Inhibition of telomerase by BIBR 1532 and related analogues

BIBR 1532 has been reported to be a potent, small molecule inhibitor of human telomerase, suggesting it as a lead for the development of anti-telomerase therapy. We confirm the ability of BIBR 1532 to inhibit telomerase and report the discovery of an equally potent analogue. Importantly, IC(50) values in cell extract are considerably higher than those previously reported using assays for purified enzyme, indicating that substantial improvement may be necessary.     

Potent inhibition of human telomerase by U-73122

Summary Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC₅₀ value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 μM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC₅₀ values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.     

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.     

Mechanism of inhibition of human KSP by ispinesib

KSP, also known as HsEg5, is a kinesin that plays an essential role in the formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. Ispinesib is the first potent, highly specific small-molecule inhibitor of KSP tested for the treatment of human disease. This novel anticancer agent causes mitotic arrest and growth inhibition in several human tumor cell lines and is currently being tested in multiple phase II clinical trials. In this study we have used steady-state and pre-steady-state kinetic assays to define the mechanism of KSP inhibition by ispinesib. Our data show that ispinesib alters the ability of KSP to bind to microtubules and inhibits its movement by preventing the release of ADP without preventing the release of the KSP-ADP complex from the microtubule. This type of inhibition is consistent with the physiological effect of ispinesib on cells, which is to prevent KSP-driven mitotic spindle pole separation. A comparison of ispinesib to monastrol, another small-molecule inhibitor of KSP, reveals that both inhibitors share a common mode of inhibition.     

Mechanism of inhibition for N6022, a first-in-class drug targeting S-nitrosoglutathione reductase

N6022 is a novel, first-in-class drug with potent inhibitory activity against S-nitrosoglutathione reductase (GSNOR), an enzyme important in the metabolism of S-nitrosoglutathione (GSNO) and in the maintenance of nitric oxide (NO) homeostasis. Inhibition of GSNOR by N6022 and related compounds has shown safety and efficacy in animal models of asthma, chronic obstructive pulmonary disease, and inflammatory bowel disease [Sun, X., et al. (2011) ACS Med. Chem. Lett. 2, 402-406]. N6022 is currently in early phase clinical studies in humans. We show here that N6022 is a tight-binding, specific, and fully reversible inhibitor of GSNOR with an IC(50) of 8 nM and a K(i) of 2.5 nM. We accounted for the fact that the NAD(+)- and NADH-dependent oxidation and reduction reactions, catalyzed by GSNOR are bisubstrate in nature in our calculations. N6022 binds in the GSNO substrate binding pocket like a competitive inhibitor, although in kinetic assays it behaves with a mixed uncompetitive mode of inhibition (MOI) toward the GSNO substrate and a mixed competitive MOI toward the formaldehyde adduct, S-hydroxymethylglutathione (HMGSH). N6022 is uncompetitive with cofactors NAD(+) and NADH. The potency, specificity, and MOI of related GSNOR inhibitor compounds are also reported.     

Synthesis, human telomerase inhibition and anti-proliferative studies of a series of 2,7-bis-substituted amido-anthraquinone derivatives

Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone were designed and synthesized. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and cytotoxicity. In the series, compounds (6, 10, 13, 16, 18, 19, 20-22, and 24) showed potent telomerase inhibitory activity, while compounds 19, 21, and 22 activated hTERT expression in normal human fibroblasts. The results indicated that 2,7-diamidoanthraquinones represent an important class of compounds for telomerase-related drug developments. Compounds 8, 16, 18, 26, and 32 were also selected by the NCI for Screening Program and demonstrated high anti-proliferative activity against 60 human cancer cell lines. Structure-activity relationships (SAR) study revealed that the test compounds with side chains two carbon spacer between amido and amine are important structural moiety for telomerase inhibition. Although the exact mechanism of how this amine group contributes to its activity is still unclear, however, the amine group in the extended arm of the bis-substituted anthraquinone might contribute to proper binding to the residues within the grove of G-quadruplex structure. Our results indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase inhibitors that have the potential to be further developed into novel anticancer chemotherapeutic agents.     

Peptidomimetics, a synthetic tool of drug discovery

The demand for modified peptides with improved stability profiles and pharmacokinetic properties is driving extensive research effort in this field. Many structural modifications of peptides guided by rational design and molecular modeling have been established to develop novel synthetic approaches. Recent advances in the synthesis of conformationally restricted building blocks and peptide bond isosteres are discussed.     

Implications of high-affinity hybridization by locked nucleic acid oligomers for inhibition of human telomerase

Oligonucleotides that contain locked nucleic acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of nucleic acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucleic acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.     

Mechanism of penicillinase inhibition by alkyl sulfates in the presence of synthetic polyeletrolytes]

Competing inhibition of Bacillus licheniformis 749/C penicillinase by alkylsulfates CnH2n+ 1OSO3N1 where n was 8--16 was studied. The values of the inhibition constants Ki of individual homologues were estimated. It was shown that stability of complex "enzyme-inhibitor" increased with lengthening of the hydrocarbon radical which was probably due to increased hydrophobic interaction of the alkyl radical with the lipophilic areas of the penicillinase active center. Inhibition in the presence of sopolymers of vinylpirrolidone with N-alkylvinylamine was studied with the aim of modeling the process of penicillinase inhibitions by alkylsulfates in the presence of the blood serum. It was shown that polyelectrolytes posessing hydrophobic substituents had an ability of reducing the activity of inhibited penicillinase, cooperative transfer of alkylsulfate molecules from the enzyme to polyelectrolyte being observed. The maximum effect was registered in the polyelectrolytes with substituents C12H25 and C16H33 which was confirmed in the experiments with the penicillinase-producing strains of staphylococci.     

Mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.     

Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides

We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect.     

Mechanism of inhibition of human natural killer activity by ultraviolet radiation

Ultraviolet radiation (UVR) has been shown to inhibit various immune functions in vivo and in vitro. We have confirmed that UVR inhibits human natural killer (NK) activity in vitro and have shown that UVR inhibits human ADCC. In this report, the mechanism by which UVR inhibits NK function was investigated by analyzing the stage at which the inhibiting activity occurs and the ability of the NK cells to release cytotoxic factors previously shown to be involved in CMC. Single cell assays in Agarose revealed that inhibition of NK activity was localized at the postbinding lethal hit stage rather than the initial recognition or binding stage of lysis. We then examined whether UV-treated cells were able to release cytotoxic factors after stimulation with target cells. As expected, stimulated cells released cytotoxic factors, yet, surprisingly, these factors were also released in the absence of stimulator cells. The spontaneous release was detectable in the supernatants as early as 30 min after UV irradiation. The lytic material examined in 48- to 72-hr viability assays was not NK specific, because lysis was obtained with a wide range of NK sensitive and resistant target cells. These results demonstrate that UVR does not alter the capacity of the cells to secrete cytotoxic material, but in fact enhances its release. Several possible mechanisms are proposed to explain the UVR-induced NK inhibition.     

Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of inhibition of human cytomegalovirus replication by oxetanocin G.

Oxetanocin G(9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine, OXT-G) is a potent and selective agent against human cytomegalovirus (HCMV). In this study we synthesized the triphosphate form of OXT-G, OXT-GTP, and examined its effect on the activities of HCMV DNA polymerase, herpes simplex type 2 (HSV-2) DNA polymerase and human DNA polymerase alpha. OXT-GTP was found to inhibit all these polymerases in a competitive manner with respect to dGTP. The Km for dGTP and the Ki for OXT-GTP of HCMV DNA polymerase were 0.86 and 0.53 mu M, respectively, while the corresponding values of DNA polymerase alpha were 2.2 and 3.6 mu M, respectively. HPLC analysis using [3H]OXT-G also revealed that OXT-G was converted to its triphosphate form 7- to 8-fold more efficiently in HCMV-infected cells than in uninfected cells. The results suggest that both the preferential phosphorylation of OXT-G in HCMV-infected cells and the preferential inhibition of HCMV DNA polymerase by OXT-GTP may contribute towards the selective activity of OXT-G against HCMV replication.     

Structural insights into drug processing by human carboxylesterase 1: tamoxifen, mevastatin, and inhibition by benzil

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.     

Mechanism for slow-binding inhibition of human leukocyte elastase by valine-derived benzoxazinones

Valine-derived benzoxazinones have been synthesized and found to be competitive, slow-binding inhibitors of human leukocyte elastase (HLE). Steady-state inhibition constants Ki are dependent on aryl substitution and reach a maximum of potency of 0.5 nM with the 5-Cl compound 6. UV-spectral data for the interaction of HLE and the unsubstituted inhibitor 3 indicate that the stable complex formed between enzyme and inhibitor is an acyl-enzyme that can either undergo ring closure, to reform intact benzoxazinone, or hydrolysis, to liberate an N-acylanthranilic acid. "Burst" kinetic data, derived from the direct observation of the interaction of HLE and 3, are consistent with results of the inhibition of catalysis experiments.     

Inhibition of microtubule assembly in vitro by TN-16, a synthetic antitumor drug

An antitumor drug, 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) inhibited the assembly of porcine brain microtubules in vitro. The assembly induced by taxol was also suppressed by the drug. However, the latter required much higher concentration of TN-16 than the former. Binding studies by means of the fluorometric method and the spun-column procedure indicate that the inhibition was caused by the reversible binding of the drug to the colchicine-sensitive site of tubulin. The affinity of TN-16 to tubulin was almost equal to that of nocodazole.