Rho-mediated signaling promotes BRAF inhibitor resistance in de-differentiated melanoma cells.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that ~50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anticancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

Design and synthesis of seco-oxysterol analogs as potential inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene transcription.

The synthesis and biological activity of a series of seco-oxysterol analogs designed to be inhibitors of transcription of the gene for 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR) are described. The compound possessing the most significant activity, [1 alpha (E),4 beta]-3-[2-(4- hydroxy-1-methylcyclohexyl)ethenyl]-alpha,alpha-dimethylbenzenepentan ol (4, U-88156), inhibited (IC50 = 10 microM) the expression of beta-galactosidase (beta-gal) in a transfected human HepG2 cell line wherein the beta-gal gene was driven by a 5 kB segment of the promoter for hamster HMGR. Furthermore, using wild-type HepG2 cells, it was shown that 10 microM 4 reduced HMGR mRNA levels by 73% while stimulating LDL-receptor activity by 47%. In the same system, the related oxysterol, 25-hydroxycholesterol (1), at 10 microM lowered both HMGR mRNA levels and LDL-receptor activity by 58% and 64%, respectively. Overall HMGR activity in wild-type HepG2 cells was inhibited 30% by 4 at 10 microM. These findings collectively demonstrate that a seco-oxysterol analog is capable of regulating HMGR gene expression and that this regulation can occur without a concomitant attenuation of the level of LDL-receptor activity.

Synthesis and hypocholesterolemic activity of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-diones, novel inhibitors of acylCoA:cholesterol O-acyltransferase.

A novel series of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione inhibitors of the enzyme acyl-CoA:cholesterol O-acyltransferase is described. A number of these derivatives were found to be potent modulators of serum lipoprotein levels in cholesterol-fed rats. Further evaluation of one of the most effective analogues confirmed that it was significantly blocking the absorption of cholesterol from the gut.

Synthesis and biological activity of aminoguanidine and diaminoguanidine analogues of the antidiabetic/antiobesity agent 3-guanidinopropionic acid.

3-Guanidinopropionic acid (1) has been demonstrated both to improve insulin sensitivity and to promote weight loss selectively from adipose tissue in animal models of non-insulin-dependent diabetes mellitus (NIDDM). However, 1 has also been shown to be a substrate for both the creatine transporter and creatine kinase, leading to marked accumulation in muscle tissue as the corresponding N-phosphate. The corresponding aminoguanidine analogue 2 was recently discovered to retain the antidiabetic activity of 1 while being markedly less susceptible to creatine-like metabolism, suggesting that it should have less potential to accumulate in muscle. Further structural modification of 2 was undertaken to investigate whether the antidiabetic potency could be augmented while maintaining resistance to creatine-like metabolism. Modifications such as alpha-alkylation, homologation, and bioisosteric replacement of the aminoguanidine all were detrimental to antidiabetic activity. However, the simple regioisomeric aminoguanidinoacetic acid 9 and diaminoguanidinoacetic acid analogue 7 were found to be equipotent to 2, leading eventually to the discovery of the significantly more potent diaminoguanidinoacetic acid regioisomers 52 and 53. Further attempts to modify the more active template represented by 52 led only to reductions in antidiabetic activity. Each of the new active analogues displayed the same resistance to creatine-like metabolism as 2. Further testing of 7, 9, and 53 in obese diabetic ob/ob mice confirmed that weight loss is induced selectively from adipose tissue, similar to the lead 1. Administration of 53 to insulin-resistant rhesus monkeys led to reductions in both fasting and post-prandial plasma glucose levels with concomitant reductions in plasma insulin levels, suggesting that the compound improved the action of endogenous insulin. Compounds 7 and 53 were selected for further preclinical development.

Synthesis and biological activity of analogues of the antidiabetic/antiobesity agent 3-guanidinopropionic acid: discovery of a novel aminoguanidinoacetic acid antidiabetic agent.

3-Guanidinopropionic acid (1, PNU-10483) has been demonstrated to both improve insulin sensitivity and to promote weight loss selectively from adipose tissue in animal models of non-insulin-dependent diabetes mellitus (NIDDM). However, 1 has also been shown to be a substrate for both the creatine transporter and creatine kinase, leading to marked accumulation in muscle tissue as the corresponding N-phosphate 4. In an effort to identify novel entities that maintain antidiabetic potency without susceptibility to creatine-like metabolism, an analogue program was undertaken to explore the effects of various structural modifications, including homologation, simple substitution, single atom mutations, and bioisosteric replacements for the guanidine and carboxylic acid. Overall, the scope of activity encompassed by the set of new analogues proved to be exceedingly narrow. Notable exceptions demonstrating equivalent or improved antidiabetic activity included the alpha-amino derivative 29, aminopyridine 47, isothiourea 67, and aminoguanidine 69. On the basis of its superior therapeutic ratio, aminoguanidine 69 was selected for preclinical development and became the foundation for a second phase of analogue work. Furthermore, in vitro studies demonstrated that 69 is markedly less susceptible to phosphorylation by creatine kinase than the lead 1, suggesting that it should have less potential for accumulation in muscle tissue than 1.

Traceless solid-phase synthesis of 1,2,4-triazoles using a novel amine resin.

[reaction: see text]. A solid-phase route to 5-aryl-3-arylthiomethyl-1,2,4-triazoles has been developed that permits the incorporation of two elements of diversity. The heterocycle was constructed upon a novel 4-benzyloxy-2-methoxybenzylamine (BOMBA) resin, from which traceless cleavage could be effected with dilute TFA. A library of 96 triazoles was produced with an average yield of 26% (84% yield per step) and an average purity of 80%. In a direct comparison, the new BOMBA resin proved to be markedly superior to Rink Amide resin for this application.

Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action.

Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.