Siquan Sun

Oxysterols direct B-cell migration through EBI2.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein–Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

TLR7/9 Antagonists as Therapeutics for Immune-Mediated Inflammatory Disorders

There is an increasing interest in ligands of nucleic acid-sensing Toll-like receptors (TLR), especially TLR7 and TLR9, for pharmacological intervention in various diseases. The TLR7 agonist imiquimod is currently used as a topical treatment for genital warts caused by human papillomavirus (HPV), actinic keratosis (AK) and superficial basal cell carcinoma. Oligodeoxynucleotides (ODN) TLR9 agonists are currently in clinical trials for use in lung cancer, as anti-viral therapy, as adjuvants and as immune modulators in asthma and allergies. TLR7/9 antagonists, such as the anti-malaria drugs chloroquine, hydroxychloroquine and quinacrine, have been used since the 1950s to treat immune-mediated inflammatory disorders (IMID) such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sjögrens syndrome. However, the use of these anti-malarials in IMID is limited due to the side effects or suboptimal efficacy. Pre-clinical animal models as well as genetic linkage studies have indicated that TLR7/9 play a pivotal role in the aforementioned as well as other IMID such as multiple sclerosis (MS), inflammatory bowl disease (IBD)/colitis and psoriasis. Recent evidence has suggested that selective, specific antagonists for TLR7 and/or 9 might be more beneficial in certain diseases, such as SLE. Thus, the use of suppressive ODN or novel small molecule TLR7/9 inhibitors with a larger safety window and differentiated selectivity may potentially have significant clinical utility in these IMID. Herein, we review efforts to develop novel TLR7/9 antagonists and the rationale for the use of such therapeutics in a variety of IMID.

Cathepsin S inhibitors: 2004 – 2010

Introduction: Cathepsin S, a lysosomal cysteine protease, plays an important role in antigen presentation. Its inhibition is expected to result in immunosuppression, making this enzyme an attractive target to potentially treat autoimmune and inflammatory diseases. Areas covered: The focus of this review is on patent literature regarding small molecule inhibitors of cathepsin S published from 2004 to April 2010. Different structure classes based on binding strategies (covalent vs non-covalent) are surveyed and listed according to warhead type and research organization. Expert opinion: Although > 40 patent applications have appeared between 2004 and 2010, the decrease in applications focusing on cathepsin S over the past 2 – 3 years may reflect a renewed interest in other cathepsins, especially cathepsin K, for which a small molecule inhibitor is currently in Phase III clinical trials.

Recent advances in the design of cathepsin S inhibitors.

Cathepsin S has been of increasing interest as a target of medicinal chemistry efforts given its role in modulating antigen-presentation by major histocompatibility class II (MHC II) molecules as well as its involvement in extracellular proteolytic activities. Inhibition of the cathepsin S enzyme reduces degradation of the invariant chain, a crucial chaperon which also blocks peptide-binding by MHC II molecules, thereby decreasing antigen presentation to CD4(+) T-cells. Extracellular cathepsin S may also be involved in angiogenesis and initiation and/or maintenance of neuropathic pain by cleavage of the membrane-bound chemokine fractalkine (CX3CL1). Cathepsin S inhibitors have thus been suggested to hold potential as therapeutics for a variety of diseases. The initial development of cathepsin S inhibitors targeted irreversible, covalent inhibitors, but more recently the focus has been on reversible inhibitors, representing both covalent modifiers of the enzyme and, of late, noncovalent inhibitors. This review details advances in cathepsin S inhibitor design as reported in the primary literature since 2006, focusing especially on structure-activity relationships of the various covalent and noncovalent inhibitor series.

Diazinones as P2 replacements for pyrazole-based cathepsin S inhibitors

A pyridazin-4-one fragment 4 (hCatS IC(50)=170 microM) discovered through Tethering was modeled into cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12 (hCatS IC(50)=430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12 bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC(50)=60 nM) and 27 (hCatS IC(50)=40 nM).

Pyrazole-based arylalkyne cathepsin S inhibitors. Part II: Optimization of cellular potency

Basic lipophilic substituents dramatically improved the cellular potency of a previously disclosed series of pyrazole-based arylalkyne cathepsin S inhibitors. The incorporation of substituted benzylamines in the para position of the arylalkyne maintained enzymatic activity (hCatS IC50=80-420 nM) and imparted cellular potency (IC50=0.8-4.0 microM). Further refinement of the morpholine portion of the pharmacophore enabled the identification of bicyclic piperidines with enhanced affinity for CatS (IC50=10-30 nM) and sub-micromolar cellular potency (JY Ii IC50=200-720 nM).

Pyrazole-based cathepsin S inhibitors with arylalkynes as P1 binding elements.

A crystal structure of 1 bound to a Cys25Ser mutant of cathepsin S helped to elucidate the binding mode of a previously disclosed series of pyrazole-based CatS inhibitors and facilitated the design of a new class of arylalkyne analogs. Optimization of the alkyne and tetrahydropyridine portions of the pharmacophore provided potent CatS inhibitors (IC50=40-300 nM), and an X-ray structure of 32 revealed that the arylalkyne moiety binds in the S1 pocket of the enzyme.

Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors: part 1.

A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crystal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submicromolar cellular potency.

Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors. Part 2: Modification of P3, P4, and P5 regions

A novel class of tetrahydropyrido-pyrazole thioether amines that display potency against human Cathepsin S have been previously reported. Here, further SAR investigations of the P3, P4, and P5 regions are described. In particular, 4-fluoropiperidine is identified as a competent P3 binding element when utilized in conjunction with a (S)-2-hydroxypropyl linker-containing P5 moiety and oxamide or sulfonamide P4 substitution.

Thioether acetamides as P3 binding elements for tetrahydropyrido-pyrazole cathepsin S inhibitors

A series of tetrahydropyrido-pyrazole cathepsin S (CatS) inhibitors with thioether acetamide functional groups were prepared with the goal of improving upon the cellular activity of amidoethylthioethers. This Letter describes altered amide connectivity, in conjunction with changes to other binding elements, resulting in improved potency, as well as increased knowledge of the relationship between this chemotype and human CatS activity.