Reversible Inhibition Mechanism Research Articles

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

Inhibitive determination of metal ions by an amperometric glucose oxidase biosensor

Abstract An amperometric glucose biosensor based on glucose oxidase immobilized in electrosynthesized poly-o-phenylenediamine was successfully applied to the determination of a wide group of heavy metals of environmental interest. The inhibition effects of Hg2+, Ag+, Cu2+, Cd2+, Pb2+, Cr3+, Fe3+, Co2+, Ni2+, Zn2+, Mn2+ and also CrO42− on glucose oxidase were studied. Collected data showed a reversible inhibition mechanism. Results about the quantitative analysis of metal ions in terms of detection limit, linear range, sensitivity and R.S.D. are discussed for each tested metal ion. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr3+ and to detect the toxic species CrO42−. Also biosensor storage stability and response reproducibility were investigated. Moreover, this study represents the first attempt of evaluating the effect of the hydrogen peroxide decomposition by metal ions on the response of an enzymatic biosensor based on the amperometric detection of the hydrogen peroxide. Experiments were performed with the aim to quantitatively evaluate, for any single metal ion, if this process is competitive with the inhibition of enzymatic reaction in the adopted experimental conditions.

How thionphosphonates inhibit activity of different cholinesterases

Analysis of mechanism of reversible inhibition of human erythrocyte acetylcholinesterase (AChE), of horse serum cholinesterase (ChE), and ChE of optical ganglia tissue of individuals of the Commander squid Berryleuthis magister from various habitat zones was studied under effect of thionphosphonates (P=S), derivatives of piperidine, morpholine, perhydroazepine as well as several heterocyclic model compounds. Data of comparative inhibitory specificity have allowed us to suggest that thionphosphonates are sorbed in the area of cholinesterase esterase center through the phosphoryl part of the inhibitor molecule, rather than through its heterocyclic grouping. An advantage in the antienzyme efficiency of thionphosphonates (P=S) over phosphonates (P=O) is revealed. Effect of the ion strength is used for analysis of contribution of the hydrophobic—hydrophilic interaction in the enzyme—inhibitor system.

Study of the mechanism of reversible inhibition of various cholinesterases by thionephosphonates on the basis of data on comparative inhibitory specificity

Study of the mechanism of reversible inhibition of various cholinesterases by thionephosphonates on the basis of data on comparative inhibitory specificity

Exploitation of the unusual thermodynamic properties of human myeloperoxidase in inhibitor design

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. It uses hydrogen peroxide and chloride to catalyze the production of hypochlorous acid (HOCl), which contributes to both bacterial killing and oxidative injury of host tissue. Thus, MPO is an interesting target for anti-inflammatory therapy. Here, based on the extraordinary and MPO-specific redox properties of its intermediates compound I and compound II, we present a rational approach in selection and design of reversible inhibitors of HOCl production mediated by MPO. In detail, indole and tryptamine derivatives were investigated for their ability to reduce compounds I and II and to affect the chlorinating activity of MPO. It is shown that these aromatic one-electron donors bound to the hydrophobic pocket at the distal heme cavity and were oxidized efficiently by compound I ( k 3), which has a one-electron reduction potential of 1.35 V. By contrast, compound II ( E°′ of the compound II/ferric couple is 0.97 V) reduction ( k 4) was extremely slow. As a consequence compound II, which does not participate in the halogenation cycle, accumulated. The extent of chlorinating activity inhibition (IC 50) was related to the k 3/ k 4 ratio. The most efficient inhibitors were 5-fluorotryptamine and 5-chlorotryptamine with IC 50 of 0.79 μM and 0.73 μM and k 3/ k 4 ratios of 386,000 and 224,000, respectively. The reversible mechanism of inhibition is discussed with respect to the enzymology of MPO and the development of drugs against HOCl-dependent tissue damage.

Development of α-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of α-ketoamide-, α-ketoacid-, α-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1′ residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different α-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3 Å crystallographic structure of cruzain bound with one of the α-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.

Role of itraconazole metabolites in CYP3A4 inhibition.

Itraconazole (ITZ) is a potent inhibitor of CYP3A in vivo. However, unbound plasma concentrations of ITZ are much lower than its reported in vitro Ki, and no clinically significant interactions would be expected based on a reversible mechanism of inhibition. The purpose of this study was to evaluate the reasons for the in vitro-in vivo discrepancy. The metabolism of ITZ by CYP3A4 was studied. Three metabolites were detected: hydroxy-itraconazole (OH-ITZ), a known in vivo metabolite of ITZ, and two new metabolites: keto-itraconazole (keto-ITZ) and N-desalkyl-itraconazole (ND-ITZ). OHITZ and keto-ITZ were also substrates of CYP3A4. Using a substrate depletion kinetic approach for parameter determination, ITZ exhibited an unbound K(m) of 3.9 nM and an intrinsic clearance (CLint) of 69.3 ml.min(-1).nmol CYP3A4(-1). The respective unbound Km values for OH-ITZ and keto-ITZ were 27 nM and 1.4 nM and the CLint values were 19.8 and 62.5 ml.min(-1).nmol CYP3A4(-1). Inhibition of CYP3A4 by ITZ, OH-ITZ, keto-ITZ, and ND-ITZ was evaluated using hydroxylation of midazolam as a probe reaction. Both ITZ and OH-ITZ were competitive inhibitors of CYP3A4, with unbound Ki (1.3 nM for ITZ and 14.4 nM for OH-ITZ) close to their respective Km. ITZ, OH-ITZ, keto-ITZ and ND-ITZ exhibited unbound IC50 values of 6.1 nM, 4.6 nM, 7.0 nM, and 0.4 nM, respectively, when coincubated with human liver microsomes and midazolam (substrate concentration < Km). These findings demonstrate that ITZ metabolites are as potent as or more potent CYP3A4 inhibitors than ITZ itself, and thus may contribute to the inhibition of CYP3A4 observed in vivo after ITZ dosing.

Herbicidal pyridyl derivatives of aminomethylene-bisphosphonic acid inhibit plant glutamine synthetase.

A series of aminomethylene-bisphosphonic acid derivatives, previously synthesized and shown to be endowed with herbicidal properties, were evaluated as potential inhibitors of plant glutamine synthetase. The cytosolic form of the enzyme was partially purified from rice cultured cells and assayed in the presence of millimolar concentrations of the compounds by means of three different assay methods, respectively measuring the hemibiosynthetic, the transferase, and the full biosynthetic reactions. Several compounds were found to exert a remarkable inhibition, with I(50) values similar to those obtained under the same conditions with a well-established inhibitor of glutamine synthetase, the herbicide phosphinothricin. Contrary to the reference compound, enzyme kinetics accounted for a reversible inhibition mechanism. The biological activity of the most active derivatives was further characterized by measuring free glutamine levels in cell suspension rice cultures following treatment with the inhibitors. Results confirmed their ability to interfere in vivo with nitrogen metabolism. A preliminary analysis of structure-activity relationship allowed it to be hypothesized that steric rather than electronic factors are responsible for the inhibitory potential of these compounds.

Effect of the substrate structure on the mechanism of reversible inhibition of cholinesterases of different origin

The mechanism of reversible inhibition of human erythrocyte acetylcholinesterase, horse blood serum butyrylcholinesterase, cholinesterase from optical ganglia of the squids, PacificTodarodes pacificus and CommodoreBerryteuthis magister, from different zones of habitation area is studied in the presence of substrates of various structures (acetylcholine, butyrylcholine, acetylthiocholine, butyrylthiocholine, phenylacetate, indophenylacetate, 2,6-dichlorophenylindophenylacetate). Tested as reversible inhibitors were tetramethylammonium iodide, tetraethylammonium iodide, choline iodide, and two derivatives of α,ω-bis(trimethylammoniommethyl)oligodimethylsiloxane dichloride. It has been revealed that the mechanism of the reversible anticholinesterase action depends essentially both on the enzyme nature and on the structures of substrate and inhibitor. The transfer from cation-containing to hydrophobic substrates increased essentially the contribution of uncompetitive component of the inhibitory constant. In the presence of butyric acid esters (butyrylcholine, butyrylthiocholine), the potency of inhibitors was lower than at hydrolysis of the corresponding acetates. The effect of the substrate structure on the mechanism of reversible inhibition was revealed to a greater extent in reactions with participation of squid cholinesterases.

A reversible monoamine oxidase inhibitor, Toloxatone: comparison of its physicochemical properties with those of other inhibitors including Brofaromine, Harmine, R40519 and Moclobemide

Summary Reversible, competitive and selective monoamine oxidase A inhibitors (MAO A Is) are an exciting new type of anti-depressants with a safe profile. The mechanism for reversible inhibition of MAO A at the molecular level is still unknown. The planar structure of most reversible MAO A Is and the well-defined acceptor power of flavin adenine dinucleotide (FAD), the cofactor of the enzyme, suggest that MAO A Is exert their inhibitory effect through charge-transfer interactions with the FAD. This hypothesis has been evaluated for Toloxatone 1 , the first reversible MAO A I marketed in France. In this work, we give evidence for the ability of other reversible MAO A Is, including Brofaromine 2 , Harmine 3 and R40519 4 to interact with the flavin cofactor in comparison with Moclobemide 5 , and we underline the physicochemical properties required for these interactions. First, the formation of a complex between each of the MAO A Is and riboflavin, a model of the flavin cofactor, is shown by electronic absorption spectroscopy. Essential electronic descriptors of MAO A Is, such as the molecular electrostatic potential and the topology of the frontier orbitals, are then calculated by the ab initio Hartree—Fock method and compared with those of previously studied Toloxatone. This confirms the electronic absorption spectroscopy results. Finally, the similarities between the different MAO A Is are underlined and an interaction model is discussed on the basis of a detailed analysis of the electronic descriptors of all the considered MAO A Is and the flavin nucleus.

A reversible monoamine oxidase inhibitor, toloxatone: spectrophotometric and molecular orbital studies of the interaction with flavin adenine dinucleotide (FAD)

Toloxatone is a monoamine oxidase A (MAO A) inhibitor, marketed as antidepressant devoid of the undesirable side effects of first-generation irreversible monoamine oxidase inhibitors (MAOIs). Its advantages arise from the reversible, competitive and specific nature of its inhibition. The mechanism for irreversible inhibition of MAO A at the molecular level is known (suicide substrate). A physicochemical study was undertaken to establish the mechanism of reversible inhibition by Toloxatone. After determination of structural and electronic properties [6], experimental and theoretical approaches were used to explore the possibility of a physical association between the eutomer R-Toloxatone and flavin, a cofactor of MAO A. For this, 2 models of flavin were used. First, the existence of a charge-transfer complex between R-Toloxatone and riboflavin was demonstrated by electron absorption spectroscopy. Second, ab initio Hartree-Fock calculations of frontier orbitals and electrostatic potentials confirm the favourable overlap of complementary electronic zones of R-Toloxatone and SCH 3-lumiflavin for a defined relative orientation.

Effect of a novel monamine oxidase‐a inhibitor, bw 1370U87, on urinary 3‐methoxy‐4‐hydroxyphenylglycol in normal volunteers

AbstractUrinary 3‐methoxy‐4‐hydroxyphenlglycol (MHPG) levels were measured in 14 normal volunteers during a Phase I study of the novel, reversible monoamine oxidase‐A (MAO‐A) inhibitor BW 1370U87 targeted for the treatment of depression. baseline MHPG excretion (day 0) showed a significant diurnal increase (n = 14, P &lt; 0.05) between 1200 and 2000 h. Oral administration of BW 1370U87 produced significant (35‐45% maximal, P &lt; 0.05) decreases from these basal levels during the same 1200‐2000 h time interval Twenty‐four hours after the dose (dose was given at 0800 h), urinary MHPG levels returned to baseline, consistent with a reversible mechanism of inhibition by BW 1370U87. Decreases in urinary MHPG did not appear to be dose‐dependent between 200‐800 mg BW 1370U87 per day, suggesting that maximal MAO‐A inhibition was achieved at the 200 mg dose. These effects on MHPG excretion may be helpful in predicting the efficacy of BW 1371370U87 in the treatment of depression. © 1993 wiley‐Liss, Inc.

A Reexamination of the Proposed Inactivation of Thyroid Peroxidase in the Rat Thyroid by Propylthiouracil*

The antithyroid drug 6-propylthiouracil (PTU) was previously shown in our laboratory to have an unexpectedly prolonged inhibitory effect on iodination in the thyroid glands of rats. Eighteen hours after injection of a relatively small dose, iodination in the thyroid remained inhibited by more than 90%. We previously suggested that the prolonged inhibitory effect might be due to inactivation of thyroid peroxidase (TPO), a reaction previously shown to occur under certain conditions in an in vitro iodinating system containing highly purified TPO. However, the analytical procedure used in our earlier study did not exclude the possibility that sufficient PTU remained in the thyroid even after 18 h to inhibit TPO-catalyzed iodination by a reversible mechanism. Development of an improved analytical procedure, based on HPLC, led us to reexamine the mechanism of the prolonged inhibitory effect of PTU on iodination in rat thyroid glands. Rats were injected with [35S]PTU (1 mumol/100 g BW), and ultrafiltrates prepared from their homogenized thyroid glands were analyzed by HPLC. The major 35S-labeled metabolites were identified as sulfate/sulfite, PTU sulfinate, and PTU sulfonate. However, even after 18 h, a significant amount of unchanged [35S]PTU was also present. The calculated mean concentration of residual PTU was 20 microM, a sufficiently high level to explain the observed inhibition of iodination on the basis of a reversible mechanism. Experiments were also performed to examine the intrathyroidal distribution of 35S at intervals after the injection of [35S]PTU. All of the oxidation products of PTU showed marked increases between 2 and 16 h after injection. Based on our view that TPO is the major mediator of intrathyroidal metabolism of PTU, this observation is inconsistent with our previous proposal that TPO is inactivated after PTU injection. The results of the present study, therefore, lead us to withdraw our previous suggestion that TPO is inactivated after injection of PTU into rats. It is more likely that inhibition of iodination by PTU in the rat thyroid involves competition between PTU and tyrosyl residues of thyroglobulin for oxidized iodine, comparable to the reversible mechanism of inhibition observed in the TPO model system.

Novel inhibitors of Nocardia erythropolis cholesterol oxidase: Screening and characterization

Nocardia erythropolis cholesterol oxidase catalyzes two distinct reactions: the oxidation of a 3-hydroxy-Δ 5-steroid to a 3-keto-Δ 5-derivative followed by the isomerization to a 3-keto-Δ 4-steroid. As this enzyme has potential applications in steroid hormone synthesis, separation of these activities would be useful. In this study, we were able to demonstrate that fenpropimorph and tridemorph were effective inhibitors of cholesterol oxidase from N. erythropolis in vitro. The isomerization reaction was the most probable target of a reversible inhibition mechanism. Tridemorph probably blocked the active site, thus lowering the apparent enzyme concentration. Fenpropimorph acted via an unknown mechanism. A tertiary nitrogen atom able to form a morpholinium ion and the two morpholine methyl groups, preferably in cis configuration, were found to be essential for inhibition. The three dimethylmorpholine side chains tested were found to be interchangeable and replacement of the oxygen atom by carbon was without any major effect. These findings suggest an inhibition mechanism in which the positively charged morpholinium ion interacts strongly with an aspartate residue in the catalytic center of the enzyme, the inhibitor molecule being correctly oriented in the active site due to its resemblance to cholesterol.