Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates.

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.

Comparative bioavailability of two losartan formulations in healthy human volunteers after a single dose administration.

OBJECTIVE: To compare the bioavailability of two potassic losartan immediate release tablet (50 mg) formulations (Losartan from Laboratórios Cristália Ltd., Brazil, as a test formulation and Cozaar from Merck Sharp & Dohme Farmacêutica Ltd., Brazil, as a reference formulation) in 25 volunteers of both sexes. MATERIAL AND METHODS: The study was conducted in an open, randomized, 2-period crossover design and a 1-week washout period. Plasma samples were obtained over a 24-hour interval. The concentrations of losartan and its active metabolite losartan acid were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with negative ion electrospray ionization using a selected ion monitoring method. From the losartan and losartan acid plasma concentrations vs. time curves the following pharmacokinetic parameters were obtained: AUClast, AUC0-inf and Cmax. RESULTS: The geometric mean and respective 90% confidence interval (CI) of Losartan/Cozaar losartan percent ratios were 92.9% (82.2-105.0%) for Cmax, 99.0% (92.5-105.9%) for AUClast, and 99.1% (92.7-105.8%) for AUC0-inf. Furthermore, the geometric mean and respective 90% CI of Losartan/Cozaar losartan acid percent ratios were 98.5% (91.5-106.0%) for Cmax, 97.9% (93.3 102.7%) for AUClast, and 98.1% (93.6-102.9%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC0-inf were within the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the potassic losartan immediate release 50 mg tablet was bioequivalent to the Cozaar immediate release 50 mg tablet, according to both the rate and extent of absorption. While there were no significant differences in the bioequivalence assessed by either losartan or losartan acid, future bioequivalence studies on losartan may be performed by quantifying losartan alone as the parent compounds are more discriminative.

In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives.

In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.

Synthesis and hydrolysis by cathepsin B of fluorogenic substrates with the general structure benzoyl-X-ARG-MCA containing non-natural basic amino acids at position X.

We synthesized one series of fluorogenic substrates for cathepsin B derived from the peptide Bz-F-R-MCA (Bz=benzoyl, MCA=7-methyl-coumarin amide) substituting Phe at the P(2) position by non-natural basic amino acids that combine a positively charged group with aromatic or aliphatic radicals at the same side chain, namely, 4-aminomethyl-phenylalanine, 4-guanidine-phenylalanine, 4-aminomethyl-N-isopropyl-phenylalanine, 3-pyridyl-alanine, 4-piperidinyl-alanine, 4-aminomethyl-cyclohexyl-alanine, 4-aminocyclohexyl-alanine, and N(im)-dimethyl-histidine. Bz-F-R-MCA was the best substrate for cathepsin B but also hydrolyzed Bz-R-R-MCA with lower efficiency, since the protease accepts Arg at S(2) due to the presence of Glu(245) at the bottom of this subsite. The presence of the basic non-natural amino acids at the P(2) position of the substrate partially restored the catalytic efficiency of cathepsin B. All the kinetic parameters for hydrolysis of the peptides described in this paper are in accordance with the structures of the S(2) pocket previously described. In addition, the substrate with 4-aminocyclohexyl-alanine presented the highest affinity to cathepsin B although the peptide was obtained from a mixture of cis/trans isomers of the amino acid and we were not able to separate them. For comparison all the obtained substrates were assayed with cathepsin L and papain.

Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids.

We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.

S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids.

We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K(i) of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P(1) position.

Clarithromycin bioequivalence study of two oral formulations in healthy human volunteers.

OBJECTIVE: To assess the bioequivalence of two tablet formulations of clarithromycin (Clamicin 500 mg from Medley Indlistria Farmaceutica, Brazil, as the test formulation, and Biaxin 500 mg from Abbott Industries, USA, as the reference formulation). METHODS: A single 500 mg oral dose of each formulation was administrated in 24 healthy volunteers of both sexes (12 males and 12 females). The study was conducted open, randomized, two-period crossover design with a 7-day interval between doses. The plasma concentrations of clarithromycin were quantified by reversed phase liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM) method. 14-hydroxyclarithromycin concentration was estimated semiquantitatively as equivalent of clarithromycin/ml. The precision of the method was evaluated using calibration curves and plasma quality control samples. The pharmacokinetic parameters calculated for both compounds included: AUC(0 - 48h), AUC(0 - infinity), Cmax, Cmax/AUC(0 - 48h), Tmax, T1/2 and Ke. RESULTS: Standard curves of clarithromycin in plasma were linear in the range of 0.05 microg x ml(-1) to 10 microg x ml(-1) (r > 0.999). The limit of quantification was 5 ng/ml. Within- and between-run plasma quality control CV were 5.8% and 15.7%, respectively. Inaccuracy within- and between-runs were 14% and 17%, respectively. 90% CI for clarithromycin geometric mean AUC(0 - 48h), AUC(0 - infinity) and Cmax ratios (test/reference) were: 8.7% - 103.1%, 89.4% - 103.7% and 85.4% - 99.6%, respectively, and for hydroxyclarithomycin were 80.3% - 108.6%, 80.1% - 110.1% and 85.4% - 112.6%, respectively. CONCLUSION: The method described for the quantification of charithomycin and its main metabolite is accurate and sensitive. Clamicin was considered bio-equivalent to Biaxin based on the rate and extent of absorption. Since these were no significant differences in the bioequivalence determined using the pharmacokinetic parameters of either clarithromycin or 14-hydroxyclarithromycin, we suggest that future bioequivalence trials of this drug may be performed by quantifying clarithromycin only.

Determination of 21-hydroxydeflazacort in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Application to bioequivalence study.

A liquid chromatographic atmospheric pressure chemical ionization tandem mass spectrometric method is described for the determination of 21-hydroxydeflazacort in human plasma using dexamethasone 21-acetate as an internal standard. The procedure requires a single diethyl ether extraction. After evaporation of the solvent under a nitrogen flow, the analytes are reconstituted in the mobile phase, chromatographed on a C18 reversed-phase column and analyzed by mass spectrometry via a heated nebulizer interface where they are detected by multiple reaction monitoring. The method has a chromatographic run time of less than 5 min and a linear calibration curve with a range of 1-400 ng ml(-1) (r>0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was < or =5.5% (10 ng ml(-1)), 1.0% (50 ng ml(-1)) and 2.7% (200 ng ml(-1)). The between-run accuracy was +/-7.1, 3.8 and 4.8% for the above concentrations, respectively. This method was employed in a bioequivalence study of two DFZ tablet formulations (Denacen from Marjan Industria e Comercio, Brazil, as a test formulation, and Calcort from Merrell Lepetit, Brazil, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 30 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual geometric mean ratio for Denacen/Calcort was 89.8-109.5% for area under the curve AUC(0-24 h) and 80.7-98.5% for Cmax. Since both the 90% CI for AUC(0-24 h) and Cmax were included in the 80-125% interval proposed by the US Food and Drug Administration, Denacen was considered bioequivalent to Calcort according to both the rate and extent of absorption.