Defined protein conjugates as signaling agents in immunoassays

BACKGROUND: Conventional methods for conjugation of macromolecules, such as antibodies and reporter groups, typically yield a mixture ranging from unconjugated starting materials to large aggregates. We explored the use of a solid-phase process to allow improved control in conjugation of macromolecules for use in immunodiagnostic reagents. METHODS: Activated components were sequentially delivered to an immobilized core protein, linking in concentric layers. For immunodiagnostic reagents, proteins with the desired signaling properties were added as interior layers and binding proteins were placed in the final surface layer. After assembly, the conjugates were released into solution by cleaving the linker holding the core protein to the support. Conjugates were prepared with use of three different reporter agents: R-phycoerythrin for microsphere fluorescence flow immunoassay, alkaline phosphatase for enzyme immunoassay, and acridinium for magnetic chemiluminescence immunoassay. For each reporter, six conjugates were prepared with various concentrations of both the reporter and an antibody directed against the alpha-subunit of thyroid-stimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. RESULTS: Products ranged in mass from approximately 1 to approximately 20 MDa. HPLC analysis of the conjugates on a gel-permeation column showed sizes and chromophore contents highly consistent with the intended structures. In appropriate assay formats, the signal generated by a conjugate increased with incubation time, then plateaued at an intensity approximately proportional to the reporter content but relatively independent of the antibody content of the conjugate. The time required to reach this maximum decreased with increasing antibody content. CONCLUSION: The high degree of structural control available with solid-phase assembly and the close correlation of structure with desired function of the resulting conjugates make this an attractive method for preparation of an important class of in vitro diagnostic reagents.     

Inhibitors of the IMPDH enzyme as potential anti-bovine viral diarrhoea virus agents

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 microM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4-800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 microM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 microM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 microM].     

Inhibitors of DNA polymerase III as novel antimicrobial agents against gram-positive eubacteria.

6-Anilinouracils are selective inhibitors of DNA polymerase III, the enzyme required for the replication of chromosomal DNA in gram-positive bacteria (N. C. Brown, L. W. Dudycz, and G. E. Wright, Drugs Exp. Clin. Res. 12:555-564, 1986). A new class of 6-anilinouracils based on N-3 alkyl substitution of the uracil ring was synthesized and analyzed for activity as inhibitors of the gram-positive bacterial DNA polymerase III and the growth of gram-positive bacterial pathogens. Favorable in vitro properties of N-3-alkyl derivatives prompted the synthesis of derivatives in which the R group at N-3 was replaced with more-hydrophilic methoxyalkyl and hydroxyalkyl groups. These hydroxyalkyl and methoxyalkyl derivatives displayed K(i) values in the range from 0.4 to 2.8 microM against relevant gram-positive bacterial DNA polymerase IIIs and antimicrobial activity with MICs in the range from 0.5 to 15 microg/ml against a broad spectrum of gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Two of these hydrophilic derivatives displayed protective activity in a simple mouse model of lethal staphylococcal infection.     

Synthesis of novel natural product-like diaryl acetylenes as hypoxia inducible factor-1 inhibitors and antiproliferative agents

The selaginellin derivatives are a type of novel natural pigments with an unusual alkynyl phenol skeleton from the genus Selaginella. Some of these natural compounds were previously reported to show important bioactivities, including anticancer activity, cardiovascular protection and phosphodiesterase-4 inhibition. We designed and synthesized fifteen biphenyl-containing diaryl acetylene derivatives mimicking the skeleton of natural alkynyl phenols. In MTT assay in cancer cells, compounds 1c, 2d, 2g, 2h, 2i and 2j exhibited potent antiproliferative activity. The evaluation of Hypoxia Inducible Factor-1 (HIF-1) pathway inhibitory activity in dual luciferase assay demonstrated that most tested compounds exhibited moderate to good activities. Compounds 1a, 2f and 2h displayed high HIF-1 inhibitory activities and relatively low cytotoxicity, demonstrating great potential as HIF-1 inhibitors. These results afford a new strategy for the discovery of new HIF-1 inhibitors and anti-proliferative agents from natural or synthetic diaryl acetylene derivatives.

A series of selaginellin analogues, biphenyl-containing diaryl acetylenes, were synthesized and evaluated for their cytotoxic and HIF-1 inhibitory activities.     

Synthesis and biological evaluation of novel quinolone derivatives dual targeting histone deacetylase and tubulin polymerization as antiproliferative agents

A strategy to develop chemotherapy agents by combining two complimentary chemo-active groups into a single molecule may have higher efficacy and fewer side effects than that of single-target drugs. In this article, we describe the synthesis and evaluation of a series of novel dual-acting levofloxacin–HDACi conjugates to target both histone deacetylase (HDAC) and tubulin polymerization. These bifunctional conjugates exhibited potent inhibitory activities against HDACs and tubulin polymerization. In docking analysis provides a structural basis for HDACs inhibition activities. Moreover, these conjugates showed selective anticancer activity that is more potent against MCF-7 compared to other four cancer cells A549, HepG2, PC-3, HeLa, but they had no toxicity toward normal cells.

Synthesis of a series of novel dual-acting levofloxacin–HDACi conjugates, which show potent inhibitory activities against HDACs, tubulin polymerization, and significant antiproliferative effect on MCF-7 cells.

Cancer is a highly complex multifactorial disease involving multiple cross-talking between signaling networks. Almost all single-target-based drugs suffer from severe toxicities or other undesirable side effects. In contrast, combination therapy, which combines multiple anticancer agents working with different mechanisms, might have superior efficacy and fewer side effects compared to single-target treatments.^1,2Histone deacetylases (HDACs) are epigenetic enzymes that are capable of removing acetyl groups from ε-amino groups of lysine residues in histone or other nonhistone proteins.³ Abnormal expression of HDACs has been observed in various types of cancer,^4–6 and these enzymes have emerged as important targets in the development of anticancer drugs. Consequently, inhibition of HDAC activity is now recognized as a powerful strategy for cancer therapy. There are 18 human HDAC isoforms categorized into four major classes: class I (HDACs 1, 2, 3, 8), class IIa (HDACS 4, 5, 7, 9), class IIb (HDACs 6 and 10), and class IV (HDAC 11) are Zn²⁺-dependent metalloenzymes, while class III (SirTs 1–7) are NDA⁺-dependent sirtuins.^7,8 Of these HDAC isoforms, only HDACs 1, 2, 3, and 6 have shown biologically relevant deacetylation ability.⁹ Specially, selective inhibition of HDAC6 may have fewer side effects than pan-HDAC and class I isoform.^10–12To date, more than twenty HDAC inhibitors have been initiated in clinical trials, and four HDAC inhibitors vorinostat (SAHA),¹³ romidepsin (FK-228),¹⁴ belinostat (PXD-101),^15,16 panobinostat (LBH-589),¹⁷ have been approved by FDA for the treatment of T-cell lymphoma, cutaneous T-cell lymphoma and multiple myeloma. However, most of them are pan-HDAC (SAHA, LBH-589) or class I selective (FK-228, PXD-101) inhibitors, which usually lead to several mild to severe side effects.^16,18,19 In addition, most of HDAC inhibitors lack visible efficacy against solid tumor,^14,20 the doses given in clinical are much higher, which severely limit their clinical utility for the treatment of broad spectrum of cancer. Therefore, preclinical evaluation of new HDAC inhibitors will need to focus on improving HDAC isoform selectivity and enhancing potency against solid tumors. One strategy may be able to ameliorate the shortcomings of current inhibitors, which is to develop a dual-acting HDAC inhibitor (HDACi) by incorporation of the surface recognition group of prototypical HDACi into other anticancer drugs, forming a single molecule that can modulate intracellular multiple targets, other than various HDAC isoforms. So far, a few examples of bifunctional HDACi-derived conjugates have been obtained.^21–26 Expansion of the diversity of such bifunctional conjugates could lead to broad acting, therapeutically viable anticancer drugs.In another aspect, fluoroquinolones (FQs) have recently been proven as an excellent class of broad-spectrum anticancer drugs against a variety of cancer cells such as bladder cancer,²⁷ non-small cell lung carcinoma,²⁸ colorectal carcinoma cells,²⁹etc. For instance, it has been demonstrated that levofloxacin (Lv) displays antiproliferative activity against various cancer cells.^30,31 Additionally, many of fluoroquinolones were potent inhibitors of tubulin polymerization and exhibited selective activity against some tumor cell types.^32,33 More importantly, fluoroquinolones have favorable pharmacokinetic profiles and good adsorption, which possess an established record of safety.³⁴ Therefore, on the basis of therapeutic effectives of aforementioned HDACi as well as fluoroquinolone, we conceived that concurrent inhibition of HDAC and tubulin polymerization would be a viable alternative approach for cancer treatment.In this work, we describe the design, synthesis and biological evaluation of novel dual-action levofloxacin–HDACi conjugates, which can be prepared conveniently by direct connection of levofloxacin with a triazole-liked SAHA (Fig. 1). The levofloxacin–SAHA conjugates (compounds 8a–c and 9a–c) of this design not only have HDACi unit but also have a second pharmacologically quinolone scaffold. Thus, they expand the exploration of bifunctional HDACi-derived conjugates. For comparison, the carboxylic acid analogues (by replacing the hydroxamic acid (–CONHOH) group with (–COOH), compounds 6a–c and 7a–c) were also prepared and evaluated for their HDAC and tubulin polymerization inhibition activity, antiproliferative activity and cell-type selectivity, etc.Open in a separate windowFig. 1Design of dual-acting levofloxacin–HDACi conjugates.     

Activities of antimicrobial agents against intracellular pneumococci

Pneumococci can enter and survive inside human lung alveolar carcinoma cells. We examined the activity of azithromycin, gentamicin, levofloxacin, moxifloxacin, penicillin G, rifampin, telithromycin, and trovafloxacin against pneumococci inside and outside cells. We found that moxifloxacin, trovafloxacin, and telithromycin were the most active, but only telithromycin killed all intracellular organisms.     

Synthesis and biological evaluation of 3-nitro-4-chromanone derivatives as potential antiproliferative agents for castration-resistant prostate cancer

A series of novel 3-nitro-4-chromanones were synthesized and their in vitro cytotoxicity was evaluated on castration-resistant prostate cancer cell (CRPC) lines using the sulforhodamine B (SRB) assay. The amide derivatives showed more potent antitumor activity than their corresponding ester derivatives. Most of the tested compounds showed less toxicity towards human fibroblasts (HAF) compared with the tumor cell lines. The optimal compound 36 possessed much more potent antiproliferative activity than the positive compound cisplatin. The colony formation, cell cycle distribution, apoptosis, transwell migration and wound healing assays of 36 were performed on CRPC cell lines.

A series of novel 3-nitro-4-chromanones were synthesized and their in vitro cytotoxicity was evaluated on castration-resistant prostate cancer cell lines.     

GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling

Tsc22d3 coding for glucocorticoid-induced leucine zipper (GILZ) was initially identified as a dexamethasone-responsive gene involved in the control of T lymphocyte activation and apoptosis. However, the physiological role of this molecule and its function in the biological activity of glucocorticoids (GCs) has not been clarified. Here, we demonstrate that GILZ interacts directly with Ras in vitro and in vivo as shown by GILZ and Ras coimmunoprecipitation and colocalization upon PMA activation in primary mouse spleen T lymphocytes and thymus cells. The analysis of GILZ mutants showed that they bound Ras through the tuberous sclerosis complex box (TSC) and, depending on the Ras activation level, formed a trimeric complex with Ras and Raf, which we previously identified as a GILZ binder. As a consequence of these interactions, GILZ diminished the activation of Ras and Raf downstream targets including ERK1/2, AKT/PKB serine/threonine kinase, and retinoblastoma (Rb) phosphorylation and cyclin D1 expression, leading to inhibition of Ras- and Raf-dependent cell proliferation and Ras-induced NIH-3T3 transformation. GILZ silencing resulted in an increase in concanavalin A-induced T cell proliferation and, most notably, inhibition of dexamethasone antiproliferative effects. Together, these findings indicate that GILZ serves as a negative regulator of Ras- and Raf-induced proliferation and is an important mediator of the antiproliferative effect of GCs.     

Design,synthesis and biological evaluation of novel nitric oxide-donating podophyllotoxin derivatives as potential antiproliferative agents against multi-drug resistant leukemia cells

Multidrug resistance remains a major obstacle for the effective treatment of carcinoma. To find new drugs for the chemotherapy of drug-resistant leukemia, in this study, two novel nitric oxide-donating podophyllotoxin derivatives were synthesized and preliminarily evaluated in vitro. Biological evaluation indicated that the more active molecule, S1, enhanced the intracellular NO level and significantly inhibited the proliferation of drug-resistant K562/VCR and K562/ADR cells with IC₅₀ values of 0.008 ± 0.001 and 0.007 ± 0.001 μM, respectively, which were similar to that of sensitive K562 cells. Furthermore, it was observed that S1 blocked the G2 phase of the K562/ADR cell cycle by disruption of the microtubule organization and inhibition of CDK1 and CDK2 expression. Meanwhile, S1 induced apoptosis of K562/ADR cells via mitochondrial depolarization and activation of caspase-3. In addition, S1 suppressed the P-gp expression, induced autophagy by regulation of Beclin1 and LC3-II, and inhibited the mTOR and STAT3 signaling in K562/ADR cells. Overall, S1 may be a promising candidate against drug-resistant leukemia.

Conjugate S1 exhibited potential antiproliferative activity against multi-drug resistant leukemia cells.     

Structure-based design,synthesis, and biological evaluation of novel piperine–resveratrol hybrids as antiproliferative agents targeting SIRT-2

A series of novel piperine–resveratrol hybrids 5a–h was designed, synthesized, and structurally elucidated by IR, and ¹H, ¹³C, and ¹⁹F NMR. Antiproliferative activities of 5a–h were evaluated by NCI against sixty cancer cell lines. Compound 5b, possessing resveratrol pharmacophoric phenolic moieties, showed a complete cell death against leukemia HL-60 (TB) and Breast cancer MDA-MB-468 with growth inhibition percentage of −0.49 and −2.83, respectively. In addition, 5b recorded significant activity against the other cancer cell lines with growth inhibition percentage between 80 to 95. New 5a–h hybrids were evaluated for their inhibitory activities against Sirt-1 and Sirt-2 as molecular targets for their antiproliferative action. Results showed that compounds 5a–h were more potent inhibitors of Sirt-2 than Sirt-1 at 5 μm and 50 μm. Compound 5b showed the strongest inhibition of Sirt-2 (78 ± 3% and 26 ± 3% inhibition at 50 μM and 5 μM, respectively). Investigation of intermolecular interaction via Hirschfeld surface analysis indicates that these close contacts are mainly ascribed to the O–H⋯O hydrogen bonding. To get insights into the Sirt-2 inhibitory mechanism, a docking study was performed where 5b was found to fit nicely inside both extended C-pocket and selectivity pocket and could compete with the substrate acyl-Lys. Another possible binding pattern showed that 5b could act by partial occlusion of the NAD⁺ C-pocket. Collectively, these findings would contribute significantly to better understanding the Sirt-2 inhibitory mechanism in order to develop a new generation of refined and selective Sirt-2 inhibitors.

A series of novel piperine–resveratrol hybrids 5a–h was designed, synthesized, and structurally elucidated by IR, and ¹H, ¹³C, and ¹⁹F NMR.     

Inhibitors of chymase as mast cell-stabilizing agents: contribution of chymase in the activation of human mast cells.

There has long been evidence that inhibitors of chymotryptic proteinases can inhibit the degranulation of rodent mast cells, but their actions on human mast cells and the contribution of mast cell chymase itself have received little attention. We investigated the ability of the selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO(2)Me and other proteinase inhibitors to inhibit chymase and cathepsin G activity, and we examined their potential to modulate the responsiveness of mast cells dispersed from human skin, lung, and tonsil tissues. IgE-dependent histamine release from skin mast cells was inhibited by up to about 80% after preincubation with Z-Ile-Glu-Pro-Phe- CO(2)Me (up to 0.1 microM), 70% with chymostatin (17 microM), and 60% with soybean trypsin inhibitor (0.5 microM). The mast cell-stabilizing properties of chymase inhibitors appeared to be greater for skin mast cells than for those from lung, whereas tonsil mast cells were relatively unresponsive. There were marked differences in the time course of responses to inhibitors, and the effect was dependent on the stimulus, with calcium ionophore-induced histamine release being unaffected. Incubation of dispersed skin, lung, or tonsil cells for up to 45 min with purified chymase failed to induce histamine release, although preincubation of cells with chymase was able to suppress IgE-dependent activation. Chymase could thus contribute to mast cell degranulation and after secretion could provide a feedback mechanism to limit this process. Nevertheless, inhibitors of chymase can be potent mast cell stabilizers, particularly in the skin.     

Ion channels and intracellular signaling proteins as potential targets for novel therapeutics for addictive and depressive disorders

Modern neuroscience is placing increased emphasis on understanding how the activity of ion channels and intracellular molecules in the central nervous system affect behavior. An improved understanding of the brain and the biological bases of conditions such as addictive and depressive disorders is important because it should ultimately enable the design of innovative treatments for these conditions. The development of rational therapies that are based on knowledge of what is different about the addicted or depressed brain would be an important advance. Here, we describe how multidisciplinary studies that combine numerous approaches (behavioral analysis, physiology, molecular biology, and genetic engineering) have begun to provide important advances that have helped to establish causal relationships between the pathophysiology of these conditions and behavior. This type of work has identified classes of molecules on the outside of cells (receptors and ion channels) that receive signals from other cells and initiate cellular events that have short-term effects on the neurons. It has also identified other classes of molecules that are inside of cells (signal transduction molecules) that can have immediate effects on cell function (e.g., ion channel phosphorylation), as well longer term effects (alterations in protein expression) that affect the ways in which neurons function within circuits. Innovative treatments that block, negate, or even reverse the extracellular or intracellular neuroadaptations resulting from exposure to drugs of abuse or stress might be more effective than current therapies because they directly target the molecular processes that cause maladaptive behaviors.     

Mutagenesis studies toward understanding the intracellular signaling mechanism of antithrombin

Summary.  Background:  Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties. Objectives:  The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site. Methods:  Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P₁). Furthermore, the roles of prostacyclin and nuclear factor (NF)-κB in modulating these effects were investigated. Results:  Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-κB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P₁ was inconsequential. Conclusions:  The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P₁-related G_i-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.     

Resistance mechanisms associated with altered intracellular distribution of anticancer agents

The resistance of tumor cells to anticancer agents remains a major cause of treatment failure in cancer patients. The term multidrug resistance (MDR) is used to define a resistance phenotype where cells are resistant to multiple drugs with no obvious structural resemblance and with different molecular targets. It is now clear that MDR is always multifactorial. The intracellular drug distribution is modified in many MDR cell lines, leading to increased drug sequestration in acidic vesicles, such as the trans-Golgi apparatus, recycling endosomes, and lysosomes, followed by transport to the plasma membrane and extrusion into the external medium. Since most anticancer agents target DNA or nuclear enzymes, sequestration of drug in cytoplasmic organelles will lead to decreased drug-target interaction and thereby, decreased cytotoxicity. Altered intracellular drug distribution is usually associated with the expression of drug efflux pumps, such as the P-glycoprotein and the multidrug resistance protein. Another common modification in MDR cells is alkalization of the intracellular pH. The relationship between these different resistance mechanisms is reviewed and a model proposed that suggests why these different resistance mechanisms are co-expressed in multiple cell lines.     

Deoxysugars as Antituberculars and Alpha-Mannosidase Inhibitors

A promising modified sugar molecule was identified which was active against multidrug-resistant (MDR) strains of Mycobacterium tuberculosis, suggesting involvement of a new target. The compound was demonstrated to be bactericidal, inhibited the growth of M. tuberculosis in mice, and targeted alpha-mannosidase as a competitive inhibitor with a K_i value of 353.9 μM.