Jean-Marie Brusq

Discovery and Characterization of Small Molecule Inhibitors of the Bet Family Bromodomains.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.

Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine

The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-β-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.

Discovery of Epigenetic Regulator I-Bet762: Lead Optimization to Afford a Clinical Candidate Inhibitor of the Bet Bromodomains.

The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure-activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.

Inhibition of TGF-β signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis

1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF‐β), a well‐known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI‐1 and collagens. The identification of GW6604 (2‐phenyl‐4‐(3‐pyridin‐2‐yl‐1H‐pyrazol‐4‐yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF‐β pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF‐β signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF‐β‐overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)‐induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC50 of 140 nM and in a cellular assay inhibited TGF‐β‐induced transcription of PAI‐1 (IC50: 500 nM). In vivo, GW6604 (40 mg kg−1 p.o.) increased liver regeneration in TGF‐β‐overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN‐induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg−1 p.o., b.i.d.), mortality was prevented and DMN‐induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP‐1 and TGF‐β were reduced by 50–75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.

From ApoA1 upregulation to BET family bromodomain inhibition: discovery of I-BET151.

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.

The Discovery of I-Bet726 (Gsk1324726A), a Potent Tetrahydroquinoline Apoa1 Up-Regulator and Selective Bet Bromodomain Inhibitor.

Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.

Effects of C-type natriuretic peptide on rat cardiac contractility

Natriuretic peptide receptors have been found in different heart preparations. However, the role of natriuretic peptides in the regulation of cardiac contractility remains largely elusive and was, therefore, studied here. The rate of relaxation of electrically stimulated, isolated rat papillary muscles was enhanced (114.4±1.4%, P<0.01) after addition of C‐type natriuretic peptide (CNP; 1 μM). Time to peak tension decreased in parallel (88±3 and 75±2 msec before and 5 min after addition of CNP, respectively, P<0.01). On the other hand, the rate of contraction slowly decreased when CNP was added to the papillary muscles. These results show that CNP displays a positive lusitropic effect associated with a negative inotropic effect. The effects of CNP were mimicked by 8‐bromo‐guanosine 3′,5′ cyclic monophosphate. Addition of CNP to isolated adult rat cardiomyocytes, induced a 25 fold increase in guanosine 3′,5′ cyclic monophosphate (cGMP) levels and stimulated the phosphorylation of phospholamban and troponin I, two proteins involved in the regulation of cardiac contractility. The levels of adenosine 3′,5′ cyclic monophosphate (cAMP) were not affected by the addition of CNP to the myocytes. The CNP‐dependent phospholamban phosphorylation was accompanied by the activation of the sarcoplasmic reticulum Ca2+‐ATPase. In summary, CNP exerts a positive lusitropic effect, in rat papillary muscles. The putative mechanism involved in the lusitropism induced by this peptide, a cGMP‐dependent enhancement of the rate of relaxation with a slowly developing negative inotropic effect, seems different to that described for catecholamines.

Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

Pharmacological inhibition of stearoyl-CoA desaturase 1 improves insulin sensitivity in insulin-resistant rat models.

Stearoyl-CoA Desaturase 1 (SCD1) is a central enzyme that catalyzes the biosynthesis of monounsaturated fatty acids from saturated fatty acids. SCD1 is an emerging target in obesity and insulin resistance due to the improved metabolic profile obtained when the enzyme is genetically inactivated. Here, we have investigated if the pharmacological inhibition of SCD1 could elicit the same profile. We have identified a small molecule, GSK993 and characterized it as a potent and orally available SCD1 inhibitor. In Zucker(fa/fa) rats, GSK993 exerted a marked reduction in hepatic lipids as well as a significant improvement of glucose tolerance. Furthermore, in a diet-induced insulin resistant rat model, GSK993 induced a very strong reduction in Triton-induced hepatic Very Low Density Lipoprotein-Triglyceride production. In addition, following a hyperinsulinemic-euglycemic clamp in GSK993-treated animals, we observed an improvement in the whole body insulin sensitivity as reflected by an increase in the glucose infusion rate. Taken together, these findings demonstrate that the pharmacological inhibition of SCD1 translates into improved lipid and glucose metabolic profiles and raises the interest of SCD1 inhibitors as potential new drugs for the treatment of insulin resistance.

Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin

Background Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy. Methods and Findings The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin. Conclusions Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.