Dear Editor,
We express our sincere gratitude to Dr. Ryu and Dr. Koo for their insightful editorial on our recently published work on Clinical and Molecular Hepatology [
1]. This editorial provides valuable comments on the current status and limitations of clinical therapeutic strategies for metabolic dysfunction-associated steatotic liver disease (MASLD), analyzes the feasibility and challenges of targeting carnitine palmitoyltransferase 1A (CPT1A) as a drug development strategy for MASLD. As the commentary emphasized, we believe that regulating the stability and degradation of CPT1A protein to reverse disorders of hepatic lipid metabolism offers a novel approach for MASLD treatment [
2].
The current pharmacotherapeutic strategies for MASLD focus on four main pathways: 1) improving insulin sensitivity (e.g., using pioglitazone) [
3]. 2) inhibiting lipid synthesis or promoting lipid efflux (e.g., with obeticholic acid) [
4]. 3) modulating the gut-liver axis and inflammatory response (e.g., with vitamin E). 4) Using GLP-1 receptor agonists (e.g., semaglutide and tirzepatide) [
5]. In 2024, resmetirom, a thyroid hormone receptor β agonist, became the first targeted therapy for non-cirrhotic MASLD approved by the US Food and Drug Administration [
6]. However, current treatment regimens for MASLD have significant limitations. Resmetirom can improve hepatic steatosis and inflammation, being considered a significant milestone in MASLD therapy; however, it is less effective at reversing early fibrosis. GLP-1 agonists can substantially reduce body weight and improve metabolic parameters, but they may cause loss of lean body mass in non-obese MASLD patients. Also, hepatological and histological benefits of these drugs vary in different demographic populations. These limitations and variations underscore the complexity of MASLD pathogenesis and emphasize the necessity of developing novel therapeutic strategies that precisely target critical hepatocellular metabolic dysfunction.
Dysfunction of mitochondrial fatty acid β-oxidation in hepatocytes is widely recognised as a key pathological mechanism of MASLD.7 Located on the outer mitochondrial membrane, CPT1A is the rate-limiting enzyme in the entry of long-chain fatty acids into the mitochondria for β-oxidation. This makes it a promising therapeutic target. Preclinical studies confirm that knocking out the Cpt1a gene specifically in hepatocytes leads to significant hepatic triglyceride accumulation, whereas CPT1A gain-of-function mutations effectively ameliorate hepatic steatosis in animal models. These findings suggest that modulating CPT1A activity could be an effective therapeutic option for MASLD. However, developing drugs that directly target CPT1A poses significant challenges. Traditional drug development approaches have focused on identifying allosteric activators of CPT1A to enhance its enzymatic activity and promote fatty acid oxidation. Despite substantial research efforts, the unique structure of the CPT1A protein and its high homology with other CPT1 isoforms (such as CPT1B and CPT1C) have thus far averted the successful development of highly selective, potent, small-molecule activators of CPT1A. These findings are just like what Dr. Ryu and Dr. Koo commented, “allosteric activation of CPT1A would provide metabolic benefit; however, despite substantial efforts, no specific CPT1A activator has been successfully developed to date”. Conversely, the pharmacotherapeutic effect of the developed CPT1A small molecule inhibitors, such as etomoxir [
8] and perhexiline [
9], showed inconsistent results with MASLD patients and exhibited strong off-target effects, causing cardiotoxicity and skeletal muscle toxicity. These results only demonstrate CPT1A is a ‘drugable’ target, but further underscore the difficulties of directly targeting the CPT1A active site, as commented by Dr. Ryu and Dr. Koo, “Despite its pivotal role in lipid catabolism, certain limitations remain in applying CPT1A as a pharmacological target for MASLD.”
How to overcome these difficulties? Dr. Ryu and Dr. Koo emphasized that “The limitations concerning drug development by targeting CPT1A highlight the importance of exploring alternative strategies, such as targeting the regulatory mechanisms that control CPT1A protein stability and degradation”. The protein degradation can be manipulated with the “Targeted protein degradation technology”, which involves forming ternary complexes between E3 ubiquitin ligases and target proteins using dual-functional degraders, such as proteolysis targeting chimera (PROTAC) molecules or molecular glues. This complex activates the ubiquitin-proteasome system degradation pathway, enabling the specific clearance of disease-associated target proteins. This strategy offers a new approach to target ‘undruggable’ proteins and overcome drug resistance. For example, NX-1607 [
10,
11] is a novel, highly potent, orally administered inhibitor of E3 ligases; it innovatively targets the E3 ligase CBLB, releasing immune cells from upstream suppression and demonstrating significant potential to overcome resistance to existing immunotherapies. NX-1607 is considered to represent a new generation of cancer immunotherapies and is currently in Phase I clinical trials. Additionally, our team has designed and developed 28 PROTAC molecules that target the RNF4 E3 ubiquitin ligase. Following a comprehensive screening of linkers and other components, one PROTAC molecule, RD12, was selected as the most effective and has demonstrated potent anti-hepatocellular carcinoma effects in both in vitro and in vivo models. The manuscript about the results is currently under review. The reports and our PROTAC results indicate the feasibility and potential of targeting CPT1A with the targeted protein degradation technology, and provide new therapeutic approaches for MASLD, as in oncology, immune disorders, neurodegenerative diseases, etc.
Dr. Ryu and Dr. Koo highlighted that “this study represents a conceptual advance by identifying Ankyrin repeat and SOCS box protein 3 (ASB3) as the first E3 ligase that mediates CPT1A degradation in the liver.” Our study provides a novel therapeutic approach for indirectly regulating the stability of the CPT1A protein. Cellular experiments demonstrate that reducing ASB3 levels inhibits lipid droplet formation, whereas increasing ASB3 levels promotes lipid accumulation. ASB3 inhibits fatty acid oxidation by ubiquitinating and degrading CPT1A. Further quantitative lysine-specific proteomics analysis revealed that ASB3 directly mediates K180 and K639 ubiquitin modifications on CPT1A, ultimately leading to its degradation. ASB3 deficiency markedly increases CPT1A levels, thereby enhancing the capacity for mitochondrial fatty acid oxidation and alleviating hepatic lipid deposition. Validation in animal models showed that hepatocyte-specific ASB3 knockout (ASB3-HKO) mice exhibited significant improvements in hepatic steatosis, fibrosis, and inflammation when fed a high-fat diet or diets containing a methionine-choline-deficient diet or a high-fat, high-fructose, and carbon tetrachloride diet. AAV-mediated CPT1A knockdown reversed the protective effects of ASB3 deficiency, indicating that ASB3’s protective mechanism operates via CPT1A. Clinical studies reveal significantly elevated ASB3 expression in liver tissue from MASLD patients, which is negatively correlated with CPT1A levels. Furthermore, ASB3 expression positively correlates with markers of insulin resistance (IRF1 and IRS1) and inflammatory factors (CRP, IL6, and TNF), indicating its role in broader metabolic dysregulation. As the editorial addressed, “Unlike direct CPT1A inhibitors such as perhexiline, which face challenges of isoform selectivity and off-target binding, targeting the degradation machinery may yield superior specificity”. The therapeutic approaches targeting E3 ligases, such as ASB3, show promise in overcoming the technical challenges associated with directly activating CPT1A enzyme activity while improving treatment specificity and safety. We acknowledged that further investigation is needed to tackle the remaining challenges regarding target selectivity, drug delivery, and long-term safety.
FOOTNOTES
-
Authors’ contribution
Manuscript drafting: Dongqin Yang. Manuscript edition and final approval: all authors.
-
Acknowledgements
We would like to thank Dr. Ryu and Dr. Koo once again for their invaluable comments.
-
Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
Ankyrin repeat and SOCS box protein 3
hepatocyte-specific ASB3 knockout
carnitine palmitoyltransferase 1A
metabolic dysfunction-associated steatotic liver disease
proteolysis targeting chimera
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