Project description:High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl) amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the 4 separate diastereomers identified the (S,S)-diastereomer (IDH125) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial SAR exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physico-chemical properties identified (S)-3-(2-(((S)-1-(5-(4-fluoro-3-methylphenyl)pyrimidin-2-yl)ethyl)amino)pyrimidin-4-yl)-4-isopropyloxazolidin-2-one (IDH889) with good in-vivo exposure and in-vivo activity in a mutant IDH1 xenograft mouse model. identified the (S,S)-diastereomer (IDH125) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial SAR exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physico-chemical properties identified (S)-3-(2-(((S)-1-(5-(4-fluoro-3-methylphenyl)pyrimidin-2-yl)ethyl)amino)pyrimidin-4-yl)-4-isopropyloxazolidin-2-one (IDH889) with good in-vivo exposure and in-vivo activity in a mutant IDH1 xenograft mouse model
Project description:Therapeutic aryl hydrocarbon receptor (AHR) modulating agents (TAMAs) gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR’s known ligand promiscuity, we generated novel TAMAs by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. We questioned whether SGA derivatives can restore the disturbed epidermal differentiation processes that are known for the key AD-associated T helper-2 cytokine, interleukin-4 (IL-4). We performed genome-wide transcriptomic analysis by bulk RNA-sequencing after co-stimulation of human epidermal equivalents with interleukin-4 (IL-4; AD-HEE) and SGA360 (SAhRM activity), SGA360f or SGA388 (coupling of SAhRM to agonist activity), or TCDD (full AHR agonist).
Project description:Cardiovascular diseases (CVD) are the leading cause of death among elderly people. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism. Herein, we investigated the role of PCSK9 in age-related CVD. Both in humans and rats, sPCSK9 correlated positively with increasing age and the development of cardiovascular dysfunction. Network analysis identified PCSK9 as an important factor in age-associated lipid alterations and it correlated positively with intima media thickness, a clinical parameter of CVD risk. PCSK9 inhibition with alirocumab effectively reduced the CVD progression in aging rats suggesting that PCSK9 plays an important role in cardiovascular aging.
Project description:Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived AQ3 from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (−38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (−89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19±0.77-fold in HLC-S127R compared to 1.38±0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.
Project description:The aim of the study was to characterize the role of PCSK9 in human beta cells. We performed siRNA-mediated knockdown of PCSK9 in human beta cell line EndoC-bH1 and compared the expression profiles against control siRNA-treated cells.
Project description:To explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion
Project description:Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of LDL cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocyte. Here, we show that PCSK9 is highly expressed in undifferentiated hiPSCs. PCSK9 inhibition in hiPSCs with the use of shRNA, CRISPR/cas9-mediated knockout or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. Indeed, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in the TGFß-R1 lysosomal degradation. Using different PCSK9 constructs we show that PCSK9 interacts with DACT2 through its CHRD domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development, beyond its canonical effect on lipid metabolism.