High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders.

Discovery of 2-aminoimidazopyridine adenosine A(2A) receptor antagonists.

A novel series of adenosine A(2A) receptor antagonists was identified by high-throughput screening of an encoded combinatorial compound collection. The initial hits were optimized for A(2A) binding affinity, A(1) selectivity, and in vitro microsomal stability generating orally available 2-aminoimidazo[4,5-b]pyridine-based A(2A) antagonist leads.

Discovery of novel quinolinone adenosine A2B antagonists.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).

Synthesis of 2-amino-5-benzoyl-4-(2-furyl)thiazoles as adenosine A(2A) receptor antagonists.

The discovery and synthesis of a series of 2-amino-5-benzoyl-4-(2-furyl)thiazoles as adenosine A(2A) receptor antagonists from a small-molecule combinatorial library using a high-throughput radioligand-binding assay is described. Antagonists were further characterized in the A(2A) binding assay and an A(1) selectivity assay. Selected examples exhibited excellent affinity for A(2A) and good selectivity versus the A(1) receptor.

Synthesis and SAR studies of trisubstituted purinones as potent and selective adenosine A2A receptor antagonists.

A series of trisubstituted purinones was synthesized and evaluated as A(2A) receptor antagonists. The A(2A) structure-activity relationships at the three substituted positions were studied and selectivity against the A(1) receptor was investigated. One antagonist 12o exhibits a K(i) of 9nM in an A(2A) binding assay, a K(b) of 18nM in an A(2A) cAMP functional assay, and is 220-fold selective over the A(1) receptor.

Synthesis and structure-activity relationships of new disubstituted phenyl-containing 3,4-diamino-3-cyclobutene-1,2-diones as CXCR2 receptor antagonists.

A series of 3,4- and 3,5-disubstituted phenyl-containing cyclobutenedione analogues were synthesized and evaluated as CXCR2 receptor antagonists. Variations in the disubstitution pattern of the phenyl ring afforded new compounds with potent CXCR2 binding affinity in the low nanomolar ranges. Moreover, two potent compounds 19 and 26 exhibited good oral pharmacokinetic profiles.

Exploring structure-activity relationships of tricyclic farnesyltransferase inhibitors using ECLiPS libraries.

The development of structure-activity relationships (SARs) relating to the function of a biological protein is often a long and protracted undertaking when using an iterative medicinal chemistry approach. High throughput screening of ECLiPS (Encoded Combinatorial Libraries on Polymeric Support) libraries can be used to simplify this process. In this paper, we illustrate how a large ECLiPS library of 26,908 compounds, based on a tricyclic core structure, was used to define a multitude of SARs for the oncogenic target, farnesyltransferase (FTase). This library, FT-2, was prepared using a split-and-pool approach in which small molecules are constructed on resin that contains tag/linker constructs to track the synthetic process [1-5] Highly defined SARs were produced from this screen that enhanced our understanding of FTase binding site interactions. The pivotal compounds culled from this library were potent in both cell-free and cell-based FTase assays, selective over the closely related enzyme, geranylgeranyltransferase I (GGTase I), and inhibited the adherent-independent growth of a transformed cell line.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Design, synthesis, and evaluation of tetrahydroquinoline and pyrrolidine sulfonamide carbamates as gamma-secretase inhibitors.

Gamma-secretase is a key enzyme involved in the production of beta-amyloid peptides which are believed to play a critical role in the onset and progression of Alzheimer's disease (AD). As such, inhibition of gamma-secretase has been an attractive approach to AD therapy. In this paper, the design, synthesis, and evaluation of tetrahydroquinoline and pyrrolidine sulfonamide carbamates as gamma-secretase inhibitors are described.

Discovery and preliminary evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin receptor antagonists.

The discovery and evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin (IP) receptor antagonists is described. Analogs disclosed showed high affinity for the IP receptor in human platelet membranes with IC50 values of 0.05-0.50 microM, demonstrated functional antagonism by inhibiting cAMP production in HEL cells with IC50 values of 0.016-0.070 microM, and exhibited significant selectivity versus other prostanoid receptors.

Pyrazolo[1,5-a]pyrimidines as orally available inhibitors of cyclin-dependent kinase 2.

Properly substituted pyrazolo[1,5-a]pyrimidines are potent and selective CDK2 inhibitors. Compound 15j is orally available and showed efficacy in a mouse A2780 xenograft model.

Guiding farnesyltransferase inhibitors from an ECLiPS library to the catalytic zinc.

Farnesyltransferase inhibitors identified from an ECLiPS library were optimized using solution-phase synthesis. X-ray crystallography of inhibited complexes was used to identify substructures that coordinate to the active site zinc. The X-ray structures were ultimately used to guide the design of second-generation analogs with FTase IC(50)s of less than 1.0 nM.

Synthesis and structure-activity relationships of 3,4-diaminocyclobut-3-ene-1,2-dione CXCR2 antagonists.

A novel series of 3,4-diaminocyclobut-3-ene-1,2-diones was prepared and found to show potent inhibitory activity of CXCR2 binding and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Microsome stability and Caco2 studies were subsequently used to show that compounds of this chemotype are predicted to have good oral bioavailability and are thus suitable for pharmaceutical development.

Discovery and SAR of 4-amino-2-biarylbutylurea MCH 1 receptor antagonists through solid-phase parallel synthesis.

-4-Amino-2-arylbutylbenzamides such as 1 were identified as micromolar MCH 1 receptor (MCH1R) antagonists via screening using a scintillation proximity assay based on [125I]-MCH binding to recombinant, human MCH1R. Subsequent lead optimization efforts using solid-phase parallel synthesis resulted in the defined structure-activity relationships and the identification of 4-amino-2-biarylbutylureas, such as 11g, as potent single digit nanomolar MCH1R antagonists.

Surfing the piperazine core of tricyclic farnesyltransferase inhibitors.

In order to fully explore structure-activity relationships at the 1- and 2-positions of the piperazine core of tricyclic farnesyltransferase inhibitors, an 11,718-member ECLiPS library was synthesized and screened in a farnesyltransferase scintillation proximity assay. A detailed description of the library and analyses of the screening data will be provided.

Discovery and SAR of biaryl piperidine MCH1 receptor antagonists through solid-phase encoded combinatorial synthesis.

An encoded combinatorial library based on aryl and biaryl piperidine scaffolds was designed and synthesized. Screening of this library resulted in the discovery of high-nanomolar biaryl piperidine-based MCH1 receptor antagonists. Follow-up optimization using a parallel synthesis provided potent, single digit nanomolar antagonists.