Inhibition of matrix metalloproteinase-9 by "multi-prong" surface binding groups

A novel strategy of blocking the active site accessibility of MMP-9 by "multi-prong" surface binding groups is described.     

A general strategy for creating "inactive-conformation" abl inhibitors

Kinase inhibitors that bind to the ATP cleft can be broadly classified into two groups: those that bind exclusively to the ATP site with the kinase assuming a conformation otherwise conducive to phosphotransfer (type I), and those that exploit a hydrophobic site immediately adjacent to the ATP pocket made accessible by a conformational rearrangement of the activation loop (type II). To date, all type II inhibitors were discovered by using structure-activity-guided optimization strategies. Here, we describe a general pharmacophore model of type II inhibition that enables a rational "hybrid-design" approach whereby a 3-trifluoromethylbenzamide functionality is appended to four distinct type I scaffolds in order to convert them into their corresponding type II counterparts. We demonstrate that the designed compounds function as type II inhibitors by using biochemical and cellular kinase assays and by cocrystallography with Abl.     

A general preparation of fluoroallylamine enzyme inhibitors incorporating a β-substituted heteroatom

A general route to β-heteroatom substituted fluoroallylamines is described. This has led to the synthesis of new time-dependent inhibitors of monoamine oxidase, diamine oxidase and γ-aminobutyric acid transaminase.     

A novel retro-reaction strategy toward designing a selective fluorescence Cu(II) chemodosimeter

A novel C9 acridane-adduct, featuring ketobenzimidazole chelate, functions as a highly selective fluorescent chemodosimeter for Cu²⁺, while other metal ions pose little interferences, if any. The signaling strategy operates via the Cu²⁺-mediated retro-reaction, generating a strongly fluorescent acridinium ion at the expense of the weakly emitting probe.     

Development of energetic pharmacophore for the designing of 1,2,3,4-tetrahydropyrimidine derivatives as selective cyclooxygenase-2 inhibitors

We present here the Energetic pharmacophore model representing complementary features of the 1,2,3,4-tetrahydropyrimidine for selective cyclooxygenase-2 (COX-2) inhibition. For the development of pharmacophore hypothesis, a total of 43 previously reported compounds were docked on active site of COX-2 enzyme. The generated pharmacophore features were ranked using energetic terms of Glide XP docking for 1,2,3,4-tetrahydropyrimidine scaffold to optimize its structure requirement for COX-2 inhibition. The thirty new 4,5,6-triphenyl-1,2,3,4-tetrahydropyrimidine derivatives were synthesized and assessed for selective COX-2 inhibitory activity. Two compounds 4B1 and 4B11 were found to be potent and selective COX-2 inhibitors. The molecular docking studies revealed that the newly synthesized compounds can be docked into COX-2 binding site and also provide the molecular basis for their activity.     

"Inject-mix-react-separate-and-quantitate" (IMReSQ) method for screening enzyme inhibitors

Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors.     

Supraphos: A supramolecular strategy to prepare bidentate ligands

Herein, we report a new strategy for the preparation of chelating bidentate ligands, which involves the mixing of two mondentate ligands functionalized with complementary binding sites. The assembly process is based on selective metal-ligand interactions employing phosphite zinc(II) porphyrins 1-6 and the nitrogen-containing phosphorus ligands b-i (Scheme 1). Only 14 monodentate ligands were utilized to generate a library of 48 palladium catalysts based on supraphos-type bidentate ligands. The characterization of rhodium complexes based on representative Supramolecular bidentate ligands and the comparison of their performance in the hydroformylation of styrene will be presented. The current library of catalysts was tested in the asymmetric palladium-catalyzed alkylation of rac-1,3-diphenyl-2-propenyl acetate, which resulted in a large variety in the observed enantioselectivity for the different catalysts. Importantly, small variations in the supraphos building blocks, lead to large differences in the enantioselectivity imposed by the catalyst, the most selective catalyst producing 97% ee.     

A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as L-cysteine, and thereby internalization of the liposomes was significantly facilitated. When L-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells.     

A model for the structure of MCM-41 incorporating surface roughness

The surface area of MCM-41 mesoporous silica, estimated by several models in the literature, is significantly less than the value derived from BET analysis of nitrogen adsorption at 77.4 K. In the past, the difference has been attributed to several reasons including the errors involved in the BET analysis of the multilayer-capillary condensation region and the heterogeneity of the walls. In the present work, we present an alternate model of MCM-41 based on molecular simulations that gives surface area values that are in closer agreement to those determined by experiment. The model incorporates bulk heterogeneity of the material, surface hydroxyls, and most importantly, physical deformations or indentations of the pore surface. The model predicts small-angle X-ray diffraction (XRD) and wide-angle X-ray scattering (WAXS) results that are consistent with experimental data as well as surface areas and pore volumes that compare favorably with published experimental results. The simulation results are consistent with the hypothesis that the interstitial space in MCM-41 is relatively amorphous despite the regular arrangement of the mesopores. The surface roughness associated with the amorphous structure increases the surface area beyond the nominal value produced by assuming smooth cylindrical pores.     

A simple strategy for charge selective biopolymer sensing

Polysaccharide-induced protein fluorescence 'turn-on' responses have been studied in the presence of similarly charged micelle at pH ～3. Quenched protein fluorescence is selectively recovered ('on' state) for bovine serum albumin (BSA) with sodium carboxy methyl cellulose (SCMC) and for pepsin (PS) with chitosan (CS) with starting ultra low concentrations of 0.04 μM and 0.008 μM respectively.     

Screening for inhibitors of histone deacetylase by incorporating a spraying method to micro-arrayed compound screening

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to micro ARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the micro ARCS format. IC(50) values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of micro ARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.     

A novel strategy for designing irreversible inhibitors of metalloproteases: acetals as latent electrophiles that interact with catalytic nucleophile at the active site

A new strategy for design of irreversible inactivators for carboxypeptidase A (CPA), a prototypic zinc protease, has been developed by exploiting the property of acetals to generate an oxacarbenium ion intermediate in the conversion into the corresponding carbonyl compounds. The design strategy is exemplified by 2-benzyl-5-alkyl-3,5-dioxapentanoic acids (1a-c). Interestingly, (R)-1b is slightly more potent than an (S)-1b as an inactivator of CPA.     

A novel strategy to assemble achiral ligands to chiral helical polyrotaxane structures

Using the achiral N,N'-bis(2-pyridylmethyl)-1,6-hexanediamine ligand bearing two end pyridyl groups as the source of conformational chirality, a novel type of TMeQ[6]-based helical polyrotaxane was prepared and characterized by X-ray crystallography and (1)H NMR spectroscopy. The chirality of the polyrotaxane was generated from twisting of the hexylidene of the N,N'-bis(2-pyridylmethyl)-1,6-hexanediamine "string" when bound within the hydrophobic cavity of TMeQ[6]. Two opposite chiral helical polyrotaxanes crystallize as a racemic compound.     

An integration strategy to measure enzyme activities for detecting irreversible inhibitors with dimethoate on butyrylcholinesterase as a model

An integration strategy was investigated to measure initial rates of horse butyrylcholinesterase (BChE) at 50.0?µmol?L^?1 butyrylthiocholine (BTCh) for detecting irreversible inhibitors as pollutants in environment and foods with dimethoate as a model. In this integration strategy: (a) if BTCh consumption within 5.0?min was >60%, BChE initial rates were derived from maximal reaction rates, estimated by an improved integrated method, according to Michaelies-Menten kinetics at 47.0?µmol?L^?1 BTCh and Michaelis-Menten constant at 94.0?µmol?L^?1; (b) or else initial rates were determined by the classical initial rate method. Thus, the differences in BChE initial rates without and after dimethoate treatment indexed final dimethoate contents in reaction mixtures to treat BChE. Results supported that this integration strategy determined BChE activities with a linear range about two magnitudes and an upper limit about twice that by the classical initial rate method alone at 2.0?mmol?L^?1 BTCh. The coefficient of variation with this integration strategy was below 5%. The difference in BChE initial rates before and after dimethoate treatment was proportional to final dimethoate contents in reaction mixtures. By enzymatic analyses, the molar contents of dimethoate extracted from polluted cabbages were consistent with the summed molar contents of dimethoate and dimethoxon by gas-chromatography. Therefore, this integration strategy was effective to detect irreversible inhibitors as pollutants in environment and foods.     

Conjugation of poor inhibitors with surface binding groups: a strategy to improve inhibition

Conjugation of surface binding groups with inhibitors for carbonic anhydrase leads to the conversion of weak inhibitors to strong inhibitors.     

A synthetic approach to F-analogues of Angiotensin converting enzyme inhibitors

Studies on the synthetic routes to F-analogues of captopril, which is known as an Angiotensin converting enzyme inhibitor, have been undertaken.     

A simple "molecular beacon"-based fluorescent sensing strategy for sensitive and selective detection of mercury (II)

A novel fluorescent sensor for the detection of Hg(2+) in aqueous media was developed. The method takes advantages of the highly selective thymine-Hg(2+)-thymine coordination and the sensitive "signal-on" structure-switching molecular beacon.