Novel 5- and 6-subtituted benzothiazoles with improved physicochemical properties: potent S1P1 agonists with in vivo lymphocyte-depleting activity.

An SAR campaign designed to increase polarity in the 'tail' region of benzothiazole 1 resulted in two series of structurally novel 5-and 6-substituted S1P(1) agonists. Structural optimization for potency ultimately delivered carboxamide (+)-11f, which in addition to possessing improved physicochemical properties relative to starting benzothiazole 1, also displayed good S1P(3) selectivity and acceptable in vivo lymphocyte-depleting activity.

Benzofuran Derivatives as Potent, Orally Active S1P1 Receptor Agonists: A Preclinical Lead Molecule for MS.

We have discovered novel benzofuran-based S1P1 agonists with excellent in vitro potency and selectivity. 1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl) azetidine-3-carboxylic acid (18) is a potent S1P1 agonist with >1000× selectivity over S1P3. It demonstrated a good in vitro ADME profile and excellent oral bioavailability across species. Dosed orally at 0.3 mg/kg, 18 significantly reduced blood lymphocyte counts 24 h postdose and demonstrated efficacy in a mouse EAE model of relapsing MS.

Discovery of a Potent, S1P3-Sparing Benzothiazole Agonist of Sphingosine-1-Phosphate Receptor 1 (S1P1).

Optimization of a benzofuranyl S1P1 agonist lead compound (3) led to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl)benzyl)azetidine-3-carboxylic acid (14), a potent S1P1 agonist with minimal activity at S1P3. Dosed orally at 0.3 mg/kg, 14 significantly reduced blood lymphocyte counts 24 h postdose and attenuated a delayed type hypersensitivity (DTH) response to antigen challenge.

Discovery of AMG 369, a Thiazolo[5,4-b]pyridine Agonist of S1P1 and S1P5.

The optimization of a series of thiazolopyridine S1P1 agonists with limited activity at the S1P3 receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and reduce the severity of experimental autoimmune encephalomyelitis in rat.

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis.

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.

Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155 -> Ala.

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 --> Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 --> Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.

Glycolysis and glucose transporter 1 as markers of response to hormonal therapy in breast cancer.

Estrogen plays a key role in the development and progression of breast cancer; hence, antiestrogens, such as tamoxifen, have a marked impact on the treatment and outcome of breast cancer patients. Estrogen-induced growth requires continuous replenishment of energy, predominantly generated by glycolysis. Previous work from this laboratory demonstrated estrogen induction and tamoxifen inhibition of glycolysis in MCF7 human breast cancer cells in vitro (Furman et al., J Steroid Biochem Mol Biol 1992;43:189-95). We present here studies of estrogen vs. tamoxifen regulation of glycolysis in orthotopic MCF7 human breast cancer xenografts in vivo. In addition we investigated mediation of this metabolic regulation through glucose transporter 1, in the same cells, in vitro, as well as in 2 other hormone-responsive human breast cancer cells. Tumor response and glycolysis were monitored noninvasively by means of magnetic resonance imaging and 13C spectroscopy, respectively. During estrogen-stimulated tumor growth (from approximately 0.5 to approximately 1.3 cm3 in 10 days), the rate of glucose metabolism through glycolysis in vivo was high at 40 +/- 4 micromole/g/min. However, treatment for 10 days with tamoxifen induced growth arrest and a concomitant decrease of 2-fold in the rate of glycolysis. In congruence, glucose transporter 1 expression was stimulated by estrogen, reaching after 72 hr a 2- to 3-fold higher level of expression relative to that in tamoxifen-treated cells. Thus, estrogen-induced changes in glycolysis appeared to be mediated via its regulation of glucose transporter 1 expression. The in vivo monitoring of glycolysis may serve as a tool to expose hormonal regulation of glucose transporter 1 expression in breast cancer tumors, as well as to assess response to hormonal therapy.

Glycolysis as a metabolic marker in orthotopic breast cancer, monitored by in vivo (13)C MRS.

Enhanced glycolysis represents a striking feature of cancers and can therefore serve to indicate a malignant transformation. We have developed a noninvasive, quantitative method to characterize tumor glycolysis by monitoring (13)C-labeled glucose and lactate with magnetic resonance spectroscopy. This method was applied in MCF7 human breast cancer implanted in the mammary gland of female CD1-NU mice and was further employed to assess tumor response to hormonal manipulation with the antiestrogen tamoxifen. Analysis of the kinetic data based on a unique physiological-metabolic model yielded the rate parameters of glycolysis, glucose perfusion, and lactate clearance in the tumor, as well as glucose pharmacokinetics in the plasma. Treatment with tamoxifen induced a twofold reduction in the rate of glycolysis and of lactate clearance but did not affect the other parameters. This metabolic monitoring can thus serve to evaluate the efficacy of new selective estrogen receptor modulators and may be further extended to improve diagnosis and prognosis of breast cancer.

Metabolic markers of breast cancer: enhanced choline metabolism and reduced choline-ether-phospholipid synthesis.

Specific genetic alterations during malignant transformation may induce the synthesis and breakdown of choline phospholipids, mediating transduction of mitogenic signals. The high level of water-soluble choline metabolites in cancerous breast tumors, relative to benign lesions and normal breast tissue, has been used as a diagnostic marker of malignancy. To unravel the biochemical pathways underlying this phenomenon, we used tracer kinetics and 13C and 31P magnetic resonance spectroscopy to compare choline transport, routing, and metabolism to phospholipids in primary cultures of human mammary epithelial cells and in MCF7 human breast cancer cells. The rate of choline transport under physiological choline concentrations was 2-fold higher in the cancer cells. The phosphorylation of choline to phosphocholine and oxidation of choline to betaine yielded 10-fold higher levels of these metabolites in the cancer cells. However, additional incorporation of choline to phosphatidylcholine was similar in both cell types. Thus, enhanced choline transport and augmented synthesis of phosphocholine and betaine are dominant pathways responsible for the elevated presence of choline metabolites in cancerous breast tumors. Uniquely, reduced levels and synthesis of a choline-ether-phospholipid may also serve as a metabolic marker of breast cancer.