350 g/week and 210 to 420 g/week for females and males respectively).
Conclusions:
The new nomenclature and diagnostic criteria are widely supported, non-stigmatising and can improve awareness and patient identification.
Unlabelled:
The principal limitations of the terms nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favour of a change in nomenclature and/or definition.
Methods:
A modified Delphi process was led by three large pan-national liver associations. Consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria.
Results:
A total of 236 panellists from 56 countries participated in four online surveys and two hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83% and 78%, respectively. 74% of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms 'non-alcoholic' and 'fatty' were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease (SLD) was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). There was consensus to change the definition to include the presence of at least one of five cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic SLD. A new category, outside pure MASLD, termed MetALD was selected to describe those with MASLD who consume greater amounts of alcohol per week (140 to 350 g/week and 210 to 420 g/week for females and males respectively).
Conclusions:
The new nomenclature and diagnostic criteria are widely supported, non-stigmatising and can improve awareness and patient identification.
The ever-increasing prevalence of noncommunicable diseases (NCDs) represents a major public health burden worldwide. The most common form of NCD is metabolic diseases, which affect people of all ages and usually manifest their pathobiology through life-threatening cardiovascular complications. A comprehensive understanding of the pathobiology of metabolic diseases will generate novel targets for improved therapies across the common metabolic spectrum. Protein posttranslational modification (PTM) is an important term that refers to biochemical modification of specific amino acid residues in target proteins, which immensely increases the functional diversity of the proteome. The range of PTMs includes phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, glycosylation, palmitoylation, myristoylation, prenylation, cholesterylation, glutathionylation, S-nitrosylation, sulfhydration, citrullination, ADP ribosylation, and several novel PTMs. Here, we offer a comprehensive review of PTMs and their roles in common metabolic diseases and pathological consequences, including diabetes, obesity, fatty liver diseases, hyperlipidemia, and atherosclerosis. Building upon this framework, we afford a through description of proteins and pathways involved in metabolic diseases by focusing on PTM-based protein modifications, showcase the pharmaceutical intervention of PTMs in preclinical studies and clinical trials, and offer future perspectives. Fundamental research defining the mechanisms whereby PTMs of proteins regulate metabolic diseases will open new avenues for therapeutic intervention.
Background aims:
NAFLD is a leading cause of liver-related morbidity and mortality. We assessed the global and regional prevalence, incidence, and mortality of NAFLD using an in-depth meta-analytic approach.
Approach results:
PubMed and Ovid MEDLINE were searched for NAFLD population-based studies from 1990-2019 survey year (last published 2022) per PRISMA. Meta-analysis was conducted using random-effects models. Bias risk assessment was per Joanna Briggs Institute. Of 2,585 studies reviewed, 92 studies (N=9,361,716) met eligibility criteria. Across the study period (1990-2019), meta-analytic pooling of NAFLD prevalence estimates and ultrasound-defined NAFLD yielded an overall global prevalence of 30.05% (95% CI 27.88-32.32%) and 30.69% (28.4-33.09), respectively. Global NAFLD prevalence increased by +50.4% from 25.26% (21.59-29.33) in 1990-2006 to 38.00% (33.71-42.49) in 2016-2019 (P<0.001); ultrasound-defined NAFLD prevalence increased by +38.7% from 25.16% (19.46-31.87) in 1990-2006 to 34.59% (29.05-40.57) (P=0.029). The highest NAFLD prevalence was in Latin America 44.37% (30.66-59.00%), then Middle East and North Africa (MENA) (36.53%, 28.63-45.22%), South Asia (33.83%, 22.91-46.79%), South-East Asia (33.07%, 18.99-51.03%), North America (31.20%, 25.86-37.08%), East Asia (29.71%, 25.96-33.76%), Asia Pacific 28.02% (24.69-31.60%), Western Europe 25.10% (20.55-30.28%). Among the NAFLD cohort diagnosed without a liver biopsy, pooled mortality rate per 1000 PY was 12.60 (6.68-23.67) for all-cause mortality; 4.20 (1.34-7.05) for cardiac-specific mortality; 2.83 (0.78-4.88) for extra-hepatic cancer-specific mortality; and 0.92 (0.00-2.21) for liver-specific mortality.
Conclusions:
NAFLD global prevalence is 30% and increasing which requires urgent and comprehensive strategies to raise awareness and address all aspects of NAFLD on local, regional, and global levels.
Background & aims:
As a global health threat, NASH has been confirmed to be a chronic progressive liver disease that is strongly associated with obesity. However, no approved drugs or efficient therapeutic strategies are valid, mainly because its complicated pathological processes is underestimated.
Approach & results:
We identified the RING-type E3 ubiquitin transferase-tripartite motif-containing protein 31 (TRIM31), a member of the E3 ubiquitin ligases family, as an efficient endogenous inhibitor of transforming growth factor-beta-activated kinase 1 (mitogen-activated protein kinase kinase kinase 7; MAP3K7), and we further confirmed that TRIM31 is an MAP3K7-interacting protein and promotes MAP3K7 degradation by enhancing ubiquitination of K48 linkage in hepatocytes. Hepatocyte-specific Trim31 deletion blocks hepatic metabolism homeostasis, concomitant with glucose metabolic syndrome, lipid accumulation, up-regulated inflammation, and dramatically facilitates NASH progression. Inversely, transgenic overexpression, lentivirus, or adeno-associated virus-mediated Trim31 gene therapy restrain NASH in three dietary mice models. Mechanistically, in response to metabolic insults, TRIM31 interacts with MAP3K7 and conjugates K48-linked ubiquitination chains to promote MAP3K7 degradation, thus blocking MAP3K7 abundance and its downstream signaling cascade activation in hepatocytes.
Conclusions:
TRIM31 may serve as a promising therapeutic target for NASH treatment and associated metabolic disorders.
Systemic metabolic syndrome significantly increases the risk of morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, no effective therapeutic strategies are available, practically because our understanding of its complicated pathogenesis is poor. Here we identify the tripartite motif-containing protein 31 (Trim31) as an endogenous inhibitor of rhomboid 5 homolog 2 (Rhbdf2), and we further determine that Trim31 directly binds to Rhbdf2 and facilitates its proteasomal degradation. Hepatocyte-specific Trim31 ablation facilitates NAFLD-associated phenotypes in mice. Inversely, transgenic or ex vivo gene therapy-mediated Trim31 gain-of-function in mice with NAFLD phenotypes virtually alleviates severe deterioration and progression of steatohepatitis. The current findings suggest that Trim31 is an endogenous inhibitor of Rhbdf2 and downstream cascades in the pathogenic process of steatohepatitis and that it may serve as a feasible therapeutical target for the treatment of NAFLD/NASH and associated metabolic disorders.
Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with various biological functions. However, the effects of Mul on liver fibrosis have not been addressed, and thus were investigated in our present study, as well as the underlying mechanisms. Here, we found that Mul administration significantly ameliorated carbon tetrachloride (CCl4)-induced liver injury and dysfunction in mice. Furthermore, CCl4-triggerd collagen deposition and liver fibrosis were remarkably attenuated in mice with Mul supplementation through suppressing transforming growth factor β1 (TGF-β1)/SMAD2/3 signaling pathway. Additionally, Mul treatments strongly restrained the hepatic inflammation in CCl4-challenged mice via blocking nuclear factor-κB (NF-κB) signaling. Importantly, we found that Mul markedly increased liver TRIM31 expression in CCl4-treated mice, accompanied with the inactivation of NOD-like receptor protein 3 (NLRP3) inflammasome. CCl4-triggered hepatic oxidative stress was also efficiently mitigated by Mul consumption via improving nuclear factor E2-related factor 2 (Nrf2) activation. Our in vitro studies confirmed that Mul reduced the activation of human and mouse primary hepatic stellate cells (HSCs) stimulated by TGF-β1. Consistently, Mul remarkably retarded the inflammatory response and reactive oxygen species (ROS) accumulation both in human and murine hepatocytes. More importantly, by using hepatocyte-specific TRIM31 knockout mice (TRIM31Hep-cKO) and mouse primary hepatocytes with Nrf2-knockout (Nrf2KO), we identified that the anti-fibrotic and hepatic protective effects of Mul were TRIM31/Nrf2 signaling-dependent, relieving HSCs activation and liver fibrosis. Therefore, Mul-ameliorated hepatocyte injury contributed to the suppression of HSCs activation by improving TRIM31/Nrf2 axis, thus providing a novel therapeutic strategy for hepatic fibrosis treatment.
Introduction:
TRIpartite motif (TRIM) proteins are important members of the Really Interesting New Gene-finger-containing E3 ubiquitin-conjugating enzyme and are involved in the progression of hepatocellular carcinoma (HCC). However, the diverse expression patterns of TRIMs and their roles in prognosis and immune infiltrates in HCC have yet to be analyzed.
Materials:
Combined with previous research, we used an Oncomine database and the Human Protein Atlas to compare TRIM family genes' transcriptional levels between tumor samples and normal liver tissues, as verified by the Gene Expression Profiling Interactive Analysis database. We investigated the patient survival data of TRIMs from the Kaplan-Meier plotter database. Clinicopathologic characteristics associations and potential diagnostic and prognostic values were validated with clinical and expressional data collected from the cancer genome atlas.
Results:
We identified TRIM28, TRIM37, TRIM45, and TRIM59 as high-priority members of the TRIMs family that modulates HCC. Low expression of TRIM28 was associated with shorter overall survival (OS) than high expression (log-rank p = 0.009). The same trend was identified for TRIM37 (p = 0.001), TRIM45 (p = 0.013), and TRIM59 (p = 0.011). Multivariate analysis indicated that the level of TRIM37 was a significant independent prognostic factor for both OS (p = 0.043) and progression-free interval (p = 0.044). We performed expression and mutation analysis and functional pathways and tumor immune infiltration analysis of the changes in TRIM factors.
Conclusion:
These data suggested that TRIM28, TRIM37, TRIM45, and TRIM59 could serve as efficient prognostic biomarkers and therapeutic targets in HCC.
Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD), characterized with hepatocellular steatosis, ballooning, lobular inflammation, fibrotic progression, and insulin resistance. NASH may progress to cirrhosis and hepatocellular carcinoma (HCC), which are the major indications for liver transplantation and the causes for mortality. Thus far, there are no approved pharmacotherapeutics for the treatment of NASH. Given the complexity of NASH pathogenesis at multifaceted aspects, such as lipotoxicity, inflammation, insulin resistance, mitochondrial dysfunction and fibrotic progression, pharmacotherapeutics under investigation target different key pathogenic pathways to gain either the resolution of steatohepatitis or regression of fibrosis, ideally both. Varieties of pharmacologic candidates have been tested in clinical trials and have generated some positive results. On the other hand, recent failure or termination of a few phase II and III trials is disappointing in this field. In face to growing challenges in pharmaceutical development, this review intends to summarize the latest data of new medications which have completed phase II or III trials, and discuss the rationale and preliminary results of several combinatory options. It is anticipated that with improved understanding of NASH pathogenesis and critical endpoints, efficient pharmacotherapeutics will be available for the treatment of NASH with an acceptable safety profile.