Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo.

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Discovery of an in vitro and in vivo potent resorcylic lactone analog of LL-Z1640-2 as anti-inflammatory lead, II.

The potent in vitro lead compound, ER-803064 (2), a MEK1 and MEKK1 inhibitor inspired from natural product LL-Z1640-2 (f152A1), was further optimized to improve in vitro and in vivo potency. The modifications on C14 position led to discovery of the lead compounds 28 and 29, which regained full in vitro potency of f152A1 and showed higher in vivo potency by iv administration.

Evaluation of a candidate anti-arthritic drug using the mouse collagen antibody induced arthritis model and clinically relevant biomarkers.

The most rigorous scenario for testing a candidate rheumatoid arthritis therapeutic would be to use clinically relevant biomarkers and readouts to monitor disease development in an animal model that has a mechanism of disease that reflects the human condition. Treatment should begin when the full spectrum of arthritic processes, including bone damage, is present. We have tried to take this approach to evaluate a novel EP4 receptor antagonist (ER-886046) for its anti-arthritic potential. This work aimed not only to test a potential drug, but to also demonstrate a strategy for performing a more clinically relevant evaluation of future candidate arthritis treatments. A variety of biomarkers including: radiographic evaluation, clinical scoring, histology analysis, F4/80 macrophage immunohistochemistry, luminol bioluminescent imaging and (99m)Tc-MDP-SPECT imaging were evaluated as disease readouts in the mouse anti-collagen antibody induced arthritis model (CAIA). CAIA mice were treated either prophylactically or therapeutically with ER-886046 and the compound's efficacy was probed using the various biomarkers and compared to the reference drugs prednisolone and celecoxib. The various biomarkers effectively measured different aspects of arthritis pathology and consistently demonstrated the efficacy of ER-886046. The compound was found to be effective even when dosed therapeutically after bone damaging processes had initiated. The results presented herein demonstrate how biomarkers and a clinically relevant experimental design can be used to evaluate a candidate therapeutic. Utilization of clinically relevant biomarkers may provide a means for more translatable pre-clinical testing of candidate therapeutics and may provide information on their mechanism of action.

Tubulin-based antimitotic mechanism of E7974, a novel analogue of the marine sponge natural product hemiasterlin.

E7974 is a synthetic analogue of the marine sponge natural product hemiasterlin. Here, we show that E7974, such as parental hemiasterlin, acts via a tubulin-based antimitotic mechanism. E7974 inhibits polymerization of purified tubulin in vitro with IC(50) values similar to those of vinblastine. In cultured human cancer cells, E7974 induces G(2)-M arrest and marked disruption of mitotic spindle formation characteristic of tubulin-targeted anticancer drugs. Extensive hypodiploid cell populations are seen in E7974-treated cells, indicating initiation of apoptosis after prolonged G(2)-M blockage. Consistent with this observation, E7974 induces caspase-3 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis. Only a short cellular exposure to E7974 is sufficient to induce maximum mitotic arrest, suggesting that E7974's antitumor effects in vivo may persist even after blood levels of the drug decrease after drug administration. Interactions of E7974 with purified tubulin were investigated using two synthetic tritiated photoaffinity analogues incorporating a benzophenone photoaffinity moiety at two different positions of the E7974 scaffold. Both analogues preferentially photolabeled alpha-tubulin, although minor binding to beta-tubulin was also detected. E7974 thus seems to share a unique, predominantly alpha-tubulin-targeted mechanism with other hemiasterlin-based compounds, suggesting that, unlike many tubulin-targeted natural products and related drugs, the hemiasterlins evolved to mainly target alpha-tubulin, not beta-tubulin subunits.

Toll-like receptor 2 antagonists. Part 1: preliminary SAR investigation of novel synthetic phospholipids.

Novel synthetic phospholipid compound 1 was discovered to be an antagonist of human toll-like receptor 2 (TLR2) signaling. In a preliminary SAR campaign we synthesized several analogues of 1 and found that considerable structural changes could be made without loss of TLR2 antagonistic activity.