Kinase inhibitor recognition by use of a multivariable QSAR model.

We have applied a retrosynthetic program to determine the scaffold and R-group chemical space seen within a library of known kinase inhibitors and non-kinase drug-like molecules. Comparison of the differences quickly revealed that kinase inhibitors are distinct in several chemical fragment and physical properties. We then applied these descriptors in a multivariable quantitative structure-activity relationship (QSAR) model with the goal to distinguish kinase inhibitors from non-kinase drug-like molecules. This model is heuristic in that it was trained over a dataset of 258 known kinase inhibitors and 230 non-kinase drug molecules. The final model recognized 98% of the training set as being kinase inhibitors and had a false positive rate of 15%. This trait for false positives was accepted out of a desire to maintain diversity and not miss possible good kinase inhibitors for screening. The model was validated by reserving a portion of the datasets as test sets, which were not included in the QSAR model building stage. This was done repetitively for different percentiles of the total dataset population. It was seen that model recognition and false positive were only slightly damaged well down to a 70% reserve (30% dataset used for QSAR model training while 70% used for reserve test set). Beyond 70%, the QSAR models were inconsistent, signifying that the training sets were inadequately diverse to represent the greater reserve test sets. We applied this model to evaluate the commercial kinase libraries available from Asinex, BioFocus, ChemDiv and LifeChemicals to facilitate purchase decisions for compounds for HTS for lead compounds. We observed that there are significant differences in populations of recognizable kinase inhibitors across the vendors analyzed, with BioFocus showing the greatest population of kinase like molecules.

The neutralization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation.

A panel of monoclonal antibodies (mAb) derived against human interferon-alpha/beta receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-alpha1, IFN-alpha2a, and IFN-beta). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-alpha2a and IFN-beta-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-alpha2a and IFN-beta binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-alpha2a and IFN-beta-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation. mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-alpha/beta to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-alpha/beta but did not inhibit IFN-alpha/beta-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation.

Soluble human lymphocyte activation gene-3 modulates allospecific T cell responses.

Lymphocyte activation gene (LAG)-3, a member of the Ig superfamily, has been characterized as an activation antigen of T cells and NK cells. LAG-3 has been proposed as an alternate ligand for HLA class II due to some sequence homology and similarities in exon-intron organization with CD4. Here, we report the functional evaluation of a soluble Ig fusion molecule of human LAG-3 (LAG-3-Ig) in T cell activation assays. Cytofluorimetry studies revealed LAG-3-Ig binding predominantly to class II-expressing cells. In functional assays, inhibition of primary allogeneic mixed lymphocyte response (MLR) and murine-human xenogeneic MLR was observed in the presence of LAG-3-Ig. Effects of LAG-3-Ig addition were not observed on mitogen-, recall antigen- or superantigen-mediated stimulation. Cytotoxic T lymphocyte effector functions were also not affected by LAG-3-Ig. Inhibition of alloresponses by LAG-3-Ig occurred within the first 24 h of activation, resulting in a strong inhibition of IL-2 production. Unlike blockade of the CD28 receptor, however, LAG-3-Ig-mediated inhibition could not be reversed by exogenous IL-2 supplementation. Cytofluorimetric analysis of the phenotype of cells exposed to LAG-3-Ig in MLR cultures revealed a decrease in IL-2 receptor expression (CD25) on CD4+ cells in all donors tested. Based on the results from these studies, we conclude that LAG-3-Ig inhibits alloresponses of naive peripheral blood lymphocytes, by blocking the activation of a subpopulation of allo reactive cells.

Elucidating the mechanism of action of the immunosuppressant 15-deoxyspergualin.

15-Deoxyspergualin (DSG) is an immunosuppressive agent currently in Phase I/II clinical trials. We have previously shown that DSG specifically interacts with Hsc70, a member of the heat shock protein 70 family. In this article we show that DSG appears to bind either kinetically or to a different site on Hsc70 from that of peptides. In addition, we show that DSG inhibits the localization of Hsp70 into the nucleus in response to heat shock. Finally, data are presented showing that there is a correlation between decreases in the transcription factor nuclear factor kappa B and kappa light chain expression in response to varying doses of DSG.

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

Deoxyspergualin inhibits kappa light chain expression in 70Z/3 pre-B cells by blocking lipopolysaccharide-induced NF-kappa B activation.

Deoxyspergualin (DSG) is a potent immunosuppressive agent that is currently undergoing clinical trials for treatment of transplant rejection, preventive of human anti-mouse Ab response, and blocking autoimmune disease progression. The mechanism of action of DSG appears to be novel, with in vivo activity attributable to the suppression of both humoral and cell-mediated immunity. In this study we investigated the effect of DSG on the induction of lg expression in the 70Z/3 murine pre-B cell line. Treatment of 70Z/3 cells with DSG for 24, 48, or 72 h before LPS or IFN-gamma induction resulted in a time-dependent inhibition of surface lgM expression, with greater than 80% inhibition observed after 72 h of pretreatment. Inhibition of surface expression was specific for lgM, as neither MHC class I nor CD45 (B220) surface expression was affected by DSG pretreatment. Cyclosporin A was ineffective at suppressing surface igM induction. DSG pretreatment results in a 10-fold reduction in LPS- or IFN-gamma-induced kappa L chain protein and mRNA expression. No change was observed in either mu or beta-actin mRNA levels. Analysis of nuclear and cytoplasmic NF-kappa B expression using electrophoretic mobility shift analysis and Western analysis, revealed that DSG blocked LPS-induced NF-kappa B nuclear translocation, but had no effect on cytoplasmic NF-kappa B levels. We conclude that DSG may act to suppress humoral immune responses by blocking the transcriptional activation of kappa L chain expression during certain stages of B cell development.

Phase I clinical and pharmacological study of suppression of human antimouse antibody response to monoclonal antibody L6 by deoxyspergualin.

Development of human antimouse antibody (HAMA) is a major limiting factor in the application of murine mAb for clinical use. A novel immunomodulatory drug, deoxyspergualin (DSG), has shown potential to suppress antimouse antibody response in preclinical model systems. We conducted a Phase I trial to determine the effect of DSG on HAMA response to murine mAb L6 administered to patients with advanced cancers (in previous trials, this antibody elicited HAMA in two-thirds of the treated patients). L6 mAb was administered at a fixed dose of 200 mg/m2 on days 1-5. DSG was administered at doses of 50 mg/m2 [dose level (dl) 1] or 150 mg/m2 (dls II and III) on days 1-7. Treatment courses were repeated every 6 weeks (dls I and II) or every 3 weeks (dl III). HAMAs were quantitated by a commercially available ELISA assay (ImmuSTRIP; anti-isotypic antibodies) and a radiometric assay (antiisotypic and anti-idiotypic antibodies). Pharmacokinetics of L6 and DSG was also studied in all consenting patients. Among 24 evaluable patients, 2 patients developed detectable HAMAs using the ELISA (one each at dls I and II) after a median follow-up of 122 days (P = 0.0001 as compared to historical controls). Even in the two patients who developed HAMA, the HAMA levels were quite low (160 and 181 ng/ml; historical experience, 70-38,744 ng/ml). The radiometric assay detected anti-L6 antibodies in 13 patients (4, 6, and 3 at dls I-III, respectively) after a median of 82 days. The median highest anti-L6 antibody level was 129 ng/ml (range, 21-2150). The highest anti-L6 antibody level at dl III was only 44 ng/ml. The results suggest suppression of anti-idiotypic response also. No clinical antitumor activity was observed, and no significant changes in T4/T8 subsets or immunoglobulins occurred (suggesting a lack of generalized immunosuppression). We conclude that DSG can suppress HAMA response to L6. A starting dose of 150 mg/m2/day is recommended for Phase II trials to confirm this observation.

Immunosuppressant deoxyspergualin inhibits antigen processing in monocytes.

Deoxyspergualin (DSG) is a novel immunosuppressive agent recently shown to bind to the constitutive heat shock protein 70, which is involved in binding and intracellular transport of antigenic peptides. In this study, we show that DSG inhibits the proliferation of PBMCs to the Ags tetanus toxoid and diphtheria toxoid, but not to the mitogens PHA and PMA/ionomycin, nor to the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. DSG's effect was specific for monocytes as preincubation of T cells with DSG did not inhibit their proliferation to monocytes pulsed with tetanus toxoid Ag for 16 h, whereas the presence of DSG during Ag pulsing of the monocytes inhibited their ability to stimulate T cell proliferation. DSG did not down-regulate the expression of MHC class II molecules by monocytes, and the inhibitory effect of DSG on T cell proliferation was not reversed by the addition of IL-2, nor by the addition of the costimulatory signals IL-1, IL-6, and anti-CD28. Studies with two human T cell clones, HA1.7 and PF5, specific, respectively, to peptides spanning amino acids 307-319 and 256-270 of influenza hemagglutinin, showed that DSG inhibited the proliferation of the clones to the native hemagglutinin molecule but minimally affected their proliferation to the peptides. These data suggest that DSG interferes with Ag processing and/or presentation.

Prevention of immunotoxin-mediated vascular leak syndrome in rats with retention of antitumor activity.

Immunotoxins are hybrid molecules composed of a cell-surface binding domain and a protein toxin moiety that together target specific cell populations for elimination. These agents represent a promising approach for the treatment of many human diseases, most notably cancer. However, it has recently become clear that many immunotoxins when used in human clinical trials induce vascular leak syndrome (VLS), restricting the administration of doses necessary to achieve good therapeutic responses. The lack of an appropriate animal model has hindered efforts to understand and prevent immunotoxin-induced VLS. We have found that in rats, intravenous administration of the single-chain immunotoxin BR96 sFv-PE40 results in symptoms that closely resemble VLS seen in human immunotoxin trials. A large fluid accumulation in the thoracic cavity was observed, along with an increase in hematocrit and body weight and a decrease in serum albumin. The VLS was apparent within 24 hr after administration of immunotoxin and was seen in both immunocompetent and athymic rats. Similar symptoms were not found in mice even at lethal doses. Prophylactic administration of the corticosteroid dexamethasone resulted in prevention of VLS and survival of rats injected with what would otherwise be lethal doses of BR96 sFv-PE40. Prophylactic treatment with dexamethasone in rats xenografted with human tumors either did not inhibit or minimally inhibited the antitumor activity of BR96 sFv-PE40. The use of prophylactic corticosteroids should be considered for immunotoxin clinical trials, since it may improve therapeutic efficacy by decreasing the dose-limiting toxicity of VLS.

Prevention of the humoral response induced by an anti-CD3 monoclonal antibody by deoxyspergualin in a murine model.

Multiple treatments with the potent immunosuppressant murine antihuman CD3 mAb OKT3 is sometimes precluded by the onset of a neutralizing humoral response mostly consisting of anti-idiotypic antibodies. A hamster antimurine CD3 monoclonal Ab, 145-2C11, shares many properties with OKT3, in particular the ability to induce a strong Ab response in mice. Deoxyspergulain (DSG), a metabolite of the antibiotic spergualin, has been shown to reduce Ab production triggered by pathogens in a variety of infectious models and against common antigens. In this study, we examined the ability of DSG to inhibit the humoral response induced by 145-2C11. DSG prevented the Ab production triggered by the anti-CD3 mAb in an Ag-specific manner and significantly reduced the Ab production in mice previously primed with 145-2C11. We showed that DSG had a long-term effect on B cells and a transient effect on T cells. In effect, DSG was found to induce a prolonged Ag-specific unresponsiveness of B lymphocytes, and to transiently reduce the capacity of T lymphocytes to deliver help to B cells, in part by reducing IL-4 production. DSG did not reduce the immunosuppressive properties of the anti-CD3 mAb. In fact, the combination of DSG with 145-2C11 prolonged the survival of allogeneic skin grafts when compared with the administration of 145-2C11 or DSG alone. Thus, the coadministration of DSG with OKT3 may be of clinical interest to reduce the humoral response triggered by the mAb.

Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90.

Deoxyspergualin (DSG), a spermidinyl, alpha-hydroxyglycyl, 7-guanidinoheptanoyl peptidomimetic, shows immunosuppressive activity. In confirmation of a recent report that immobilized methoxyDSG selectively retains the heat shock protein Hsc70, we report here quantitative binding of DSG and analogs to both Hsc70 and the 90-kDa heat shock protein Hsp90. We have utilized affinity capillary electrophoresis to obtain Kd values for DSG and analogs, and stimulation of the ATPase activity of Hsc70 to obtain Km values for DSG, that are comparable and corroborative. Kd values are 4 microM for DSG binding to Hsc70 and 5 microM for DSG binding to Hsp90. Two active analogs, methoxy- and glycylDSG, bind with similar affinities. Glyoxylylspermidine and des(aminopropyl)DSG, two inactive metabolites, have much reduced affinity for Hsc70 and Hsp90. These data validate binding of these novel immunosuppressant agents to these molecular chaperones, at concentrations in the range of pharmacologically active doses, and indicate that further characterization of Hsc70 and/or Hsp90 as potential targets for DSG is warranted.

Deoxyspergualin--a novel immunosuppressant.

DSG appears to have a unique, although as yet undefined, mechanism of action and may be a useful immunosuppressive agent. Because DSG is effective in reducing preformed antibodies in the xenograft situation, it may have a significant advantage in ABO-incompatible grafts in transplant recipients with high panel-reactive antibodies. Most important, DSG may have a definitive role in immunosuppressive therapy for pancreatic grafts.

15-Deoxyspergualin: a novel immunosuppressive drug with clinical potential.

The studies discussed in this review suggest that DSG is a potent immunosuppressive agent, with at least some of its activity due to its direct effects on macrophages and B cells. The effects of DSG on macrophages include inhibition of IL-1, chemiluminescence, expression of MHC Class I antigen on the surface, and development of MAF. The agent may well interfere with antigen processing and the discovery of its binding to the heat-shock protein 70 may shed some light on this area. The effects of DSG on B lymphocytes include inhibition of surface Ig expression and B cell differentiation. In addition, however, DSG exhibits some effects on both B and T lymphocytes and is markedly active in blocking both the primary and secondary cytotoxic T cell and antibody-producing cell generation. The agent is relatively nontoxic at the doses in which it exerts these marked effects. Thus, an overall assessment of DSG shows that it may provide immune suppression at levels different from those of immunosuppressive agents already available. Advances in human organ transplantation have for the most part occurred stepwise with the introduction of progressively improved techniques of immunosuppression. Tissue-matching, organ preservation, techniques of transplantation, and surgical post-operative care of organ transplant recipients have all improved over the 30 years since organ transplantation began, but have not been the major factors in the improved survival of both patients and grafts seen at the present time. The initial advance in immunosuppression which made the first organ transplants possible was the finding of the immunosuppressive capabilities of azathioprine (Imuran) and the ability to reverse acute rejection crisis with prednisone. Anti-thymocyte or anti-lymphocyte globulin, introduced in 1966, achieved variable results, with some groups reporting excellent patient and graft survival using this agent. Others using different variations of this drug showed no improvement in results. By the mid-1970s, transplant results had improved at a number of units, with graft survivals of kidney and heart recipients in the 70 percent range at 1 to 2 years. No major progress was reported in most units, however, until the introduction in 1978 of cyclosporine. A major effect of cyclosporine was to create a universal improvement in graft survival in all units surveyed around the world with a 70 to 75 percent one-year cadaver kidney graft survival, representing the basic standard for clinical results.(ABSTRACT TRUNCATED AT 400 WORDS)