Guifen Xu

Identification and characterization of a non-retinoid ligand for retinol-binding protein 4 which lowers serum retinol-binding protein 4 levels in vivo

Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes.

Structure guided design of a series of sphingosine kinase (SphK) inhibitors.

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each others loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Sphingosine Kinase Activity Is Not Required for Tumor Cell Viability

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors.

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.

Investigation of collision-induced dissociations involving odd-electron ion formation under positive electrospray ionization conditions using accurate mass

Collision induced dissociation (CID) has been extensively used for structure elucidation. CID in the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) modes has been found to generate mostly even-electron fragment ions while it has been occasionally reported to form odd-electron free radical ions. However, the structural requirements and the fragmentation mechanisms for free-radical CIDs have not been well characterized in the literature. For this purpose, we studied a series of aromatic and non-aromatic compounds such as sulfonamides, N-aryl amides, tert-butyl-substituted aromatic compounds, aryl alkyl ethers, and O-alkyl aryl oximes using the LTQ and LTQ Orbitrap linear ion trap mass spectrometers. The accurate measurement of the fragment ion masses established the unambiguous assignment of the fragment structures resulting from the test compounds. Our results showed that free radical fragmentation is structure dependent and is to a large extent correlated with the neighboring groups in the structures that stabilize the newly formed free radical ions.

Further Studies with the 2-Amino-1,3-thiazol-4(5H)-one Class of 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors: Reducing Pregnane X Receptor Activity and Exploring Activity in a Monkey Pharmacodynamic Model

A series of compounds containing the 2-amino-1,3-thiazol-4(5H)-one core were found to be potent inhibitors of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). One of our lead compounds from this series activated the human nuclear xenobiotic receptor, pregnane X receptor (PXR). To try and mitigate the PXR activity, we prepared analogues of our lead series that contained polar groups on the right-hand side of the thiazolone. Several analogues containing amides or alcohols appended to the C-5 position of the thiazolone showed a significant reduction in PXR activity. Through these structure-activity efforts, a compound containing a tert-alcohol group off the C-5 position, analogue (S)-33a, was found to have an 11beta-HSD1 Ki = 35 nM and negligible PXR activity. Compound (S)-33a was advanced into a pharmacodynamic model in cynomolgus monkeys, where it inhibited adipose 11beta-HSD1 activity after being orally administered.

Application of automated dried blood spot sampling and LC-MS/MS for pharmacokinetic studies of AMG 517 in rats

BACKGROUND The use of dried blood spot (DBS) sampling technique is of particular interest for drug discovery pharmacokinetic studies due to the small blood volume requirement. In addition, automated blood sampling is an attractive approach for rat pharmacokinetic studies as animal handling work is minimized. The goal of this study was to use an automated DBS sampler for automated blood collection and spotting onto DBS paper for pharmacokinetic studies in rats. AMG 517, a potent and selective vanilloid receptor antagonist, was dosed to rats (n = 3) intravenously and blood samples were collected at nine time points over a 24 h period using the automated DBS sampler. After drying, storage and shipment, the DBS samples were extracted and analyzed by LC-MS/MS. RESULTS The developed bioanalytical method for the analysis of DBS samples had good accuracy and precision within the context of a discovery, non-GLP analysis. The concentration-time data and pharmacokinetic parameters generated from automated spotted samples were very similar to those derived from manually spotted DBS samples. The manual DBS data were also comparable to plasma data after correction for blood-to-plasma ratio. CONCLUSION The automated DBS sampling is a promising technique for rodent pharmacokinetic studies and will improve the efficiency and quality of DBS sampling.

Design of 1-piperazinyl-4-arylphthalazines as potent Smoothened antagonists

The Hedgehog (Hh) signaling pathway regulates cell proliferation and differentiation in developing tissues, and abnormal activation of the Hh pathway has been linked to several tumor subsets. As a transducer of Hh signaling, the GPCR-like protein Smoothened (Smo) is a promising target for disruption of unregulated Hh signaling. A series of 1-amino-4-arylphthalazines was developed as potent and orally bioavailable inhibitors of Smo. A representative compound from this class demonstrated significant tumor volume reduction in a mouse medulloblastoma model.

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.