Fatty acids are essential for cell survival as they serve as key structural components of cell membranes and important signalling molecules. Fatty acids are also the most calorically dense form of energy storage with the conversion of excess glucose into fatty acids protecting against glucotoxicity and providing a much larger energy reserve than glycogen for times of nutrient scarcity. Given the vital roles of fatty acids, cells have evolved mechanisms to maintain them at adequate levels. This includes mechanisms to take up exogenous fatty acids but also to generate fatty acids from alternative carbon sources through a series of enzymatic reactions, a process highly conserved across phyla known as de novo lipogenesis (DNL)1.

DNL is initiated when excess substrate availability, relative to cellular energy demands, leads to increases in mitochondrial citrate, which is exported from mitochondria into the cytosol by the mitochondrial citrate/isocitrate carrier (CIC; also known as CTP and SLC25A1) (Fig. 1). This cytosolic citrate is then converted into fatty acids by a series of biosynthetic reactions catalysed by ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC; also known as ACACA) and fatty acid synthase (FAS; also known as FASN). The expression of these enzymes differs across tissues and stages of development (for example, proliferation or quiescence). Expression and activity are also acutely and chronically regulated through transcriptional control and post-translational modifications that are linked to nutritional status (for example, fasting and feeding) and substrate availability (for example, fatty acids suppress DNL) (Box 1).

Fig. 1: Overview of DNL.
figure 1

A series of coordinated enzymatic reactions takes place during fatty acid biosynthesis. Typically, pyruvate produced by glycolysis is converted in the mitochondrion into acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle to produce citrate. In conditions of carbohydrate excess, citrate is exported to the cytosol by the citrate/isocitrate carrier (CIC) and is broken down to acetyl-CoA and oxaloacetate (OAA) by ATP-citrate lyase (ACLY). Acetyl-CoA is subsequently carboxylated by acetyl-CoA carboxylase (ACC) to generate malonyl-CoA, which is considered the first committed metabolic intermediate in fatty acid synthesis. Utilizing seven malonyl-CoA molecules and one acetyl-CoA primer, the synthesis of palmitate (16:0 fatty acid) is completed by repeating a cycle of condensation, reduction, condensation and dehydration catalysed by fatty acid synthase (FAS). An alternative carbon source of de novo lipogenesis (DNL) is acetate, which can be produced de novo from glucose through non-enzymatic and enzymatic reactions. Acetyl-CoA synthetase 2 (ACSS2) catalyses the reaction of acetate and CoA to form acetyl-CoA, which is subsequently used for fatty acid biosynthesis. With hypoxia or CIC deficiency another alternative pathway for DNL is reductive carboxylation of glutamine via cytosolic isocitrate dehydrogenase 1 (IDH1) and mitochondrial IDH2. αKG, α-ketoglutarate; ACP, acyl carrier protein; mKDH, mitochondrial ketoacid dehydrogenase; nKDH, nuclear ketoacid dehydrogenase; ROS, reactive oxygen species.

Although DNL is vital to maintain whole-body and cellular homeostasis, chronic elevations are associated with the development of a broad spectrum of diseases and disorders including cardiovascular disease (CVD)2,3, nonalcoholic fatty liver disease (NAFLD)4,5, type 2 diabetes (T2D)5,6, numerous cancers7,8, viral infections9,10, autoimmune diseases11,12, acne vulgaris13, neurodegeneration14 and ageing15. This suggests that pharmacological inhibition may be beneficial across multiple disease areas (Box 2; Supplementary Fig. 1). Several natural products have been identified as inhibitors of DNL and these have been adopted as a cornerstone for the development of synthetic inhibitors that display improved bioavailability, efficacy and specificity. Recently, some of these compounds have reached clinical stage development and have been approved for the treatment of hyperlipidaemia16 or are in late-stage development for NAFLD and oncology.

However, there are still many important questions that remain to be answered as it is currently unclear whether systemic or organ-specific inhibition of DNL should be targeted and what degree of inhibition is necessary to avoid potential side effects such as defects in fetal development17, platelet production18 or muscle dysfunction19. It is also unclear under what conditions inhibition of the classical DNL pathway may be bypassed by the scavenging of alternative carbon sources, through acetyl-CoA synthetase20,21, ketoacid dehydrogenase22 or isocitrate dehydrogenases23,24,25, and therefore whether combination therapies or changes in diet may be required. Although there are several important targets that indirectly influence lipogenesis (for example, fructokinase, glucokinase or the glucagon receptor, sterol regulatory element-binding protein 1 (SREBP1) and liver X receptor (LXR))26,27 or are involved in downstream lipid processing (for example, SCD1, DGAT1 and DGAT2)28,29, here, we specifically review recent advances in the development of inhibitors that directly target core components within the lipogenic pathway, namely, citrate/isocitrate carrier (CIC), ATP-citrate lyase (ACLY), ACC and fatty acid synthase (FAS).

Physiological and pathological roles of DNL enzymes

The physiological regulation of DNL is complex, differing widely between cell types and with nutritional status. As discussed in this Review, studies from mice genetically lacking Slc25a1, Acly, Acaca and Fas have confirmed the importance of DNL in lipogenic tissues such as liver and adipose tissue, but also revealed unexpected biological consequences in cell types thought to have limited capacity for DNL. These findings indicate a much broader role for DNL in regulating the production of lipids under a wider array of physiological functions than initially appreciated (Fig. 2). Although a detailed description of the complex physiology that regulates lipogenesis (reviewed in refs30,31) is not within the scope of this Review, an overview of the key physiological and pathological roles of DNL enzymes is provided below. Importantly, the information obtained from genetic studies provides crucial insight into both the opportunities and challenges of developing DNL inhibitors.

Fig. 2: Tissue-specific actions of DNL.
figure 2

Important insights into distinct actions of citrate/isocitrate carrier (CIC) and de novo lipogenesis (DNL) enzymes in various tissues have been postulated from studies that employed animal models that lack one of these core components of the DNL pathway. The actions of each enzyme is colour coded: CIC-mediated effects are in blue boxes, ATP-citrate lyase (ACLY) in purple boxes, acetyl-CoA carboxylase (ACC) in green boxes and fatty acid synthase (FAS) in yellow boxes. NA, not available.

Energy intake and expenditure

Activity of the DNL pathway appears to regulate energy intake and expenditure. For example, mice that lack Fas have reduced food intake32, whereas food intake is increased in mice that lack Acc33. Mechanistically, the divergent effects of FAS and ACC on food intake can potentially be explained through differential effects on malonyl-CoA, which has been shown to suppress food intake: FAS inhibition increases malonyl-CoA, whereas ACC inhibition reduces it. Consistent with changes in malonyl-CoA, stereotactic delivery of malonyl-CoA decarboxylase (MCD) into the hypothalamus increases food intake34, whereas food intake is reduced in mice with constitutively active ACC1 and ACC2 isoforms35. These data suggest that inhibition of FAS suppresses food intake whereas inhibition of ACC stimulates food intake.

In both mice and humans, brown adipose tissue (BAT) is switched on in response to increases in nutrients (diet-induced thermogenesis) or cold (adaptive thermogenesis)36,37. The activation of BAT in rodents and humans increases glucose uptake; however, recent studies have found that surprisingly, the primary function of this glucose is not to support glycolysis but instead to fuel DNL38. Consistent with this concept, both cold exposure and high carbohydrate availability increase DNL within BAT39, suggesting that BAT primarily utilizes endogenous triglycerides to fuel thermogenesis. Paradoxically, reductions in adipose tissue Fas leads to reductions in body mass40 due to enhanced local sympathetic nervous system activity, which causes the browning of the white fat41. These data suggest that counterintuitively inhibiting DNL in adipose tissue may increase whole-body energy expenditure. Future studies are required to selectively inhibit ACLY, ACC and FAS within BAT and white adipose tissue (WAT) to directly quantify the importance of the DNL pathway for regulating futile cycling and energy expenditure.

Lipid deposition: NAFLD–NASH

Individuals with nonalcoholic fatty liver disease (NAFLD) have increased rates of DNL and this is a major factor contributing to increased lipid deposition4,5. Consistent with these observations, the expression of CIC, ACLY, ACC and FAS are increased in the liver of patients with NAFLD or nonalcoholic steatohepatitis (NASH)42. In mice fed a high-fat diet (HFD), liver-specific deletion of the Slc25a1 gene reduces liver steatosis43. In ob/ob mice fed a high-carbohydrate diet, transient genetic inhibition of ACLY using small interfering RNA (siRNA) reduces liver lipid content44. Similarly, when Acly is selectively removed from hepatocytes of adult mice with NASH induced by a high-fat and high-fructose diet, there are reductions in liver steatosis (G.R.S., unpublished observations). By contrast, lifelong inhibition of liver Acly in mice fed high levels of fructose does not lower liver fat, potentially because of upregulation of acetate production by the gut microbiome and compensatory upregulation of acetate-CoA synthetase 2 (ACSS2) (ref.45). However, it should be noted that the high levels of fructose used in this study did not promote obesity, NAFLD or NASH45; thus, the therapeutic relevance of the findings is currently unclear.

Genetic inhibition of liver Acc1 (ref.46) or both Acc1 and Acc2 (refs47,48) lowers liver fat in mice fed a high-carbohydrate diet independently of changes in adiposity. Conversely, mice with constitutively active ACC1 and ACC2 isoforms, owing to lack of AMP-activated protein kinase (AMPK) inhibitory phosphorylation, develop greater steatosis and fibrosis than controls when fed a high-carbohydrate diet49. These data indicate that inhibition of ACC can exert positive effects on lowering steatosis and fibrosis. However, an important consequence of inhibiting liver ACC is the development of hypertriglyceridaemia owing to reductions in liver polyunsaturated fatty acids, which increase SREBP1c and the expression of glycerol-3-phosphate acyltransferase (GPAT), the rate-limiting enzyme for triglyceride synthesis48. Although most studies have indicated positive effects of ACC inhibition on liver steatosis, in one study genetic inhibition led to increases in liver lipids attributed to hyperacetylation of mitochondrial proteins, potentially due to accumulation of acetyl-CoA and lower fatty acid oxidation50. The phenotype of these liver-specific ACC null mice was similar to that of mice lacking FAS, which also have increased steatosis owing to reductions in fatty acid oxidation51. These studies in ACC-null and FAS-null mice highlight complex interactions that may limit therapeutic application in NAFLD and NASH.

Insulin sensitivity: type 2 diabetes

Insulin resistance is a hallmark of type 2 diabetes (T2D). Rates of DNL in the liver are inversely correlated with hepatic and whole-body insulin sensitivity in humans5,52. Liver-specific deletion of Slc25a1 improves glucose tolerance in mice fed a control carbohydrate diet or HFD43. siRNA-mediated suppression of ACLY expression reduces fasting glucose and improves glucose tolerance in ob/ob mice fed a high-carbohydrate diet44. Similar observations are made when ACLY is selectively deleted from hepatocytes of mice fed a high-fat and high-fructose diet for 16 weeks (G.R.S., unpublished observations). Genetic inhibition of ACC2 in the muscle53 or ACC1 and ACC2 in the liver47 leads to improvements in muscle and liver insulin sensitivity, respectively, whereas mice with constitutively active ACC1 and ACC2 isoforms have worse insulin resistance49,54. These data indicate that inhibition of DNL and/or upregulation of fatty acid oxidation within muscle and liver of mice can improve insulin sensitivity.

In addition to muscle and liver, adipose tissue is also crucial for regulation of insulin sensitivity55,56. In rodents, WAT has rates of DNL comparable to those of liver during the postprandial period57. However, with obesity, rates of adipose tissue DNL decline in both rodents58 and humans59, despite only modest reductions in insulin-simulated glucose uptake60. Recent studies indicate that this reduction in adipose tissue DNL occurs rapidly and continues to decline with worsening obesity and insulin resistance and is associated with reductions in the expression of ACLY, ACC, FAS61 and carbohydrate-responsive element-binding protein-β (ChREBPβ)62, suggesting that inhibition of adipose tissue DNL may be detrimental to whole-body insulin sensitivity. Consistent with this concept, when ACLY fat-specific null mice are challenged with a high-sucrose diet, female mice develop a lipodystrophy-like phenotype that is associated with hepatic lipid accumulation and insulin resistance63. Similar observations are also observed in ACC1 fat-specific null mice64. These data indicate an important role for adipose tissue DNL in maintaining whole-body insulin sensitivity, potentially by protecting the liver from developing steatosis. Additional studies characterizing transient knock-down of ACLY and ACC in fully differentiated adipocytes of obese adult mice, rather than lifelong deletion20,63,64, could better inform the potential protective role of adipose tissue DNL in insulin resistance.

The inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain euglycaemia is a hallmark of T2D. Available evidence suggests that ACC may be important for pancreatic β-cell function, as deletion of ACC impairs glucose-stimulated insulin secretion ex vivo and insulin tolerance in vivo65. Mechanistically, ACC is abundant in β-cells, whereas FAS is expressed at low levels, leading to a rise in malonyl-CoA levels following glucose stimulation66. As a result, fatty acid oxidation is inhibited by elevated malonyl-CoA, increasing the availability of cytosolic long-chain fatty acyl-CoA (LCFA-CoA) for lipid signalling to cellular processes involved in insulin secretion67. In addition, recent studies have found that ACC is important for promoting β-cell mass in mice65. These data suggest that inhibition of ACC in pancreatic islets may lead to impaired insulin secretion and reduced β-cell mass that could contribute to T2D.

Cardiovascular disease

Elevations in liver DNL lead to increases in plasma VLDL and LDL, major risk factors of cardiovascular disease (CVD) mortality68,69. Recent studies have found that incident heart failure and CVD mortality2,70 are positively associated with increases in fatty acids derived from DNL. Consistent with this concept, genetic variants in ACLY are associated with reduced plasma LDL and decreased cardiovascular events3. Similarly, polymorphisms of ACLY and ACC are associated with lower triglycerides following dietary fish oil supplementation71. Surprisingly, to the best of our knowledge, no studies have examined the effects of genetic inhibition of liver Acly, Acc or Fas on established models of atherosclerotic development. However, given the effects of ACC inhibition in promoting hypertriglyceridaemia48, it might be anticipated to enhance atherosclerosis. By contrast, a deficiency in liver ACLY lowers triglycerides in mice (G.R.S., unpublished observations). The reasons for the opposing effects of ACLY and ACC inhibition on circulating triglycerides is unclear; however, it could be linked to the concomitant suppression of cholesterol synthesis unique to ACLY inhibition and/or the reciprocal effect that ACLY and ACC inhibition has on levels of acetyl-CoA, which may be important to support protein acetylation. These data indicate that while ACC inhibition may be effective at reducing liver steatosis, it has detrimental effects on CVD risk profile that would need to be managed with other lipid-lowering therapies such as fish oil71, DGAT inhibition72 or PPARα agonists such as fenofibrate73.

Macrophages are also crucial for the development of atherosclerotic CVD as they take up and store lipid, triggering inflammation within the atherosclerotic lesion. The activity of ACLY74 and FAS75 is increased in atherosclerotic plaques of mice and humans, suggesting that inhibition may be beneficial. Consistent with this concept, genetic inhibition of Acly74 or Fas76 within macrophages of ApoE mice reduces atherosclerosis. Surprisingly, there have been no studies examining the effects of genetic deletion of macrophage ACC on atherosclerosis.


DNL is constitutively active in many cancer cells and contributes most of the intracellular lipid mass77. It is now well recognized that common genetic mutations (for example, in the gene encoding p53 (ref.78) or the gene encoding PTEN79) and growth factor signalling (epidermal growth factor80, HER2 (ref.81) and keratinocyte growth factor82, ERK1/ERK2 MAPKs82,83) enhance the activity of SREBP1 leading to upregulation of lipogenic genes. In addition to transcriptional control, enhanced glucose uptake — common in glycolytic tumours — increases citrate availability, which allosterically activates ACLY and ACC. Mutations in PTEN, which lead to constitutive activation of AKT, also increase the activity of ACLY through phosphorylation at Ser454, while at the same time enhancing ACC activity by suppressing AMPK84. Similarly, mutations in LKB1 reduce activating AMPK phosphorylation, thereby increasing ACC activity and cell proliferation85. Thus, tumours use multiple overlapping mechanisms to enhance DNL.

Genetic evidence that supports a crucial role for DNL enzymes in regulating tumorigenesis has been obtained from multiple studies involving knock-down of ACLY7,86,87, ACC88 and FAS89,90 in a wide variety of distinct tumour types (reviewed in refs91,92). Inhibition of DNL also increases sensitivity to standards of care such as radiation93, androgen deprivation94 chemotherapies or tyrosine kinase inhibitors such as sorafenib95. Survival analysis examining the relationship between DNL-expressing genes also supports an important relationship across different tumour types96. These data suggest that inhibiting DNL may exert favourable effects in multiple types of cancer.

Infection and immunity

Inflammation and metabolism are intimately linked, as the maintenance of cellular defence systems and removal of pathogens is an energetically demanding process. Upon activation, immune cells undergo metabolic reprogramming to meet the energy demand for cell proliferation, differentiation and cytokine production97. One of the metabolic switches that occurs during these processes is the induction of DNL98,99. It is known that immune cells that subsume a pro-inflammatory role (M1-like) use aerobic glycolysis and have high rates of DNL, whereas anti-inflammatory immune cells (M2-like) use predominantly oxidative metabolism and have low rates of DNL. Although this is an oversimplification of an extremely complicated non-binary relationship, it does provide a general context for understanding the link between inflammation and metabolism100. The triggers for immune activation vary, but findings in chronic metabolic diseases such as obesity, NASH and CVD have found an important role for microbial products such as lipopolysaccharide (LPS) and ectopic lipid accumulation (cholesterol and fatty acids)101.

Both ACC and ACLY regulate immune function. The inhibition of ACLY inhibits LPS-induced inflammation102 in some but not all studies74 and is important for mediating the anti-inflammatory effects of IL-4 (ref.103) and IL-2 (ref.104). In human and mouse naive T cells, deletion of ACC1 restrains the development of pro-inflammatory T helper 17 (TH17) cells and promotes the formation of anti-inflammatory regulatory T cells; a finding that translates in vivo into the attenuation of TH17-mediated autoimmune disease98 and infection-associated intestinal inflammation105. ACC1 is also crucial in obesity-induced TH17 cell differentiation in humans12. Deletion of ACC1 also reduces antigen-specific clonal expansion of cytotoxic CD8+ T cell populations during Listeria infection106. Interestingly, this increase in DNL is exploited by many viruses for the formation of their replication complex, and as a result, inhibition of SREBP, ACLY, ACC1 and FAS reduces viral replication9,10,107. These studies suggest that inhibiting DNL may be effective at reducing inflammation in chronic disease settings such as obesity and NASH and may exert positive effects to reduce viral replication. However, whether this may be exploited therapeutically without suppressing host immunity, which requires activation of DNL to promote an M1 phenotype, remains to be determined.


Neural differentiation occurs throughout life108 and is strongly associated with upregulation of DNL in neural stem and progenitor cells14. The inactivation of FAS and ACC reduces neurodifferentiation14, and this has been linked to neurodegenerative diseases, including multiple sclerosis109, Parkinson disease110 and Alzheimer disease111. Interestingly, individuals with a variant of FAS (FAS-R1819W) have cognitive disorders112, a finding that has been shown to be consistent in rodents overexpressing this variant113. In addition to neural stem and progenitor cells, DNL is central for the proper morphological formation and function of neurons. FAS is highly expressed in neurons and is crucial for dendrite branching and function114. DNL is also essential for myelination, as lipids comprise a large portion of the myelin membrane115. Recent studies have demonstrated that FAS is necessary for the correct onset of myelination and proper myelin growth by Schwann cells in the peripheral nervous system116 and oligodendrocytes in the central nervous system109. These data suggest that inhibition of DNL in the nervous system, not only in fetal development but also in adulthood, could potentially promote the progression of neurodegenerative disease.


Regulation and structure

CIC is encoded by the SLC25A1 gene and is ubiquitously expressed, with highest expression in liver, reproductive organs, gastrointestinal tract and adipose tissue (see Related links). It is located within the inner mitochondrial membrane, and primarily catalyses the efflux of tricarboxylates such as citrate and isocitrate in exchange for tricarboxylates, dicarboxylates and phosphoenolpyruvate117. LCFA-CoAs inhibit CIC in a reversible manner, competitive with citrate118, while acetylation can increase allosteric activation by citrate119 (Fig. 3).

Fig. 3: Physiological regulation of DNL.
figure 3

Regulatory mechanisms of de novo lipogenesis (DNL) involve allosteric regulation, covalent modifications and transcriptional changes. Allosteric activators include citrate, glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) while oxaloacetate (OAA) and long-chain fatty acyl (LCFA)-CoAs are allosteric inhibitors. Regulatory phosphorylation is facilitated by several enzymes including AMP-activated protein kinase (AMPK), AKT, branched-chain α-keto dehydrogenase kinase (BDK), glycogen synthase kinase 3 (GSK3) and protein kinase A (PKA), whereas caspase 10 and constitutive photomorphogenic 1 (COP1) facilitate the degradation of ATP-citrate lyase (ACLY) and fatty acid synthase (FAS), respectively. Transcriptional modifications are regulated by two major transcription factors, sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate-responsive element-binding protein (ChREBP). Additional transcription factors, such as liver X receptor (LXR) are also implicated in the transcriptional regulation to varying degrees of importance depending on the cell type. ACC, acetyl-CoA carboxylase; CIC, citrate/isocitrate carrier; FAS, fatty acid synthase; PPP, pentose phosphate pathway; TCA, tricarboxylic acid; X5P, xylulose 5-phosphate.

Little is known about how the structural components of CIC are implicated in physiological regulation of activity. Structurally, eukaryote CIC is composed of three homologous domains, each of which form two hydrophobic membrane-spanning α-helices that are connected by hydrophilic loops that span from the intermembrane space to the mitochondrial matrix117 (Fig. 4). There are at least two citrate binding sites, residing at different depths within the membrane bilayer and serving as the binding site of CIC inhibitors. Residues within sites 1 and 2 form six and eight hydrogen bonds with citrate, respectively120. CIC site 1 is kinetically accessible to anions from the inner surface and determines specificity to internal substrate as it moves through the CIC121. After binding to site 1, citrate is transferred to site 2, before being released into the intermembrane space where it then diffuses through a voltage-dependent anion transport channel within the mitochondrial outer membrane, into the cytoplasm.

Fig. 4: Structural domains and binding sites of DNL inhibitors with chemical structures of the most advanced inhibitors.
figure 4

Lipogenesis inhibitors interact with one or more druggable sites of the enzyme to exhibit an inhibitory effect. Linear organization and model representation of each enzyme are shown with known inhibitor binding sites in colours. Inhibitors with known enzyme binding sites are colour coded with their respective interaction sites. a | Citrate/isocitrate carrier (CIC) inhibitors: compounds that bind to citrate binding site 1 are shown in light blue and those that bind to citrate binding site 2 in orange. The chemical structure of CTPI-2 is shown. b | ATP-citrate lyase (ACLY) inhibitors: compounds that interact with the CoA binding site are highlighted in red and those that interact with citrate binding site in dark blue. The chemical structure of bempedoic acid is shown. c | Acetyl-CoA carboxylase (ACC) inhibitors: compounds that target the biotin carboxylase (BC) domain are highlighted in green and those that target the carboxyl transferase (CT) domain are highlighted in violet. Chemical structures of clinical stage inhibitors are shown. d | Fatty acid synthase (FAS) inhibitors: inhibitors that bind to the β-ketoacyl synthase (KS) domain are highlighted in purple, inhibitors that bind to malonyl-acetyl transferase (MAT) are in blue, inhibitors that bind to enoyl reductase (ER) are in green, inhibitors that bind to β-ketoreductase (KR) in yellow and those that bind to thioesterase (TE) in orange. The chemical structure of TVB-2640 is shown. BCCP, biotin carboxyl carrier protein; BTC, benzenetricarboxylate; CCL, citryl-CoA lyase; CCS, citryl-CoA synthetase; DH, dehydratase; HCA, (−)-hydroxycitric acid; TMD, transmembrane domain. Part a CIC structure adapted from P53007, CC BY 4.0; part b ACLY structure adapted from PDB ID 6POF, CC BY 1.0; part c ACC structure adapted from PDB ID 5CSK, CC BY 1.0; part d FAS structure adapted from PDB ID 2VZ8, CC BY 1.0.

Pharmacological inhibitors

The first-generation CIC inhibitor to have a higher affinity for the transporter than any substrate was benzenetricarboxylate (BTC)117 (Table 1; Supplementary Table 1), which inhibits the CIC in a mixed competitive and uncompetitive manner117,122. BTC is structurally similar to citrate and primarily interacts with citrate binding site 2 (ref.122) (Fig. 4). Although this inhibitor has been widely used for structural and functional characterization of the CIC, potential binding of the inhibitor to other citrate binding proteins has limited its therapeutic development.

Table 1 CIC inhibitors

CTPI-1, also known as compound 792949, is a next-generation competitive CIC inhibitor identified via in silico screening of commercially available small molecules. It has a slightly higher affinity for CIC than BTC does and binds to residues from both citrate binding sites simultaneously122. This inhibitor was identified in yeast CIC and later it was found to exhibit suboptimal binding for human CIC, potentially owing to a key amino acid difference in the citrate binding sites between the species123.

Attempts to optimize compounds specific for human CIC led to the identification of CTPI-2, which exhibited a 20-fold improvement in binding affinity relative to CTPI-1 and inhibited citrate transport at lower concentrations123. Furthermore, it has shown several favourable effects in preclinical models as discussed below, indicating the potential of the inhibitor for development.

Therapeutic indications

Metabolic diseases

Lowering cytosolic citrate would be expected to inhibit DNL by reducing both substrate availability and allosteric activation of ACLY and ACC124,125. Consistent with this concept, BTC lowers triglyceride content in primary hepatocytes126 (Supplementary Fig. 1). CIC expression is increased in the liver of people with NASH, and CTPI-2 treatment of HFD-fed mice reversed steatohepatitis and liver injury, with a concomitant reduction in serum cholesterol and triglycerides43. In addition, CTPI-2 reduced fasting glucose and normalized glucose tolerance and insulin sensitivity, potentially through targeting gluconeogenesis. However, as CTPI-2 also lowered body mass through mechanisms that are not understood, this may have contributed to improvements in glucose homeostasis. BTC acutely inhibits glucose-stimulated insulin secretion in INS1 cells and islets127; however, in vivo effects on pancreatic islets have not been evaluated. These data suggest that inhibition of CIC may exert favourable effects on obesity, NAFLD and T2D. Future studies are needed to determine the mechanisms that contribute to weight loss and whether this is required for the beneficial effects on liver steatosis and glucose homeostasis.

One potential mechanism by which CIC inhibitors may exert positive effects in metabolic disease independently of reductions in body mass, may involve inhibition of inflammation. Inflammatory stimuli such as LPS, TNFα and IFNγ, elicit metabolic reprogramming in macrophages and natural killer cells that leads to increases in CIC expression, suggesting that inhibition may exert anti-inflammatory effects128,129. In cultured macrophages, CTPI-1 inhibits inflammatory responses induced by TNFα and IFNγ128. Similarly, in obese mice CTPI-2 blocks inflammatory macrophage infiltration into the liver and adipose tissue and this is accompanied by a decrease in circulating pro-inflammatory cytokines43. These data suggest that inhibiting CIC may reduce inflammation and this may exert a positive effect on metabolic diseases.


As CIC expression is increased in several different cancer cells130 and inhibition of CIC blunts cell growth131, CIC has emerged as an attractive target for anticancer drug development. All three CIC inhibitors have been found to exert anticancer activity in cultured cancer cell lines123,132,133 and reduce tumour growth in vivo132,133. Future studies are needed to evaluate the effects of these inhibitors in clinical settings.


Regulation and structure

ACLY expression is highest in adipose and liver and lowest in skeletal muscle (see Related links). Although ACLY is predominantly a cytosolic enzyme, it is also found within the nucleus (see Related links). The activation of ACLY involves four steps: first, Mg–ATP binding, and phosphorylation of His760, which catalyses the formation of an enzyme-bound citryl-phosphate, followed by a CoA attack and formation of a citryl-CoA intermediate and finally, citryl-CoA cleavage into final products acetyl-CoA and oxaloacetate (OAA). This reaction is allosterically activated by the glycolytic intermediates glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), with the latter being more potent134 (Fig. 3). Citrate also allosterically activates ACLY while the products of the reaction, acetyl-CoA and OAA, inhibit enzyme activity124.

In addition to allosteric activation, early studies identified that insulin135 and glucagon136 increase Ser/Thr phosphorylation on ACLY (Fig. 3), with subsequent studies establishing that Thr446 and Ser450 are phosphorylated by glycogen synthase kinase 3 (GSK3)137, and Ser454 is phosphorylated by protein kinase A (PKA)136 and the ‘insulin-stimulated kinase’138 that was subsequently identified as AKT (also known as PKB)139. mTORC2 was also suggested to phosphorylate ACLY Ser454 (ref.8), an effect shown to require AKT140. More recently, branched-chain ketoacid dehydrogenase kinase was also found to phosphorylate Ser454, thus linking ACLY activity with amino acid availability and insulin resistance141. Although initial studies found that Ser454 phosphorylation had minimal effects on ACLY activity142, further work established that phosphorylation of this residue decreased sensitivity to allosteric activation by F6P and G6P134. Subsequent studies in various cell lines have confirmed the important effects of this phosphorylation event in enhancing ACLY activity8,104,143. In addition, ACLY is regulated by cleavage of the enzyme at a post-transcriptional level144.

The structural underpinnings of the chemical reactions mediated by ACLY are still not fully understood124,145. ACLY consists of two modules, ACLY-A and ACLY-B, which are present as separate domains (heteromeric) in prokaryotes, but are linked (homomeric) in animals146 to form a functional tetrameric structure124 (Fig. 4). Each monomer consists of an N-terminal citryl-CoA synthetase (CCS) module, containing CCSβ and CCSα regions, and a C-terminal citryl-CoA lyase (CCL) domain145. The ACLY-A module forms the CCSβ region, while ACLY-B forms the remaining domains of the monomer, with Ser/Thr phosphorylation occurring within a linker region between the modules145. Recent studies have provided a detailed structural basis for the multistep catalytic mechanism of ACLY, showing that citrate binds to the CCSβ region, while the CCSα region serves as a CoA binding site124. An additional CoA binding site is in the CCL domain124,145; however, it is controversial whether binding at this site is required for acetyl-CoA formation.

Pharmacological inhibitors

ACLY is an attractive drug target owing to its strategic position at the nexus of fatty acid, cholesterol and carbohydrate metabolism. As such, several ACLY inhibitors have been identified on the basis of their ability to mediate concomitant inhibition of fatty acid and cholesterol synthesis, while activating fatty acid β-oxidation.

The first identified and extensively studied inhibitor of ACLY was (−)-hydroxycitric acid (HCA), a derivative of citric acid found in tropical plants Garcinia cambogia and Hibiscus subdariffa147. HCA inhibits ACLY by competing with citrate147, which results in concomitant suppression of DNL and cholesterol biosynthesis148 (Table 2; Supplementary Table 2). However, HCA possesses poor physicochemical properties and was later found to allosterically activate ACC149. Although numerous attempts were made to improve on the HCA scaffold, these efforts were unsuccessful as off-target effects continued to be observed150.

Table 2 ACLY inhibitors

Additional screening of natural products identified several other ACLY inhibitors, including purpurone151, a series of anthrones and anthraquinones derived from Penicillium sp.152, radicicol, a 14-membered macrolide originally isolated from Monosporium bonorden153, and cucurbitacin B, a natural bioactive compound abundant in cucumber154. Another series of inhibitors was developed on the chemical scaffold of the natural product emodin155. Although many of these compounds suppressed ACLY activity in biochemical assays, their specificity for ACLY and mechanisms of action remained unresolved156.

Several inhibitors were designed on the basis of an alternative targeting strategy aimed to disrupt the formation of a stable citryl-CoA intermediate bound to the active site. SB-201076 was a promising compound that demonstrated activity against purified rat ACLY, but was inactive in cell-based DNL assays owing to poor cell permeability157. Improved cell permeability was achieved by generating the lactone prodrug analogue, SB-204990, which undergoes hydrolysis and activation once inside the cell to yield the active metabolite, SB-201076 (ref.158). However, development of this series was halted before clinical development. More recently, in silico screening identified four subtypes of furans and benzofurans that inhibit ACLY by also binding to the citrate binding domain159.

A novel chemical scaffold discovered by high-throughput screening (HTS) led to the identification of BMS-303141, a 2-hydroxy-N-arylbenzenesulfonamide160. This compound inhibits ACLY, but also ACC1 and ACC2, although nearly 100-fold less potently160. Building on the BMS-303141 chemical scaffold, Nimbus Therapeutics used rational computer-aided design to develop a new series of ACLY inhibitors. The most potent compound, NDI-091143, inhibited human ACLY competitively with respect to citrate. The use of cryo-electron microscopy revealed an unexpected allosteric mechanism of inhibition whereby NDI-091143 bound next to the citrate binding site in a hydrophobic cavity, resulting in an extensive conformational change that prevented citrate binding to the enzyme. Although no cell-based or in vivo data were reported, the identification of this novel allosteric mechanism provides a new approach to discover novel ACLY inhibitors with improved drug-like properties161.

One of the first synthetic fatty acid-like ACLY inhibitors was MEDICA 16. This compound was designed by modifying long-chain dicarboxylic fatty acids to generate ββ′-methyl-substituted α,ω-dicarboxylic acids, with the aim of maintaining the lipid-regulating properties of natural long-chain fatty acids while improving drug-like properties by preventing β-oxidation and enzymatic esterification162. MEDICA 16 inhibited ACLY competitively with citrate163 and ACC competitively with acetyl-CoA and ATP164. A similar dicarboxylic acid, 3-thiadicarboxylic acid, was synthesized by replacing the dimethyl substitution in the β-position with a sulfur atom. This molecule inhibited ACLY and FAS, resulting in reduced levels of plasma triglycerides and cholesterol165. However, neither molecule advanced beyond preclinical studies.

Using a phenotypic screen based on inhibiting fatty acid and sterol synthesis in primary rat liver cells, Esperion Therapeutics discovered a liver-specific inhibitor, ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid, also known as bempedoic acid and ESP-55016 (ref.166). Studies have established that bempedoyl-CoA potently inhibits recombinant human ACLY competitively with CoA and that the prodrug (bempedoic acid) is inactive167. Importantly, conversion into the CoA conjugate is dependent on very long-chain acyl-CoA synthetase (ACSVL1/FATP2/SCL27A2), an enzyme highly expressed in the liver, but not in most other tissues including skeletal muscle, thus enabling the liver-specific actions of bempedoic acid167. And while bempedoyl-CoA also inhibits ACC166 and activates AMPKβ1-containing heterotrimers167, the relevance of these activities has not been demonstrated, since potency towards ACC is relatively weak and bempedoic acid continues to suppress liver DNL even in the absence of the AMPKβ1 isoform167.

Therapeutic indications


Preclinical studies in rodents found that HCA reduces body mass through a mechanism related to caloric restriction168,169, but this effect is not observed in humans170,171 (Supplementary Fig. 1). Bempedoic acid and BMS-303141, two of the better-characterized ACLY inhibitors, have strengthened a potential connection between weight loss and ACLY, with both reducing body weight gain and adiposity independently of changes in food intake in preclinical models160,166,167,172. Importantly, recent evidence has emerged from pooled analyses of clinical trials that bempedoic acid elicits modest weight loss in humans173. Studies examining the potential mechanisms by which ACLY inhibitors exert weight loss are warranted.

NAFLD–NASH and type 2 diabetes

Liver lipids and insulin sensitivity are directly linked; as such there is a very close connection between NAFLD and T2D. MEDICA 16 reduced liver lipid content162, hepatic glucose production, and improved peripheral insulin sensitivity174,175 in several distinct rodent models of obesity-induced insulin resistance. Bempedoic acid also reduced hepatic triglycerides and markers of inflammation in Ldlr−/− mice fed a diet high in fat and cholesterol. Importantly, the liver lipid-lowering effects of bempedoic acid are independent of liver AMPK activation167. In multiple mouse models, bempedoic acid also reduced fasting glucose, fasting insulin and glucose intolerance, suggesting improvements in insulin sensitivity172. Importantly, these effects appear to be translated to humans, as a meta-analysis of randomized trials suggests that bempedoic acid reduces new incidence or worsening of diabetes176. Whether bempedoic acid is effective at reversing NASH and fibrosis remains to be determined.

Cardiovascular disease

Given its dual impact on cholesterol and fatty acid biosynthesis, pharmacological ACLY inhibition has been studied for CVD. Early studies in animal models demonstrated the broad lipid-lowering effects of MEDICA 16 (refs177,178) on circulating levels of cholesterol and triglycerides and associated beneficial effects on vascular and myocardial lesions178. Studies of BMS-303141 and SB-204990 further support ACLY as a target for hyperlipidaemia, as both compounds effectively lowered circulating triglycerides and cholesterol in animal models of hyperlipidaemia158,160. Bempedoic acid also suppresses hepatic cholesterol and fatty acid biosynthesis172 and its hypolipidaemic actions have been demonstrated in hyperlipidaemic hamsters172, obese Zucker rats166 and in mice deficient for ApoE167 or the LDL receptor179 in which atherosclerosis is also reduced. In humans, bempedoic acid promotes dose-dependent LDL-cholesterol lowering effects as monotherapy, and when combined with a statin or ezetimibe180. Unlike rodents, in which bempedoic acid has a profound effect in reducing both plasma triglycerides and cholesterol, the primary effect of bempedoic acid in humans appears to be a reduction in plasma LDL-cholesterol180. The lack of effect on plasma triglycerides may potentially be due to lower liver DNL in humans compared with rodents. In addition to lowering LDL-cholesterol, bempedoic acid also reduces several plasma markers associated with atherosclerotic CVD such as total cholesterol, non-HDL cholesterol, plasma apoB, LDL particle numbers and high-sensitivity C-reactive protein180. More recently, bempedoic acid has been demonstrated to be a safe and effective therapeutic option for patients on maximally tolerated statin therapy181 or patients intolerant of statins182. Bempedoic acid was recently approved for patients with heterozygous familial hypercholesterolaemia (HeFH) (USA and Switzerland), established atherosclerotic CVD (ASCVD) (USA and Switzerland) who need additional LDL-cholesterol lowering, as an adjunct to diet and maximally tolerated statins (USA, EU, Switzerland and UK), and alone or with other lipid-lowering therapies in patients who are statin-intolerant or for whom a statin is contraindicated (EU and UK). The effect on cardiovascular morbidity and mortality has not been established.


Substantial evidence supports the positive effects of pharmacological inhibition of ACLY using SB-204990 and BMS-303141 in cultured cancer cells94. However, despite the robust in vitro evidence, there is surprisingly few data supporting the concept that pharmacological inhibition of ACLY may be effective in vivo. For example, although early studies of HCA showed antitumour effects183, target specificity and suppression of food consumption and body mass confounded interpretation of the primary mechanism of action. Similarly, SB-204990 reduced tumour growth in several distinct xenograft models; however, weight loss was again a confounding variable and no direct effect on metabolic reprogramming within the tumour was reported86. More recently, bempedoic acid has been demonstrated to inhibit hepatocellular carcinoma in mice, but given the requirement for bempedoic acid to be converted into a CoA by ASCVL1, it is currently unclear whether this is due to direct tumour effects or is secondary to inhibition of ACLY in the liver87. Future studies in mouse models examining the effects of pharmacological ACLY inhibitors in conjunction with measures of target engagement within the tumour (for example, suppression of DNL) are needed to evaluate whether pharmacological inhibition of ACLY may be effective in cancer.


Regulation and structure

In mammals, there are two isoforms of ACC, ACC1 (also known as ACCα) and ACC2 (also known as ACCβ). ACC1 is ubiquitously expressed (see Related links), whereas ACC2 is predominantly found in skeletal muscle, breast, adipose and liver (see Related links). ACC isoforms exhibit amino acid sequence similarity of 82% and 76% in their biotin carboxylase (BC) and carboxyl transferase (CT) domains, respectively184, with the major difference being an N-terminal extension in ACC2 resulting in a higher molecular weight compared with ACC1 (ref.184). Although both ACC isoforms are expressed in the cytosol, it was hypothesized that the N-terminal extension in ACC2 might facilitate a preferential role (compared with ACC1) in controlling fatty acid oxidation, because of proximity to carnitine palmitoyltransferase 1, which is allosterically inhibited by malonyl-CoA184. Genetic evidence supports significant overlap between ACC isoforms in regulating fatty acid oxidation and DNL47,49, suggesting that the relative importance of each isoform in regulating DNL and fatty acid oxidation is related to tissue-specific expression profiles rather than cellular localization.

The mechanisms linking tricarboxylic acid (TCA) cycle intermediates with increases in DNL were first uncovered in 1962 when it was found that citrate stimulated the activity of ACC125 (Fig. 3). Subsequent studies identified that activation was mediated by promoting polymerization of the enzyme, an effect inhibited by LCFA-CoAs185. In addition to allosteric control, ACC is inactivated by phosphorylation186, which is sufficient to overcome allosteric activation under physiological concentrations of citrate187 (Fig. 3). This suggested that phosphorylation, not allosteric control, may be the predominant mechanism regulating ACC activity. ACC is phosphorylated and inhibited by the AMPK at Ser79 (ref.188). ACC2 activity is similarly inhibited by phosphorylation at a homologous site (Ser221). In hepatocytes, mutations in both ACC1 and ACC2 were necessary to exert maximal effects on DNL and fatty acid oxidation189. Interestingly, a recent study found that LCFA-CoAs directly activate AMPK190, thus enacting a bimodal mechanism involving both allosteric and covalent inhibition of ACC activity, to inhibit DNL and increase fatty acid oxidation.

The ACC enzyme consists of three domains — BC, biotin-containing carboxyl carrier protein (BCCP) and CT — which are assembled in a single chain in most eukaryotes including mammals (Fig. 4). In eukaryotes, ACC functions as dimers and higher oligomers with the BC and CT domain dimers located at the top and bottom of the structure, respectively, while the BCCP is located within the CT active site191. The ACC reaction involves two steps: the first is ATP-dependent carboxylation of a biotin moiety by the BCCP, followed by transfer of a carboxyl group from biotin to acetyl-CoA to produce malonyl-CoA, within the CT192. Recent structural studies have detailed the dynamic interactions that occur between polymerization state and filament structures upon exposure to citrate and palmitoyl-CoA, effectively locking the enzyme into catalytically competent or incompetent conformational states, respectively193. Phosphorylation at Ser79 in ACC1, and presumably also Ser221 in ACC2, also induces a large conformational change involving the BC dimer interface, which promotes dissociation of the dimer and inactivation194. Thus, both allosteric regulation of ACC by citrate and palmitoyl-CoA and phosphorylation by AMPK regulate enzyme activity through alterations in conformational state188,193.

Pharmacological inhibitors

Inhibition of ACC lowers malonyl-CoA, which is also an allosteric inhibitor of carnitine palmitoyl transferase 1, the rate-limiting enzyme that controls the flux of fatty acids into the mitochondria for β-oxidation. Thus, ACC inhibition represents an attractive approach to simultaneously suppress DNL and increase fatty acid oxidation. Given the embryonic lethality associated with ACC1 inhibition, differential tissue-specific expression profiles and potential compensation by the alternative isozyme, isozyme-specific (ACC1 or ACC2) and nonspecific (ACC1 and ACC2) inhibitors, as well as tissue-selective inhibitors, have been pursued.

The first generation of ACC inhibitors to be studied were soraphen A and TOFA (5-(tetradecyloxy)-2-furancarboxylic acid). Soraphen A is a macrocyclic polyketide, originally isolated from the soil myxobacterium Sorangium cellulosum for its potent antifungal activity. It was later identified as an inhibitor of yeast and rat ACC1 (ref.195). In eukaryotes soraphen A binds to the BC domain, allosterically inhibiting enzyme activity by disrupting oligomerization; however, this does not occur in prokaryotes owing to large structural differences within the BC domain196 (Table 3; Supplementary Table 3). TOFA is intracellularly converted into an ester, TOFyl-CoA, which acts as an ACC inhibitor but also reduces cholesterol synthesis197, suggesting that it may inhibit ACLY. Given limited bioavailability and lack of specificity, neither agent was commercially developed.

Table 3 ACC inhibitors

Pfizer identified a series of isozyme-nonspecific ACC inhibitors through HTS which, following a series of optimizations, led to CP-640186 (ref.198). The crystal structure of the yeast CT domain of ACC in complex with CP-640186 indicates that the compound has tight associations with the active site of the enzyme, blocking biotin binding to the CT domain199. Taisho Pharmaceutical200 and Takeda Pharmaceuticals201 subsequently developed derivatives of CP-640186; however, these compounds had relatively poor metabolic stability and, while subsequent reductions in lipophilicity led to more favourable pharmacokinetics202, and the introduction of a 7-methoxy group led to greater potency and metabolic stability203, none of these compounds entered clinical testing. Also binding to the CT domain is WZ66, a compound identified via structure-based drug design studies by China Pharmaceutical University, which inhibits recombinant human ACC1 and ACC2 in cells and in rodents204.

Several pan-ACC inhibitors have recently entered clinical testing in humans. Studies by Merck revealed MK-4074 as a liver-selective ACC inhibitor that dose-dependently inhibited DNL and lowered liver lipids in both rodent and humans48 (Table 3; Supplementary Table 3). Although the exact binding site of the compound has not been published, it does not appear that clinical development is continuing, potentially owing to the observed induction of hypertriglyceridaemia48. Following on from studies with CP-640186, Pfizer developed PF-05221304, as a selective, orally bioavailable and reversible ACC inhibitor that is preferentially distributed to the liver205, thereby avoiding potential toxicity related to the inhibition of platelet formation18 and developmental defects17. Phase II clinical studies with PF-05221304 alone or in combination with a DGAT inhibitor, PF-06865571, in NAFLD–NASH have been completed72.

In contrast to PF-05221304, which binds to the CT domain, Nimbus Therapeutics developed several potent ACC inhibitors by focusing on compounds that bind to the BC domain, blocking dimerization. Briefly, using crystal structure of the human ACC2 BC domain in complex with soraphen A and subsequent optimization of noncovalent interactions with the dimerization site, they developed a reversible ACC1 and ACC2 inhibitor, ND-630 (ref.206). In contrast to previous ACC inhibitors, this compound disrupted subunit dimerization by mimicking the effects of AMPK phosphorylation206. A second compound of this series, ND-646, exhibited similar potency and mode of inhibition to ND-630, but was developed to be more broadly distributed among peripheral tissues207. The third compound, ND-654, was modified to allow for enhanced hepatic uptake189. ND-630, also known as GS-0976 (Firsocostat), entered clinical development and is currently in phase II clinical trials for NASH.

In addition to the pan-ACC inhibitors described above, isozyme-specific ACC inhibitors have been developed. Takeda identified novel monocyclic derivatives208 and carboxamide derivatives209 that demonstrated specificity for ACC1 over ACC2. Abbott Laboratories developed a series of ACC2-specific inhibitors, one of the most potent and selective being A-908292, which exhibited more than 1,000-fold selectivity against ACC2 compared with ACC1. This compound dose-dependently lowered malonyl-CoA in muscle but not liver of rodents210. However, a preliminary safety assessment showed neurological and cardiovascular side effects that were resolved by replacement of the alkyne moiety with a heteroaryl linker211. Building on this series, Boehringer Ingelheim Pharma and Shionogi research laboratory also identified a series of new molecules that selectively inhibited ACC2 compared with ACC1 without toxicity212,213. To the best of our knowledge, none of these agents has entered clinical testing.

Therapeutic indications

NAFLD–NASH and type 2 diabetes

Significant preclinical evidence supports the benefits of ACC inhibition on liver steatosis, but they have also revealed potential on-target liabilities. In rodents, soraphen A lowered steatosis and this was associated with improved insulin sensitivity214 (Supplementary Fig. 1). However, these improvements in insulin sensitivity did not translate to reductions in blood glucose, as consistent with studies in ACC null mice, ACC inhibition reduced insulin secretion from pancreatic β-cells215. To avoid this problem, TOFA derivatives such as WZ66 were developed that have a preferential liver distribution, reducing hepatic steatosis and hepatic stellate cell activation in mice with diet-induced obesity204. CP-640186 also lowers hepatic steatosis and insulin resistance in mice fed a HFD; however, this may be secondary to reductions in adiposity216. Lastly, short-term treatment of db/db mice with a selective ACC2 inhibitor, (S)-9c, reduced muscle malonyl-CoA levels and intramyocellular lipids while long-term treatment increased muscle glucose uptake and glucose tolerance while reducing HbA1c, postprandial glucose and plasma triacylglycerol levels212. These studies suggest that inhibition of ACC is associated with reductions in liver steatosis and modest improvements in glycaemic control.

Given the promising effect of studies evaluating the effects of genetic and pharmacological inhibition of ACC on liver steatosis, several new-generation ACC inhibitors have advanced to clinical trials in NAFLD–NASH. In a phase I clinical study, PF-05175157 reduced DNL and increased whole-body fatty acid utilization217. However, subsequent clinical trials were terminated owing to extra-hepatic activity leading to reduced platelet count18,205. The liver-optimized inhibitor, PF-05221304, was subsequently shown to inhibit DNL, stimulate fatty acid oxidation and reduce triglyceride accumulation in primary human hepatocytes, along with reducing DNL, hepatic steatosis, fibrosis and immune cell activation in preclinical models218. Preclinical studies designed to assess the level of liver targeting achieved with PF-05221304 showed a maximal reduction of 82% in hepatic DNL compared with up to 33% reductions in lung and bone marrow219. In phase I clinical trials, PF-05221304 inhibited hepatic DNL in a dose-dependent manner without affecting platelet count, consistent with its more liver-targeted actions205. And in phase II clinical trials for NAFLD, PF-05221304 dose-dependently reduced liver fat by up to 65% at the highest dose while also lowering HbA1c (ref.72).

Studies using the liver-specific inhibitor MK-4074 in rodents and humans have supported the therapeutic potential of potent ACC inhibition, but they have also raised additional questions about on-target liabilities. In a phase I clinical trial, MK-4074 reduced hepatic steatosis by 36% after 4 weeks of treatment48. In a preclinical model of NASH, the compound decreased liver fibrosis by 19% in addition to reducing liver fat220. However, clinical development of MK-4074 has been discontinued.

ND-630 is preferentially distributed to liver206 and shows favourable effects in both preclinical and clinical studies. ND-630 dose-dependently reduces hepatic steatosis and hyperinsulinaemia in rodents206. An independent group has confirmed these effects on hepatic steatosis in animals fed a Western diet and also observed reductions in fibrosis221. In line with these studies, ND-630 inhibits hepatic stellate cell activation to reduce fibrogenic activity222. In a phase I clinical study, ND-630/GS-0976 was well tolerated and inhibited hepatic DNL in subjects with obesity and/or overweight223. In an open labelled, non-placebo-controlled study, patients with NASH treated with ND-630 had a median decrease of 22% in hepatic DNL and a significant reduction in hepatic fat and liver stiffness after 12 weeks of treatment4. In a subsequent larger randomized, placebo-controlled study, ND-630 treatment reduced hepatic steatosis by 21%224. ND-630 has recently been tested in combination with the apoptosis signal-regulating kinase 1 (ASK1) inhibitor selonsertib and farnesoid X receptor (FXR) agonist cilofexor in patients with bridging fibrosis or compensated cirrhosis, with combinations showing greater improvement in NASH activity than ND-630 alone225. Phase II studies with ND-630 and the GLP1R-agonist semaglutide are currently in progress.

Cardiovascular disease

Circulating triglycerides are an important risk factor for CVD, and in preclinical studies in mice, several ACC inhibitors, such as CP-640186 (ref.216), A-908292 (ref.226), (S)-9c212, ND-630 (ref.206) and ND-654 (ref.189) lowered plasma triglyceride levels. However, in humans, ACC inhibitors dose-dependently increase plasma triglycerides and/or VLDL. As originally described for MK-4074 (ref.48), but also observed with PF‐05221304 (ref.72), this increase in circulating triglycerides is likely mediated by the effects of decreased liver polyunsaturated fatty acids to stimulate SREBP1c activation, leading to increased GPAT and VLDL secretion48. ND-630 also increases plasma triglycerides224, an effect associated with reduced triglyceride clearance227. To avoid this adverse effect, preclinical studies have been undertaken that combine ACC inhibitors with agents that reduce plasma triglycerides such as omega-3 fatty acids48 and PPARα agonists such as fenofibrate227. Similarly, DGAT2 inhibition also blocked the effects of PF‐05221304, to increase liver SREBP1c and circulating triglycerides while exerting additive effects on liver fibrosis in rodent models72. Importantly, in phase II clinical trials, DGAT2 inhibition also mitigated PF‐05221304-induced increases in serum triglycerides and ApoaC3 (ref.72). These data suggest that combination strategies may be an effective means to improve the cardiovascular risk profile of ACC inhibitors in patients with NASH.


Studies of early ACC inhibitors, soraphen A228 and TOFA229 demonstrated inhibitory effects on multiple cancer types. Although the contribution of ACC inhibition to the effects of these early inhibitors is unknown, the anti-tumorigenesic effect of ACC inhibition has been supported by more recent studies using inhibitors with improved potency and specificity. For example, CP-640186 reduced the growth of human lung cancer cells230, while ND-646 and ND-654 reduced tumour burden in mouse models of non-small-cell lung cancer207 and hepatocellular carcinoma189, respectively, when used alone and in combination with existing standards of care.


Regulation and structure

Human FAS is highly expressed in adipose tissues and reproductive organs (see Related links). In contrast to ACLY and ACC, few allosteric and covalent mechanisms regulating the activity of FAS have been described (Fig. 3). In yeast, phosphorylation of Ser1140, Ser1640 and Ser1827 are associated with increased 18:0-CoA production231, while in a breast cancer cell line, FAS is phosphorylated at Tyr66 when in complex with human epidermal growth factor receptor 2 (ref.232). In addition, FAS is degraded by E3 ubiquitin ligase COP1 in the presence of adaptor protein SHP2 and deubiquitinated by ubiquitin-specific protease 2a233 (Fig. 3). These data suggest that under some conditions and cell types, FAS may be regulated by covalent and ubiquitylation mechanisms; however, it appears that the regulation of FAS primarily occurs through transcriptional control.

FAS has evolved structurally and functionally different variants that are generally divided into two types based on the organization of their catalytic units — type 1 and type 2 FAS234,235. In eukaryotes, the multifunctional complex of type 1 FAS is expressed in the cytosol and regulated by only one gene234. Type 2 FAS is present in mitochondria and functions completely independently of the cytosolic type 1 FAS235. Type 1 FAS typically generates only palmitate, whereas type 2 FAS is able to produce diverse fatty acids, including unsaturated fatty acids236. Structurally, mammalian FAS is a homodimer protein that consists of two multifunctional polypeptides, containing seven functional domains and two non-enzymatic or pseudo domains in each chain237, all of which are required for fatty acid biosynthesis (Fig. 4).

Pharmacological inhibitors

Several natural products and their derivatives have been found to inhibit FAS. Cerulenin, an antifungal antibiotic originally isolated from Cephalosporium caerulens inhibits FAS in both mammals and bacteria238. And although cerulenin inhibits FAS by binding covalently to the active cysteine thiol in the β-ketoacyl synthase (KS) domain (Fig. 4), it also inhibits HMG-CoA synthetase activity238, blocking sterol synthesis. The reactivity and lack of specificity of cerulenin led to the development of the synthetic analogue C75, which binds to the KS239 and thioesterase (TE) domains of human FAS240.

Several novel small-molecule FAS inhibitors have been developed that inhibit the TE domain. Orlistat, also known as tetrahydrolipstatin, is a derivative of lipstatin, a naturally occurring inhibitor of pancreatic lipase, and was found to also inhibit FAS through irreversible binding to the TE domain of the enzyme241. Owing to poor oral bioavailability and metabolic instability, several reformulation efforts were initiated, including a hydrophilic nanoparticle delivery system242, poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles243 and folate receptor-targeted micellar nanoparticles244; however, none of these has moved into clinical development. Fasnall, a thiophenopyrimidine derivative also targets cofactor binding sites in multiple domains and in pharmacokinetic and toxicity studies shows no toxic effects245. More recently, triazole urea-based substitutions to Fasnall, led to the identification of MP-ML-24-N1, which inhibits FAS through the TE domain and has good cellular permeability246. Lastly, IPI-9119 irreversibly inhibits the TE domain by promoting acetylation of the catalytic serine residue and has shown high potency and selectivity against FAS as well as good pharmacological properties247.

FAS inhibitors that target the β-ketoacyl reductase (KR) domain have also been developed with some recently entering clinical testing. GSK identified GSK2194069 using an HTS approach based on compound competition with NADPH for binding in the KR domain248. Inhibitor-bound X-ray crystal structures showed that the compound forms hydrogen bonds with residues Ser2021 and Thyr2034, while studies in various cell lines indicated good cellular activity towards inhibiting DNL248. Using a similar approach, Boehringer Ingelheim Pharma GmbH & Co identified BI-99179 as a potent and selective inhibitor for human FAS that is also assumed to bind to the KR domain249. BI-99179 showed high metabolic stability in rat and human microsomes and high oral bioavailability in rats249. Lastly, Sagimet Biosciences developed TVB-3166, an orally bioavailable, reversible, potent and selective inhibitor of FAS that also likely inhibits the KR domain250. A related but more advanced compound, TVB-2640, is under clinical development. Recently, Forma Therapeutics reported that FT-4101 inhibited human FAS by also targeting the KR domain251 (Table 4; Supplementary Table 4).

Table 4 FAS inhibitors

Therapeutic indications


The link between FAS inhibition and reductions in body weight is not well understood. Initial observations that cerulenin induced weight loss in both lean252 and obese253 mice suggested that effects may be primarily related to toxicity rather than inhibition of FAS. However, subsequent studies showed that C75 also had a profound effect on body weight254,255 (Supplementary Fig. 1), suggested that effects may involve an increase in energy expenditure and suppression of appetite. Although the FAS inhibitor orlistat has been approved for weight management in obesity it is important to note that owing to poor bioavailability the inhibition of pancreatic lipase, not FAS, is the primary mechanism by which this agent induces weight loss. Importantly, data from selective FAS inhibitors such as TVB-2640 do not support a link with weight loss.

NAFLD–NASH and type 2 diabetes

Early studies demonstrated that treatment with C75 decreases hepatic steatosis256 and blood glucose in mouse models of obesity252,257. These findings supported the development of TVB-2640, which was found to lower DNL by up to 90% in individuals with obesity and insulin resistance258. Phase II clinical studies are currently underway in patients with NASH. FT‐4101 has been tested in humans with obesity, where it suppressed DNL and lowered steatosis251. However, clinical development has ceased for reasons that have not been disclosed. Although these studies support a potential therapeutic benefit of inhibiting FAS in NASH, results from the ongoing phase II studies will be imperative to better understand safety and efficacy.


Although cerulenin has beneficial effects in various cancer cell lines and xenograft tumour models259,260,261, reactivity and the profound weight loss caused by this compound limited its potential. Antitumour effects of C75 were also demonstrated in various cancer cell lines and xenograft tumours alone261 and in combination with radiotherapy93. In addition, orlistat exhibits cytostatic and cytotoxic effects in tumour cells, and nanoparticle technology has been used to improve bioavailability and subsequent cytotoxicity in xenografts241,243,244. Studies have been conducted with more-specific inhibitors that strengthen FAS as an anticancer target. Fasnall was shown to inhibit breast cancer245 and prostate cancer cell growth262, and GSK2194069 inhibits the proliferation of non-small-cell lung cancer cell lines248. IPI-9119 (ref.247) and TVB-3166 (ref.250) also inhibit cancer cell proliferation in several distinct preclinical models. Interestingly, recent studies have found that owing to limited exogenous fatty acids within the brain, metastasis of breast cancer is highly sensitive to FAS inhibition by TVB-3166 and BI-99179 (ref.263). Phase I clinical trials with TVB-2640 have been completed in healthy subjects and in patients with solid malignant tumours264, and phase II studies in multiple cancers are underway. Results from these studies will be important to better understand the potential therapeutic effects of FAS inhibition in cancer.


In the past 50 years tremendous progress has been made in understanding the biochemical mechanisms and physiological significance of DNL in regulating cellular metabolism and whole-body energy homeostasis. Important steps along the way have included biochemical identification of the key metabolic intermediates in the conversion of glucose to fatty acids, the molecular cloning of the key enzymes regulating the process and the discovery of crucial allosteric and covalent mechanisms that regulate flux through the pathway. Subsequent studies in genetically modified mice revealed the physiological role of DNL, broadening our understanding of the complex connections that exist between multiple cellular processes and tissues well beyond the simple storage of excess calories in adipose tissue. Meanwhile, advances in structural biology laid the foundation for the molecular underpinnings by which natural products and new-generation small molecules inhibited enzyme activity. Collectively, this information led to an explosion in the number of novel small molecules that have been developed and tested in preclinical models for cardiometabolic disease, cancer and other indications. Although these compounds vary in their molecular target, chemical structure and physicochemical properties, the common action in which DNL is inhibited supports the notion that it is beneficial for the prevention and treatment of a broad spectrum of diseases. However, the development and testing of this new generation of highly selective and efficacious small molecules has also revealed several surprising new biological insights that have important implications for safety, efficacy and the need for combination therapies as they progress into clinical trials.

Safety-related side effects

There are several safety concerns associated with inhibiting DNL. The first and most significant to be identified was related to embryonic development. Mice homozygous deficient for ACLY265, ACC1 (ref.266) or FAS267 die during embryonic development, and CIC deficiency is characterized by severe neurodevelopmental disorders268. The ACC inhibitor PF-05175157 also induces developmental toxicity (growth retardation and dysmorphogenesis associated with disrupted midline fusion) in rats and rabbits17. Thus, avoiding systemic inhibition of DNL is essential to safeguard embryonic development.

Another major concern with inhibiting DNL in lipogenic organs such as liver and adipose tissue is that this simply redirects carbon into other parts of the body where toxicity may be even greater. Experimental evidence of this effect was first established with the ACC inhibitor MK-4074, which dose-dependently increased SREBP1c and serum triglycerides; an effect that was shown to be ‘on-target’ as mice genetically lacking ACC in the liver had a similar phenotype48. Subsequent studies with ND-630 (ref.224) and PF‐05221304 (ref.205) in humans with NASH revealed similar effects. Importantly, increases in serum triglycerides by PF‐05221304 can be inhibited by co-administration of a DGAT2 inhibitor72. Interestingly, inhibiting ACC by activating AMPK269,270 lowers serum triglycerides and cholesterol effects. Similar results are also observed with the inhibition of ACLY173,181,182. A greater understanding of the mechanism behind these differential responses to inhibiting liver DNL will be important for alleviating potential adverse events.

The upstream and downstream consequences associated with blockade at different steps in the DNL pathway, leading to the accumulation of metabolic intermediates, must also be considered. For example, inhibition of FAS leads to a build-up of malonyl-CoA. This increase in malonyl-CoA as a result of FAS inhibition impairs physiological and pathological angiogenesis via the malonylation of mTOR271. Malonylation has also been implicated in various metabolic pathways272, histone modifications273 and immune cell reprogramming274, suggesting that FAS inhibition may exhibit broader biological effects than the anticipated DNL inhibition. CIC inhibition may also pose safety concerns, as a deficiency of CIC increases mitochondrial citrate and isocitrate and this is associated with severe neurometabolic effects268. Lastly, ACLY inhibition, which lowers acetyl-CoA, may affect the acetylation profile of many different histones, potentially having a wide array of differential effects on gene expression profiles and epigenetic programming275. Thus, it is pivotal to consider the effects of metabolic intermediates when developing DNL inhibitors as a therapeutic approach.

Bypassing DNL inhibition/compensatory pathways

The concept of cellular metabolic flexibility is another key concern for DNL inhibitors. Metabolism is fluid and interconnected: a block in one pathway enhances flux through alternative pathways. This has been studied most intensely in cancer in which plasticity is a hallmark of chemoresistance. For example, in response to reduced flux from glucose to acetyl-CoA under hypoxic conditions, mutations in KRAS have been shown to provide cancer cells with increased ability to scavenge exogenous lipids, thereby diminishing dependence on DNL276. The reciprocal relationship is also observed whereby inhibiting exogenous fatty acid uptake by blocking CD36 enhances the efficacy of the FAS inhibitor C75 (ref.277). Similarly, in breast cancer brain metastasis, where exogenous fatty acids are limited, inhibiting FAS exerts greater lethality compared with the liver where exogenous fatty acids are abundant263. Inhibition of ACLY can also be bypassed in some cancer cells278 and in the liver of mice fed a high-fructose diet45 through upregulation of ACSS2. These data suggest that depending on the nutritional context and cell types involved, for DNL inhibitors to be highly effective it may be necessary to also target compensatory mechanisms. For example, might it be important for people being treated with an ACLY inhibitor to limit their alcohol and fructose intake to maximize effects? Alternatively, might an individual having their cancer treated with a FAS inhibitor need to limit fatty foods? Despite these compelling data in preclinical models, data generated in clinical settings with respect to ACLY inhibition for LDL lowering173,181,182, ACC inhibitors for NASH48,72,205,224 and FAS inhibition for cancer264 suggest that the degree of DNL inhibition achieved may be sufficient to overcome any compensatory pathways. However, further studies investigating this will be important.

Systemic/organ-specific inhibition

A significant challenge with the clinical development of DNL inhibitors has been the discovery of differential responses of certain cells between rodents and humans. An example of this occurred with the clinical development of the systemic ACC inhibitor PF-05175157, which was found to have no toxicity in rodents but dose escalation studies in people revealed reduced platelet count18,205. Subsequent studies established that the reduction in platelet count was due to ACC inhibition within bone marrow, which impaired megakaryocyte maturation18. This finding demonstrated a requirement for DNL in humans but not rodents, which was especially surprising given that DNL in tissues such as liver and adipose tissue is much higher in rodents than in humans279. Importantly, these effects have been avoided with new-generation liver-targeted ACC inhibitors ND-630 (ref.224) and PF‐05221304 (ref.205). Similarly, genetic inhibition of ACLY in muscle and adipose tissue results in muscle weakness and lipodystrophy, respectively, but has been avoided by the development of the liver-targeted prodrug bempedoic acid167. Liver-specific inhibition of DNL may also be helpful to avoid the potential detrimental effect of adipose tissue DNL inhibition on whole-body insulin sensitivity, given that adipose tissue DNL directly correlates with insulin sensitivity61,280. Lastly, FAS inhibition is associated with alopecia, an effect that has been reduced with the new generation of liver-directed FAS inhibitors TVB-2640 (ref.264) and FT-4101 (ref.251). Thus, data presented to date suggest that inhibition of FAS, ACC and ACLY can be safely achieved through liver targeting; however, whether chronically inhibiting DNL in other organs or cell types can be safely achieved requires further study.


Over the past decade, many challenges have been addressed leading to DNL inhibitors entering clinical development for cancer, CVD, NASH and acne vulgaris. In the era of single-cell analysis of cellular metabolism, an especially exciting avenue of research will be to determine whether there may be specific cell types in which DNL is upregulated under different pathological conditions. The identification of these potential cell types may lead to cell-specific DNL inhibitors that could be applied during certain windows of development for alleviating or potentially preventing disease. Another important consideration will be related to balancing efficacy and safety as enhanced potency may lead to greater compensatory upregulation and/or toxicity. An example of this is seen with the ACC inhibitors whereby inhibition of DNL closely parallels concomitant increases in circulating triglycerides and inhibition of platelets. As DNL is transiently upregulated after a meal, one way to potentially avoid upregulation of compensatory pathways and/or toxicity may involve the use of inhibitors with a shorter half-life so that DNL is maintained at basal levels. Ultimately, whether DNL inhibitors are safe and effective enough to be used as a monotherapy or will be used in combination with other therapies to enhance efficacy or offset liabilities, as has been proposed for ACC inhibitors in NASH, will depend on clinical trial results. In this regard, findings with the ACLY inhibitor bempedoic acid are encouraging as they suggest that chronic inhibition of DNL in the liver is safe when used alone or with other standards of care. Whether DNL inhibitors that target other cell types and organ systems become widely adopted as a cornerstone for treating other disease indications beyond CVD and NASH remains to be determined.