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We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of5% or95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

[Image: see text] Bruton’s tyrosine kinase (BTK) has been shown to play a key role in the pathogenesis of autoimmunity. Therefore, the inhibition of the kinase activity of BTK with a small molecule inhibitor could offer a breakthrough in the clinical treatment of many autoimmune diseases. This Letter describes the discovery of BMS-986143 through systematic structure–activity relationship (SAR) development. This compound benefits from defined chirality derived from two rotationally stable atropisomeric axes, providing a potent and selective single atropisomer with desirable efficacy and tolerability profiles.

Protein kinases are intensely studied mediators of cellular signaling. While traditional biochemical screens are capable of identifying compounds that modulate kinase activity, these assays are limited in their capability of predicting compound behavior in a cellular environment. Here, we aim to bridge target engagement and compound-cellular phenotypic behavior by utilizing a bioluminescence resonance energy transfer (BRET) assay to characterize target occupancy within living cells for Bruton's tyrosine kinase (BTK). Using a diverse chemical set of BTK inhibitors, we determine intracellular engagement affinity profiles and successfully correlate these measurements with BTK cellular functional readouts. In addition, we leveraged the kinetic capability of this technology to gain insight into in-cell target residence time and the duration of target engagement, and to explore a structural hypothesis.

Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fce receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a member of the Tec family of kinases. BTK plays an essential role in B cell receptor (BCR)-mediated signaling as well as Fcγ receptor signaling in monocytes and Fce receptor signaling in mast cells and basophils, all of which have been implicated in the pathophysiology of autoimmune disease. As a result, inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as lupus and rheumatoid arthritis. This article details the structure–activity relationships (SAR) leading to a novel series of highly potent and selective carbazole and tetrahydrocarbazole based, reversible inhibitors of BTK. Of particular interest is that two atropisomeric centers were rotationally locked to provide a single, stable atropisomer, resulting in enhanced potency and selectivity as well as a reduction in safety liabilities. With significantly enhanced potency and selectivity, excellent in vivo properties an...

Incorporation of a suitably-placed electrophilic group transformed a series of reversible BTK inhibitors based on carbazole-1-carboxamide and tetrahydrocarbazole-1-carboxamide into potent, irreversible inhibitors. Removal of one ring from the core of these compounds provided a potent irreversible series of 2,3-dimethylindole-7-carboxamides having excellent potency and improved selectivity, with the additional advantages of reduced lipophilicity and molecular weight.

Bruton’s tyrosine kinase (BTK) regulates critical signal transduction pathways involved in the pathobiology of rheumatoid arthritis (RA) and other autoimmune disorders. BMS-986142 is a potent and highly selective reversible small molecule inhibitor of BTK currently being investigated in clinical trials for the treatment of both RA and primary Sjogren’s syndrome. In the present report, we detail the in vitro and in vivo pharmacology of BMS-986142 and show this agent provides potent and selective inhibition of BTK (IC50 = 0.5 nM), blocks antigen receptor-dependent signaling and functional endpoints (cytokine production, co-stimulatory molecule expression, and proliferation) in human B cells (IC50 ≤ 5 nM), inhibits Fcγ receptor-dependent cytokine production from peripheral blood mononuclear cells, and blocks RANK-L-induced osteoclastogenesis. Through the benefits of impacting these important drivers of autoimmunity, BMS-986142 demonstrated robust efficacy in murine models of rheumatoid arthritis (RA), including collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). In both models, robust efficacy was observed without continuous, complete inhibition of BTK. When a suboptimal dose of BMS-986142 was combined with other agents representing the current standard of care for RA (e.g., methotrexate, the TNFα antagonist etanercept, or the murine form of CTLA4-Ig) in the CIA model, improved efficacy compared to either agent alone was observed. The results suggest BMS-986142 represents a potential therapeutic for clinical investigation in RA, as monotherapy or co-administered with agents with complementary mechanisms of action.

Bruton’s tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure–activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide 6 (BMS-935177) was selected to advance into clinical development.

The synthesis, structure-activity relationships (SAR), and biological results of pyridyl-substituted azaindole based tricyclic inhibitors of IKK2 are described. Compound 4m demonstrated potent in vitro potency, acceptable pharmacokinetic and physicochemical properties, and efficacy when dosed orally in a mouse model of inflammatory bowel disease.

A new series of tricyclic-based inhibitors of IKK have been derived from an earlier lead compound. The synthesis and structure-activity relationships (SAR) are described. Compound 4k inhibited TNF production in rats stimulated with LPS.

The design and synthesis of a novel series of oxazole-, thiazole-, and imidazole-based inhibitors of IkappaB kinase (IKK) are reported. Biological activity was improved compared to the pyrazolopurine lead, and the expedient synthesis of the new tricyclic systems allowed for efficient exploration of structure-activity relationships. This, combined with an iterative rat cassette dosing strategy, was used to identify compounds with improved pharmacokinetic (PK) profiles to advance for in vivo evaluation.

We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC50 = 0.30 μM), which however was considerably less potent against IKK-1 (IC50 = 4.0 μM). In order to further explore the SAR around the imidazoquinoxaline tricyclic structure of 1, we prepared a series of tetracyclic analogues (7, 13, and 18). The synthesis and biological activities of these potent IKK inhibitors are described.

Objective The transcription of several cytokines, cell adhesion molecules, and enzymes involved in the inflammatory and destructive mechanisms of rheumatoid arthritis is dependent on nuclear factor κB (NF-κB). Because IκB kinase (IKK) is critical in transducing the signal-inducible activation of NF-κB, we examined whether the highly selective and orally bioavailable IKK inhibitor BMS-345541 is efficacious against collagen-induced arthritis (CIA) in mice. Methods Arthritis in DBA/1LacJ male mice was induced by subcutaneous immunization with bovine type II collagen on day 0 and day 21. BMS-345541 was administered perorally daily, either prophylactically (before disease onset) or therapeutically (after disease onset). Clinical assessment of the incidence and severity of disease was conducted throughout the study, and histologic evaluation was performed at the time of study termination (day 42). Results When administered prophylactically, BMS-345541 (in a dose range of 10–100 mg/kg) was effective, in a dose-dependent manner, in reducing the incidence of disease and inhibiting clinical signs of disease. Histologic evaluation of the joints showed that both inflammation and joint destruction were blocked by the IKK inhibitor. Message levels of interleukin-1β in the joints were also dose-dependently inhibited in the mice that received BMS-345541. Dose-dependent efficacy in terms of both disease severity and histologic end points was observed with the therapeutic dosing regimen of BMS-345541, with use of the 100-mg/kg dose resulting in resolution of disease. Conclusion IKK plays a key role in CIA in mice, and inhibitors of this enzyme represent a promising target for the development of novel agents to treat rheumatoid arthritis and other inflammatory diseases. BMS-345541 represents the first example of an inhibitor of IKK that has antiinflammatory activity in vivo.

Gangliosides of macrophages are potent immunoregulatory molecules. A monoclonal antibody directed at human macrophage gangliosides (25F4) inhibits macrophage migration with relative specificity. Recent reports suggested that greater expression of Gm1 in mononuclear cells accompanies advanced HIV infection, although others failed to demonstrate any differences in vitro. We purified gangliosides from blood monocyte-derived macrophages obtained from HIV-infected adults. Densitometric analysis of chromatograms demonstrated no differences in relative quantities of any macrophage gangliosides among all HIV-positive and -negative donors. Antibody 25F4 showed equivalent ELISA reactivity with purified macrophage gangliosides of HIV-positive and -negative donors. However, intact macrophages of HIV donors with CD4+ cell counts

Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties.

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognize those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration in an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

The identification of a potent series of IKK-β selective inhibitors based on an imidazothienopyrazine template and the oral efficacy of one such analog (22j) in the LPS-induced TNF-α release mouse model are described.

It has previously been shown that BMS-345541 [4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline], a highly-selective inhibitor of IkappaB kinase (IKK), blocks both inflammation and joint destruction in murine collagen-induced arthritis. Although this agent has been shown to inhibit nuclear factor-kappaB-dependent cytokine expression in mice, we examined whether the inhibitor directly inhibits cytokine-driven metalloproteinase expression and cartilage degradation. In SW-1353 human chondrosarcoma cells, BMS-345541 inhibited interleukin-1 (IL-1)-dependent expression of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 in a concentration-dependent manner. IL-1 treatment failed to induce and BMS-345541 did not inhibit the expression of aggrecanases ADAMTS-4 (a disintegrin and metalloproteinase domain with thrombospondin motif) and ADAMTS-5, as well as the tissue inhibitor of metalloproteinase-3. In bovine cartilage explant cultures stimulated with IL-1 to induce aggrecan and collagen degradation over 3 weeks of culture, BMS-345541 was effective in inhibiting the degradation of both aggrecan and collagen. Secreted ADAMTS-4 was not inhibited by BMS-345541 in these explants, whereas ADAMTS-5 secretion was blocked in the same concentration range that inhibited aggrecan degradation. The ability of the IKK inhibitor to block aggrecan and collagen degradation through suppression of metalloproteinase expression, coupled with its ability to block inflammatory cytokine production, shows IKK to be a promising target for the development of novel agents to treat arthritic diseases.

The signal-inducible phosphorylation of serines 32 and 36 of I kappa B alpha is critical in regulating the subsequent ubiquitination and proteolysis of I kappa B alpha, which then releases NF-kappa B to promote gene transcription. The multisubunit I kappa B kinase responsible for this phosphorylation contains two catalytic subunits, termed I kappa B kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC(50) = 0.3 microm, IKK-1 IC(50) = 4 microm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of I kappa B alpha in cells (IC(50) = 4 microm) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappa B in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and interleukin-6 in THP-1 cells with IC(50) values in the 1- to 5-microm range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of I kappa B alpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor alpha following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappa B activation in mice and represents an important tool for investigating the role of IKK in disease models.