CS27109, A Selective Thyroid Hormone Receptor-β Agonist Alleviates Metabolic-Associated Fatty Liver Disease in Murine Models

Background/Aim Thyroid hormone receptor-β (THR-β) agonists play crucial roles in dyslipidemia and metabolic associated fatty liver disease (MAFLD). We developed a novel oral and liver-targeted THR-β agonist, CS27109, and evaluated its efficacy in the treatment of metabolic disorders. Materials and Methods We evaluated in vitro and in vivo efficacy and/or safety of CS27109 along with MGL3196 (a phase III THR-β agonist). Results CS27109 showed pronounced activity and selectivity to THR-β and favorable PK properties, which was equivalent to MGL3196. In the hamster model, animals treated with a high dose of CS27109 showed equivalent reductions in serum TC and LDL-c with groups treated with MGL3196. In the rat model, CS27109 and MGL3196 reduced serum ALT, TC, TG, LDL-c, liver weight ratio, and liver steatosis. CS27109 simultaneously decreased liver TG and TC, and MGL3196 additionally reduced AST. In the mouse model, CS27109 dose-dependently reduced serum AST, ALT, liver inflammation, and NAS score, and also downregulated TC, LDL-c, liver steatosis, and fibrosis, but not in a dose-dependent manner. MGL3196 revealed an equivalent effect with CS27109 in that model. CS27109 also exhibited tolerable toxicity to the heart. Conclusions CS27109 shows comparative in vitro and in vivo efficacy with MGL3196, suggesting its potential therapeutic application in the treatment of MAFLD such as dyslipidemia and steatohepatitis.


Introduction
Metabolic-associated fatty liver disease (MAFLD), a nomenclature, was recently proposed by experts to integrate the accuracy of understanding patient heterogeneity with the so-called nonalcoholic fatty liver disease (NAFLD) [1][2][3]. Tis terminology refects a heterogeneous phenotype caused by the pathological accumulation of triglycerides and other lipids in hepatocytes, leading to steatohepatitis, liver fbrosis, cirrhosis, and eventually hepatocellular carcinoma. MAFLD is also associated with obesity, dyslipidemia, type 2 diabetes mellitus (T2DM), and insulin resistance [4][5][6]. Exercise and diet control are considered efective ways to prevent MAFLD, but no drugs have been approved to treat this disease, especially in patients who develop severe steatohepatitis and liver fbrosis [7]. Drugs targeting glycolipid metabolism alone or with other mechanistic candidates have been emerging as potential strategies to overcome chronic disease, one of these drugs is thyroid hormone receptor-β (THR-β) agonists [8][9][10].
Tyroid hormones (THs) are secreted by the thyroid and play a role in mediating growth, development, and metabolism [11,12]. Te thyroid hormone receptor (THR) forms a complex with TH in a ligand-dependent way to regulate gene expression downstream [13]. Dysfunction or abnormity of this complex results in a variety of diseases, including especially obesity and metabolic disorders [14,15].
Two THR isoforms are reported, thyroid hormone receptorα (THR-α) and thyroid hormone receptor-β (THR-β), in which THR-α is expressed mainly in the brain and heart and THR-β is highly expressed in the liver [16,17]. In terms of MAFLD, the TH/THR axis regulates liver triglyceride and cholesterol metabolism via enhancing lipid mobilization and increasing free fatty acid transportation [18]. However, the endogenous active TH form, 3,5,3′-triiodothyronine (T3), triggers both THRs simultaneously, and thyromimetics without selectivity display side efects on the heart, muscle, and bone due to the activation of THR-α [19,20]. Terefore, developing THR-β agonists with high potency and specifcity is a potential strategy to target MAFLD with minimal side efects.
Several THR-β agonists with oral bioavailability and liver-targeted properties have been developed and showed preclinical efcacy in NAFLD and nonalcoholic steatohepatitis (NASH) models [21][22][23][24][25][26]. Recently, two THR-β agonists, MGL3196 (Resmetirom) and VK2809, have progressed to phase III clinical trials for the treatment of NASH and hyperlipidemia [19,27]. VK2809 is a precursor of KB-141 that was terminated due to side efects such as increased heart rate, manifesting a reduction in serum and liver TG without afecting heart function. MGL3196 is an oral and liver-targeted THR-β agonist with pronounced activity and selectivity towards THR-β. According to the randomized, double-blind, phase 3b MAESTRO-NAFLD-1 trial, Resmetirom has the potential to be the frst approved drug for patients with NASH. In addition, both drugs also show efcacy in alleviating lipid disorder and NASH in preclinical studies [28,29]. However, diarrhea and increased bowel movements are the main side efects observed in patients receiving Resmetirom. Terefore, we still need to develop new compounds with higher selectivity to THR-β and eliminate side efects.
Consequently, we designed and developed a new compound named CS27109 with a brand new molecular structure. Tis compound exhibited considerable in vitro activity and selectivity to THR-β, similar to MGL3196. In this study, we focus on the in vivo efcacy of CS27109 in three diferent animal models to test its potential for treating MAFLD along with MGL3196. Each animal model represents diferent aspects of MAFLD. Our results indicate that CS27109 showed comparative efcacy with MGL3196 in these models, suggesting that it is a potential candidate to target MAFLD such as hypercholesterolemia and steatohepatitis.

Compounds.
Both CS27109 and MGL3196 were synthesized at Shenzhen Chipscreen Biosciences Co., Ltd. with over 99% purity. Compounds were dissolved in sterile DMSO and 0.2 Carboxymethylcellulose sodium (CMC-Na) and 0.1% Tween 80 (solvent) for in vitro and in vivo studies, respectively. T3 was purchased from MedChemExpress (Hycultec GmbH, Beutelsbach, Germany) and dissolved in sterile DMSO for in vitro testing.

hERG Test. hERG test was performed by Ice Bioscience
Co., Ltd. (Beijing, China) using the QPatch HTX method according to the manufacturer's instruction. 5 doses of CS27109 were tested, including 0.3, 1, 3, 10, and 10 mm, each one with a duplicate. Te tested values (current) were normalized to the negative control (0.1% DMSO), and IC 50 was calculated based on the normalized data.

Pharmacokinetic
Studies. PK in the mouse: 6 C57/B6 mice _ aged 6-7 weeks were evenly divided into two groups and administered 10 mg/kg CS27109 or MGL3196 once by oral gavage, respectively. Blood was collected for each mouse 0.5, 1, 2, 3, 4, 6, 8, 24, and 30 hours (H) after compound administration. Ten, the serum was isolated and an extractant (containing 200 ng/ml methanol) was added to the serum. Samples were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to the standard procedure. PK in the rat: 8 SD rats _ aged 5-6 weeks were evenly divided into two groups and administered 10 mg/kg CS27109 or MGL3196 once by oral gavage, respectively. Blood was collected for each mouse 0.5, 1, 2, 4, 6, 8, 10, and 24H after compound administration. Ten, the serum was isolated, and an extractant (containing 200 ng/ml methanol) was added to the serum. Samples were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to the standard procedure.
Tissue distribution: 12 SD rats _ aged 5-6 weeks were evenly divided into two groups and administered 10 mg/kg CS27109 or MGL3196 once by oral gavage, respectively. Half of mice for each group (n � 3) were sacrifced 2H and 8H after drug administration, respectively. Blood, heart, and liver were collected at each time point. Blood was processed and detected as mentioned previously. Te heart and liver were homogenized and added extractant. Te protein of tissue samples was precipitated by vortex oscillation, and the supernatant was collected and determined by LC-MS/MS procedures.

Animal Studies.
All animal studies were carried out adhering to the Guiding Principles in the Care and Use of Animals approved by the Council of Chengdu Chipscreen Pharmaceutical Ltd. All animals were housed in our animal facility (temperature, 20-25°C; humidity, 40-70%; 12/12hour light/dark cycle). Schematic representation of animal studies was illustrated in Figure 1.
Hamster study: 58 golden hamsters _ aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 10 animals were kept with a diet of food control as normal control, and the other 48 animals were fed 40% fat and 1.25% cholesterol (HFD-CHOL, D12108C, Research diet) as a model group. After 4 weeks of feeding, the animals in the model group were randomly assigned to 5 subgroups based on the serum TC level: model (n � 10), MGL3196 10 mg/kg (n � 10), CS27109 3 mg/kg (n � 10), CS27109 10 mg/kg (n � 9), and CS27109 30 mg/kg (n � 9). Animals in the normal and model group were given solvent by oral gavage. All drugs, including solvent, were administered consecutively for 4 weeks and once a day. After sacrifce, blood was collected and serum was isolated for the AST, ALT, TG, TC, LDL-c, and HDL-c test.
SD rat study: 58 SD rats _ aged 5-6 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. 9 animals were kept with a food control diet as normal control, and the other 49 animals were fed 40% fat, 1.25% cholesterol and 0.5% sodium cholate (HFD, D12109C, SYSE BIO, Changzhou, China) as a model group. After 6 weeks of feeding, the animals in the model group were randomly assigned to 5 subgroups based on the serum TC level: model (n = 9), MGL3196 15 mg/kg (n = 10), CS27109 5 mg/kg (n = 10), CS27109 15 mg/kg (n = 10), and CS27109 45 mg/kg (n = 10) (Figure 1(b)). Animals in the normal and model groups were given solvent by oral gavage. All drugs, including solvent, were administered consecutively for 4 weeks and once a day. After sacrifce, blood was collected and serum was isolated for the AST, ALT, TG, TC, LDL-c, and HDL-c test, and livers and hearts were weighed to calculate the liver weight ratio and the heart weight ratio, respectively. Te right lobe of the liver of each rat was harvested for the TG and TC test (A110-1-1 and A111-1-1, Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) according to the manufacturer's instructions. Te left lobe of the liver was collected for hematoxylin-eosin staining (H and E staining) to quantify liver steatosis, infammation, and ballooning.
C57/B6 mouse study: 61 C57/B6 mice _ aged 6-7 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Six animals were kept with a chow diet as normal control, and the other 55 animals were fed 60% fat diet (Western diet, D12492, Research diet) as the model group. After 12 weeks of feeding, the animals in the model group were administered 1 ml/kg of carbon tetrachloride (CCL4, 1 : 4 dissolved in olive oil) by intraperitoneal injection three times a week (4 weeks in total) and simultaneously randomly assigned to 5 subgroups: model (n = 11), MGL3196 10 mg/kg (n = 11), CS27109 3 mg/kg (n = 11), CS27109 10 mg/kg (n = 10), and CS27109 30 mg/kg (n = 12) (Figure 1(c)). Animals in the normal and model groups were  given solvent by oral gavage. All drugs, including solvent, were administered consecutively for 4 weeks and once a day. After sacrifce, blood was collected and serum was isolated for the AST, ALT, TG, TC, LDL-c, HDL-c, and CK tests, and the livers were weighed to calculate the liver weight ratio. Te left lobe of the liver of each mouse was collected for hematoxylin-eosin staining (H and E staining) and sirius red staining.
2.6. Pathological Studies of the Liver. Te liver samples were fxed in 10% formalin and embedded in parafn using standard methods. Te 4 μm parafn sections were then assessed for liver histology by H and E staining and liver fbrosis by sirius red staining. Te stained sections were panoramic scanned with a NanoZoomer Digital Pathology scanner (S210, Hamamatsu, Japan), and then observed with diferent visual felds at diferent magnifcations, and scored for diferent liver lobes in the sections. Te total NAS score represents the sum of the scores for steatosis (0-3), lobular infammation (0-3), and ballooning (0-2), and ranges from 0-8 [30]. VIS7.0 software was used to analyze the area of liver fbrosis in the panoramic scanning SR staining sections, and the percentage of liver fbrosis area in the whole section area was calculated.

Statistical Analysis.
Data are expressed as the mean ± SD and the GraphPad Prism 8.0 software (GraphPad Software Inc, CA, USA) were used for statistical analysis. One-way analysis of variance (ANOVA) was used for comparison between groups. Te Bonferroni test was used for data with homogeneous variance, and the Dunnett test was used for data with uneven variance. p < 0.05 indicates that the difference is statistically signifcant.

CS27109
has Favorable PK Properties. We then conducted PK studies of CS27109 and MGL3196 in both mouse and rat at 10 mg/kg. Results showed that comparative PK properties were observed in mouse between CS27109 and MGL3196 (Figure 2(d)), while MGL3196 exhibited slightly higher PK properties in rat compared with CS27109 ( Figure 2(e)). We further characterized the tissue distribution of both drugs in the rat. Results showed that the pattern of tissue distribution between the two compounds was diferent, which drug concentration was increased for CS27109 and decreased for MGL3196 from 2H to 8H in three tested organs (Table 2). Te average of concentration liver/heart for MGL3196 and CS27109 was 22.325 and 26.69, respectively, indicating both drugs were liver-targeted.
MGL3196 was studied in several preclinical models, in which 10 mg/kg was an efective dose frequently used in mouse models. Terefore, we chose this dose for MGL3196 as a positive control and for CS27109 as a medium dose in mouse studies. We then expanded doses (range 3-30 mg/kg) to check a dose-response pattern for CS27109. Te AUC of CS27109 in mouse was 1.5-fold higher than that in rat, so we selected a dose range from 5 to 45 mg/kg in the rat model study.

CS27109 Improves Dyslipidemia in the HFD-CHOL
Hamster Model. In vitro and PK results suggest that CS27109 should exhibit pharmacodynamical efcacy in animal studies. To achieve this, we established three diferent animals to test the efcacy of both compounds. Te frst is an HFD-CHOL hamster model fed a high-fat and highcholesterol diet for 8 weeks in total (Figure 1(a)). Tis model was characterized by an increase in serum TG, TC, and LDL-c level (Table 3), which resembles hyperlipidemia and hypercholesterolemia in human patients. In addition, serum AST and ALT were also elevated in this model compared with the normal control, indicating a possible liver injury (Table 3).
After 4-week treatments, CS27109 dose-dependently reduced serum TC and LDL-c with a signifcant efect in the medium and high-dose groups but not in the low-dose group (Table 3). Similarly, MGL3196 10 mg/kg also lowered the serum TC and LDL-c level and showed an equivalent efect with high-dose CS27109 (30 mg/kg). Additionally, CS27109 reduced serum AST in a dose-dependent manner but only with a signifcant efect in the high-dose group (Table 3). Interestingly, none of the CS27109 group exhibited serum TG reduction even with a dose-dependent trend, which is inferior to MGL3196. Neither CS27109 nor MGL3196 decreased serum ALT (Table 3). However, lowand high-dose CS27109 was also found to reduce serum HDL-c, which is still in the normal psychological range. Collectively, CS27109 and MGL3196 potentiate the amelioration of dyslipidemia, especially in the regulation of serum TC and LDL-c.

CS27109 Improves Lipid Metabolism and Liver Steatosis in HFD Rat
Model. Te SD rat with HFD was reported to be a suitable model for the study of dyslipidemia and liver steatosis study [31]. In our study, we found that serum TC was dramatically elevated in rats even after two weeks of HFD feeding (data not shown). Tis study lasts 10 weeks in total, in which the duration of drug administration is 4 weeks (Figure 1(b)). Compared with the control of the chow diet (the normal group), the animals in the model group demonstrated an extremely increased serum TC and LDL-c, but not TG and HDL-c (Table 4), which is consistent with hypercholesterolemia in human disease. Furthermore, liver TC but not TG was also elevated in the model group, according to the serum result. Furthermore, serum AST and ALT also increased dramatically in the model group, (e) Figure 2: Chemical structure, THR-β activity, and PK properties of CS27109: (a) chemical structure of several oral and liver-targeted THR-β agonists, including KB-141, VK2809, MGL3196, and CS27109, (b) THR-α and THR-β activity for each compound were determined by a luciferase reporter gene assay, (c) hERG test of CS27109 was conducted by the QPatch HTX, (d) mouse, and (e) rat PK studies for CS27109 and MGL3196.    (Table 4). Te enlarged liver is also a characteristic of this model, resulting in a higher liver weight ratio compared with the normal control (Table 4). After 4-week drug administration, three doses of CS27109 signifcantly reduced serum ALT, TG, TC, and LDL-c (Table 4), in which TC and LDL-c were dosedependent. Compared with MGL3196, CS27109 exhibited a comparative reduction in serum TC and LDL-c at the same dosage. However, MGL3196 reduced serum AST and TG more than for the three groups of CS27109. In accordance with the serum test, CS27109 also reduced liver TC and TG content, while MGL3196 did not exert a signifcant efect on neither index (Table 4). Furthermore, CS27109 as well as MGL3196 dramatically reduced the liver weight ratio. Only CS27109 30 mg/kg increased the heart weight ratio but was still close to the normal group, suggesting that neither CS27109 nor MGL3196 adversely afected cardiac function in this model (Table 4).
Subsequently, we assessed liver pathology by H and E staining among diferent groups of treatments (Figure 3(a)). As shown, animals in the model group exhibited higher liver steatosis, infammation, and ballooning than the normal group, especially liver steatosis (Figures 3(b)-3(d)). Te NAS score reached 5, which is characterized as a mild MASH model (Figure 3(e)). As expected, both CS27109 and MGL3196 lessened steatosis, but not infammation and ballooning scores, in which CS27109 showed a dosedependent reduction in steatosis. However, both drugs slightly but not signifcantly increased the ballooning score, implying possible liver toxicity for both drugs, which needs to be elucidated in the further study. CS27109 also dosedependently reduced liver infammation, but without a signifcant efect compared with the model group. Statistically, only a high dose of CS27109 decreased the NAS score due to its robust efcacy in regulating liver steatosis (Figure 3(e)). However, MGL3196 exhibited a tendency to reduce the NAS score, but without signifcance.

CS27109 Attenuates Liver Infammation and Prevents
Fibrosis in the WD + CCL4 Mouse Model. Most murine models with HFD even after long-term feeding feature as steatohepatitis, but rarely liver fbrosis. Chemical reagents such as CCL4 can induce liver injury and fbrosis in diferent murine models [26]. Furthermore, animals that received WD and CCL4 simultaneously are similar to the corresponding human disease. To validate this model, we fed C57/ B6 male mice with WD for 12 weeks and then injected CCL4 three times a week for 4 weeks (Figure 1(c)). After sacrifce, mice in the model group showed a tremendous increase in serum ALT and AST (Table 5) compared with the normal control. Furthermore, serum TC and LDL-c were also signifcantly elevated, but serum HDL-c was reduced in the model group (Table 5). However, serum TG and CK level were not afected between the two groups. Sadly, the liver weight ratio did not increase in this model (Table 5), which was diferent from the HFD only models.
After 4-week drug administration, medium and high doses CS27109, as well as MGL3196 dramatically reduced serum ALT and AST, in which CS27109 showed a dosedependent pattern (Table 5). Serum TC and LDL-c were also signifcantly reduced in 4 drug-treated groups compared with the model group, close to the normal group (Table 5). Neither drug afected serum TG, CK, or liver weight ratio.
We further examined hepatitis and liver fbrosis of this model by pathological studies (Figure 4(a)). According to the reported studies, our model also displayed severe liver infammation (Figure 4(c)) and fbrosis (Figure 4(f )) with mild steatosis and ballooning (Figures 4(b) and 4(d)). After drug intervention, we observed a notable reduction in liver steatosis and infammation in MGL3196 and CS27109 highdose groups as compared with the model group (Figures 4(b) and 4(c)). However, only the low-dose group CS27109 signifcantly reduced liver ballooning (Figure 4(d)). Furthermore, the NAS score which combines scoring of liver steatosis, infammation, and ballooning was dramatically decreased in all drug-treated groups, in which the highdosed CS27019 group showed equivalent efcacy with the MGL3196 treated group (Figure 4(e)). Furthermore, three doses of CS27109 and MGL3196 also prominently reduced the area of the fbrosis in relation to the model group (Figure 4(f )). Collectively, we suggest both compounds are capable to attenuate liver infammation and prevent fbrosis.

Discussion
Te NAFLD was coined by Ludwig to distinguish it from alcoholic fatty liver disease (AFLD) characterized by excessive Table 4: Biochemical, organ/body weight ratio, and liver TC and TG test in HFD SD rat model after 4-week drug intervention. * and # represents normal vs. model and drugs vs. model, respectively. * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001, # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001. L/B (%) stands for liver/body weight ratio and H/B stands for heart/body weight ratio. International Journal of Endocrinology drug intake [32]. Tis terminology has been used for decades, but its accuracy has been challenged due to its complexity and heterogeneity, which impede clinical management and pathogenic understanding. To solve this problem, MAFLD was proposed to better refect current knowledge of the disease, and a consensus was reached in 2020 by an international expert panel. Tis new defnition links liver disease closer to metabolic disorders and simplifes the diagnostic process used by NAFLD [33]. In other words, MAFLD emphasizes the role of metabolic dysfunction in it instead of exclusion of signifcant alcohol intake or other chronic liver diseases. Tis renaming will further increase the prevalence of MAFLD and prompt disease awareness, research, and clinical management. Terefore, we urge researchers to use this MAFLD nomenclature instead of NAFLD or NASH which is still popular and used in other articles.
As MAFLD was recently proposed, there is few research on the development of animal models, particularly under this terminology, but tons of studies were found based on NAFLD or NASH [34,35]. Terefore, we refer to the NAFLD or NASH animal models, as they also share some common features with MAFLD. We focus on the metabolic   and hepatic disorders of our drug, and key characteristics of MAFLD. To achieve this goal, we chose three diferent animal models representing diferent aspects of the disease: dyslipidemia, steatohepatitis, and fbrosis. Te golden hamster model with HFD-CHOL illustrates the human disease of hypertriglyceridemia and hypercholesterolemia, in which serum TG and TC along with LDL-c are tremendously elevated. Serum AST and ALT are also dramatically increased, indicating that liver injury in this model may be due to lipotoxicity through excessive lipid intake. Te hamster was reported to be more similar to human lipid metabolism than other rodents such as rat and mouse [36]. To develop a model with both dyslipidemia and steatohepatitis that frequently occur in humans, we fed SD rats with longer term (10 weeks) and an extra 0.5% cholate in HFD. Together with cholesterol, cholate was reported to induce the progressive development of liver steatosis and infammation in 6-24 weeks [37]. We confrm this observation in our study, demonstrating elevated serum TC and LDL-c, pronounced liver steatosis, mild infammation, and ballooning. However, HFD alone rarely induced liver fbrosis in murine models even with a longer time (more than 20 weeks). In order to shorten research time, chemicals such as CCL4 and streptozotocin (STZ) are often introduced into the HFD system to accelerate liver injury and fbrosis [35]. In a previous study, WD + CCL4 resulted in stage 3 fbrosis at 12 weeks and hepatocellular carcinoma development at 24 weeks in mice [38]. Intriguingly, these models shared dysregulated molecular pathways and immunologic characteristics closely with the human disease by transcriptomic analysis. We verifed this model according to the literature with similar observations. In detail, our model shows an extraordinary increase in serum AST and ALT, indicating a severe liver injury. Tis abnormality is confrmed by the pathological study, demonstrating high scores of liver infammation, ballooning, and fbrosis. However, lipoproteins such as TC and LDL-c in the blood are only signifcantly increased, but the fold changes are not comparable with those in the hamster and rat models, which may be due to the toxicity of CCL4, which restricts food intake and lipogenesis.
Lifestyle interventions such as diet control and exercise are the main strategies for the treatment of patients with MAFLD, but most patients with advanced stage of the disease unwillingly implement such changes [39]. Terefore, pharmacological interventions remain highly demanded. Dozens of targets are reported to play diferent roles in MAFLD progression, in which THR-β mediates cholesterol metabolism and excretion through bile acids. Moreover, THR-β agonists alleviate liver injury including infammation and fbrosis by reducing lipotoxicity and liver fat. Safety and efcacy are two aspects of an ideal drug candidate, where MGL3196, the frst phase III THR-β agonist, already showed promising efcacy and tolerable safety. To compete with MGL3196, we designed CS27109, a new compound with high potency and selectivity towards THR-β, and tested in vitro and in vivo efcacy of both compounds.
In general, CS27109 shows equivalent efcacy with MGL3196 in improving features of MAFLD such as dyslipidemia and steatohepatitis. By further analysis, CS27109 only dose-dependently reduces serum TC and LDL-c in all animal models, which is in line with its mechanism of cholesterol regulation [39]. Some other parameters are signifcantly modulated by CS27109 in one murine model, but not in others. For example, CS27109 medium and high doses reduce serum ALT in the rat and mouse model, but not in the hamster model. Another example is that the high dose of CS27109 reduces the liver infammation score in the mouse but not in the rat model, even showing a tendency in it. Tese data indicate that more information needs to be combined to determine drug efcacy. Compared with MGL3196, CS27109 showed an equivalent reduction in serum TC and LDL-c, but an inferior reduction in serum TG, suggesting its potential for hypercholesterolemia. In the WD + CCL4 model, simultaneously intervention with CCL4 and compounds suggests that CS27109 potentially prevents progression from liver steatohepatitis to fbrosis, whether its therapeutic efect in this model is still undefned. In our another study (manuscript is submitting to another journal), we evaluated combination therapy between CS27109 and CS17919 (a ASK1 inhibitor developed by our team) in a similar model in which CCL4 induction was started at week 8 (8 weeks in total) and drug intervention began 4week after CCL4 induction. In this study, we found CS27109 alone was sufcient to reverse liver fbrosis and achieve a synergistic efect with CS17919. Terefore, CS27109 might also hold potential for the treatment of metabolic-related fbrosis.

Conclusion
In summary, we designed a novel THR-β agonist CS27109 with potent in vitro activity and selectivity. CS27109 also showed favorable PK properties and was liver-targeted. Furthermore, CS27109 exhibited equivalent serum reduction in TC and LDL-c, mitigation of steatohepatitis, and prevention of liver fbrosis with MGL3196. Tese fndings indicate that CS27109 plays a potential role in treating of MAFLD.

Data Availability
Data will be made available on request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.