Structure-based virtual screening of novel tubulin inhibitors and their characterization as anti-mitotic agents

Microtubule cytoskeletons are involved in many essential functions throughout the life cycle of cells, including transport of materials into cells, cell movement, and proper progression of cell division. Small compounds that can bind at the colchicine site of tubulin have drawn great attention because these agents can suppress or inhibit microtubule dynamics and tubulin polymerization. To find novel tubulin polymerization inhibitors as anti-mitotic agents, we performed a virtual screening study of the colchicine binding site on tubulin. Novel tubulin inhibitors were identified and characterized by their inhibitory activities on tubulin polymerization in vitro. The structural basis for the interaction of novel inhibitors with tubulin was investigated by molecular modeling, and we have proposed binding models for these hit compounds with tubulin. The proposed docking models were very similar to the binding pattern of colchicine or podophyllotoxin with tubulin. These new hit compound derivatives exerted growth inhibitory effects on the HL60 cell lines tested and exhibited strong cell cycle arrest at G2/M phase. Furthermore, these compounds induced apoptosis after cell cycle arrest. In this study, we show that the validated derivatives of compound 11 could serve as potent lead compounds for designing novel anti-cancer agents that target microtubules.     

Targeting Eph receptors with peptides and small molecules: progress and challenges

The Eph receptors are a large family of receptor tyrosine kinases. Their kinase activity and downstream signaling ability are stimulated by the binding of cell surface-associated ligands, the ephrins. The ensuing signals are bidirectional because the ephrins can also transduce signals (known as reverse signals) following their interaction with Eph receptors. The ephrin-binding pocket in the extracellular N-terminal domain of the Eph receptors and the ATP-binding pocket in the intracellular kinase domain represent potential binding sites for peptides and small molecules. Indeed, a number of peptides and chemical compounds that target Eph receptors and inhibit ephrin binding or kinase activity have been identified. These molecules show promise as probes to study Eph receptor/ephrin biology, as lead compounds for drug development, and as targeting agents to deliver drugs or imaging agents to tumors. Current challenges are to find (1) small molecules that inhibit Eph receptor-ephrin interactions with high binding affinity and good lead-like properties and (2) selective kinase inhibitors that preferentially target the Eph receptor family or subsets of Eph receptors. Strategies that could also be explored include targeting additional Eph receptor interfaces and the ephrin ligands.     

Modulation of microtubule dynamics by drugs: a paradigm for the actions of cellular regulators

Microtubules are intrinsically dynamic polymers. Two kinds of dynamic behaviors, dynamic instability and treadmilling, are important for microtubule function in cells. Both dynamic behaviors appear to be tightly regulated, but the cellular molecules and the mechanisms responsible for the regulation remain largely unexplored. While microtubule dynamics can be modulated transiently by the interaction of regulatory molecules with soluble tubulin, the microtubule itself is likely to be the primary target of cellular molecules that regulate microtubule dynamics. The antimitotic drugs that modulate microtubule dynamics serve as excellent models for such cellular molecules. Our laboratory has been investigating the interactions of small drug molecules and stabilizing microtubule-associated proteins (MAPs) with microtubule surfaces and ends. We find that drugs such as colchicine, vinblastine, and taxol, and stabilizing MAPs such as tau, strongly modulate microtubule dynamics at extremely low concentrations under conditions in which the microtubule polymer mass is minimally affected. The powerful modulation of the dynamics is brought about by the binding of only a few drug or MAP molecules to distinct binding sites at the microtubule surface or end. Based upon our understanding of the well-studied drugs and stabilizing MAPs, it is clear that molecules that regulate dynamics such as Kin 1 and stathmin could bind to a large number of distinct tubulin sites on microtubules and employ an array of mechanisms to selectively and powerfully regulate microtubule dynamics and dynamics-dependent cellular functions.     

Hybrid molecules between distamycin A and active moieties of antitumor agents

The DNA minor groove is an attractive target for the design and development of molecules able to specifically recognize predetermined DNA sequences. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicle for the delivery of alkylating functions to DNA targets. Selectivity for specific sequences may be of particular importance in affecting the activity of regulatory genes (oncogenes and tumor suppressor genes). Recent work on a number of hybrid compounds, in which known antitumor compounds or simple active moieties of known antitumor agents have been tethered to distamycin frame or hairpin polyamides derived from distamycin, is reviewed. The DNA alkylating and growth inhibition activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methyl pyrrolic rings and the type of the alkylating unit tethered to the oligopyrrolic frame.     

Effects of cytochalasin B on cell movements and chemoattractant-elicited actin changes of Dictyostelium

The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB-binding sites per cell detectable after cell breakage and preparation of Triton-insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25-45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3-5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G-actin, that the first (3-5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis.     

The challenge of multidrug resistance: actual strategies in the development of novel antibacterials

Bacterial resistance against established antibiotics is becoming an increasingly important global healthcare problem. Despite enormous efforts, the number of therapeutically useful compounds that emerge from chemical derivatisation programs, which aim at circumventing mechanisms of resistance, is continuously decreasing and no truly novel class of compound has been introduced into therapy for nearly four decades. Hopes are now set on a thorough elucidation of bacterial cell functions to identify new bacterial target sites, and on the development of novel compounds with alternative modes of action. The pursuit of these strategies is rendered possible by employment of biotechnologically based methods such as in vivo modification of biosynthetic routes in antibiotic-producing organisms, large-scale screening assays with isolated bacterial targets, the molecular profiling of bacterial genomes and proteomes, and the development and clinical use of biochips as diagnostic tools for rapid identification and characterization of pathogenic strains. As one of the most promising class of compounds known to date with unique modes of action that escape most known mechanisms of resistance, peptic agents have recently came under the focus of anti-infective research, just as extracellular signalling molecules (autoinducer) have emerged as new bacterial target sites.The mentioned antibiotic preparations Augmentan, Klacid, Rulid, Synercid and Ketek are registered trade names of GlaxoSmithKline, Abbott and Aventis, respectively.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.     

Effects of estramustine, a new anti-microtubule drug, on the induction of casein gene expression by prolactin

Estramustine, a new anti-microtubule drug, was added to the culture medium of rabbit mammary explants with lactogenic hormones. In the absence of the drug, prolactin with insulin and cortisol stimulated DNA synthesis and it induced beta-casein and beta-casein mRNA accumulation in the tissue. As opposed to other anti-microtubule agents such as colchicine, estramustine was unable to prevent prolactin actions. An examination of the mammary cells by immunofluorescence revealed that the microtubule network was significantly altered under the influence of estramustine. These data indicate that the integrity of microtubules is not required for prolactin to deliver its message to the mammary cell. These data also suggest that other anti-microtubule drugs such as colchicine which prevent prolactin action act through their binding to tubulin molecule unrelated to microtubule structures.     

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.     

A comparative study of inhibition of acetylcholinesterase,trypsin, neuropathy target esterase,and spleen cell activation by structurally related organophosphorus compounds

Organophosphorus (OP) compounds can bind to and inactivate several target molecules other than acetylcholinesterase (AChE). In the present study, five sets of structurally related organophosphorus compounds were used to evaluate the relationships between organophosphorus binding sites of AChE, neuropathy target esterase (NTE), trypsin, and the target molecule(s) involved in inhibition of splenocyte activation by OP compounds. The concentration of each OP compound required to inhibit enzyme activity or splenocyte activation by concanavalin A by 50% was determined. The pattern of IC₅₀ values indicated that AChE, trypsin, NTE, and the molecule(s) involved in inhibition of splenocyte activation are distinct with regard to patterns of inhibition by OP compounds. However, there was a striking similarity in the patterns of inhibition for trypsin and NTE with substantial differences for only 2 of 20 compounds. This pattern suggests similarity in the active sites of these molecules. There were also similarities in the IC₅₀ patterns for lymphocyte activation and trypsin or NTE activity. However, the correlation was not as strong as between NTE and trypsin, and the data suggested the possibility of multiple target molecules for inhibition of splenocyte activation by OP compounds. More importantly, there was essentially no correlation between the pattern of IC₅₀ values for AChE and splenocyte activation. This strongly suggests that acetylcholine and AChE of the type found in the brain are not important in the regulation of splenocyte activation by concanavalin A.     

Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library

High-throughput fluorescent intercalator displacement (HT–FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT–FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.     

Polyreactivity of natural antibodies: Exchange by HL-fragments

The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.     

Pathogens, toxins, and lipid rafts

Summary The plasma membrane is not a uniform two-dimensional space but includes various types of specialized regions containing specific lipids and proteins. These include clathrin-coated pits and caveolae. The existence of other cholesterol- and glycosphingolipid-rich microdomains has also been proposed. The aim of this review is to illustrate that these latter domains, also called lipid rafts, may be the preferential interaction sites between a variety of toxins, bacteria, and viruses and the target cell. These pathogens and toxins have hijacked components that are preferentially found in rafts, such as glycosylphosphatidylinositol-anchored proteins, sphingomyelin, and cholesterol. These molecules not only allow binding of the pathogen or toxin to the proper target cell but also appear to potentiate the toxic action. We briefly review the structure and proposed functions of cholesterol- and glycosphingolipid-rich microdomains and then describe the toxins and pathogens that interact with them. When possible the advantage conferred by the interaction with microdomains will be discussed.Abbreviation GPI glycosylphosphatidylinositol     

Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.     

Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus.

Microbial organisms secrete antibiotics that cause the selective destruction of specific target cells. Although the mode of action is known for many antibiotics, the mechanisms by which these molecules are directed specifically to their target cells hitherto have not been described. Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that cleaves staphylococcal peptidoglycans in general but that is directed specifically to Staphylococcus aureus target cells. The sequence element sufficient for the binding of the bacteriocin as well as of hybrid indicator proteins to the cell wall of S.aureus consisted of 92 C-terminal lysostaphin residues. Targeting to the cell wall of S.aureus occurred either when the hybrid indicator molecules were added externally to the bacteria or when they were synthesized and exported from their cytoplasm by an N-terminal leader peptide. A lysostaphin molecule lacking the C-terminal targeting signal was enzymatically active but had lost its ability to distinguish between S.aureus and S.simulans cells, indicating that this domain functions to confer target cell specificity to the bacteriolytic molecule.     

Microtubule-targeting agents are clinically successful due to both mitotic and interphase impairment of microtubule function

Microtubules undergo continual dynamic changes in mitotic cells as the mitotic spindle forms and is broken down and in interphase cells where they play a central role in intracellular trafficking, cell signaling, cell migration, and angiogenesis. Compounds that target the microtubule have been hugely successful in the clinic as chemotherapeutics, and this success is likely due to their ability to target cells regardless of their cell cycle stage. Additionally, new generation antibody-conjugated microtubule-targeting agents are improving the targeting of these drugs to tumors. Microtubule-targeting agents have been shown to have anti-angiogenic and vascular-disrupting properties as well as effects on cellular migration, intracellular trafficking, and cell secretion. There are a number of these compounds in development that target the vasculature, and different formulations of clinically used drugs are being developed to take advantage of these anti-angiogenic properties. Microtubule-targeting agents have also been shown to have the potential to treat neurodegenerative diseases, such as Alzheimer’s disease. Thus, drugs that target the microtubule will continue to have a major impact in oncology not only as anti-mitotics but also as potent inhibitors of interphase functions, and in future may also prove to be effective in reducing the consequences of neurodegenerative disease.     

Binding of a Sialic Acid-recognizing Lectin Siglec-9 Modulates Adhesion Dynamics of Cancer Cells via Calpain-mediated Protein Degradation

Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs are well known, signals transduced via interaction between Siglecs and sialyl compounds on their counterreceptors into target cells have not been reported to date. In this study, we found that an astrocytoma cell line, AS, showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS cells regrew as co-cultured cells with Siglec-9-deficient cells. They also showed increased motility and invasiveness upon Siglec-9 binding. In immunoblotting, rapid degradation of focal adhesion kinase (FAK) and related signaling molecules such as Akt, paxillin, and p130Cas was observed immediately after the co-culture. Despite degradation of these molecules, increased p-Akt was found at the front region of the cytoplasm, probably reflecting increased cell motility. Calpain was considered to be a responsible protease for the protein degradation by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by Siglec-9 binding to its counterreceptors via sialylglycoconjugates, leading to the modulation of adhesion kinetics of cancer cells. Thus, this might be a mechanism by which cancer cells utilize Siglec-9-derived signals to escape from immunosurveillance.