Slow-binding Inhibitor Research Articles

Slow-binding inhibition of soybean lipoxygenase-1 by luteolin

Luteolin, isolated from the seeds of Perilla frutescens (perilla seeds), inhibited the peroxidation of linoleic acid catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC(50) of 5.0 M (1.43 μg/mL) noncompetitively. The progress curves for an enzyme reaction indicate that luteolin shows slow binding kinetics. Both the initial velocity and steady-state rate in the progress curve were decreased with increasing the concentration of luteolin. The kinetic parameters, which described the inhibition by luteolin, were evaluated by nonlinear regression fits.

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CLpro and PLpro, viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1–7) act as time dependent inhibitors of PLpro, but no improved inhibition was observed following preincubation with the 3CLpro. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC50=0.7μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.

N-O-Isopropyl sulfonamido-based hydroxamates: Kinetic characterisation of a series of MMP-12/MMP-13 dual target inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases known to play key roles in extracellular matrix (ECM) breakdown disorders, such as the two main forms of arthritis, rheumatoid arthritis (RA) and osteoarthritis (OA). MMP-13 (collagenase 3) is the leading MMP involved in cartilage degradation through its particular ability to cleave type-II collagen and as such plays a pivotal role in the pathogenesis of these diseases. Here we report the kinetic characterisation of N-O-isopropyl sulfonamido-based hydroxamates, potent inhibitors of MMP-13 and MMP-12, bearing different P1 and P1′ substituents. One of these compounds proved to be a potent (4≤Ki≤5nM) slow-binding inhibitor towards MMP-13 and MMP-12, with very favourable low association (104M−1s−1) and dissociation constants (10−4s−1). Moreover, this compound exhibited a good selectivity for MMP-13 and MMP-12 over MMP-1, MMP-3, MMP-7, MMP-8 and, even to a minor extent, MMP-2. A molecular-docking study carried out using the experimentally-derived X-ray crystal structure of MMP-12 (PDB ID: 3F17) revealed critical hydrogen bonding of the hydroxamate and the sulfonamide moieties with key active site residues. Since also MMP-12 is involved in RA, this MMP-13/MMP-12 dual target inhibitor could be a valid candidate for the treatment of this pathology.

1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were synthesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight binding inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model of inhibition, demonstrating either an encounter complex or a condensation product for the slow and slow-tight binding inhibitors, respectively.

Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1–6) and three shikonofurans (7–9) from Lithospermum erythrorhizon. The nine isolated compounds 1–9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7–9) were greater than shikonin derivatives (1–6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC50=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1–6) were of almost equal efficiency with IC50 value of 40μM and shikonofurans (7–9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism.

Kinetic characterization of a slow-binding inhibitor of Bla2: thiomaltol

The increasing prevalence of drug resistant bacteria is a pandemic problem. Metallo-β-lactamases (MBLs) are one of the main causes of drug resistance due to hydrolysis of β-lactam antibiotics. Thus, the development of effective inhibitors of MBLs remains urgent. The compound thiomaltol was used as a lead compound to investigate its ability to inhibit metallo-β-lactamase from Bacillus anthracis (Bla2), which causes anthrax. Kinetic evaluation with nitrocefin as a substrate indicates that thiomaltol inhibits Bla2 in a time-dependent manner with an IC50 value of 290 µM after 20 min preincubation. Progress curve analysis and reversibility tests suggest that thiomaltol is a reversible, slow-binding inhibitor with a Ki of 85 ± 30 µM. Furthermore, studies on the modality of inhibition and in silico analysis indicate thiomaltol to be a competitive inhibitor. The results demonstrate that thiomaltol is a promising lead compound for slow binding inhibitor design of Bla2.

Phosphonate Analogs of 2-Oxoglutarate Perturb Metabolism and Gene Expression in Illuminated Arabidopsis Leaves

Although the role of the 2-oxoglutarate dehydrogenase complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf disks with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-OGDHC. Our results indicate that the reduced respiration rates are associated with changes in the regulation of metabolic and signaling pathways leading to an imbalance in carbon-nitrogen metabolism and cell homeostasis. The inducible alteration of primary metabolism was associated with altered expression of genes belonging to networks of amino acids, plant respiration, and sugar metabolism. In addition, by using isothermal titration calorimetry we excluded the possibility that the changes in gene expression resulted from an effect on 2-oxoglutarate (2OG) binding to the carbon/ATP sensing protein PII. We also demonstrated that the 2OG degradation by the 2-oxoglutarate dehydrogenase strongly influences the distribution of intermediates of the tricarboxylic acid (TCA) cycle and the GABA shunt. Our results indicate that the TCA cycle activity is clearly working in a non-cyclic manner upon 2-OGDHC inhibition during the light period.

Selenosemicarbazones as potent cruzipain inhibitors and their antiparasitic properties against Trypanosoma cruzi

The cysteine protease cruzipain is an essential T. cruzienzyme and one of the few validated drug targets for Chagas disease. Thiosemicarbazones have been described as cruzipain inhibitors. While searching for new antichagasic drugs, we synthesized a series of selenosemicarbazone analogs and demonstrated that the isosteric replacement of the sulfur atom with selenium resulted in an enhancement of the cysteine protease inhibitory effect. Three selenosemicarbazones were characterized enzymatically and proved to be reversible, slow-binding inhibitors for the Z-Phe-Arg-AMC substrate. Their KI values were in the low nM range (3.7 to 29.7 nM), suggesting a strong interaction with the enzyme, approaching a tight binding definition. All selenosemicarbazones tested showed better activities against epimastigotes than Benznidazole, the currently used drug, with IC50 values ranging from 1.2 to 5.9 μM (Bnz IC50 = 12.5 μM). Three of these compounds showed a better Selectivity Index (SI) than Benznidazole. These compounds also displayed activity against the infective intracellular amastigote form at low micromolar range. Overall, our results support the role of these novel organoselenium compounds as promising lead candidates for further drug development studies.

Piperidine azasugars displaying competitive α-rhamnosidase inhibition and their kinetic mechanism

Azasugars derived from L-alanine and L-serine were screened for inhibitory activity against α-rhamnosidase. The enantiomers of 1,6-dideoxynojirimycin (ent-1,6-dDNJ) (1) and (2S,3R)-2-(hydroxymethyl)piperidin-3-ol (5) showed highly specific and potent inhibition against α-rhamnosidase with K i values of 4.2 and 16.6 μM, respectively. Structure of the best inhibitor features the same stereochemical configuration as L-rhamnose at C2, C3, and C4 centers. In kinetic studies, both compounds exhibited competitive inhibition behavior. Compound 1 manifested simple reversible slow-binding inhibition with the following kinetic parameters: k 3=1.17 nM−1 min−1, k 4 = 5.96 ×10−3 min−1, and K i app =5.1 mM.

Kinetic and in silico analysis of the slow-binding inhibition of human poly(A)-specific ribonuclease (PARN) by novel nucleoside analogues

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based glucopyranosyl nucleoside analogues with adequate tumour-inhibitory effect could effectively inhibit PARN. In the present study we dissect the mechanism of a more drastic inhibition of PARN by novel glucopyranosyl analogues bearing uracil, 5-fluorouracil or thymine as the base moiety. Kinetic analysis showed that three of the compounds are competitive inhibitors of PARN with Ki values in the low μM concentration and significantly lower (11- to 33-fold) compared to our previous studies. Detailed kinetic analysis of the most effective inhibitor, the uracil-based nucleoside analogue (named U1), revealed slow-binding behaviour. Subsequent molecular docking experiments showed that all the compounds which inhibited PARN can efficiently bind into the active site of the enzyme through specific interactions. The present study dissects the inhibitory mechanism of this novel uracil-based compound, which prolongs its inhibitory effect through a slow-binding and slow-release mode at the active site of PARN, thus contributing to a more efficient inhibition. Such analogues could be used as leading compounds for further rationale design and synthesis of efficient and specific therapeutic agents. Moreover, our data reinforce the notion that human PARN can be established as a novel molecular target of potential anti-cancer agents through lowering mRNA turnover rates.

Time-dependent slowly-reversible inhibition of monoamine oxidase A by N-substituted 1,2,3,6-tetrahydropyridines

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.

Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl‐ACP reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (Ki) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.

Screening and rank ordering of reversible mechanism‐based inhibitors of Hepatitis C virus NS3 protease using electrospray ionization mass spectrometry

An affinity-selection study using size exclusion chromatography (SEC) combined with off-line electrospray ionization mass spectrometry (ESI-MS) was performed on libraries of peptidic α-ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 µM) was incubated with equimolar amounts (100 µM) of 49 reversible mechanism-based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme-inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease-inhibitor complexes by SEC spin columns. The eluate of the SEC was immediately analyzed by direct-infusion ESI-MS. An enzyme-inhibitor complex, with a molecular mass corresponding to the NS3 protease binding to the preferred inhibitor, SCH212986, was the only molecular species detected. By increasing the molar ratio of HCV NS3 protease to inhibitors to 1:2 while keeping the inhibitors' concentration constant, the complex of the second most tightly bound inhibitor, SCH215426, was also identified. Although the potencies of these inhibitors were virtually un-measurable by kinetic assays, a rank order of CVS4441 > SCH212986 > SCH215426 was deduced for their inhibition potencies by direct competition experiment with CVS4441 (K(i)*>80 µM). As discussed in the article, through judicious application of this strategy, even large libraries of fairly weak, reversible and slow-binding inhibitors could be rapidly screened and rank ordered to provide critical initial structure-activity insights.

Identification and Characterization of a Trypsin Inhibitor from Fagopyrum tataricumSeeds.

This study was aimed at investigating the purification and identification of serine protease inhibitors, F. tataricum trypsin inhibitor (FtTI) from tartary buckwheat (Fagopyrum tataricum) seeds. The FtTI was isolated by anion exchange chromatography, affinity chromatography, and centrifugal ultrafiltration. Under reducing and nonreducing conditions, an SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with a molecular mass of approximately 14kDa. The two isoforms of FtTI were confirmed by the mass spectrometric profile where the two peaks corresponded to 11.487 and 13.838kDa. The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. The molecule of FtTI consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Asp66-Arg67 bond. The Ki value was calculated using the equation for slow tight binding inhibition which was 1.6nM for trypsin. FtTI retained its inhibitory activity over a wide range of pH (3-10) and temperature (20-80°C). FtTI can be rapidly inactivated by the combination of high temperature and high pressure. An analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor Ι family. Furthermore, FtTI exhibited a strong inhibitory activity against phytopathogenic fungi.

HIV-1 Integrase Strand Transfer Inhibitors Stabilize an Integrase–Single Blunt-Ended DNA Complex

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC 50 values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN–single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20–25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.

GSK256066, an Exceptionally High-Affinity and Selective Inhibitor of Phosphodiesterase 4 Suitable for Administration by Inhalation: In Vitro, Kinetic, and In Vivo Characterization

Oral phosphodiesterase (PDE) 4 inhibitors such as roflumilast have established the potential of PDE4 inhibition for the treatment of respiratory diseases. However, PDE4 inhibitor efficacy is limited by mechanism-related side effects such as emesis and nausea. Delivering the inhibitor by the inhaled route may improve therapeutic index, and we describe 6-({3-[(dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy) phenyl]amino}-3-quinolinecarboxamide (GSK256066), an exceptionally high-affinity inhibitor of PDE4 designed for inhaled administration. GSK256066 is a slow and tight binding inhibitor of PDE4B (apparent IC(50) 3.2 pM; steady-state IC(50) <0.5 pM), which is more potent than any previously documented compound, for example, roflumilast (IC(50) 390 pM), tofimilast (IC(50) 1.6 nM), and cilomilast (IC(50) 74 nM). Consistent with this, GSK256066 inhibited tumor necrosis factor α production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes with 0.01 nM IC(50) (compared with IC(50) values of 5, 22, and 389 nM for roflumilast, tofimilast, and cilomilast, respectively) and by LPS-stimulated whole blood with 126 pM IC(50). GSK256066 was highly selective for PDE4 (>380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and >2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (>17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED(50) values of 1.1 μg/kg (aqueous suspension) and 2.9 μg/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED(50) 9.3 μg/kg). Thus, GSK256066 has been demonstrated to have exceptional potency in vitro and in vivo and is being clinically investigated as a treatment for chronic obstructive pulmonary disease.

Synthesis of bi-substrate state mimics of dihydropteroate synthase as potential inhibitors and molecular probes

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillusanthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.

Hydroxycoumarin Derivatives: Novel and Potent α-Glucosidase Inhibitors

A novel class of hydroxycoumarin derivatives were found to be potent α-glucosidase inhibitors. Their syntheses were reported and the structure-activity relationship was established. Kinetic enzymatic assays indicated that compound 10 was a slow-binding and noncompetitive inhibitor with a Ki value of 589 nM, while compound 11 was a competitive inhibitor with a Ki value of 4.810 μM. Among all hydroxycoumarin derivatives studied, compounds 10 and 11 exhibited the highest activities, were specific inhibitors of α-glucosidase, and could be exploited as the lead compounds for the development of potent α-glucosidase inhibitors. Compounds 10 and 11 were also selected for further discussion for the mechanism of enzymatic inhibition.

A Novel Competitive Class of α‐Glucosidase Inhibitors: (E)‐1‐Phenyl‐3‐(4‐Styrylphenyl)Urea Derivatives

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo- and regiocontrolled. Here, we show that readily accessible achiral (E)-1-phenyl-3-(4-strylphenyl)ureas are potent competitive α-glucosidase inhibitors. A systematic synthesis study shows that the 1-phenyl moiety on the urea is critical for ensuring competitive inhibition, and substituents on both terminal phenyl groups contribute to inhibition potency. The most potent inhibitor, compound 12 (IC(50)=8.4 μM, K(i)=3.2 μM), manifested a simple slow-binding inhibition profile for α-glucosidase with the kinetic parameters k(3)=0.005256 μM(-1) min(-1), k(4)=0.003024 min(-1), and K(i)(app) =0.5753 μM.

Physical trapping of HIV-1 synaptic complex by different structural classes of integrase strand transfer inhibitors.

Raltegravir is an FDA approved inhibitor directed against human immunodeficiency virus type 1 (HIV-1) integrase (IN). In this study, we investigated the mechanisms associated with multiple strand transfer inhibitors capable of inhibiting concerted integration by HIV-1 IN. The results show raltegravir, elvitegravir, MK-2048, RDS 1997, and RDS 2197 all appear to encompass a common inhibitory mechanism by modifying IN-viral DNA interactions. These structurally different inhibitors bind to and inactivate the synaptic complex, an intermediate in the concerted integration pathway in vitro. The inhibitors physically trap the synaptic complex, thereby preventing target DNA binding and thus concerted integration. The efficiency of a particular inhibitor to trap the synaptic complex observed on native agarose gels correlated with its potency for inhibiting the concerted integration reaction, defined by IC(50) values for each inhibitor. At low nanomolar concentrations (<50 nM), raltegravir displayed a time-dependent inhibition of concerted integration, a property associated with slow-binding inhibitors. Studies of raltegravir-resistant IN mutants N155H and Q148H without inhibitors demonstrated that their capacity to assemble the synaptic complex and promote concerted integration was similar to their reported virus replication capacities. The concerted integration activity of Q148H showed a higher cross-resistance to raltegravir than observed with N155H, providing evidence as to why the Q148H pathway with secondary mutations is the predominant pathway upon prolonged treatment. Notably, MK-2048 is equally potent against wild-type IN and raltegravir-resistant IN mutant N155H, suggesting this inhibitor may bind similarly within their drug-binding pockets.