Elliott S. Klein

Retinoid-dependent Recruitment of a Histone H1 Displacement Activity by Retinoic Acid Receptor

Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones. However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes. Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described. In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin. We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors. Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR. These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events.

Synthesis and biological activity of retinoic acid receptor-α specific amides

Abstract Retinoids are analogues of all- trans -retinoic acid, a powerful hormone that mediates many fundamental biological processes. Cancer and other serious hyperproliferative diseases are attractive therapeutic targets for retinoids, but the therapeutic use of retinoids is limited due to severe toxicity. We report here the design of retinoid receptor-α specific ligands with growth inhibitory activity in breast cancer cell lines, and which do not cause the cutaneous toxicity associated with the currently available nonselective retinoid agonists.

Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton’s Tyrosine Kinase

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton’s tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.

Differential RXR & RAR activity of stilbene retinoid analogs bearing thiazole and imidazole carboxylic acids

Retinoids are hormones which activate two distinct families of nuclear receptors: the retinoic acid receptors (RARα,β,γ) and the retinoid X receptors (RXRα,β,γ). We report thiazole and imidazole substituted stilbene analogs that have RXR specific or pan-agonist activity and discuss the structural features that impart these differential activation properties to compounds of this class.

Phenylcyclohexene and phenylcyclohexadiene substituted compounds having retinoid antagonist activity.

Retinoids are natural and synthetic analogues of the hormone retinoic acid. Systemic retinoid agonist therapy is usually associated with toxic side effects, such as mucocutaneous toxicity, which may be alleviated by the use of topical retinoid antagonists. We report the synthesis and biological activity of a new series of potent, RAR-specific antagonists substituted with phenylcyclohexene and phenylcyclohexadiene groups.

Highly selective inhibition of Bruton’s tyrosine kinase attenuates skin and brain disease in murine lupus

BackgroundSystemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects different end organs, including skin and brain. We and others have previously shown the importance of macrophages in the pathogenesis of cutaneous and neuropsychiatric lupus. Additionally, autoantibodies produced by autoreactive B cells are thought to play a role in both the skin and central nervous system pathologies associated with SLE.MethodsWe used a novel inhibitor of Bruton’s tyrosine kinase (BTK), BI-BTK-1, to target both macrophage and B cell function in the MRL-lpr/lpr murine model of SLE, and examined the effect of treatment on skin and brain disease.ResultsWe found that treatment with BI-BTK-1 significantly attenuated the lupus associated cutaneous and neuropsychiatric disease phenotypes in MRL/lpr mice. Specifically, BI-BTK-1 treated mice had fewer macroscopic and microscopic skin lesions, reduced cutaneous cellular infiltration, and diminished inflammatory cytokine expression compared to control mice. BTK inhibition also significantly improved cognitive function, and decreased accumulation of T cells, B cells, and macrophages within the central nervous system, specifically the choroid plexus.ConclusionsDirected therapies may improve the response rate in lupus-driven target organ involvement, and decrease the dangerous side effects associated with global immunosuppression. Overall, our results suggest that inhibition of BTK may be a promising therapeutic option for cutaneous and neuropsychiatric disease associated with SLE.

N-Arylsulfonyl-α-amino carboxamides are potent and selective inhibitors of the chemokine receptor CCR10 that show efficacy in the murine DNFB model of contact hypersensitivity

Compound 1 ((4-amino-3,5-dichlorophenyl)-1-(4-methylpiperidin-1-yl)-4-(2-nitroimidazol-1-yl)-1-oxobutane-2-sulfonamido) was discovered to be a 690nM antagonist of human CCR10 Ca2+ flux. Optimization delivered (2R)-4-(2-cyanopyrrol-1-yl)-S-(1H-indol-4-yl)-1-(4-methylpiperidin-1-yl)-1-oxobutane-2-sulfonamido (eut-22) that is 300 fold more potent a CCR10 antagonist than 1 and eliminates potential toxicity, mutagenicity, and drug-drug-interaction liabilities often associated with nitroaryls and anilines. eut-22 is highly selective over other GPCRs, including a number of other chemokine receptors. Finally, eut-22 is efficacious in the murine DNFB model of contact hypersensitivity. The efficacy of this compound provides further evidence for the role of CCR10 in dermatological inflammatory conditions.

BTK inhibition ameliorates kidney disease in spontaneous lupus nephritis

Lupus nephritis is a common disease manifestation of SLE, in which immune complex deposition and macrophage activation are important contributors to disease pathogenesis. Brutons tyrosine kinase (BTK) plays an important role in both B cell and FcgammaR mediated myeloid cell activation. In the current study, we examined the efficacy of BI-BTK-1, a recently described irreversible BTK inhibitor, in the classical NZB × NZW F1 (NZB/W) and MRL/lpr spontaneous mouse models of SLE. NZB/W mice were randomly assigned to a treatment (0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg) or control group and began treatment at 22 weeks of age. The experimental setup was similar in MRL/lpr mice, but with a single treated (10 mg/kg, beginning at 8-9 weeks of age) and control group. A separate experiment was performed in the MRL/lpr strain to assess the ability of BI-BTK-1 to reverse established kidney disease. Early treatment with BI-BTK-1 significantly protected NZB/W and MRL/lpr mice from the development of proteinuria, correlating with significant renal histological protection, decreased anti-DNA titers, and increased survival in both strains. BI-BTK-1 treated mice displayed a significant decrease in nephritis-associated inflammatory mediators (e.g. LCN2 and IL-6) in the kidney, combined with a significant inhibition of immune cell infiltration and accumulation. Importantly, BI-BTK-1 treatment resulted in the reversal of established kidney disease. BTK inhibition significantly reduced total B cell numbers and all B cell subsets (immature, transitional, follicular, marginal zone, and class switched) in the spleen of NZB/W mice. Overall, the significant efficacy of BI-BTK-1 in ameliorating multiple pathological endpoints associated with kidney disease in two distinct murine models of spontaneous lupus nephritis provides a strong rationale for BTK inhibition as a promising treatment approach for lupus nephritis.

OP0164 Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton's Tyrosine Kinase

Background Lupus nephritis (LN) causes significant morbidity and mortality in SLE patients. Autoantibody-containing immune complexes (ICs) can activate complement cascades and Fc receptors on resident and infiltrating cells within the kidney, thus promoting inflammation. Brutons tyrosine kinase (BTK) is a tyrosine kinase important for B cell development, Fc receptor signaling, and macrophage polarization. Objectives To assess the role of BTK in the pathogenesis of nephritis in an inducible model of LN, and to evaluate the therapeutic potential of BTK inhibition. Methods A novel, highly selective, and potent (mouse whole blood CD69 IC50=13±2 nM) BTK inhibitor, BI-BTK-1 (Boehringer Ingelheim), was tested in female 129 sv/J mice (10 weeks of age) injected with nephrotoxic serum (NTS), an experimental model which closely mimics LN. Mice pre-immunized with rabbit IgG (Day 0) were administered NTS containing rabbit anti-mouse glomerular antibodies (Day 5), inducing a severe IC-mediated crescentic glomerulonephritis. Mice that did not receive the NTS transfer were used as healthy controls. Mice were treated once daily with vehicle alone or BI-BTK-1 (0.3–10 mg/kg, n=16/group), before the transfer of NTS as a prophylactic assessment, or after the transfer of NTS as a therapeutic assessment. Results NTS-challenged mice treated with BI-BTK-1 exhibited a statistically significant, dose responsive protection from kidney disease. Compared to vehicle treated mice, NTS-challenged mice treated with 10 mg/kg BI-BTK-1 had significantly less proteinuria (1220 mg/dl vs 10 mg/dl, respectively, p<0.0005), serum creatinine (0.74 mg/dl vs 0.48 mg/dl, respectively, p<0.03), and BUN (82 mg/dl vs 25 mg/dl, respectively, p<0.03) at the end of study (Day 11). Histology assessment confirmed marked renal protection in the BI-BTK-1 treatment groups. BI-BTK-1 treatment resulted in decreased recruitment of inflammatory monocytes from the splenic reservoir, and a decrease in infiltrating IBA-1+ cells as well as C3 deposition within the kidney. RNA-seq revealed that more than 500 genes were modulated by BTK treatment, with further analysis highlighting many of the modulated pathways being related to inflammation and glomerular injury in LN. Serum profiling and selective kidney gene expression analyses by Q-PCR revealed that BTK inhibition was associated with a significant decrease in the levels of key LN-relevant inflammatory cytokines and chemokines. Importantly, delayed treatment with 3 mg/kg of BI-BTK-1in mice with established proteinuria was able to reverse proteinuria and improve renal histopathology. Conclusions Our results further confirm the important role for BTK activation in the pathogenesis of immune complex-mediated nephritis, and highlight BTK as a valuable therapeutic target. Taken together with previously published studies and our preliminary results in spontaneous lupus models, these findings further strengthen the rationale for selective BTK inhibition as a promising approach to the treatment of LN. Disclosure of Interest S. Chalmers: None declared, J. Doerner: None declared, T. Bosanac Employee of: Boehringer Ingelheim, S. Khalil Employee of: Boehringer Ingelheim, D. Smith Employee of: Boehringer Ingelheim, C. Harcken Employee of: Boehringer Ingelheim, J. Dimock Employee of: Boehringer Ingelheim, E. Der: None declared, L. Herlitz: None declared, D. Webb Employee of: Boehringer Ingelheim, E. Seccareccia Employee of: Boehringer Ingelheim, D. Feng Employee of: Boehringer Ingelheim, J. Fine Employee of: Boehringer Ingelheim, M. Ramanujam Employee of: Boehringer Ingelheim, E. Klein Employee of: Boehringer Ingelheim, C. Putterman Grant/research support from: Boehringer Ingelheim