Inhibitors of BTK and ITK: state of the new drugs for cancer, autoimmunity and inflammatory diseases.

BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and T cell development, with loss-of-function mutations causing X-linked agammaglobulinemia and susceptibility to severe, frequently lethal, Epstein-Barr virus infection, respectively. Over the last few years, considerable efforts have been made in order to develop small-molecule inhibitors for these kinases to treat lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The rationale is that even if complete lack of BTK or ITK during development causes severe immunodeficiency, inactivation after birth may result in a less severe phenotype. Moreover, therapy can be transient or only partially block the activity of BTK or ITK. Furthermore, a drug-induced B cell deficiency is treatable by gamma globulin substitution therapy. The newly developed BTK inhibitor PCI-32765, recently renamed Ibrutinib, has already entered several clinical trials for various forms of non-Hodgkin lymphoma as well as for multiple myeloma. Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity. ITK inhibitors are still under the early developmental phase, but it can be expected that such drugs will also become very useful. In this study, we present BTK and ITK with their signalling pathways and review the development of the corresponding inhibitors.

The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species.

Cytoplasmic protein-tyrosine kinases (PTKs) are enzymes involved in transducing a vast number of signals in metazoans. The importance of the Tec family of kinases was immediately recognized when, in 1993, mutations in the gene encoding Bruton's tyrosine kinase (Btk) were reported to cause the human disease X-linked agammaglobulinemia (XLA). Since then, additional kinases belonging to this family have been isolated, and the availability of full genome sequences allows identification of all members in selected species enabling phylogenetic considerations. Tec kinases are endowed with Pleckstrin homology (PH) and Tec homology (TH) domains and are involved in diverse biological processes related to the control of survival and differentiation fate. Membrane translocation resulting in the activation of Tec kinases with subsequent Ca2+ release seems to be a general feature. However, nuclear translocation may also be of importance. The purpose of this essay is to characterize members of the Tec family and discuss their involvement in signaling. The three-dimensional structure, expression pattern and evolutionary aspects will also be considered.

Nucleocytoplasmic shuttling of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

Signalling of Bruton's tyrosine kinase, Btk.

Bruton's tyrosine kinase, which is encoded by the BTK gene, is a cytoplasmic protein tyrosine kinase (PTK) crucial for B-cell development and differentiation. It belongs to the Tec family of PTKs containing several domains that are characteristic of signalling molecules. In humans, mutations that disrupt the function of this gene lead to the classical XLA syndrome (X-linked agammaglobulinaemia), a primary immunodeficiency mainly characterized by lack of mature B cells as well as low levels of immunoglobulins. In contrast, animal models of this disease such as the xid mice display profoundly milder XLA phenotype. BTK phosphorylation and activation in response to engagement of the B-cell receptor (BCR) by antigen is a dynamic process whereby a variety of proteins interact with each other and recruit signalling molecules resulting in a physiological response such as B-cell proliferation and antibody production. The main players, however, that participate in the intracellular downstream cascade have not yet been identified and are therefore under intense scrutiny in several laboratories. This review discusses certain aspects of BTK activation following receptor stimulation by agonists and how this event is translated into the biochemical signals within the cell that eventually lead to nuclear responses.

Missense mutations affecting a conserved cysteine pair in the TH domain of Btk.

Tec family protein tyrosine kinases have in their N-terminus two domains. The PH domain is followed by Tec homology (TH) domain, which consists of two motifs. The first pattern, Btk motif, is also present in some Ras GAP molecules. C-terminal half of the TH domain, a proline-rich region, has been shown to bind to SH3 domains. Mutations in Bruton's tyrosine kinase (Btk) belonging to the Tec family cause X-linked agammaglobulinemia (XLA) due to developmental arrest of B cells. Here we present the first missense mutations in the TH domain. The substitutions affect a conserved pair of cysteines, residues 154 and 155, involved in Zn2+ binding and thereby the mutations alter protein folding and stability.

Partial purification of a cyanobacterial membrane protein with amino terminal sequence similarity to the N-methylphenylalanine pilins.

Cyanobacterial pilin was extracted from Synechococcus 6301 membranes using a detergent mixture comprising 1% Triton X-100, 1% Thesit and 0.5% dodecyl beta-D-maltoside. Partial purification of pilin from the crude extract was achieved by a single-step purification applying the Rotofor isoelectric focusing system. Up to 100-fold purification of pilin from the crude extract was achieved in a single run. SDS-PAGE analysis showed Synechococcus 6301 pilin migration with an apparent molecular weight of 11 kDa. The amino terminal sequence of the first 28 amino acid residues was identified. Alignment of the predicted sequence showed a 60-80% identity with amino terminal sequences of pilins from pathogenic gram-negative bacteria (enterobacteria). The apparent mass of Synechococcus 6301 pilin was, however, lower. The amino terminus of Synechococcus 6301 pilin, as with other pilins, has a high content of hydrophobic amino acids.

Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.

Proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: purification, characterization, and reconstitution.

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4 mumol of PPi hydrolyzed min-1 mg-1 at pH 7.5 and 20 degrees C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56,000. The oligomeric structure of the H(+)-PPi synthase is discussed.

Comparison of the contribution from different energy-linked reactions to the function of a membrane potential in photosynthetic bacteria.

The steady-state membrane potentials generated by light, PP(i), ATP or the reverse transhydrogenase reaction were studied in chromatophores from two different phototrophic bacteria, Rhodospirillum rubrum and Rhodopseudomonas viridis. The membrane potentials generated by the different energy-linked reactions were evaluated by a tetraphenylboron(TPB(-)) ion-selective electrode. The generated by light was estimated to be 110 mV and 50 mV in R. rubrum and Rps. viridis chromatophores, respectively. In the dark, PP (i), ATP and reversed transhydrogenase generated membrane potentials in R. rubrum and Rps. viridis chromatophores 50, 60 and 35 mV, and 14, 35 and 25 mV,respectively. The effect of magnesium ion on the membrane potential generated by different energy-linked reactions was also studied. The induced by different energy-generating reactions in R. rubrum and Rps. viridis chromatophores and the possible relationship to the chromatophore structures are discussed.

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria.

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PPi) in three phototrophic bacteria. Formation of PPi was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PPi synthesis in Rps. viridis was 0.15 µmol PPi formed/(min*µmol Bacteriochlorophyll) at 23°C. The synthesis of PPi was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PPi synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PPi after short light flashes was also studied. The yield of PPi after a single 1 ms flash was equivalent to approximately 1 µmol PPi/500 µmol Bacteriochlorophyll. In Rps. viridis chromatophores, PPi was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.

delta pH driven energy-linked NAD+ reduction in Rhodospirillum rubrum chromatophores.

An artificial proton gradient provided sufficient energy to drive reverse electron transport from succinate to NADH:ubiquinone oxidoreductase in chromatophores isolated from Rhodospirillum rubrum. The pH gradient created was able to reduce NAD+. In chromatophores, the optimal rate of NAD+ reduction was about 0.4-0.45 mumol NADH formed/min.mumol bacteriochlorophyll at delta pH 3. The presence of oligomycin was an obligate factor in the assay in order to observe the maximal rate of NAD+ reduction. The rate of NADH formation was dependent on the size of the induced pH gradient. The total NADH formed had a threshold value for the imposed delta pH. The effect of different inhibitors and uncouplers was demonstrated. Comparison between ATP, PPi, and light with the pH jump driven NAD+ reduction rate was studied.

The coupling between protonmotive force and the NAD(P)+ transhydrogenase in chromatophores from photosynthetic bacteria.

1. The activity of NAD(P)+ transhydrogenase in chromatophores of Rhodobacter capsulatus relaxed from a high rate during illumination to a lower rate after darkening with a half-time of approximately 100 ms. 2. The dissipative ionic current flowing across the chromatophore membrane was increased in the presence of transhydrogenase substrates. This is attributed to proton current through the transhydrogenase enzyme. Subject to the assumption that transhydrogenase does not conduct in the absence of nucleotide substrates, the ratio of protons translocated across the membrane per hydride ion transferred was 0.4 +/- 0.5. Within the error and uncertainities in the calibration procedure, this ratio may be consistent with a stoichiometry of one but higher values seem unlikely. The ratio of hydride ion transferred in the transhydrogenase to electrons transferred through the cyclic electron transport system was approximately 0.2. 3. The Kappm values for the transhydrogenase substrates were determined for chromatophores in illuminated and darkened suspensions over a range of pH. These values are discussed in relation to the equivalent parameters reported for mitochondria transhydrogenase [Rydstrom, J. (1977) Biochim. Biophys. Acta 255, 9641-9646] and were used to calculate the concentrations of substrates which effectively saturate the enzyme. 4. At substrate concentrations which were in excess of 8 X Kappm the dependence of transhydrogenase rate on the value of the membrane potential (zero pH gradient) was determined at pH 6.3, 6.9, 7.6 and 9.0. The relation was similar at pH 6.9 and 7.6. At alkaline pH the apparent threshold in the relation became more prominent as it was shifted to slightly higher values of membrane potential. At acid pH a shift in the opposite direction diminished the apparent threshold and saturation at high membrane potential became more dominant. We use these data in an attempt to discriminate between two models of energy transduction: (a) the driving force exerted by the membrane potential is mediated by a pH gradient formed through the operation of a proton well in the transhydrogenase; (b) the membrane potential increases a rate constant for charge translocation through transhydrogenase by decreasing the effective height of the Eyring barrier for charge transfer across the membrane through the enzyme. The second model leads to a more simple description than the first of the pH dependence of transhydrogenase rate on membrane potential.4+ transhydrogenase activity in chromatopho

Diethylstilbestrol is a potent inhibitor of the H (+) (-)PPase but not of the H (+) (-)ATPase of Rhodospirillum rubrum chromatophores.

In RhodospiriUum rubrum chromatophores, diethylsrilbestrol inhibits the photoinduced synthesis of ATP and PPi by the membrane-bound H +-ATPase and H +-PPase, respectively. 50% inhibition of ATP synthesis is obtained at 8 µM diethylsrilbestroi in the presence of 0.13 µM BChi, while Is0 for the PPi formation is 20 µM diethylstilhestrol at the same chromatophore concentration. Diethylstilbestroi also inhibits the hydrolyricactivity of the H +-PPase, both in the membrane-bound and in the solubilized and purified state.Inhibition to 50% is already attained at 3 µM diethyistilbestrol in chromatopbores when 1 µM FCCP ispresent and the BChl-concentrarion is 0.62 µM. The hydrolysis by the solubilized enzyme has an /50 of 5 µM when 5 µg protein/ml is used. In contrast to the PPi-hydrolysis, the ATPase activity of thechromatophores shows a small activation at low diethylstiihestroi concentration and becomes inhibited at higher concentrations. Also, solubilized FoFI-ATPase is activated to a small extent by diethyisrilbestrol at the concentrations tested. At low concentrations of BChl, the inhibitory action of diethyistilhestrol on ATP and PPI synthesis can be reversed by addition of bovine serum albumin. The time dependence and inhibition dependence on the energy state of the membrane and on the BChl concentration are examined for the ATP synthesis. The mechanism of inhibition by diethylsrilbestrol is discussed.

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores.

Photosynthetic formation of inorganic pyrophosphate (PP(i)) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP(i) synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP(i) synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP(i) synthesis much less. Even at low rates of electron transport a significant rate of PP(i) synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPI synthesis is inhibited. It is shown that PP(i) synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.

Synthesis of pyrophosphate coupled to the reverse energy-linked transhydrogenase reaction in Rhodospirillum rubrum chromatophores.

Chromatophores from the photosynthetic bacterium Rhodospirillum rubrum contain a membrane-bound transhydrogenase catalyzing the transfer of a hydride ion between NADH and NADP⁺. The reverse reaction, i.e. reduction of NAD⁺ by NADPH, can furnish sufficient energy (ΔµH⁺) to drive the phosphorylation of inorganic orthophosphate (P(i)) to pyrophosphate (PP(i)). The rate of PP(i), synthesis is 50 nmol PP(i) formed/min per µmol Bchl which is 5% of the rate of light-induced PP(i), synthesis. PP(i), synthesis is inhibited by both the H+-PPase inhibitor fluoride and the specific transhydrogenase inhibitor palmitoyl-CoA. The effects of both DCCD and uncouplers on the system provide additional evidence that the ΔµH⁺ generated by the reverse transhydrogenase reaction drives PP(i), synthesis. The rate of PP(i), synthesis can be partially inhibited by the addition of NADP⁺, a substrate of the forward energy-consuming reaction. The ΔµH⁺ generated can also be used to drive ATP synthesis by the H⁺-ATPase, but at a lower rate than the PP(i), synthesis.