GM1 ( IC50 = 14 nM ); glucosylceramide (GlcCer) synthase; GL1 synthase; glucosylceramide synthase
Genz-123346 targets glucosylceramide synthase (GCS), also known as GL1 synthase, which is a key enzyme in the glycosphingolipid biosynthesis pathway. By inhibiting GCS, it blocks the conversion of ceramide to glucosylceramide, thereby reducing the production of downstream glycosphingolipids such as GM1, GM3, and Gb3. This inhibition has downstream effects on cellular signaling pathways, including the Akt-mTOR pathway, as evidenced by reduced phosphorylation of Akt and ribosomal protein S6 in treated cells. The compound also acts as a substrate for multi-drug resistance efflux pumps such as P-glycoprotein (P-gp, ABCB1).

Cells exposed to nontoxic concentrations of Genz-123346 and other GCS inhibitors can increase the ability of cytotoxic anti-cancer agents to kill tumor cells. Genz-123346 and a few other GCS inhibitors serve as substrates for efflux pumps that are resistant to multiple drugs, like P-gp (ABCB1, gP-170). The primary cause of Genz-123346's chemosensitization in cell lines chosen to overexpress P-gp or that express P-gp endogenously is its effects on P-gp function[2]. Genz-123346(Genz) is an autophagy flux enhancer[3].
In vitro, Genz-123346 inhibits GM1 ganglioside synthesis with an IC50 of 14 nM, demonstrating significantly higher potency compared to miglustat, which inhibits GCS with an IC50 of 32 µM. In cell lines engineered to overexpress P-glycoprotein (P-gp) or naturally expressing P-gp, the chemosensitization effects of Genz-123346 are primarily attributed to its impact on P-gp function. Exposure of cells to Genz-123346 and other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. Genz-123346 also promotes autophagy flux. It does not inhibit α-glucosidase or glucocerebrosidase and has little effect on 1-O-acylceramide synthase activity.

Genz-123346 decreased glucose and A1C levels and enhanced glucose tolerance in the Zucker diabetic fatty rat. Additionally, drug therapy maintained the animals' capacity to secrete insulin and stopped the loss of pancreatic beta-cell function. Treatment with Genz-123346 normalized A1C levels and enhanced glucose tolerance in the diet-induced obese mouse. The medication has been demonstrated to have an oral bioavailability of 10% in mice and 30% in rats, with a half-life in plasma of 30 to 60 minutes[1]. Genz-123346 treatment reduces renal GlcCer and GM3 levels in a dose-dependent manner, effectively inhibiting cystic disease. A direct effect of Genz-123346 on the Akt-mTOR signaling pathway is observed, with reduced phosphorylation of Akt and ribosomal protein S6[4].
In vivo, oral administration of Genz-123346 in Zucker diabetic fatty rats significantly reduced glucose and HbA1c levels while enhancing glucose tolerance. Drug treatment also preserved pancreatic beta-cell function and maintained insulin secretion capacity. In diet-induced obese mice, Genz-123346 normalized HbA1c levels and improved glucose tolerance. In WT mice treated with Genz-123346 (0.11% final concentration in regular chow) for two weeks, renal Gb3 levels were reduced by approximately 50% compared to control mice. The compound also produced a dose-dependent reduction of renal GlcCer and GM3 levels, translating into effective inhibition of cystic disease in mouse models of polycystic kidney disease.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.[2]
The in vitro enzymatic activity of Genz-123346 is assessed using cell-free glucosylceramide synthase assays. Recombinant or purified GCS enzyme is incubated with its substrate, ceramide, and UDP-glucose in the presence of varying concentrations of the inhibitor. The production of glucosylceramide (GL1) or downstream ganglioside GM1 is measured using chromatographic or radiometric methods. The IC50 value of 14 nM for GM1 inhibition is determined from dose-response curves. Additional selectivity profiling against other glycosidases such as α-glucosidase, glucocerebrosidase, and 1-O-acylceramide synthase can be performed using similar enzyme activity assays.

Inhibition of glucosylceramide synthase stimulates autophagy flux in neurons[3]
In this study, researchers identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol and Genz-123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT-mammalian target of rapamycin (mTOR) signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on-target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant α-synuclein in neurons, it does so, according to our data, through autophagy-independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease. Inhibition of GlcCer synthase enhances autophagy by inhibiting AKT-mTOR signaling, and increases the number and size of lysosomal/late endosomal structures. Furthermore, inhibition of GlcCer synthase decreased levels of mutant α-synuclein in neurons, which may represent a potential therapeutic target for Parkinson's disease.
To evaluate the cellular effects of Genz-123346, various cell lines are treated with the compound at concentrations typically ranging from nanomolar to low micromolar levels. The inhibition of glycosphingolipid synthesis is measured by quantifying the levels of GL1, GM1, GM3, or Gb3 using LC-MS/MS or thin-layer chromatography. The effects on cell viability, proliferation, and apoptosis are assessed using standard assays such as MTT, Annexin V/PI staining, or caspase activity assays. The modulation of signaling pathways, particularly the Akt-mTOR pathway, is evaluated by Western blotting using phospho-specific antibodies against Akt and ribosomal protein S6. Autophagy flux can be assessed by measuring LC3-II accumulation and p62 degradation.

Rats: In water, Genz-123346 dissolves. After receiving Genz-123346 (75 mg/kg) for six weeks, Zucker diabetic fatty rats are fasted for the entire night. The fasted rats are put under anesthesia and given five human insulin shots into their hepatic portal veins the next morning. Two minutes after injection, the liver and quadriceps muscle are removed and instantly frozen in liquid nitrogen. The immunoprecipitated insulin receptor By using immunoblotting, the immunoprecipitates are examined[1].
Mice: For eight weeks, C57BL/6 mice are given a high-fat (45% of kcal) diet. Obese mice with similar body weight gain, insulin, and glucose levels are placed in the treated or control groups. After that, the mice are given water or Genz-123346 every day for ten weeks[1].

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Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.[4]
In vivo studies with Genz-123346 typically involve oral administration to animal models, including Zucker diabetic fatty rats, diet-induced obese mice, and mouse models of polycystic kidney disease. The compound is often administered via oral gavage or mixed into chow at concentrations such as 0.11% final concentration. Treatment duration may range from several days to several weeks. Efficacy endpoints include measurements of blood glucose and HbA1c levels, glucose tolerance tests, and quantification of renal glycosphingolipid levels. In polycystic kidney disease models, kidney weight, cyst volume, and histopathology are assessed. Pharmacodynamic markers such as Akt and S6 phosphorylation are measured in target tissues.

The oral bioavailability of Genz-123346 is approximately 10% in mice and 30% in rats. The plasma half-life is 30 to 60 minutes. The compound is soluble in DMSO (50 mg/mL) and can be formulated for in vivo administration using vehicles such as 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. Powder formulations should be stored at -20°C for up to 3 years, and stock solutions can be stored at -80°C for up to 1 year. The molecular weight of Genz-123346 free base is 418.57 g/mol, with a molecular formula of C24H38N2O4.

Exposure of cells to Genz-123346 at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. The compound has been evaluated for potential toxicity in cell-based assays, where it shows cytotoxic effects only at higher concentrations. According to safety data sheets, Genz-123346 may cause skin irritation and serious damage to eyes, and thermal decomposition may produce toxic gases such as carbon monoxide, carbon dioxide, and nitrogen oxides. It should be handled with appropriate personal protective equipment and standard laboratory safety precautions.

[1]. Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes. Diabetes. 2007 May;56(5):1210-8.

[2]. The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function. Int J Oncol. 2011 Mar;38(3):701-11.

[3]. Inhibition of glucosylceramide synthase stimulates autophagy flux in neurons. J Neurochem. 2014 Jun;129(5):884-94

[4]. Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models. Nat Med. 2010 Jul;16(7):788-92.

Previous studies have shown that glycosphingolipids can regulate insulin receptor activity, and transgenic mouse studies have also suggested a link between changes in the levels of various gangliosides and the development of insulin resistance. This study demonstrates that glycosphingolipid synthesis inhibitors can improve glycemic control and enhance insulin sensitivity in two different diabetic animal models. In the Zucker diabetic obese rat model, the glucosylceramide synthase inhibitor (1R,2R)-nonanoic acid [2-(2',3'-dihydrobenzo[1,4]dioxin-6'-yl)-2-hydroxy-1-pyrrolidine-1-ylmethylethyl]-amide-1-tartrate (Genz-123346) reduced blood glucose and glycated hemoglobin (A1C) levels and improved glucose tolerance. This treatment also prevented the loss of pancreatic β-cell function commonly seen in Zucker diabetic obese rats and maintained the animals' insulin secretion capacity. In diet-induced obese mice, Genz-123346 treatment normalized A1C levels and improved glucose tolerance. Phosphorylation state analysis of the insulin receptor and its downstream effector molecules showed enhanced insulin signaling in the muscles of treated Zucker diabetic obese rats and diet-induced obese mice. These results suggest that inhibiting glycosphingolipid synthesis can significantly improve insulin sensitivity and glucose homeostasis, and therefore may represent a new approach to treating type 2 diabetes. [3]
Genz-123346 is a research compound that has been used to study the role of glycosphingolipids in insulin resistance, type 2 diabetes, and polycystic kidney disease. It has demonstrated efficacy in improving glycemic control and preserving beta-cell function in diabetic animal models. The compound's ability to inhibit GCS and reduce glycosphingolipid accumulation has also been explored in the context of neurological disorders. Genz-123346 is not a clinically approved drug and is intended for research use only.

C24H38N2O4

418.5695

418.283

C, 68.87; H, 9.15; N, 6.69; O, 15.29

491833-30-8

Genz-123346; 943344-58-9

23652732

White to off-white solid powder

1.1±0.1 g/cm3

623.9±55.0 °C at 760 mmHg

331.1±31.5 °C

0.0±1.9 mmHg at 25°C

1.539

4.14

2

5

12

30

498

2

O([H])[C@]([H])(C1C([H])=C([H])C2=C(C=1[H])OC([H])([H])C([H])([H])O2)[C@@]([H])(C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O

JMNXWOFCUJJYEO-HYBUGGRVSA-N

InChI=1S/C24H38N2O4/c1-2-3-4-5-6-7-10-23(27)25-20(18-26-13-8-9-14-26)24(28)19-11-12-21-22(17-19)30-16-15-29-21/h11-12,17,20,24,28H,2-10,13-16,18H2,1H3,(H,25,27)/t20-,24-/m1/s1

N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]nonanamide

Genz123346; Genz-123346; Genz-123346; 491833-30-8; Genz-123346 free base; Genz-123346 (free base); 8JW4ZYR2CT; N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)nonanamide; N-[(1R,2R)-1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl]nonanamide; Genz123346;Genz 123346

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~84 mg/mL (~200.7 mM)
Ethanol: ~84 mg/mL

Solubility in Formulation 1: ≥ 3 mg/mL (7.17 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (7.17 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3 mg/mL (7.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)