Discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3CL protease

A diversified library of peptide anilides was prepared, and their inhibition activities against the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline, l-phenylalanine and 4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL protease with K(i) = 0.03 muM. The molecular docking experiment indicates that the P1 residue of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.     

Discovery of a novel family of SARS-CoV protease inhibitors by virtual screening and 3D-QSAR studies

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3C-like protease (3CL(pro) or M(pro)) is an attractive target for the development of anti-SARS drugs because of its crucial role in the viral life cycle. In this study, a compound database was screened by the structure-based virtual screening approach to identify initial hits as inhibitors of SARS-CoV 3CL(pro). Out of the 59,363 compounds docked, 93 were selected for the inhibition assay, and 21 showed inhibition against SARS-CoV 3CL(pro) (IC(50) 相似文献   

Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.     

Inhibition of endothelial-bound angiotensin converting enzyme, in vivo.

1. We determined apparent Ki constants of two inhibitors, captopril and CL242,817, for pulmonary endothelial-bound angiotensin converting enzyme (ACE) in anaesthetized rabbits. [3H]-benzoyl-Phe-Ala-Pro was used as the substrate. The apparent kinetic parameters Km and Amax (product of Vmax and microvascular plasma volume) were measured, as was the ratio (Amax/Km) (measured under first order reaction conditions) before and 30s after the i.v. administration of captopril 10 nmol kg-1 or CL242,817, 35 nmol kg-1. 2. Under mixed order reaction conditions, ([S] greater than or equal to Km), apparent Km values increased from 12.2 +/- 1.9 microM to 32.9 +/- 3.3 microM (P less than 0.05) in the captopril-treated rabbits and from 9.3 +/- 2.3 microM to 45.8 +/- 9.8 microM (P less than 0.05) in the CL242,817-treated rabbits, indicative of competitive inhibition. However, apparent Amax values decreased from 10.3 +/- 2.1 to 4.5 +/- 0.8 mumol min-1 (P less than 0.05) and 8.9 +/- 1.7 to 4.8 +/- 0.5 mumol min-1 (P less than 0.05), respectively. 3. Under first order reaction conditions ([S] much less than Km), the Amax/Km ratio decreased from 763 +/- 100 to 125 +/- 38 ml min-1 (P less than 0.05) and 1009 +/- 149 to 126 +/- 44 ml min-1 (P less than 0.05) in the captopril- and CL242,817-treated groups respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and kinetic studies of protease substrates containing the 1-methyl-6-aminoquinolinium ion as a fluorogenic leaving group

Several sensitive substrates for porcine pancreatic elastase, chymotrypsin, and trypsin were prepared that utilize the permanently charged, fluorogenic cation 1-methyl-6-aminoquinoline (MAQ+) as the leaving group. Kinetic rates for the hydrolysis of substrates were determined fluorimetrically and compared with analogues having 6-aminoquinoline (6-AQ) as an uncharged leaving group. It was found that substrates containing the quaternized leaving group generally have a higher kcat/Km ratio. An exception to this trend was noted with a trypsin substrate, Bz-DL-Arg-MAQ+. During the course of this investigation, several significant advantages of the MAQ+ ion as a fluorogenic leaving group in protease substrates were found: (a) its appearance can be measured fluorimetrically using wavelengths of light that result in its maximal fluorescence, while under these conditions, the unhydrolyzed substrate is essentially nonfluorescent, (b) it confers a high degree of water solubility to hydrophobic peptides, thereby eliminating the need for organic cosolvents to dissolve substrates, and (c) quaternized substrates can be prepared readily and in good yield from the corresponding 6-(peptidylamido)quinolines. These positively charged synthetic fluorogenic substrates are, therefore, useful probes for investigating the steric and electronic properties of the active-site environment of proteolytic enzymes.     

Phosphoramidate pronucleotides: a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins

To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (kcat/Km) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) for adenosine tryptamine phosphoramidate monoester were found to be 107 M-1 s-1 for hHint1 and 106 M-1 s-1 for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2'-OH group of adenosine monophosphate and the active site residue, Asp43, the second-order rate constant (kcat/Km) for thymidine tryptamine phosphoramidate was found to be 3-4 orders of magnitude smaller than that for uridine tryptamine phosphoramidate for hHint1 and 2 orders of magnitude smaller than that for E. coli hinT. Ara-A tryptamine phosphoramidate was, however, shown to be a good substrate with a specificity constant (kcat/Km) only 10-fold lower than the value for adenosine tryptamine phosphoramidate. Consequently, nucleoside phosphoramidates containing unhindered primary amines and either an alpha or beta 2'-OH group should be easily bioactivated by Hints with efficiencies rivaling those for the 5'-monophosphorylation of nucleosides by nucleoside kinases. The differential substrate specificity observed for human and E. coli enzymes represents a potential therapeutic rationale for the development of selective antibiotic phosphoramidate pronucleotides.     

NADH-dependent reduction of sulphamethoxazole hydroxylamine in dog and human liver microsomes

1. Reduction of hydroxylamine drug metabolites by NADH-dependent hydroxylamine reductase (NDHR) has been suggested to be involved in the pathogenesis of idiosyncratic sulphonamide toxicity in humans. The dog represents a naturally occurring clinical model for sulphonamide toxicity in humans. he purpose of these studies, therefore, was to characterize the presence of hepatic NADH-dependent hydroxylamine reductase activity in the dog and to compare this activity with that found in humans. 2. NDHR activity was characterized by the presence of two enzymes in both dog and human liver microsomes, with comparable estimates of Km (Km1 = 75 microM, Km2 = 404 microM in dog; Km1 = 69 microM, Km2 = 503 microM in human). Estimates of maximal velocity were significantly, but not dramatically, higher for dog NDHR (Vmax1 = 2.09 nmole mg(-1) min(-1) Vmax2 = 4.58 nmole mg(-1) min(-1) compared with human NDHR (Vmax1 = 0.42 nmole mg(-1) min(-1), Vmax2 = 1.56 nmole mg(-1) min(-1)). NDHR in dog, as in humans, preferred NADH to NADPH, was more active at pH 6.3 than at 7.4 and was not inhibited by carbon monoxide, azide, anaerobic conditions, the CYP substrate inhibitors tolbutamide, dextromethorphan, or erythromycin, or antibodies directed against CYP2C, CYP2D or CYP3A. 3. It is concluded that two forms of NDHR are present in dog and humans with similar biochemical characteristics. Although NDHR activity has been attributed to a CYP2D isoform in pig, there is no evidence for involvement of CYP450 in the reduction of sulphamethoxazole hydroxylamine in either dogs or humans.     

Development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease 3CL(pro)

Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CL(pro) (M(pro)), which is highly conserved among all coronaviruses. Here 3CL(pro) derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CL(pro) with K(i)'s as low as 300 nM. The crystal structure of the complex of one of the compounds with 3CL(pro) indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.     

Spectrophotometric kinetic studies and substrate inhibition of hog kidney histaminase activity by an improved 2,4-dinitrophenylhydrazine method

We improved the experimental procedure for the measurement of hog kidney histaminase activity using histamine as a substrate on the basis of a spectrophotometric estimation of the 2,4-dinitrophenylhydrazone of imidazole acetaldehyde and studied the steady-state kinetics to obtain the basic data for further investigations of the oxidative deamination of histamine. The initial and mean velocities of the enzymatic reaction were calculated and plotted against the amount of enzyme. It was found that the initial velocity increased linearly. The time t alpha necessary to reach the extent of reaction alpha was calculated and plotted against the reciprocal of the enzyme concentration eO. It was found that t alpha was linearly proportional to 1/eO. From Lineweaver-Burk plots, inhibition by high concentration of substrate was evident, and the v-pS curve was bell-shaped, with a pS maximum at 3.2. Km and V were obtained: Km = 7.7 x 10(-5) M, V = 0.0026 mumol/min (0.00075 mumol/min/mg protein). It was concluded that our DNP method was useful for the measurement of hog kidney histaminase activity using histamine as a substrate, basic steady-state kinetic studies and further investigations of substrate inhibition and inhibitory effect.     

Human cytochrome P450 2A13 efficiently metabolizes chemicals in air pollutants: naphthalene, styrene, and toluene

Human P450 2A13 is the most efficient enzyme for catalyzing the metabolism of nicotine and metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). It is conceivable that P450 2A13 also metabolizes chemicals in air pollutants because this enzyme is highly expressed in the respiratory tract. In this study, we investigated the possibility that P450 2A13 can metabolize naphthalene, styrene, and toluene, which are included in air pollutants as well as tobacco smoke, although they were known to be metabolized by P450 1A2 or 2E1. We found that P450 2A13 catalyzed 1- and 2-naphthol formations from naphthalene with higher intrinsic clearances ( kcat/ Km) (3.1- and 2.2-fold, respectively) than P450 1A2 and also more efficiently catalyzed the styrene 7,8-oxide formation from styrene and the benzylalcohol formation from toluene than P450 2E1. The overlapping substrate specificity of P450 2A13 with P450 2E1 was supported by the finding that P450 2A13 catalyzed chlorzoxazone 6-hydroxylation (8-fold higher value of kcat/ Km) and p-nitrophenol 2-hydroxylation (19-fold higher value of kcat/ Km), which are marker activities of P450 2E1. Thus, we found that P450 2A13 metabolizes diverse environmental chemicals and has overlapping substrate specificities of P450 1A2 and 2E1, suggesting that P450 2A13 plays important roles in the local metabolism of environmental chemicals in the respiratory tract related to toxicity or carcinogenicity.     

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.     

Metabolic conversion of fluorenone oxime to phenanthridinone by hepatic enzymes.

Fluorenone oxime is converted to phenanthridinone by enzymes present in rat liver homogenates. The reaction is analogous to the chemical Beckman rearrangement. The oxime-amide rearrangement enzyme is localized primarily in the microsomes, with some activity in the cytosol. The reaction requires reduced nicotinamide adenine dinucleotide phosphate and observes Michaelis-Menten kinetics. The reaction is relatively slow (Vmax = 7.75 +/- 2.01 nmoles of phenanthridinone formed/100 mg of liver/15 min), but the enzyme reaches maximum velocity at relatively low substrate concentrations (Km = 3.90 +/- 1.85 x 10(-5) M). The reaction is strongly competitively inhibited by 1-decylimidazole (KI = 3.75 +/- 1.77 X 10(-7) M) and inhibited to a lesser extent by the chelating agents bipyridyl (KI = 1.33 +/- 0.21 X 10(-3) M) and ethylenediamine tetraacetate (KI = 1.00 +/- 0.28 X 10(-3) M) and the sulfhydryl binding agent p-chloromercuribenzoate (KI = 2.71 +/- 0.07 X 10(-4) M). Studies also suggest that the reaction mechanism does not involve initial enzymatic substrate esterification through acetylation, glucuronidation, phosphorylation, or sulfation.     

Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P.

A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.     

A new trend to determine biochemical parameters by quantitative FRET assays

Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1–10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K_d), enzymatic velocity (kcat) and Km. We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.     

Characterization of a tyrosylprotein sulfotransferase in human liver

Sulfation of tyrosyl residue(s) has been found to be a post-translational modification that precedes the secretion of many biologically active proteins or peptides. In the present paper, we report on the characterization of human liver tyrosylprotein sulfotransferase (TPST), the enzyme responsible for sulfation of tyrosine in proteins. Using poly Glu,Ala,Tyr (6:3:1; EAY) as the model substrate, human liver TPST was recovered in the microsomal fraction after differential centrifugation. This enzyme displayed a pH optimum of 6.4 and was stimulated approximately 2.5-fold in the presence of 0.5% non-ionic detergents, such as Lubrol-PX and Triton X-100. The divalent cation Mn2+ was required for enzymatic activity and produced maximal activation at 30 mM, whereas other divalent cations, including Mg2+ and Co2+, failed to enhance sulfoconjugation at this concentration. Using the optimized assay condition, the apparent Km for EAY was found to be approximately 1.5 microM, with significant substrate inhibition at EAY concentrations above 2 microM. The 16 amino acid peptide of the C-terminus of C4 possessed an apparent Km of approximately 2.1 microM. Using EAY as a substrate, TPST activity was measured in liver samples from ten organ donors to detect the variability of this enzyme among human subjects. The activity in the male group (1.065 +/- 0.074 pmol/min/mg) was significantly (P less than 0.005) higher than that of the female group (0.662 +/- 0.158 pmol/min/mg), suggesting that TPST activity may be regulated, in part, by sex hormones.     

Multiple carriers for uptake of [3H]estradiol-17 beta(beta-D-glucuronide) in isolated rat hepatocytes

The transport of the cholestatic steroid glucuronide, 3H-estradiol-17 beta-(beta-D-glucuronide) (E2 17G), was examined in isolated female rat hepatocytes over a broad substrate concentration range (0.1-100 microM). Two different carrier systems were identified with the following kinetic parameters: Km1 = 4.54 microM; Vmax1 = 0.149 nmol/min/mg protein; Km2 = 149 microM; Vmax2 = 0.641 nmol/min/mg protein. Taurocholate and testosterone glucuronide selectively and competitively inhibited [3H]-E2 17G uptake at the high affinity site. Ki values calculated for taurocholate (43 microM) and testosterone glucuronide (28 microM) indicated that these two inhibitors were relatively weak competitors for this E2 17G transport site. Conversely, E2 17G inhibited [3H]taurocholate uptake into isolated hepatocytes (Ki = 43 microM). Bromosulfophthalein (10 microM) inhibited uptake of 0.5-50 microM [3H]-E2 17G by 55-85%, whereas morphine glucuronide (100 microM) had no significant effect on uptake of [3H]-E2 17G at these concentrations. The effects of taurocholate, testosterone glucuronide, bromosulfophthalein, and morphine glucuronide on [3H]-E2 17G uptake into isolated rat hepatocytes correlated with the ability of these agents to inhibit binding of [3H]-E2 17G to specific sites in rat liver plasma membranes. These data support the postulate that the two [3H]-E2 17G binding sites identified in female rat liver plasma membranes represent two distinct organic anion carriers and indicate that the high affinity site for [3H]-E2 17G represents a carrier that is shared by organic anions and bile acids.     

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.     

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease

Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chemical database containing 58,855 compounds followed by the testing of potential compounds for SARS-CoV M(pro) inhibition leads to two hit compounds. The core structures of these two hits, defined by the docking study, are used for further analogue search. Twenty-one analogues derived from these two hits exhibited IC50 values below 50 microM, with the most potent one showing 0.3 microM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV M(pro) were solved by X-ray crystallography. They bind to the protein in a distinct manner compared to all published SARS-CoV M(pro) complex structures. They inhibit SARS-CoV M(pro) activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.     

A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.

Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.     

Stereochemical aspects of phenylethanolamine analogues as substrates of phenylethanolamine N-methyltransferase

Phenylethylamines and phenylethanolamines represent two major classes of ligands for the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT;EC 2.1.1.28). Phenylethylamines are usually competitive inhibitors and the isomers with the relative configuration as in (2S)-amphetamine (1) and (2S)-2-aminotetralin (3) are better inhibitors than their enantiomers. Phenylethanolamines are usually substrates of PNMT and the enzyme prefers the 1R isomers, such as (1R)-phenylethanolamine (5), in this class. Optically active norephedrines (7 and 8), norpseudoephedrines (9 and 10), and 2-amino-1-tetralols (13-16) were used to study the stereochemical requirements of phenylethanolamines for PNMT active site binding. Although the norephedrines (7 and 8) and the norpseudoephedrines (9 and 10) were poorer ligands for PNMT than were the 2-amino-1-tetralols (13-16), (1R,2S)-(-)-norephedrine (7) showed some activity as a PNMT substrate (Km = 1310 microM, Vmax = 0.22, 100 x Vmax/Km = 0.017). In the 2-amino-1-tetralols (13-16), the isomers with the 2S configuration (13 and 15) showed higher affinity to PNMT (13, Km = 4.5 microM; 15, Ki = 4.6 microM) and those with the 1R configuration (13 and 16) were substrates for the PNMT-catalyzed methyl transfer (13, Km = 4.5 microM, Vmax = 0.16, 100 x Vmax/Km = 3.6; 16, Km = 195 microM, Vmax = 0.12, 100 x Vmax/Km = 0.062); the combination of 1R and 2S configurations, such as in (1R,2S)-2-amino-1-tetralol (13), was required for a good substrate. These stereochemical requirements derived from the norephedrines (7 and 8), the norpseudoephedrines (9 and 10), and the 2-amino-1-tetralols (13-16) complement those for phenylethylamines (1-4) and for phenylethanolamines (5 and 6) and strongly suggest that phenylethylamine inhibitors bind to PNMT in the same orientation as do phenylethanolamine substrates.