Site-directed Irreversible Inhibitors Research Articles

The role of TG2 in ECV304-related vasculogenic mimicry

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.

X-ray crystal structure and mechanism of action of oestrone 3-O-sulphamate, a synthetic active site-directed inhibitor of oestrone sulphatase

Oestrone 3-O-sulphamate, wherein a sulphamate moiety is a sulphate group surrogate relative to oestrone sulphate, is the most potent oestrone sulphatase inhibitor developed to date and inhibits oestrone sulphatase in a time- and concentration-dependent manner, showing that it is acting as an active site-directed irreversible inhibitor. It also inhibits dehydroepiandrosterone sulphatase, the enzyme which regulates the synthesis of androstenediol. Oestrone 3-O-sulphamate has also been shown to inhibit these enzymes in-vivo. We report here its crystal structure and the synthesis and inhibitory activites of analogues in which the 3-O atom is replaced by other heteroatoms (N and S). Oestrone 3-N-sulphamate and oestrone 3-S-sulphamate were found to inhibit placental microsome oestrone sulphatase weakly, i.e. 53% and 12%, respectively, at 50 μM (compared with >99% for oestrone 3-O-sulphamate), but none of these compounds appears to behave as a time-dependent inhibitor.

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes: a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Inactivation of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue in Corynebacterium glutamicum, by (R)- and (S)-Oxirane-2-carboxylate: Analysis and Implications

(R)- and (S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the (R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the (R) and (S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by (R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the (R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

Inactivation of recombinant human steroid sulfatase by KW-2581

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC 50 of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner ( k inact, 0.439 min −1; K i, app , 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.

Using a Small Molecule Inhibitor of Peptide: N-Glycanase to Probe Its Role in Glycoprotein Turnover

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.

Reactions of trans-3-chloroacrylic acid dehalogenase with acetylene substrates: consequences of and evidence for a hydration reaction.

Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic Acid

Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.

Mechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity.

4,7-Dioxosebacic acid (4,7-DOSA) is an active site-directed irreversible inhibitor of porphobilinogen synthase (PBGS). PBGS catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors such as heme, vitamin B(12), and chlorophyll. 4,7-DOSA was designed as an analogue of a proposed reaction intermediate in the physiological PBGS-catalyzed condensation of two molecules of 5-aminolevulinic acid. As shown here, 4,7-DOSA exhibits time-dependent and dramatic species-specific inhibition of PBGS enzymes. IC(50) values vary from 1 microM to 2.4 mM for human, Escherichia coli, Bradyrhizobium japonicum, Pseudomonas aeruginosa, and pea enzymes. Those PBGS utilizing a catalytic Zn(2+) are more sensitive to 4,7-DOSA than those that do not. Weak inhibition of a human mutant PBGS establishes that the inactivation by 4,7-DOSA requires formation of a Schiff base to a lysine that normally forms a Schiff base intermediate to one substrate molecule. A 1.9 A resolution crystal structure of E. coli PBGS complexed with 4,7-DOSA (PDB code ) shows one dimer per asymmetric unit and reveals that the inhibitor forms two Schiff base linkages with each monomer, one to the normal Schiff base-forming Lys-246 and the other to a universally conserved "perturbing" Lys-194 (E. coli numbering). This is the first structure to show inhibitor binding at the second of two substrate-binding sites.

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure–activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1–9 ) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1–9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme–inhibitor adduct (Ki), the inactivation constant of the enzyme–inhibitor adduct (k2), and the bimolecular inhibition constant (ki) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1–9 and the Hammett substituent constant (σ), are observed, and the reaction constants (ρs) are −1.36, 0.35 and −1.01, respectively. Therefore, the above reaction may form a positive charged enzyme–inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors

4,4′-Biphenyl-di- N-butylcarbamate ( 1), ( S)-1,1′-bi-2-naphthyl-2,2′-di- N-butylcarbamate ( S- 2), ( S)-1,1′-bi-2-naphthyl-2- N-butylcarbamate-2′-butyrate ( S- 3), 2,2′-biphenyl-di- N-butylcarbamate ( 4), 2,2′-biphenyl-2- N-octadecylcarbamate-2′- N-octylcarbamate ( 5), 2,2′-biphenyl-2- N-octadecylcarbamate-2′- N-phenylcarbamate ( 6), 2,2′-biphenyl-2- N-butylcarbamate-2′-butyrate ( 7), 2,2′-biphenyl-2- N-butylcarbamate-2′-ol ( 8), 2,2′-biphenyl-2- N-octylcarbamate-2′-ol ( 9), ( R)-1,1′-bi-2- N-naphthyl-2-butylcarbamate-2′-ol ( R- 10), and glyceryl-1,2,3-tri- N-butylcarbamate ( 11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1– 3 and 7– 10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S- 2, S- 3, and R- 10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4– 9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4– 6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4– 6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable π-π interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8– 10 are more potent than carbamates S- 2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.

Enantiomeric inhibitors of cholesterol esterase and acetylcholinesterase

Enantiomers of N-methyl- N,α-methylbenzylbutyramide ( 1), 1-butyl-3-methyl-3′-α-methylbenzylurea ( 2), 1,2,3,4-tetrahydro-1-naphthyl- N-butylcarbamate ( 3), 1,1′-bi-2-naphthyl-2,2′-di- N-butylcarbamate ( 4), 1,1′-bi-2-naphthyl-2-ol-2′- N-butylcarbamate ( 5), and 1,1′-bi-2-naphthyl-2-butyrate-2′- N-butylcarbamate ( 6) are inhibitors of porcine pancreatic cholesterol esterase-catalyzed hydrolysis of 4-nitrophenyl butyrate and of electric eel acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine in the presence of 5,5′-dithiobis-2-nitrobenzoate. For competitive inhibitors, values of the inhibition constant ( K i) and the enantiomeric ratio ( E comp.) are investigated. For active site-directed irreversible inhibitors, values of the inhibition constant ( K i), the carbamylation constant ( k 2), the bimolecular rate constant ( k i), and the enantiomeric ratio ( E) are investigated. Toward both enzymes, compounds 1 are poor competitive inhibitors ( K i=10 2–10 4 μM) but have good enantioselectivities ( E comp.=10–50, the preference for R). R- 2 and S- 2 are competitive inhibitors of acetylcholinesterase with K i=26 and 80 μM, respectively (the preference for R) but are active site-directed irreversible inhibitors of cholesterol esterase with k i=4 and 16 M −1 sec −1, respectively (the preference for S). For those competitive inhibitions, both leaving group hydrophilic and hydrophobic binding sites of cholesterol esterase or both anionic substrate binding site and peripheral anionic binding site of acetylcholinesterase bind to N, N-methyl-α-methylbenzyl disubstituted amide parts of these inhibitors and the enzyme does not catalyze the hydrolysis of these inhibitors. The opposite stereopreference ( S) for the inhibition of cholesterol esterase by compounds 2 may be due to the fact that N, N-methyl-α-methylbenzyl disubstituted amide parts of these inhibitors bind to the alkyl chain binding site of the enzyme. Compounds 3– 6 are active site-directed irreversible inhibitors of cholesterol esterase ( k i=1–13 000 M −1 s −1) and peripheral anionic binding site-directed irreversible inhibitors of acetylcholinesterase ( k i=1.7–1300 M −1 s −1). Compounds 3 have low enantioselectivities ( E=1.3–1.4) for both enzymes. The stereopreference for atropisomers 4 and 6 is S-form toward both enzymes ( E=2–30) and is identical to that of cholesterol esterase-catalyzed hydrolysis of 1,1′-bi-2-naphthyl-2,2′-diacylate. This stereopreference ( S) may be due to the fact that the butyryl group or one of two butylcarbamate groups of S-atropisomers binds more effectively to the leaving group hydrophobic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase than that of R-atropisomers. The opposite stereopreference ( R) for atropisomers 5 toward both enzymes may be due to a favorable interaction between the hydroxyl group of the inhibitors and the leaving group hydrophilic binding site of cholesterol esterase or the peripheral anionic binding site of acetylcholinesterase.

Crystal Structure of 4-Oxalocrotonate Tautomerase Inactivated by 2-Oxo-3-pentynoate at 2.4 Å Resolution: Analysis and Implications for the Mechanism of Inactivation and Catalysis,

The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivated by the active site-directed irreversible inhibitor 2-oxo-3-pentynoate (2-OP) has been determined to 2.4 A resolution. The structure of the enzyme covalently modified at Pro-1 by the resulting 2-oxo-3-pentenoate adduct is nearly superimposable on that of the free enzyme and confirms that the active site is located in a hydrophobic region surrounding Pro-1. Both structures can be described as a trimer of dimers where each dimer consists of a four-stranded beta-sheet with two antiparallel alpha-helices on one side. Examination of the structure also reveals noncovalent interactions between the adduct and two residues in the active site. The epsilon and eta nitrogens of the guanidinium side chain of Arg-39" from a neighboring dimer interact respectively with the C-2 carbonyl oxygen and one C-1 carboxylate oxygen of the adduct while the side chain of Arg-61' from the same dimer as the modified Pro-1 interacts with the C-1 carboxylate group in a bidentate fashion. An additional interaction to the 2-oxo group of the adduct is provided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-butynoate is a more potent irreversible inhibitor of 4-oxalocrotonate tautomerase than is 2-OP suggest that Arg-39" and the ordered water molecule polarize the carbonyl group of 2-OP which facilitates a Michael reaction between Pro-1 and the acetylene compound. On the basis of the crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.

Conformationally restricted carbamate inhibitors of horse serum butyrylcholinesterase

Conformationally restricted carbamate inhibitors, exo-2-norbornyl- N-butylcarbamate ( 1), endo-2-norbornyl- N-butylcarbamate ( 2), 1-adamantyl- N-butylcarbamate ( 3), and 2-adamantyl- N-butylcarbamate ( 4) as active site-directed irreversible inhibitors of horse serum butyrylcholinesterase are investigated for values of the dissociation constant ( K I), the carbamylation constant (k 2), and the bimolecular rate constant (k i). Compound 1 is the most potent inhibitor of the enzyme and the values of K I and k i are 20 nM and 1.1×10 5 M −1sec −1.

4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy.

4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta 1, residues 2-8; beta 2, residues 39-45), a beta-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The beta 1 strand is parallel to the beta 2 strand of the same subunit on the basis of cross stand NH(i)-NH(j) NOEs in a 2D 15N-edited 1H-NOESY spectrum of hexameric 4-OT containing two 15N-labeled subunits/hexamer. The beta 1 strand is also antiparallel to another beta 1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the beta-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.

Atropisomeric carbamoyl type inhibitors of pancreatic cholesterol esterase

Atropisomers of 1,1′-bi-2-naphthyl-2,2′-di- N-butylcarbamate ( 1), 1,1′-bi-2-naphthyl-2- N-butylcarbamate-2′-ol ( 2), and 1,1′-bi-2-naphthyl-2- N-butylcarbamate-2′-butyrate ( 3) as active site-directed irreversible inhibitors of pancreatic cholesterol esterase are investigated for values of the dissociation constant (K I), the carbamylation constant (k 2), and the bimolecular rate constant (k i).

Linear free energy relationships of the inhibition of pancreatic cholesterol esterase by 4-Nitrophenyl-N-alkylcarbamate

4-Nitrophenyl-N-alkylcarbamates (1) as active site-directed irreversible inhibitors of pancreatic cholesterol esterase are investigated for values of the dissociation constant (Ki), the carbamylation constant (k2), and the bimolecular rate constant (ki). Linear free energy relationships between −logKi, logk2, or logki and polar substituent constant (σ∗) are observed. Taft's Es steric constants are not important in these correlationships. Multiple linear relationships of −logKi, logk2, or logki are observed on Charton equation (logk = ασI + βσR + ψν + h).

Hammett analysis of the inhibition of pancreatic cholesterol esterase by substituted phenyl-N-butylcarbamate

Substituted phenyl-N-butylcarbamates ( 1) as active site-directed irreversible inhibitors of pancreatic cholesterol esterase are investigated for values of the dissociation constant (K I), the carbamylation constant (k 2), and the bimolecular rate constant (k i). Linear free energy relationships between-logK I, logk 2, or logk i and substituent constant (σ) are observed.