Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters

n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction.     

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.     

A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor.

Using DNA synthesis technology we constructed two synthetic DNAs, designated syn-uPA-DNA and mut-uPA-DNA. Syn-uPA-DNA contains the complete coding sequence of human presecretion form of single-chain u-PA. Mut-uPA-DNA was derived from syn-uPA-DNA by deleting 18 base pairs coding for amino acids Arg179-Ser184. Each synthetic DNA was inserted into a bovine papilloma viral genome-based expression vector to obtain expression in mouse cells. The results indicate that both syn-uPA and mut-uPA proteins are secreted predominantly in single-chain form. The single-chain form of both enzymes can be completely converted to two-chain form by treatment with plasmin. Both enzymes are as active as natural urokinase (std-uPA) isolated from urine. Analysis of enzymatic activity indicates that under conditions where syn-uPA and std-uPA are completely inhibited by endothelial-type plasminogen activator inhibitor (PAI-1), mut-uPA retains 90% activity. In identical experiments with placental-type PAI (PAI-2), mut-uPA retains 80% activity. Syn-uPA is capable of forming a approximately 100-kDa complex with PAI, whereas mut-uPA can not. PAI-treated mut-uPA has kinetic properties similar to untreated syn-uPA or std-uPA. Overall, the data indicate that amino acids Arg179-Ser184 function at least in part as a binding site for PAI. Resistance to PAI inhibition may increase the potency of mut-uPA as a thrombolytic agent.     

Influence of elastin on the inhibition of leucocyte elastase by alpha 1-proteinase inhibitor and bronchial inhibitor. Potent inhibition of elastin-bound elastase by bronchial inhibitor.

We have investigated the effect of human lung elastin on the inhibition of human leucocyte elastase by human alpha 1-proteinase inhibitor and bronchial inhibitor. Elastin was unable to dissociate the elastase-inhibitor complexes during the 150 min of the elastolysis reaction. When elastase was added to mixtures of elastin and alpha 1-proteinase inhibitor, it was fully bound to the latter. The competition between elastin and bronchial inhibitor was also in favour of the latter, but a 1.5 molar excess of inhibitor over elastase was required to achieve total binding of the enzyme. About 25% of elastin-bound elastase was found to be resistant to the inhibitory effect of alpha 1-proteinase inhibitor. The major isoenzyme and the mixture of the three minor isoenzymes of elastase exhibited similar behaviour. By contrast, bronchial inhibitor was as efficient in inhibiting the elastin-bound elastase as it was in inhibiting the free enzyme. This inhibitor was also able to inhibit fully the fraction of elastin-bound elastase that was resistant to alpha 1-proteinase inhibitor. We also describe a rapid procedure for the isolation of gram quantities of alpha 1-proteinase inhibitor.     

Selective inhibition of glycosyltransferases by bivalent imidazolium salts

Galactosyltransferases (GalTs) extend the glycan chains of mammalian glycoproteins by adding Gal to terminal GlcNAc residues, and thus build the scaffolds for biologically important glycan structures. We have shown that positively charged bivalent imidazolium salts in which the two imidazolium groups are linked by an aliphatic chain of 20 or 22 carbons form potent inhibitors of purified human β3-GalT5, using GlcNAcβ-benzyl as acceptor substrate. The inhibitors are not substrate analogs and also inhibited a selected number of other glycosyltransferases. These bis-imidazolium compounds represent a new class of glycosyltransferase inhibitors with potential as anti-cancer and anti-inflammatory drugs.     

Potent inhibition of checkpoint kinase activity by a hymenialdisine-derived indoloazepine

The marine sponge metabolite hymenialdisine is a potent inhibitor of a variety of kinases including MEK-1, GSK-3 beta, and CK1. In addition, hymenialdisine and debromohymenialdisine exhibit inhibition of the G(2) cell cycle checkpoint at micromolar concentrations. We report herein the potent inhibition of cell cycle kinase Chk2 by the indolic-hymenialdisine indoloazepine 1 (IC(50)=8 nM).     

Potent inhibition of sperm motility by palytoxin

Palytoxin, produced by a stationary marine animal and one of the most toxic substances known, was used as a spear poison in ancient Hawaii to cause death by cardiovascular contracture. We report here that the motility of hamster caudal epididymal (HCE) and other sperm can be inhibited by as little as 10⁻¹³ M palytoxin in a time-dependent manner. This inhibition manifested itself as a loss in flagellar amplitude, often accompanied by an increase in beat frequency, resulting in a loss of forward progression and ultimately cessation of movement. Similar effects were observed in sperm from guinea pigs, rabbit, cattle, sea urchins and man. Preincubation with palytoxin did not prevent the induction of motility from quiescence in HCE sperm when free calcium ion was added. However, regardless of the timing of palytoxin addition this very vigorous motility disappeared shortly after it appeared. These, plus earlier observations showing palytoxin did not cause lysis under similar conditions or inhibit the progressive motility of demembranated sperm axoneme preparations, suggest both that this large molecule acts via the plasma membrane to cause its exceedingly toxic effects and that spermatozoa may be useful for the investigation of the mechanism of action of palytoxin.     

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate

Summary. Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.     

Potent and selective inhibition of membrane-type serine protease 1 by human single-chain antibodies

Specific human antibodies targeting proteases expressed on cancer cells can be valuable reagents for diagnosis, prognosis, and therapy of cancer. To this end, a phage-displayed antibody library was screened against a cancer-associated serine protease, MT-SP1. A protein inhibitor of serine proteases that binds to a defined surface of MT-SP1 was used in an affinity-based washing procedure. Six antibodies were selected on the basis of their ELISA profiles and ability to serve as useful immunological reagents. The apparent K(i), indicative of the potency of the antibodies at inhibiting human MT-SP1 activity, ranged from 50 pM to 129 nM. Two of the antibodies had approximately 800-fold and 1500-fold selectivity when tested against the most homologous serine protease family member, mouse MT-SP1, that exhibits 86.6% sequence identity. Surface plasmon resonance was used as an independent means of determining the binding constants of the six antibodies. Association rates were as high as 1.15 x 10(7) s(-)(1) M(-)(1), and dissociation rates were as low as 3.8 x 10(-)(4) s(-)(1). One antibody was shown to detect denatured MT-SP1 with no cross reactivity to other family members in HeLa or PC3 cells. Another antibody recognized the enzyme in human prostate tissue samples for immunohistochemistry analysis. The mode of binding among the six antibodies and the protease was analyzed by competition ELISA using three distinctly different inhibitors that mapped the enzyme surface. These antibodies constitute a new class of highly selective protease inhibitors that can be used to dissect the biological roles of proteolytic enzymes as well as to develop diagnostic and therapeutic reagents.     

Potent inhibition of human influenza H5N1 virus by oligonucleotides derived by SELEX

New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections.     

Identification of human glutaminyl cyclase as a metalloenzyme. Potent inhibition by imidazole derivatives and heterocyclic chelators

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 +/- 1 and 295 +/- 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 +/- 0.02, 6.93 +/- 0.03, and 5.60 +/- 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical interventi...     

Potent enzyme-activated inhibition of aromatase by a 7 alpha-substituted C19 steroid

Enzyme-activated inhibitors of aromatase would result in effective medicinal agents for modulating estrogen-dependent processes and thus may be useful in controlling reproductive processes and in treating estrogen-dependent diseases such as breast and endometrial cancer. A potential enzyme-activated inhibitor of aromatase, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD), was synthesized and examined in vitro with placental aromatase. Under initial velocity conditions, 7 alpha-APTADD exhibited high affinity for the enzyme and is a potent inhibitor of aromatase with an apparent Ki of 9.9 +/- 1.0 nM and with a Km for androstenedione of 52.5 +/- 5.9 nM. This inhibitor produced a rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, while no inactivation of aromatase activity was observed in the absence of NADPH. Protection of aromatase from inactivation was observed when the substrate, androstenedione, was included in the incubation mixture containing enzyme, inhibitor, and NADPH. On the other hand, nucleophilic trapping agents such as cysteine did not protect the enzyme from inactivation by 7 alpha-APTADD. Additionally, second enzyme pulse experiments demonstrated identical rates of inactivation, suggesting that the enzyme-activated inhibitor was not being released from the active site of the enzyme. The apparent Kinact for 7 alpha-APTADD is 159 +/- 21 nM and represents the inhibitor concentration required to produce a half-maximal rate of inactivation. The half-time of inactivation at infinite inhibitor concentration was 1.38 +/- 0.92 min and is the most rapid enzyme-activated aromatase inhibitor reported to date. Thus, 7 alpha-APTADD is a potent enzyme-activated inhibitor of aromatase, exhibiting high affinity and rapid inactivation. This inhibitor will be useful in probing the biochemistry of aromatase and should also serve as an effective medicinal agent for the treatment of estrogen-dependent cancers.     

The inhibition of human leukocyte elastase by basic pancreatic trypsin inhibitor

The effects of 30-min intravenous infusions of ethanol (about 50 mm blood concentration), acetaldehyde (about 100 μm blood concentration), and acetate (equimolar dose to acetaldehyde) were studied in normal and adrenalectomized rats. Blood glucose, plasma free fatty acids (FFA), plasma immunoreactive insulin, and glucagon and hepatic glycogen concentrations were measured. Ethanol itself in the presence of 4-methylpyrazole (4-MP) produced no marked changes in the parameters measured. Its infusion without 4-MP reduced plasma insulin by 35% in the normal rats, but not in the adrenalectomized rats, with no simultaneous changes in blood glucose. Acetaldehyde infusion produced hyperglycemia and relatively slight hyperinsulinemia in the normal rats, but not in the adrenalectomized rats. Equimolar acetate was not as potent a stimulator of glycogenolysis as acetaldehyde. Plasma FFA concentrations were markedly reduced by ethanol (without 4-MP), acetaldehyde and acetate both in the normal and adrenalectomized rats, but in the presence of 4-MP ethanol was without effect. The results indicate that metabolites of ethanol (mostly acetaldehyde) produced during ethanol oxidation in vivo are responsible for the stimulation of glycogenolysis through the release of catecholamines from the adrenal glands. The ethanol-induced decrease in plasma FFA is also attributable to the metabolites of ethanol, acetaldehyde having a more potent depressing action than acetate. The mode of inhibition of lipolysis is not related to hormonal factors.     

Potent inhibition of human leukocyte elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamide derivatives

The design, synthesis, and in vitro biochemical evaluation of a class of mechanism-based inhibitors of human leukocyte elastase (HLE) that incorporate in their structure a 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold with appropriate recognition and reactivity elements appended to it is described. The synthesized compounds were found to be efficient, time-dependent inhibitors of HLE. The interaction of the inhibitors with HLE is postulated to lead to the formation of a highly reactive N-sulfonyl imine (a Michael acceptor) that arises from an enzyme-induced sulfonamide fragmentation cascade. Subsequent reaction ultimately leads to the formation of a relatively stable acyl enzyme. The results cited herein demonstrate convincingly the superiority of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold over other scaffolds (e.g., saccharin) in the design of inhibitors of (chymo)trypsin-like serine proteases.     

Potent inhibition of macrophomate synthase by reaction intermediate analogs

Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I50 1.3-8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I50 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (-)-Enantiomers of the diacids showed 30 times stronger activity (I50 0.34, 0.41 mM) than (+)-ones. The I50/Km values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.