Synthesis and biological activity of L-tyrosine-based PPARgamma agonists with reduced molecular weight.

A series of PPARgamma agonists were synthesized from L-tyrosine that incorporated low molecular weight N-substituents. The most potent analogue, pyrrole (4e), demonstrated a K(i) of 6.9nM and an EC(50) of 4.7nM in PPARgamma binding and functional assays, respectively. Pyrrole (4e), which is readily synthesized from L-tyrosine methyl ester in four steps, also demonstrated in vivo activity in a rodent model of Type 2 diabetes.

Identification of a series of PPAR gamma/delta dual agonists via solid-phase parallel synthesis.

We have developed a general solid-phase synthesis for identification of PPAR ligands. Synthesis of a 480-member library led to the identification of a potent PPAR gamma/delta dual agonist 23. Compound 23 showed good plasma exposure in rats and demonstrated antihyperglycemic and antihyperlipidemic efficacy in diabetic fatty Zucker rats.

Identification of a series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with activity on PPARalpha, PPARgamma, and PPARdelta.

A series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with dual agonist activity on PPARalpha and PPARgamma is described. Several of these compounds also showed partial agonist activity on PPARdelta. Resolution of one analogue showed that PPARalpha and PPARgamma activity resided in mainly one enantiomer, whereas PPARdelta activity was retained in both enantiomers.

A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the alpha (NR1C1) and gamma (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the delta (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARdelta agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARdelta agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.

Potent dipeptide inhibitors of the pp60c-src SH2 domain.

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Peptide ligands of pp60(c-src) SH2 domains: a thermodynamic and structural study.

Thermodynamic measurements, structural determinations, and molecular computations were applied to a series of peptide ligands of the pp60(c-src) SH2 domain in an attempt to understand the critical binding determinants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-ray crystallographic studies on 12 peptide-SH2 domain complexes. The peptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine replacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents either Glu, Gln, or NH2. Dipeptide analogs which featured X as either hexanolamine or heptanolamine were able to pick up new hydrogen bonds involving their hydroxyl groups within a predominantly lipophilic surface cavity. However, due to internal strain as well as the solvent accessibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the case where a reversible covalent thiohemiacetal was formed between a formylated phosphotyrosine analog and the thiol side chain of Cys-188, deltaS was 25.6 cal/(mol K) lower than for the nonformylated phosphotyrosine parent. Normal mode calculations show that the dramatic decrease in entropy observed for the covalent thiohemiacetal complex is due to the inability of the phosphotyrosine moiety to transform lost rotational and translational degrees of freedom into new vibrational modes.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS: A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS: The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

Water soluble inhibitors of topoisomerase I: quaternary salt derivatives of camptothecin.

Eleven water soluble 7-substituted quaternary ammonium salt derivatives of 10,11-(methylenedioxy)- and 10,11-(ethylenedioxy)-(20S)-camptothecin were synthesized via the Friedlander reaction followed by nucleophilic displacement with an aromatic amine. All of these compounds were more potent than camptothecin in the in vitro cleavable complex assay. These inherently charged camptothecin derivatives were cytotoxic against three different human tumor cell lines (SKOV3, an ovarian adenocarcinoma; SKVLB a multidrug resistant ovarian adenocarcinoma; and HT-29, a colon carcinoma). A selected group of five compounds was evaluated in the nude mouse HT-29 xenograft model. Two of these quaternary salts (17 and 18) were more efficacious than Topotecan in delaying tumor growth. In an extended in vivo model, 18 demonstrated tumor regression.

Rigid analogs of camptothecin as DNA topoisomerase I inhibitors.

Substituted 8-ethyl-2-(2-oxo-1,2-dihydroindol-3-ylidene)-8-hydroxy-2,3,5,8- tetrahydro-6-oxa-3a-azacyclopenta[b]naphthalene-1,4,7-triones were synthesized and evaluated as topoisomerase I inhibitors in an in vitro cleavable complex assay. The activity of these compounds may be attributed to their rigid, planar geometry, and an attempt was made to correlate the SAR in this series to known attributes of camptothecin.

In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues.

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.