Discovery of potent and liver-targeted stearoyl-CoA desaturase (SCD) inhibitors in a bispyrrolidine series

Inhibition of stearoyl-CoA desaturase (SCD) activity represents a potential novel mechanism for the treatment of metabolic disorders including obesity and type II diabetes. To circumvent skin and eye adverse events observed in rodents with systemically-distributed SCD inhibitors, our research efforts have been focused on the search for new and structurally diverse liver-targeted SCD inhibitors. This work has led to the discovery of novel, potent and structurally diverse liver-targeted bispyrrolidine SCD inhibitors. These compounds possess suitable cellular activity and pharmacokinetic properties to inhibit liver SCD activity in a mouse pharmacodynamic model.     

2-Aryl benzimidazoles: human SCD1-specific stearoyl coenzyme-A desaturase inhibitors

A series of potent, benzimidazole-based SCD inhibitors which demonstrate selectivity for the hSCD1 enzyme over the hSCD5 isoform are described. The compounds possess suitable cellular activity and pharmacokinetic properties which render them capable of inhibiting liver SCD activity in a mouse pharmacodynamic assay. These 2-aryl benzimidazoles may serve as valuable tools for studying selective hSCD1-inhibition.     

Plasma-based approach to measure target engagement for liver-targeting stearoyl-CoA desaturase 1 inhibitors

A positive correlation between stearoyl-CoA desaturase (SCD)1 expression and metabolic diseases has been reported in rodents and humans. These findings indicate that SCD1 is a promising therapeutic target for the chronic treatment of diabetes and dyslipidemia. The SCD1 enzyme is expressed at high levels in several human tissues and is required for the biosynthesis of monounsaturated fatty acids, which are involved in many biological processes. Liver-targeted SCD inhibitors were designed to pharmacologically manipulate SCD1 activity in the liver to avoid adverse events due to systemic inhibition. This article describes the development of a plasma-based SCD assay to assess the level of SCD inhibition, which is defined in this article as target engagement. Essentially, animals are dosed with an exogenous deuterated tracer (d7-stearic acid) as substrate, and the converted d7-oleic acid product is measured to monitor SCD1 inhibition. This study reveals that this plasma-based assay correlates with liver SCD1 inhibition and can thus have clinical utility.     

Discovery of 1-(4-phenoxypiperidin-1-yl)-2-arylaminoethanone stearoyl-CoA desaturase 1 inhibitors

A series of novel stearoyl-CoA desaturase 1 (SCD1) inhibitors were identified by scaffold design based on known SCD1 inhibitors. Large structural changes were made leading to multiple analogs with comparable or improved potency. This approach is valuable for generation of proprietary compounds without conducting a costly high-throughput screening.     

Discovery of piperidine-aryl urea-based stearoyl-CoA desaturase 1 inhibitors

A series of structurally novel stearoyl-CoA desaturase1 (SCD1) inhibitors has been identified via molecular scaffold manipulation. Preliminary structure–activity relationship (SAR) studies led to the discovery of potent, and orally bioavailable piperidine-aryl urea-based SCD1 inhibitors. 4-(2-Chlorophenoxy)-N-[3-(methyl carbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited robust in vivo activity with dose-dependent desaturation index lowering effects.     

Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.     

High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells

Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is ∼5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G₁/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis.     

A novel assay of cellular stearoyl-CoA desaturase activity of primary rat hepatocytes by HPLC

The stearoyl-CoA desaturase (SCD) activity is involved in regulation of metabolism, energy storage, and membrane fluidity. However, only few cellular assays have been developed. We describe a simple and robust method to quantitate SCD activity and its inhibition in primary rat hepatocytes. Hepatocytes assimilate stearic acid, with or without modification by SCD, into its lipid pool. To measure the extent of this conversion primary rat hepatocytes were cultivated 4 h or overnight with [1-¹⁴C]18:0 and extracellular fatty acids were washed out. Total cell lipids were then hydrolyzed and extracted. Recoveries of 18:0 were secured with a modified Folch method by addition of 0.1% Triton X-114 to the samples. The extracted fatty acids were dissolved in 85% ethanol and separated by reverse phase HPLC, which took 10 min including column recovery time. [1-¹⁴C]18:0 and [1-¹⁴C]18:1(n9) were detected and quantified by on-line flow scintillation analysis. Incubation of the cells with SCD inhibitors resulted in decreased ratios of 18:1/18:0 in dose-dependent manners. The improvements enabled us to establish a novel robust assay based solely on HPLC analysis of cellular SCD activity, which was developed in 12-well format.     

Conversion of systemically-distributed triazole-based stearoyl-CoA desaturase (SCD) uHTS hits into liver-targeted SCD inhibitors

It has been demonstrated that once-a-day dosing of systemically-distributed SCD inhibitors leads to adverse events in eye and skin. Herein, we describe our efforts to convert a novel class of systemically-distributed potent triazole-based uHTS hits into liver-targeted SCD inhibitors as a means to circumvent chronic toxicity.     

Bicyclic heteroaryl inhibitors of stearoyl-CoA desaturase: from systemic to liver-targeting inhibitors

Optimization of a lead thiazole amide MF-152 led to the identification of potent bicyclic heteroaryl SCD1 inhibitors with good mouse pharmacokinetic profiles. In a view to target the liver for efficacy and to avoid SCD1 inhibition in the skin and eyes where adverse effects were previously observed in rodents, representative systemically-distributed SCD1 inhibitors were converted into liver-targeting SCD1 inhibitors.     

Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives

Conjugated linoleic acid (CLA) has been reported to decrease stearoyl-CoA desaturase (SCD) activity by decreasing mRNA expression. This investigation was designed to determine whether structurally related compounds of CLA have a direct inhibitory effect on SCD activity. Trans-10,cis-12 CLA had strong inhibitory activity on SCD while cis-9,trans-11, and trans-9,trans-11 isomers had no effect. Trans-10 octadecenoate was not inhibitory, whereas cis-12 octadecenate was inhibitory, but not as effective as trans-10,cis-12 CLA. Of the oxygenated derivatives, 9-peroxy-cis/trans-10, trans-12 octadecadienoate was a more effective inhibitor than trans-10,cis-12 CLA, whereas 9-hydroxy-trans-10, cis-12 octadecadienoate was less effective. Interestingly, cis-11 octadecadienoate and cis-12 octadecen-10-ynoate were slightly inhibitory. However, trans-9 and trans-11 octadecenoates, and trans-9,cis-12 octadecadienoate were all inactive under test condition, as were linoleate, oleate, and arachidonate. Derivatives of CLA acid modified to alcohol, amide or chloride were all inactive. A cis-12 double bond appears to be a key structural feature for inhibiting SCD activity, especially when coupled with a trans-10 double, whereas a cis-11 double bond is less effective.     

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Identification and characterization of murine SCD4, a novel heart-specific stearoyl-CoA desaturase isoform regulated by leptin and dietary factors

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Thus far, three isoforms of SCD (SCD1, SCD2, and SCD3) have been identified and characterized. Regulation of the SCD1 isoform has been shown to be an important component of the metabolic actions of leptin in liver, but the effects of leptin on SCD isoforms in other tissues have not been investigated. We found that although the mRNA levels of SCD1 and SCD2 were not affected by leptin deficiency in the hearts of ob/ob mice, the SCD activity and levels of monounsaturated fatty acids were increased, implying the existence of another SCD isoform. This observation has led to the cDNA cloning and characterization of a fourth SCD isoform (SCD4) that is expressed exclusively in the heart. SCD4 encodes a 352-amino acid protein that shares 79% sequence identity with the SCD1, SCD2, and SCD3 isoforms. Liver X receptor alpha (LXR alpha) agonists and a high carbohydrate fat-free diet induced SCD4 expression, but unlike SCD1, SCD4 expression was not repressed by dietary polyunsaturated fatty acids. SCD4 mRNA levels were elevated 5-fold in the hearts of leptin-deficient ob/ob mice relative to wild type controls. Treatment of ob/ob mice with leptin decreased mRNA levels of SCD4, whereas levels of SCD1 and SCD2 were not affected. Furthermore, in the hearts of SCD1-deficient mice, SCD4 mRNA levels were induced 3-fold, whereas the levels of SCD2 were not altered. The current studies identify a novel heart-specific SCD isoform that demonstrates tissue-specific regulation by leptin and dietary factors.     

Novel inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase

Novel bisindolyl-cycloalkane indoles resulted from the reaction of aliphatic dialdehydes and indole. As bisindolyl-natural alkaloid compounds have recently been reported as inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase (PK), we tested our novel compounds as MRSA PK inhibitors and now report first inhibiting activities. We discuss structure–activity relationships of structurally varied compounds. Activity influencing substituents have been characterized and relations to antibacterial activities of the most active compounds have been proved.     

Biological activity and preclinical efficacy of azetidinyl pyridazines as potent systemically-distributed stearoyl-CoA desaturase inhibitors

Potent and orally bioavailable SCD inhibitors built on an azetidinyl pyridazine scaffold were identified. In a one-month gDIO mouse model of obesity, we demonstrated that there was no therapeutic index even at low doses; efficacy in preventing weight gain tracked closely with skin and eye adverse events. This was attributed to the local SCD inhibition in these tissues as a consequence of the broad tissue distribution observed in mice for this class of compounds. The search for new structural scaffolds which may display a different tissue distribution was initiated. In preparation for an HTS campaign, a radiolabeled azetidinyl pyridazine displaying low non-specific binding in the scintillation proximity assay was prepared.     

Substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles as Src kinase inhibitors

A series of substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles has been prepared as Src kinase inhibitors. Optimal activity is observed with compounds that have basic amines attached via the para position of the 7-phenyl ring, and a hydrogen atom at the C-6 position. The best compounds are low nanomolar inhibitors of Src kinase, and have potent activity against Src-transformed fibroblast cells.