Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.

The cardiac ATP-sensitive potassium (KATP) channel is thought to be a complex composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the sulfonylurea receptor (SUR2). This channel is activated during myocardial ischemia and protects the heart from ischemic injury. We examined the transcriptional expression of these genes in rats with myocardial ischemia. 60 min of myocardial regional ischemia followed by 24-72 h, but not 3-6 h, of reperfusion specifically upregulated Kir6.1 mRNA not only in the ischemic (approximately 2.7-3.1-fold) but also in the nonischemic (approximately 2.0-2.6-fold) region of the left ventricle. 24 h of continuous ischemia without reperfusion also induced an increase in Kir6.1 mRNA in both regions, whereas 15-30 min of ischemia followed by 24 h of reperfusion did not induce such expression. In contrast, mRNAs for Kir6.2 and SUR2 remained unchanged under these ischemic procedures. Western blotting demonstrated similar increases in the Kir6.1 protein level both in the ischemic (2.4-fold) and the nonischemic (2.2-fold) region of rat hearts subjected to 60 min of ischemia followed by 24 h of reperfusion. Thus, prolonged myocardial ischemia rather than reperfusion induces delayed and differential regulation of cardiac KATP channel gene expression.

Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety.

It is well known that the subdomain III A (site II) of human serum albumin (HSA) binds a variety of endogenous and exogenous substances. However, the nature of the microenvironment of the binding site remains unclear. Ketoprofen (KP), an arylpropionic acid NSAID which contains a benzophenone moiety, was used as a photoaffinity labeling agent to label the binding region. Subsequent CNBr cleavage of the photolabeled HSA revealed that the 11.6 kDa and 9.4 kDa fragments contained most of the incorporated radioactivity. Competition experiments showed that the 11.6 kDa fragment contains the common binding region for site II ligands. This fragment was redigested with Achromobacter lyticus protease I (AP-I) and the amino acid sequence of the photolabeled peptide was determined to be XCTESLVNRR, which corresponds to the sequence 476C-485K of HSA. The complete amino acid sequence of the corresponding AP-I digested HSA peptide encompasses residues 476 to 499, which form helices 5 and 6 of subdomain III A. The HSA-Myr X-ray crystallography data showed that helix 5 is involved to the least extent in ligand binding. A docking model provided further support that helix 6 represents the photolabeled region of KP.

Photochemical localization of the semotiadil binding region within the cardiac Ca2+ channel alpha1 subunit. Comparison with the skeletal muscle counterpart.

We have previously identified the binding region of a new Ca2+ antagonist semotiadil in the skeletal muscle Ca2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51-4700 inhibited [3H]PN200-110 binding to cardiac membrane preparations with IC50 values of 13-20 microM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94-1.0 for the cardiac channels compared to 0.63-0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [3H]D51-4700 was photo-incorporated in several polypeptides but only the alpha1 subunit of the Ca2+ channel was photolabeled in a specific manner. Antibody mapping of the [3H]D51-4700-labeled alpha1 subunit with several anti-peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys1461 and Lys1529. This region encompasses the labeled site of skeletal muscle, but contains several non-identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca2+ channels.

Azidobutyryl clentiazem, a new photoactivatable diltiazem analog, labels benzothiazepine binding sites in the alpha 1 subunit of the skeletal muscle calcium channel.

[3H]Azidobutyryl clentiazem, a new photoactivable diltiazem derivative, has a higher binding affinity than azidobutyryl diltiazem. It can be covalently incorporated into the alpha 1 subunit of the skeletal muscle calcium channel by UV irradiation, which allows the benzothiazepine binding site to be determined. The photolabeled alpha 1 subunit and its proteolytic fragments were analyzed with a panel of sequence-directed antibodies. The results suggest that the linker region between segment S5 and S6 of domain IV is involved in benzothiazepine binding. This site is different from the dihydropyridine and verapamil binding sites.

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells.

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.

Structural characterization of the dihydropyridine receptor-linked calcium channel from porcine heart.

Ca(2+)-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac alpha 1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle alpha 2 delta subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca(2+)-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (approximately 270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (alpha 1) is responsible for photoaffinity labeling with [3H]diazepine, since minor photolabeled approximately 270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (alpha 2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with alpha 1 through disulfide linkage and the other was not covalently associated with any other component.(ABSTRACT TRUNCATED AT 250 WORDS)

CD36, a member of class B scavenger receptor family, is a receptor for advanced glycation end products.

Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Nitric oxide inhibits the proliferation of murine microglial MG5 cells by a mechanism involving p21 but independent of p53 and cyclic guanosine monophosphate.

We investigated the effect of nitric oxide (NO) on the proliferation of microglial MG5 cells established from p53-deficient mice. Cells were treated with bacterial lipopolysaccharide and interferon-gamma, and expression of inducible NO synthase (iNOS) and p21/waf1, a cyclin-dependent kinase inhibitor protein which is a critical downstream effector of p53, was investigated by RNA blot and immunoblot analyses. iNOS mRNA was induced 2 h after treatment and increased with time up to 24 h. p21 mRNA was expressed at a low level in untreated cells and increased with a kinetics similar to that for iNOS mRNA. iNOS and p21 proteins were also induced. An NO donor SNAP induced p21 mRNA and protein. SNAP inhibited incorporation of [(3)H]thymidine in MG5 cells in a dose-dependent manner. 8-Bromo-cGMP neither induced p21 mRNA nor inhibited [(3)H]thymidine incorporation. These results suggest that NO inhibits the proliferation of MG5 cells by induction of p21, which occurs independent of p53 and cGMP.

Intracerebral microinjection of interleukin-4/interleukin-13 reduces ß-amyloid accumulation in the ipsilateral side and improves cognitive deficits in young amyloid precursor protein 23 mice.

We previously reported that the anti-inflammatory cytokine interleukin (IL)-4 induced selective clearance of oligomeric ß-amyloid (Aß(1-42)) in rat primary type 2 microglial cells. For the present study, we investigated whether IL-4 and IL-13 could activate microglial cells to induce Aß clearance in vivo and improve cognitive deficits in APP23 mice, which are amyloid precursor protein transgenic mice. We administered an intracerebral microinjection of a mixture of IL-4 and IL-13 or of saline vehicle into one hemisphere of APP23 mice and their wild-type littermates, 4.5 and 9 months old, after which we evaluated the effects of these treatments on spatial learning and memory by Morris Water Maze test and on accumulated amounts of Aß. The cytokine injection significantly improved memory deficits of 4.5-month-old APP23 mice, but did not do so in 9-month-old APP23 mice, even though similar Aß reductions were observed in both age groups of APP23 mice in the ipsilateral neocortex. The cytokine injection improved memory impairment of 9-month-old wild-type (WT) mice in the probe trial. Immunohistochemical analysis of the 4.5-month-old APP23 mice revealed the presence of increased numbers of microglial cells at 2 days after the cytokine injection. In addition to induced CD36 expression in the activated microglia, increased expression of neprilysin, mainly in neurons, suggested that the cytokines improved the cognitive deficits via degradation and clearance of intra- and extraneuronal Aß peptides, of buffer-extractable nonplaque form. Double immunostaining also revealed that most of the activated microglia had the M2-like phenotype. This unique mechanism of IL-4/IL-13-induced clearance of Aß may provide an additional strategy to prevent and/or cure Alzheimer's disease at early stage.

Identification of binding sites for calcium channel antagonists.

The binding sites of three typical calcium channel antagonists, 1, 4-dihydropyridines, benzothiazepines and phenylalkylamines, were successfully identified within the primary structures of calcium channels using a photoaffinity labeling technique. The results confirm pharmacological observations of the three antagonists that had been proposed to interact allosterically with each other. We briefly review the results and discuss the future prospects.

Synthesis of diazipine and [3H]diazipine: novel dihydropyridines as photoaffinity probes of calcium channels.

Diazipine and [3H]diazipine were synthesized as new 1,4-dihydropyridine photoaffinity ligands containing a phenyldiazirine group. After simple high performance liquid chromatography separation, both compounds were purified in good overall yields. [3H]Diazipine (21.2 Ci/mmol) was synthesized in two steps from commercially available [3H]ethanolamine. Diazipine competitively inhibited [3H]PN200-110 binding to the calcium channel of cardiac membranes with high affinity.

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme.

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.

Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products.

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Photochemical identification of transmembrane segment IVS6 as the binding region of semotiadil, a new modulator for the L-type voltage-dependent Ca2+ channel.

To identify the binding domain of a new Ca2+ antagonist semotiadil on L-type Ca2+ channels from skeletal muscle, photolabeling was carried out by using an azidophenyl derivative of [3H]semotiadil. Photoincorporation was observed in several polypeptides of membrane triad preparations; the only specific photoincorporation was in the alpha1 subunit of the Ca2+ channel. After solubilization and purification, the photolabeled alpha1 subunit was subjected to proteolytic and CNBr cleavage followed by antibody mapping. Specific labeling was associated solely with the region of transmembrane segment S6 in repeat IV. Quantitative immunoprecipitation was found in the tryptic and the Lys-C/Glu-C fragments of 6.6 and 6.1 kDa, respectively. Further CNBr cleavage of the Lys-C digests produced two smaller fragments of 3.4 and 1.8 kDa that were included in the tryptic and Lys-C/Glu-C fragments. The smallest labeled fragments were: Tyr1350-Met1366 and Leu1367-Met1381 containing IVS6, a possible pore-forming region. The data suggest that semotiadil binds to a region that is overlapped with but not identical to those for phenylalkylamines, dihydropyridines and benzothiazepines. The present study also provides evidence that region IV represents an important component of a binding pocket for Ca2+ antagonists.

Effects of hydroxyl radical and sulfhydryl reagents on the open probability of the purified cardiac ryanodine receptor channel incorporated into planar lipid bilayers.

We examine the effect of hydroxyl radical on the ion permeability of the ryanodine receptor, a calcium releasing channel of sarcoplasmic reticulum. The cardiac ryanodine receptor, purified from pig heart, was reconstituted to proteoliposomes and then incorporated into a planar bilayer membrane. A single channel activity with a conductance of 724 pS in 900/200 mM (cis/trans) KC1 and an ion selectivity of PK:PCl = 1:0.08 was observed. These characteristics are similar to those observed by the incorporation of the channel through sarcoplasmic reticulum vesicles. Hydroxyl radicals chemically generated by the reaction of H2O2 and Cu(ethylenediamine)2 at the cis compartment increased the open probability of the channel. Treatment with SH oxidizing reagents from the cis compartment also increased the open probability, and dithiothreitol, a SH reducing agent, reversed the effect. These findings suggest that hydroxyl radicals react with some SH groups of the ryanodine receptor and increase the Ca2+ release from sarcoplasmic reticulum through the ryanodine receptor.

[Glibenclamide inhibits cholesterol metabolism in macrophage].

Sulfonylureas are generally used in the therapeutic treatment of non-insulin-dependent diabetes mellitus. Little is known, however, whether they also affect the lipid metabolism. Using glibenclamide (GB), a typical sulfonylurea, we have investigated its effects on the lipid metabolism in macrophages, J774 and phorbol ester-treated THP-1 cells. In the whole-cell assay system for cholesteryl ester (CE) accumulation that is induced by addition of chemically modified low-density lipoprotein (LDL), such as Ac-LDL and ox-LDL, GB effectively inhibited the CE accumulation of J774 cells in dose-dependent manners. The inhibition was resulted from increase in free cholesterol but not from change in total cholesterol amount. The results suggest that GB acts on acyl-CoA: cholesterol acyltransferase (ACAT) and inhibits its activity. To confirm the possibility, we then tested GB by another assay system using ACAT that was solubilized from the cells and reconstituted into the liposome composed of phosphatidyl choline- cholesterol. GB inhibition was not so much effective as those by CI-976 and NTE-122, known ACAT inhibitors, but the inhibition was complete in the presence of 100 microM GB. Using cell homogenates of PMA-stimulated THP-1 cells, GB also inhibited the ACAT activity to the level of undifferentiated THP-1 cells. These results indicate that GB acts as ACAT inhibitor but the chemical structure is quite different from the conventional ACAT inhibitors, suggesting it can be a seed to generate potential ACAT inhibitors which do not exhibit toxicity in adrenal gland.

Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel.

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.

Na+,K(+)-ATPase inhibiting activity of cardiac glycosides from Erysimum cheiranthoides.

We previously reported the isolation of eleven new cardiac glycosides called cheiranthosides I-XI together with two known ones (olitoriside and erysimoside) from the seeds of Erysimum cheiranthoides L. The glycosides were evaluated for their inhibitory activity against Na+,K(+)-ATPase by comparing with typical cardiac glycosides. Two of them, cheiranthoside III and VIII, showed high inhibiting activity which was equivalent to that of digitoxin. Cheiranthoside XI containing a rhamnopyranosyl digitoxopyranosyl moiety and a carboxyl group showed the lowest activity which was similar to that of the inactive aglycone, strophanthidin. Some characteristics in the structure-activity relationship are also discussed.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione: a versatile radioligand targeting GS-X pumps with the ability of photoaffinity labeling.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione (GIF-0017) as a biochemical probe targeting the ATP-dependent organic anion transporters GS-X pumps was synthesized by the reaction of [35S]glutathione and excess 4-(5-bromo)pentylbenzophenone under alkaline conditions, with the radiochemical yield of 24-33% after HPLC purification. Photolysis of the mixture of [35S]GIF-0017 and plasma membrane vesicles prepared from the MRP1 cDNA-transfected LLC-PK1 cells resulted in radio-labeling of a 180-kDa membrane protein. Immunoprecipitation and western blotting using an anti-MRP1 monoclonal antibody confirmed that the [35S]GIF-0017-labeled protein was the MRPI/GS-X pump.