Indolyl-naphthyl-maleimides as potent and selective inhibitors of protein kinase C-α/ß.

The indolyl-naphthyl maleimide 7 is a potent inhibitor of the classical PKC isotypes α,ß and shows excellent selectivity over the novel PKC isotypes δ,ε,η,θ and other kinases belonging to the AGC family. The SAR around 7 as well as the physico-chemical characteristics of selected derivatives and their activity in T and B cell activation and proliferation assays are discussed.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

A discrete affinity-driven elevation of ZAP-70 kinase activity initiates negative selection.

CONTEXT: Although ZAP-70 is required for T-cell development, it's unclear how this kinase controls both positive and negative selection. OBJECTIVE AND METHODS: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs). RESULTS: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface. DISCUSSION: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface. CONCLUSIONS: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.

The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation.

There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K(i) values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKC catalytic activity and selectively affected both the canonical nuclear factor-kappaB and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody- and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.

Fingerprints of CD8+ T cells on human pre-plasma and memory B cells.

Differentiation of B cells is a stringently controlled multi-step process, which is still incompletely understood. Here we identify and characterize a rare population of human B cells, which surprisingly carry CD8AB on their surface. Existence of such cells was demonstrated both in tonsils and in human apheresis material. Gene expression profiling and real time PCR detected however no CD8A or CD8B message in these cells. Instead, we found that surface CD8 was hijacked from activated CD8+ T cells by a transfer process that required direct cell-to-cell contact. A focused transcriptome analysis at single cell level allowed the dissection of the CD8 positive B cell population. We found that the affected cells are characteristically of the CD27+CD200- phenotype, and consist of two discrete late-stage subpopulations that carry signatures of activated memory B like cells, and early plasmablasts. Thus, there is only a restricted time window in the differentiation process during which B cells can intimately interact with CD8+ T cells. The findings point to a novel link between the T and B arms of the adaptive immune system, and suggest that CD8+ T cells have the capability to directly shape the global antibody repertoire.

Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2.

Interleukin-2 (IL-2) immunotherapy is an attractive approach in treating advanced cancer. However, by binding to its IL-2 receptor α (CD25) subunit, IL-2 exerts unwanted effects, including stimulation of immunosuppressive regulatory T cells (Tregs) and contribution to vascular leak syndrome. We used a rational approach to develop a monoclonal antibody to human IL-2, termed NARA1, which acts as a high-affinity CD25 mimic, thereby minimizing association of IL-2 with CD25. The structure of the IL-2-NARA1 complex revealed that NARA1 occupies the CD25 epitope of IL-2 and precisely overlaps with CD25. Association of NARA1 with IL-2 occurs with 10-fold higher affinity compared to CD25 and forms IL-2/NARA1 complexes, which, in vivo, preferentially stimulate CD8+ T cells while disfavoring CD25+ Tregs and improving the benefit-to-adverse effect ratio of IL-2. In two transplantable and one spontaneous metastatic melanoma model, IL-2/NARA1 complex immunotherapy resulted in efficient expansion of tumor-specific and polyclonal CD8+ T cells. These CD8+ T cells showed robust interferon-γ production and expressed low levels of exhaustion markers programmed cell death protein-1, lymphocyte activation gene-3, and T cell immunoglobulin and mucin domain-3. These effects resulted in potent anticancer immune responses and prolonged survival in the tumor models. Collectively, our data demonstrate that NARA1 acts as a CD25-mimobody that confers selectivity and increased potency to IL-2 and warrant further assessment of NARA1 as a therapeutic.

A locally active antiinflammatory macrolide (MLD987) for inhalation therapy of asthma.

One of the characteristic features of asthma is a persistent pulmonary inflammation, with increased numbers of eosinophils and activated T-lymphocytes in the airways. T-helper cells of the Th2 phenotype play a pivotal role in the pathogenesis of asthma, and they are believed to orchestrate the asthmatic response by releasing a wide repertoire of cytokines. Herein, we describe the design, synthesis, and evaluation in models of allergic asthma of a locally active T-cell modulator, MLD987 (1). Compound 1 is a potent immunosuppressant that inhibits the activation, proliferation, and release of cytokines from T-cells with IC(50) values in the low nanomolar range. In a Brown-Norway rat model of allergic asthma, 1, when given into the airways by intratracheal administration (ED(50) = 1 mg/kg) or by inhalation (ED(50) = 0.4 mg/kg), potently reduced the influx of leukocytes into bronchoalveolar lavage fluid samples obtained from antigen-challenged animals. In contrast, 1 had an appreciably weaker activity in this model when given orally or intravenously. Pharmacokinetic evaluation in rat and rhesus monkey showed that 1 had both a low oral (2-4%) and a low pulmonary (7%, monkey) bioavailability. These findings are consistent with a local site of action of the compound and rule out that its antiinflammatory activity in the lung was caused by systemically absorbed material, which had been swallowed during inhalation or which had entered the circulation via the airways. Local administration and the metabolically soft structure of 1, which favors rapid systemic metabolism to less immunosuppressive metabolite 2, are the main reasons for the low exposure and weak systemic activity of the compound. Administration of a locally active compound such as 1, by inhalation, should reduce systemic side effects. Our results indicate that 1 has the potential to serve as an alternative to inhaled glucocorticosteroids for the long-term therapy of asthma of all grades of severity.

Structure-activity relationship and pharmacokinetic studies of sotrastaurin (AEB071), a promising novel medicine for prevention of graft rejection and treatment of psoriasis.

Protein kinase C (PKC) isotypes have emerged as key targets for the blockade of early T-cell activation. Herein, we report on the structure-activity relationship and the detailed physicochemical and in vivo pharmacokinetic properties of sotrastaurin (AEB071, 1), a novel maleimide-based PKC inhibitor currently in phase II clinical trials. Most notably, the preferred uptake of sotrastaurin into lymphoid tissues is an important feature, which is likely to contribute to its in vivo efficacy.

The critical role of kinase activity of interleukin-1 receptor-associated kinase 4 in animal models of joint inflammation.

OBJECTIVE: We have previously reported that the kinase activity of interleukin-1 receptor-associated kinase 4 (IRAK-4) is important for Toll-like receptor and interleukin-1 receptor signaling in vitro. Using mice devoid of IRAK-4 kinase activity (IRAK-4 KD mice), we undertook this study to determine the importance of IRAK-4 kinase function in complex disease models of joint inflammation. METHODS: IRAK-4 KD mice were subjected to serum transfer-induced (K/BxN) arthritis, and migration of transferred spleen lymphocytes into joints and cartilage and bone degradation were assessed. T cell response in vivo was tested in antigen-induced arthritis (AIA) by measuring the T cell-dependent antigen-specific IgG production and frequency of antigen-specific T cells in the spleen and lymph nodes. T cell allogeneic response was tested in vitro by mixed lymphocyte reaction (MLR). RESULTS: Lipopolysaccharide-induced local neutrophil influx into subcutaneous air pouches was impaired in IRAK-4 KD mice. These mice were also protected from inflammation in the K/BxN and AIA models, as shown by reduced swelling of joints. Histologic analysis of joints of K/BxN serum-injected mice revealed that bone erosion, osteoclast formation, and cartilage matrix proteoglycan loss were reduced in IRAK-4 KD mice. Assessment of T cell response by MLR, by frequency of antigen-specific clones, and by production of antigen-specific IgG did not reveal substantial differences between IRAK-4 KD and wild-type mice. CONCLUSION: These results demonstrate that IRAK-4 is a key component for the development of proarthritis inflammation, but that it is not crucial for T cell activation. Therefore, the kinase function of IRAK-4 appears to be an attractive therapeutic target in chronic inflammation.

Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of protein kinase C isotypes.

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

Anti-LFA-1 monotherapy prevents neointimal formation in a murine model of transplant intimal hyperplasia.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is the pre-eminent cause of late cardiac allograft failure. It is characterized by a concentric intimal hyperplasia, which we designate transplant intimal hyperplasia (TIH). To date, blockade of the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) has been shown to be effective in preventing TIH in experimental models of transplantation, but only when combined with other immunosuppressants. In this study we explored the impact of monotherapy against LFA-1 in a carotid artery allograft model of TIH. METHODS: B10A(2R) (H-2(h2)) mice were used as donors and C57BL/6 (H-2(b)) mice used as recipients. The recipients were treated with a monoclonal antibody against LFA-1alpha (M17/4) or isotype-matched control immunoglobulin. Grafts were harvested after 35 days and analyzed by histomorphometry and immunohistochemistry. Blood samples were taken and analyzed by differential cell count and alloantibody levels. RESULTS: We found that treatment with M17/4 resulted in a significant reduction in TIH compared with controls. Immunostaining revealed that LFA-1alpha blockade inhibited CD45+ leukocyte infiltration, prevented intimal smooth muscle cell (SMC) proliferation, and preserved the medial SMC population. Finally, we demonstrated a reduction in the serum alloantibody titer in the group treated with anti-LFA-1alpha when compared with controls. CONCLUSIONS: We have demonstrated for the first time that LFA-1alpha blockade on its own can prevent development of TIH in an experimental model. The concept of modulating LFA-1alpha-mediated leukocyte migration and T-cell activation may therefore be of relevance to clinical cardiac transplantation and, as such, represents a potential target for therapeutic intervention against clinical CAV.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

Pimecrolimus: absorption, distribution, metabolism, and excretion in healthy volunteers after a single oral dose and supplementary investigations in vitro.

The absorption and disposition of pimecrolimus, a calcineurin inhibitor developed for the treatment of inflammatory skin diseases, was investigated in four healthy volunteers after a single oral dose of 15 mg of [(3)H]pimecrolimus. Supplementary information was obtained from in vitro experiments. Pimecrolimus was rapidly absorbed. After t(max) (1-3 h), its blood concentrations fell quickly to 3% of C(max) at 24 h, followed by a slow terminal elimination phase (average t(1/2) 62 h). Radioactivity in blood decreased more slowly (8% of C(max) at 24 h). The tissue and blood cell distribution of pimecrolimus was high. The metabolism of pimecrolimus in vivo, which could be well reproduced in vitro (human liver microsomes), was highly complex and involved multiple oxidative O-demethylations and hydroxylations. In blood, pimecrolimus was the major radiolabeled component up to 24 h (49% of radioactivity area under the concentration-time curve(0-24) h), accompanied by a large number of minor metabolites. The average fecal excretion of radioactivity between 0 and 240 h amounted to 78% of dose and represented predominantly a complex mixture of metabolites. In urine, 0 to 240 h, only about 2.5% of the dose and no parent drug was excreted. Hence, pimecrolimus was eliminated almost exclusively by oxidative metabolism. The biotransformation of pimecrolimus was largely catalyzed by CYP3A4/5. Metabolite pools generated in vitro showed low activity in a calcineurin-dependent T-cell activation assay. Hence, metabolites do not seem to contribute significantly to the pharmacological activity of pimecrolimus.

Structure of human cyclophilin A in complex with the novel immunosuppressant sanglifehrin A at 1.6 A resolution.

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 A resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA.SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA.SFA complexes is the molecular species mediating the immunosuppressive effect.

Sanglifehrin-cyclophilin interaction: degradation work, synthetic macrocyclic analogues, X-ray crystal structure, and binding data.

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.