Application of PROTAC in Metabolic Diseases

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Metabolic diseases refer to diseases caused by metabolic issues, resulting from factors such as metabolic disorders and hypermetabolism. These mainly include hypertension (HTN), type 2 diabetes mellitus (T2DM), hyperlipidemia (HLD), obesity, and non-alcoholic fatty liver disease (NAFLD). Multiple diseases among them may co-occur, share common risk factors, and are associated with an increased risk of disability, cancer, and premature death.

Studies have shown that the incidence of all metabolic diseases increased from 2000 to 2019, with the greatest burden observed in countries with higher average incomes, education levels, and fertility rates. Currently, the treatment of these diseases mainly relies on small-molecule drugs and targeted biologics. While these therapies demonstrate significant efficacy, they face limitations in terms of drug resistance, side effects, cost, and applicability, which indicates a need for new treatment methods.

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising technology in recent years, offering advantages such as targeting undruggable targets, high efficiency, and overcoming drug resistance. Advances have been made in applying PROTACs to metabolic disease-related targets such as HMGCR, PNPLA3, LXR, PI3K, and PDEδ.

PROTACs Targeting HMGCR

Cholesterol is the most abundant sterol molecule in mammals and is a fundamental component of cell membranes. Cholesterol homeostasis is crucial for normal physiological functions, and abnormalities in cholesterol metabolism are closely associated with the development of cardiovascular diseases, neurodegenerative diseases, and cancers.

Cholesterol synthesis primarily occurs in the liver through nearly 30 enzymatic reactions that convert acetyl-CoA into cholesterol. Among these steps, the mevalonate pathway mediated by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) is the key step in de novo cholesterol synthesis. Thus, the expression and regulation of HMGCR are vital for maintaining cholesterol homeostasis in the body.

(A) HMGCR inhibition by statins leads to the compensatory upregulation of HMGCR. (B) Illustration of PROTAC-mediated HMGCR degradation. (Luo, Guoshun, et al. 2021)

HMGCR is a classical therapeutic target for statins in the prevention and treatment of cardiovascular diseases. However, statins can lead to compensatory upregulation of HMGCR protein and cause adverse effects, including skeletal muscle damage, highlighting the need for additional therapeutic strategies.

In 2020, Li et al. designed and synthesized a series of PROTAC molecules by linking atorvastatin with CRBN to target the degradation of HMGCR, a complex transmembrane protein located in the endoplasmic reticulum. Through a series of optimizations, they developed compound P22A, which effectively promotes the degradation of endogenous HMGCR protein. In Huh7 cells, HMGCR upregulation induced by P22A was significantly lower than that induced by atorvastatin. P22A also activates the sterol regulatory element-binding protein (SREBP) pathway and blocks cholesterol biosynthesis, providing a novel strategy for lowering cholesterol levels and treating related diseases.

Luo et al. developed a novel class of PROTACs targeting HMGCR by linking lovastatin with a VHL ligand. Among these, PROTAC 21c effectively degrades HMGCR in HepG2 cells and forms a stable ternary complex. Additionally, its corresponding orally bioavailable lactone, 21b, demonstrated favorable plasma exposure.

Further in vivo studies on 21b revealed potent HMGCR degradation and effective cholesterol-lowering effects in diet-induced hypercholesterolemic mice, highlighting a promising strategy for the treatment of hyperlipidemia and related diseases.

(A) Structures of the HMGCR inhibitors lovastatin (1) and simvastatin (2) together with their active forms (3 and 4). (B) Cocrystal structure of HMGCR catalytic domain complexed with simvastatin acid (4) (PDB: 1HW9). The conjugated site is indicated by a red arrow. (C) Structures of the E3 ligase ligands pomalidomide (5) and VH032 (6). (D) A general scheme for the design of HMGCR-targeting PROTAC probes. (Guoshun Luo, et al., 2021)

PROTACs Targeting PNPLA3

Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a lipid raft protein primarily expressed in the liver and adipose tissue. It exhibits lipolytic enzyme activity toward triglycerides (TG) composed of monounsaturated and polyunsaturated fatty acids and has potential transacylase activity for polyunsaturated fatty acids (PUFAs) in phospholipids. It is responsible for transferring unsaturated fatty acids from triglycerides to phospholipids.

The role of PNPLA3 in health and disease (Piero Pingitore, et al., 2019)

Studies have shown that variants of PNPLA3 increase the risk of metabolic-associated fatty liver disease (MAFLD), primarily because mutations in the PNPLA3 gene can result in the substitution of isoleucine with methionine at position 148. This change reduces lipase activity, impedes the export of fats from the liver, and leads to fat accumulation, ultimately causing disease.

Another study indicated that the PNPLA3 gene is associated with the occurrence of kidney disease. Researchers found that after adjusting for traditional renal risk factors and NAFLD histology, the PNPLA3 GG genotype significantly increased the risk of chronic kidney disease (CKD) and proteinuria, as well as elevated urinary neutrophil gelatinase-associated lipocalin (u-NGAL) levels. Therefore, the degradation of PNPLA3 presents a potential therapeutic intervention strategy.

To determine whether the accumulation of PNPLA3 on liver lipid droplets (LDs) is a cause or consequence of steatosis, BasuRay et al. designed and synthesized a PNPLA3 degrader (PROTAC 3), which contains a hydroxyproline derivative with high affinity for the E3 ligase VHL. This compound is coupled with chloromethyl phenyl and covalently binds with a modified bacterial haloalkane dehalogenase (Halo).

They treated QBI-293A cells expressing halo-tagged PNPLA3 constructs with increasing concentrations of PROTAC 3, and found a significant reduction in PNPLA3 levels. Additionally, they tested the effects of PROTAC 3 treatment in mice expressing PNPLA3 (WT) and PNPLA3 (148 M), confirming that PROTAC-mediated degradation reduced PNPLA3 (148 M) levels and improved fatty liver disease (FLD) associated with the expression of the mutant protein.

PROTAC-mediated degradation of Halo-PNPLA3(148M) reduces liver TGs. (A) A schematic of the experiment. Adapted with permission from ref. 28. Copyright 2015 American Chemical Society. (B) Recombinant AAVs were used to express Halo-tagged PNPLA3 constructs under the control of a liverspecific promoter (thyroxine-binding globulin). Female mice (n = 3 to 7 per group, aged 12 wk) were injected with AAVs (1.25 × 10 11 GCs) expressing Halo alone, Halo-PNPLA3(WT), or Halo-PNPLA3(148M). After 2 wk on an HSD, the mice were treated with vehicle alone (2.5% DMSO in 0.9% NaCl) or PROTAC3 (4.8 mg/kg) for 2 wk (three doses per wk) and then killed after 3 d of dietary synchronization. Livers were harvested for lipid analysis and for isolation of LDs as described in SI Appendix, Methods. Bars represent mean ± SEM values. *P < 0.05, ***P < 0.001. (Soumik BasuRay, et al., 2019)

PROTACs Targeting LXR

Liver X receptors α and β (LXRα and LXRβ) are nuclear receptors that play a crucial role in the transcriptional regulation of lipid metabolism. The transcriptional activity of LXR is induced when cellular cholesterol levels rise.

Liver X Receptors may be related to various pathological conditions. (Komati, Rajesh, et al. 2017)

LXR binds to and regulates the gene expression of proteins involved in cholesterol absorption, transport, efflux, excretion, and conversion to bile acids. Abnormalities in LXR function can lead to the development of related diseases.

LXR is increasingly recognized as a potential therapeutic target for treating vascular and metabolic diseases, neurological disorders, and cancers driven by lipid metabolism.

In 2021, Xu et al. discovered a class of LXR-targeting PROTACs, such as GW3965-PEG5-VH032 (3), which rely on the VHL E3 ligase to effectively degrade LXRβ protein.

The representative synthetic route for VH032-based PROTACs, GW3965-(PEG3–PEG6)-VH032 (Xu, H., et al. 2021)

PROTACs Targeting PI3K

PI3K (phosphoinositide 3-kinase) is a phosphatidylinositol kinase that possesses serine/threonine kinase activity, primarily phosphorylating the hydroxyl group at the 3' position of phosphatidylinositide (PtdIns).

PI3K plays a crucial role in regulating key processes such as cell growth, metabolism, proliferation, and apoptosis. The PI3K gene has a high mutation frequency in many tumors, and its aberrant activation is closely linked to the development and progression of malignant tumors.

The pathogenesis of type 1 (T1D) and type 2 diabetes (T2D) both ultimately lead to the decline of β-cell mass and function. Modulation of PI3K/Akt signalling can act as a therapeutic strategy to counteract β-cell loss. In T1D, autoreactive lymphocytes and pro-inflammatory cytokines (such as IL1β, TNF and IFNγ) drive β-cell destruction. In T2D, β-cells undergo compensatory expansion and increased insulin secretion in response to hyperlipidaemia and insulin resistance in the tissues, thus causing β-cell exhaustion and death. In both conditions, there is a decrease in β-cell mass and insulin secretion. This decline can be counteracted by activation of the PI3K/Akt pathway, which has been shown to promote β-cell proliferation, survival and metabolism. ( Biorender)

In 2022, Camaya et al. explored the modulation of the PI3K/Akt signaling pathway as a novel therapeutic strategy to enhance β-cell function and survival, suggesting that the PI3K/Akt signaling pathway is a promising therapeutic target for type 1 diabetes (T1D) and may develop into an emerging therapy for this condition.

In 2022, Ma et al. synthesized a series of degradation agents targeting PI3K kinases by linking PI3K inhibitors with VHL ligands, and they studied their biological activities. Most compounds exhibited significant anti-proliferative activity in HeLa cells, with compound HL-8 demonstrating anti-cancer potential at 10 μM. Furthermore, HL-8 was able to significantly degrade PI3K kinases at the same concentration [8].

In 2024, Rao et al. utilized PROTAC technology for the first time to achieve subtype-selective degradation of class I phosphoinositide 3-kinases (PI3K). For example, ZM-PI05 selectively degraded the PI3K p110α subtype in various breast cancer cells and downregulated the p85 regulatory subunit while inhibiting the non-catalytic functions of PI3K independently of the p110 catalytic subunit.

As a result, ZM-PI05 exhibited stronger anti-proliferative activity in breast cancer cells compared to the PI3K inhibitor copanlisib.

PROTACs Targeting PDEδ

PDEδ (isoprenylated protein delivery protein) is a lipid-binding protein that forms diffusible complexes with isoprenylated OS proteins, facilitating the membrane localization of isoprenylated Ras proteins.

Small molecule inhibitors targeting the isoprenyl-binding site of PDEδ have proven invaluable in analyzing PDEδ-mediated biological processes, such as the cellular transport of KRas. However, allosteric inhibitors that release PDEδ from Arl2/3 GTPases have limited their applications.

Winzker et al. designed a class of PROTAC compounds based on PDEδ inhibitors that can effectively and selectively reduce PDEδ levels in cells.

The application of PDEδ PROTACs increased the gene expression of lipid metabolism-related enzymes mediated by sterol regulatory element-binding proteins (SREBPs), which was accompanied by elevated levels of cholesterol precursors. This finding provides the first evidence that PDEδ function plays a role in regulating enzymes of the mevalonate pathway.

Metabolic syndrome specifically refers to a complex group of metabolic disorders, and the number of patients with metabolic diseases is increasing globally each year, creating an urgent need for new drugs and technologies.

Currently, there are dozens of PROTACs in clinical research worldwide, addressing a growing variety of disease types, including cancer, autoimmune diseases, and metabolic disorders. As a technology, PROTACs have made significant progress in targeting metabolic disease-related pathways, providing new options for an increasing number of patients.

PROTAC Design for Target Proteins

References:

1. Lebwohl D, Kay A, Berg W,Baladi JF and Zheng J. Progression-free survival: gaining on overall survivalas a gold standard and accelerating drug development. Cancer J 2009; 15: 386-394
2. Choi JH, Ahn MJ, Rhim HC, KimJW, Lee GH, Lee YY and Kim IS. Comparison of WHO and RECIST criteria forresponse in metastatic colorectal carcinoma. Cancer Res Treat 2005; 37: 290-293
3. McLeod C, Norman R, Litton E, Saville BR, Webb S and Snelling TL. Choosing primary endpoints for clinical trials of health care interventions. Contemp Clin Trials Commun 2019; 16: 100486
4. Anagnostou V, Yarchoan M, Hansen AR, Wang H, Verde F, Sharon E, Collyar D, Chow LQM and Forde PM. Immuno-oncology trial endpoints: capturing clinically meaningful activity. Clin Cancer Res 2017; 23: 4959-4969
5. Gyawali B, Hey SP and Kesselheim AS. Evaluating the evidence behind the surrogate measures included in the FDA's table of surrogate endpoints as supporting approval of cancer drugs. EClinicalMedicine 2020; 21: 100332.
6. Barile JP, Reeve BB, Smith AW,Zack MM, Mitchell SA, Kobau R, Cella DF, Luncheon C and Thompson WW. Monitoringpopulation health for healthy people 2020: evaluation of the NIH PROMIS(R)global health, CDC healthy days, and satisfaction with life instruments. QualLife Res 2013; 22: 1201-1211.