Probing the Peptidylglycine α‐Hydroxylating Monooxygenase Active Site with Novel 4‐Phenyl‐3‐butenoic Acid Based Inhibitors

Specific inhibition of the copper‐containing peptidylglycine α‐hydroxylating monooxygenase (PHM), which catalyzes the post‐translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure–activity study of new compounds derived from a well‐known PHM substrate analogue, the olefinic compound 4‐phenyl‐3‐butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2 d , for example, bears a meta‐benzyloxy substituent, and exhibits better inhibition features (K_i=3.9 μM , k_inact/K_i=427 M ^?1 s^?1) than the parent PBA (K_i=19 μM , k_inact/K_i=82 M ^?1 s^?1). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.     

Dipeptidyl Enoates As Potent Rhodesain Inhibitors That Display a Dual Mode of Action

Dipeptidyl enoates were prepared through a high‐yielding two‐step synthetic route. They have a dipeptidic structure with a 4‐oxoenoate moiety as a warhead with multiple reactive sites. Dipeptidyl enoates were screened against rhodesain and human cathepsins B and L, and were found to be potent and selective inhibitors of rhodesain. Among them (S,E)‐ethyl 5‐((S)‐2‐{[(benzyloxy)carbonyl]amino}‐3‐phenylpropanamido)‐7‐methyl‐4‐oxooct‐2‐enoate ( 6 ) was the most potent, with an IC₅₀ value of 16.4 nM and k_inact/K_i=1.6×10⁶ M ^?1 s^?1 against rhodesain. These dipeptidyl enoates display a reversible mode of inhibition at very low concentrations and an irreversible mode at higher concentrations. Inhibition kinetics data, supported by docking studies, suggest a dual mode of action via attack of cysteine thiolate at two reactive positions.     

Covalent Inhibition of New Delhi Metallo‐β‐Lactamase‐1 (NDM‐1) by Cefaclor

Covalent irreversible inhibitors can successfully treat antibiotic‐resistant infections by targeting serine β‐lactamases. However, this strategy is useless for New Delhi metallo‐β‐lactamase (NDM), which uses a non‐covalent catalytic mechanism and lacks an active‐site serine. Here, NDM‐1 was irreversibly inactivated by three β‐lactam substrates: cephalothin, moxalactam, and cefaclor, albeit at supratherapeutic doses (e.g., cefaclor K_I=2.3±0.1 mM ; k_inact=0.024±0.001 min^?1). Inactivation by cephalothin and moxalactam was mediated through Cys208. Inactivation by cefaclor proceeded through multiple pathways, in part mediated by Lys211. Use of a cefaclor metabolite enabled mass spectrometric identification of a +346.0735 Da covalent adduct on Lys211, and an inactivation mechanism is proposed. Lys211 was identified as a promising “handhold” for developing covalent NDM‐1 inhibitors and serves as a conceptual example for creating covalent inhibitors for enzymes with non‐covalent mechanisms.     

Covalent Inhibition of the Lymphoid Tyrosine Phosphatase

Covalent inhibitors of lymphoid tyrosine phosphatase (LYP) were identified from a screen of the NIH Molecular Libraries Small Molecules Repository (MLSMR). Both of the two lead compounds identified have phosphotyrosine‐mimetic benzoic acid moieties as well as electrophilic acrylonitrile groups. Inhibition kinetics of both compounds are consistent with covalent modification of the enzyme, with nanomolar K_I and reciprocal millisecond k_inact values, representing the best efficiency ratios (k_inact/K_I) among currently reported covalent LYP inhibitors. Covalent inhibitors can provide longer efficacy and better selectivity than more conventional noncovalent inhibitors, and these lead compounds are an important step toward the development of protein tyrosine phosphatase (PTP)‐targeted covalent therapeutic compounds.     

Highly potent and selective 4,4'‐biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamate inhibitors of Pseudomonas species lipase

4,4'‐Biphenyl‐4‐acylate‐4'‐N‐n‐butylcarbamates ( 1–8 ) are synthesized and characterized as highly potent and selective pseudo‐substrate inhibitors of Pseudomonas species lipase. Thus, the n‐butylcarbamate moieties of the inhibitors bind to the first acyl chain binding site (ACS) of the enzyme. Therefore, the ester moieties of the inhibitors may bind to the second ACS of the enzyme, due to the linear 4,4'‐biphenyl moiety of the inhibitors. –logK_i, logk₂, and logk_i values of carbamates 1–8 are multiply linearly correlated with the Taft steric constant (E_S) and the Hansch hydrophobicity constant (π), but not with the Taft substituent constant (σ*). For –logK_i, logk₂, and logk_i correlations, values of δ are 0.8, 0.34, and 1.0, respectively, and values of ψ are 1.0, 0.4, and 1.3, respectively. Positive δ and ψ values for these correlations indicate that the second ACS of the enzyme prefers to bind to small and hydrophobic ester groups of the inhibitors. Among carbamates 1–8 , carbamate 3 , with a K_i value of 2.5 nM, is the most potent inhibitor.     

A Bisbenzamidine Phosphonate as a Janus‐faced Inhibitor for Trypsin‐like Serine Proteases

A hybrid approach was applied for the design of an inhibitor of trypsin‐like serine proteases. Compound 16 [(R,R)‐ and (R,S)‐diphenyl (4‐(1‐(4‐amidinobenzylamino)‐1‐oxo‐3‐phenylpropan‐2‐ylcarbamoyl)phenylamino)(4‐amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus‐faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second‐order rate constant (k_inac/K_i) of 59 500 M ^?1 s^?1. With human matriptase, 16 showed preference for a reversible mode of inhibition (IC₅₀=2.6 μM ) as indicated by linear progress curves and enzyme reactivation.     

Aromatase and dual aromatase-steroid sulfatase inhibitors from the letrozole and vorozole templates

Concurrent inhibition of aromatase and steroid sulfatase (STS) may provide a more effective treatment for hormone‐dependent breast cancer than monotherapy against individual enzymes, and several dual aromatase–sulfatase inhibitors (DASIs) have been reported. Three aromatase inhibitors with sub‐nanomolar potency, better than the benchmark agent letrozole, were designed. To further explore the DASI concept, a new series of letrozole‐derived sulfamates and a vorozole‐based sulfamate were designed and biologically evaluated in JEG‐3 cells to reveal structure–activity relationships. Amongst achiral and racemic compounds, 2‐bromo‐4‐(2‐(4‐cyanophenyl)‐2‐(1H‐1,2,4‐triazol‐1‐yl)ethyl)phenyl sulfamate is the most potent DASI (aromatase: IC₅₀=0.87 nM ; STS: IC₅₀=593 nM ). The enantiomers of the phenolic precursor to this compound were separated by chiral HPLC and their absolute configuration determined by X‐ray crystallography. Following conversion to their corresponding sulfamates, the S‐(+)‐enantiomer was found to inhibit aromatase and sulfatase most potently (aromatase: IC₅₀=0.52 nM ; STS: IC₅₀=280 nM ). The docking of each enantiomer and other ligands into the aromatase and sulfatase active sites was also investigated.     

Thiuram Disulfides as Pseudo‐irreversible Inhibitors of Lymphoid Tyrosine Phosphatase

We screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat T cells, whereas diethyldithiocarbamate failed to inhibit LYP at the concentrations tested. Compound 13 , an N‐(2‐thioxothiazolidin‐4‐one) analogue, was found to be the most potent LYP inhibitor in this series, with an IC₅₀ value of 3 μM . Compound 13 inhibits LYP pseudo‐irreversibly, as evidenced by the time‐dependence of inhibition, with a K_i value of 1.1 μM and a k_inact value of 0.004 s^?1. The inhibition of LYP by compound 13 could not be reversed significantly by incubation with glutathione or by prolonged dialysis, but could be partially reversed by incubation with dithiothreitol. Compound 13 also inhibited LYP activity in Jurkat T cells.     

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis‐galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (K_d). This has led to high‐affinity LecA ligands with K_d values in the low nanomolar range (K_d=22 nm for the best one).     

Multiparameter Kinetic Analysis for Covalent Fragment Optimization by Using Quantitative Irreversible Tethering (qIT)

Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure–activity relationships through the simultaneous determination of K_i, k_inact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.     

E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain‐like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule‐associated serine proteases. Its exopeptidase activity is structurally explained by the so‐called exclusion domain, which blocks the active‐site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)‐trans‐epoxysuccinyl‐L ‐leucylamido‐3‐methylbutane (E‐64c) (k₂/K_i=140±5 M ^?1 s^?1) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′–S2′ area with its leucine‐isoamylamide moiety. Furthermore, structure–activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n‐butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56 000±1700 M ^?1 s^?1).     

Ozone Inactivation of Pseudomonas aeruginosa,Escherichia coli,Shigella sonnei and Salmonella typhimurium in Water.

Based on the properties of ozone as a strong germicidal agent, inactivation kinetics of Pseudomonas aeruginosa, Escherichia coli, Shigella sonnei and Salmonella typhimurium towards ozone in water were studied. The values of 90% inactivation (t₉₀) obtained varied from 0.20 minutes (2.4 mg/L, Escherichia coli ATCC 25922) to 8.33 minutes (0.39 mg/L, Pseudomonas aeruginosa wild strain). First order inactivation kinetics with respect to both the concentrations of ozone and microorganisms were found, resulting an overall second order inactivation kinetics. The ATCC strains showed to be the most sensitive toward ozone among all. Meanwhile, the environmental isolation of Pseudomonas aeruginosa was the most resistant and Escherichia coli the most sensitive wild strain. The longest time required to achieve total inactivation was 35 minutes.     

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

We have investigated 4‐halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4‐chloropyridine with amino acids and thiols was ranked with respect to common covalent protein‐modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment‐sized 4‐halopyridines inactivated human dimethylarginine dimethylaminohydrolase‐1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off‐pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2‐fluoromethyl‐substituted 4‐chloropyridines demonstrated that the pK_a and k_inact/K_I values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4‐Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.     

Development of Novel Benzodiazepine-Based Peptidomimetics as Inhibitors of Rhodesain from Trypanosoma brucei rhodesiense

Starting from the reversible rhodesain inhibitors 1 a – c , which have K_i values towards the target protease in the low-micromolar range, we have designed a series of peptidomimetics, 2 a – g , that contain a benzodiazepine scaffold as a β-turn mimetic; they are characterized by a specific peptide sequence for the inhibition of rhodesain. Considering that irreversible inhibition is strongly desirable in the case of a parasitic target, a vinyl ester moiety acting as Michael-acceptor was introduced as the warhead; this portion was functionalized in order to evaluate the size of corresponding enzyme pocket that could accommodate this substituent. With this investigation, we identified an irreversible rhodesain inhibitor (i. e., 2 g ) with a k_2nd value of 90 000 M⁻¹ min⁻¹ that showed antitrypanosomal activity in the low-micromolar range (EC₅₀=1.25 μM), this may be considered a promising lead compound in the drug-discovery process for treating human African trypanosomiasis (HAT).     

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

Isoprenoid biosynthesis is an important area for anti‐infective drug development. One isoprenoid target is (E)‐1‐hydroxy‐2‐methyl‐but‐2‐enyl 4‐diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H⁺/2e^? reduction. IspH contains a 4 Fe?4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe?4 S clusters by η¹ coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H₂O, O₂) binding. Based on these ideas, we used in silico methods to find drug‐like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a K_i value of ≈500 nm against Pseudomonas aeruginosa IspH.     

Biodegradation kinetics of benzene,methyl tert‐butyl ether,and toluene as a substrate under various substrate concentrations

Owing to the complexity of conventional methods and shortcomings in determining kinetic parameters, a convenient approach using the nonlinear regression analysis of Monod or Haldane type nonlinear equations is presented. This method has been proven to provide accurate estimates of kinetic parameters. The major work in this study consisted of the testing of aromatic compound‐degrading cultures in batch experiments for the biodegradation of benzene, methyl tert‐butyl ether (MTBE), and toluene. Additionally, batch growth data of three pure cultures (i.e., Pseudomonas aeruginosa YAMT421, Ralstonia sp. YABE411 and Pseudomonas sp. YATO411) isolated from an industrial petrochemical wastewater treatment plant under aerobic conditions were assessed with the nonlinear regression technique and with a trial‐and‐error procedure to determine the kinetic parameters. The growth rates of MTBE‐, benzene‐, and toluene‐degrading cultures on MTBE, benzene, and toluene were significant. Monod's model was a good fit for MTBE, benzene and toluene at low substrate concentrations. In contrast, Haldane's equation fitted well in substrate inhibition concentration. Monod and Haldane's expressions were found to describe the results of these experiments well, with fitting values higher than 98%. The kinetic parameters, including a maximum specific growth rate (µ_m), a half‐saturation constant (K_s), and an inhibition constant (K_i), were given. Copyright © 2007 Society of Chemical Industry     

Disruption of ergosterol biosynthesis,growth, and the morphological transition in <Emphasis Type="Italic">Candida albicans</Emphasis> by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain

The sterol substrate analog 25-thialanosterol and its corresponding sulfonium salt were evaluated for their ability to serve as antifungal agents and to inhibit sterol methyltransferase (SMT) activity in Candida albicans. Both compounds inhibited cell proliferation, were fungistatic, interrupted the yeastlike-form to germ-tube-form transition, and resulted in the accumulation of zymosterol and related Δ²⁴-sterols concurrent with a decrease in ergosterol, as was expected for the specific inhibition of SMT activity. Feedback on sterol synthesis was evidenced by elevated levels of cellular sterols in treated vs. control cultures. However, neither farnesol nor squalene accumulated in significant amounts in treated cultures, suggesting that carbon flux is channeled from the isoprenoid pathway to the sterol pathway with minor interruption or redirection until blockage at the C-methylation step. Activity assays using solubilized C. albicans SMT confirmed the inhibitors impair SMT action. Kinetic analysis indicated that 25-thialanosterol inhibited SMT with the properties of a time-dependent mechanismbased inactivator K _i of 5 =gmM and apparent k _inact of 0.013 min⁻¹, whereas the corresponding sulfonium salt was a reversible-type transition state analog exhibiting a K _i of 20 nM. The results are interpreted to imply changes in ergosterol homeostasis as influenced by SMT activity can control growth and the morphological transition in C. albicans, possibly affecting disease development.     

Zur physikalisch‐chemischen Charakterisierung von 5‐Amino‐1‐aryl‐1H‐tetrazolen: Elektronische Molekülparameter aus der thermischen Isomerisierung zu 5‐Arylamino‐1H‐tetrazolen

The known thermal isomerization of 5‐amino‐1‐aryl‐1H‐tetrazoles ( A ) into corresponding 5‐arylamino‐1H‐tetrazoles ( HB ) was used to derive physicochemical parameters characterizing the electronic substituent effect on isomerism and dissociation equilibria. For a series of 26 tetrazoles A as starting materials the equilibrium constants (pK_i) of isomerization in boiling ethylene glycol at 197 °C and the dissociation constants (pK_a) of the NH‐acidic tetrazoles HB were determined by potentiometric titration of rapidly cooled equilibrium mixtures in water and ethanol/water with KOH at 25 °C. The pK values are closely correlated with Hammett′s electronic substituent constants σ and can be used as electronic molecule parameters in QSAR or QSPR (QSAR = quantitative structure‐activity relationship; QSPR = quantitative structure‐property relationship) studies.     

Adsorption characteristics of n-propanol on silica gel by gas-chromatography

A pulse-response chromatographic method was used to measure a reversible equilibrium adsorption constant and a rate constant for irreversible adsorption of n-propanol on silica gel particles of various sizes, at 1 atm in the 175–250°C range.For the first time reversible and irreversible adsorption was observed using chromatographic techniques after passing through the column for 48 hr a flow of n-propanol with nitrogen as carrier gas.Values of K_A and k_i were calculated at 175, 200 and 250°C and from these values the heat of adsorption for reversible sites and activation energy for irreversible sites were calculated.     

Zur physikalisch‐chemischen Charakterisierung von 5‐Amino‐1‐aryl‐1H‐tetrazolen: Kinetik der Verteilung im Zweiphasensystem Octan‐1‐ol/Wasser

The kinetics of distribution of 27 5‐amino‐1‐aryl‐1H‐tetrazoles in the two‐phase system octan‐1‐ol/water were investigated UV/Vis‐spectrophotometrically at various temperatures. Studies on relationships between the obtained firstorder rate constants (logk₁, logk₂) and the hydrophobicity of the tetrazoles described by their partition coefficients (logP) show a nearly constant rate of transport from the aqueous to the organic phase (k₁) above logP = 1,5 while the reverse rate (k₂) strongly depends on hydrophobicity. In the whole logP range investigated the kinetic behaviour can be described by bilinear relationships between logk and logP corresponding to known kinetic models for distribution processes in two‐layer systems.