A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

Abstract

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

Introduction

Cardiovascular diseases are a common cause of mortality being responsible for an estimated 17.8 million deaths in 2017¹. The cost of treating heart disease and stroke is expected to be greater than one trillion dollars by the year 2035².

Familial hypercholesterolemia (FH) is an inherited disease that increases the risk of developing cardiovascular disease and is characterized by elevated plasma low-density lipoprotein-cholesterol (LDL-C) levels. The prevalence of heterozygous FH (HeFH) is between 1/200 and 1/500 people and of homozygous FH (hoFH) between 1/160,000 and 1/300,000³. FH is primarily caused by mutations in the low-density lipoprotein receptor (LDLR). The LDLR is an integral membrane protein that binds LDL particles from the circulation and mediates endocytosis^4,5. LDL-cholesterol is cleared from the serum predominantly through LDLR uptake and catabolism by hepatocytes.

Statins are the primary treatment for hypercholesterolemia. They act by inhibiting HMG-CoA reductase, reducing cholesterol synthesis, and increasing LDLR expression in hepatocytes which leads to enhanced LDL-C clearance⁶. Although statins are effective in most populations, more than 20% of individuals show an inadequate response to statin treatment^7,8,9, and two thirds of coronary artery disease patients fail to maintain cholesterol goals when using a statin alone¹⁰. Unfortunately, because statins act primarily by increasing expression of the LDLR, most hoFH patients are resistant to statin treatment. Recently, two antibodies to PCSK9, alirocumab and evolocumab, have been approved by the FDA for treatment of hypercholesterolemia. Like statins these treatments increase the steady-state level of the LDLR, promoting hepatic uptake of LDL¹¹. Unfortunately, the use of PCSK9 inhibitors is expensive, limiting their widespread use and, depending on the specific mutations, can be ineffective against hoFH¹¹.

Alternative drugs have recently become available to treat hoFH by controlling the production of LDL and its precursor VLDL by the hepatocytes. Lomitapide acts by inhibiting microsomal triglyceride transfer protein (MTP, MTTP). Inhibition prevents co-translational loading of triglyceride onto Apolipoprotein B (apoB), which is the central protein component of VLDL, and results in its proteolytic degradation⁶. Mipomersen is an antisense oligonucleotide that specifically binds to APOB mRNA to block protein translation¹². Both Lomitapide and Mipomersen showed a substantial reduction of serum LDL and were approved to treat hoFH patients. Despite their effectiveness in lowering VLDL production, both treatments can increase hepatic fat and transaminase and can cause severe liver damage¹³. Therefore, there is a need to develop pharmaceuticals to lower VLDL in a broad population of patients including those with hoFH.

Attempts to develop small molecules to treat metabolic liver diseases have been hampered by the lack of an efficacious and reproducible in vitro model. Hepatoma cells do not accurately recapitulate hepatic disease phenotypes due to their tumorigenic origin¹⁴. Although primary hepatocytes show promising physiological characteristics, their limited availability, lot-to-lot variability, and difficulty in maintaining phenotypic function under simple culture conditions, reduce their suitability for high throughput screening¹⁵. Human iPSC-derived hepatocyte-like cells (iPSC-hepatocytes) have rapidly transformed the study of liver development and disease^16,17. The capacity to produce large numbers of human hepatocyte-like cells derived from patients, allows investigators to perform high-throughput drug screens to discover therapies for monogenic liver diseases^18,19,20.

In an effort to identify compounds for the potential treatment of hypercholesterolemia, we performed a high throughput screen using human iPSC-hepatocytes. We screened the South Carolina Compound Collection (SC³), a proprietary small molecule library that contains 130,000 fully annotated drug-like molecules. Compounds that reduced apoB in the culture medium were further tested in hoFH patient-derived iPSC-hepatocytes to identify candidates that act independently of the LDLR pathway. In contrast to MTP inhibitors, lead compounds had no effect on intracellular lipid accumulation and did not impact steady-state intracellular apoB protein levels. The compounds were not only effective in cultured cells, but also lowered serum cholesterol, triglyceride, apoB and Lp(a) levels in liver-humanized mice. Through this study we have identified a class of molecules that have potential to provide a treatment for patients with FH or primary hypercholesterolemia that is unresponsive to conventional therapeutics.

Results

Small molecule screen to identify reduced apoB production by human iPSC-derived hepatocyte-like cells

To identify compounds that reduce apoB-containing lipoproteins such as VLDL by hepatocytes, we used the stepwise differentiation of human iPSCs to hepatocyte-like cells (iPSC-hepatocytes)^21,22. iPSC-hepatocytes recapitulate many aspects of human liver function, including the robust and stable secretion of VLDL^19,23. ApoB accumulation in the culture medium, provides a reliable biomarker for the identification of compounds with the potential to reduce the production of VLDL by the liver because apoB is the central protein component of all VLDL particles²⁴. We have previously validated an Enzyme-linked immunosorbent assays (ELISA)-based approach for identifying compounds that reduce secreted apoB (Z′ = 0.73)²⁵. Therefore, we measured the level of apoB in the culture medium as the primary assay.

Human iPSCs were differentiated to hepatocyte-like cells in a 96-well monolayer format. The level of apoB was measured by ELISA before and after treatment with 10,000 small molecules from the SC³ representative set (Fig. 1a). The SC³ library contains ~130,000 proprietary drug-like molecules with an average MW of 400 g/mol. The initial screening dose was 2 µg/ml (average 5 µM), which is within the range of the established acceptable dose of a high throughput screen²⁶. The post-drug: pre-drug ratio of apoB in the culture medium was determined for each compound and normalized with the DMSO-treated group (Fig. 1b). The identification of hits was then determined by z-score analysis (Fig. 1c)²⁷. Small molecules with a z score of ≤–2.75 were identified as primary hits. Using these parameters, we identified 46 compounds (SC3-01 through SC3-46) with an apoB ratio ranging from 0.82 to 0.2…