Structural features determining the intestinal epithelial permeability and efflux of novel HIV-1 protease inhibitors.

The primary aim of this study was to identify structural features that alter the intestinal epithelial permeability and efflux in a series of novel HIV-1 protease inhibitors (PIs). Eleven PIs were selected containing a tertiary alcohol in a transition-state mimicking scaffold, in which two substituents (R(1) and R(2) ) were varied systematically. Indinavir was selected as a reference compound. The apical-to-basolateral permeability was investigated in 2/4/A1 and Caco-2 monolayers. In addition, the basolateral-to-apical permeability was investigated in the Caco-2 monolayers and the efflux ratios were calculated. The absence of active drug transport processes in 2/4/A1 cells allowed identification and modeling of structural elements affecting the passive permeability. For instance, small aromatic R(1) substituents and a small (bromo-) R(2) substituent were associated with a high passive permeability. Efflux studies in Caco-2 cells indicated that amide-substituted neutral hydrophobic amino acids, such as valine and leucine, in the R(1) position, reduced the apical-to-basolateral transport and enhanced the efflux. We conclude that our investigation revealed structural features that alter the intestinal epithelial permeability and efflux in the series of PIs and hope that these results can contribute to the synthesis of PIs with improved permeability and limited efflux properties.

In vitro ADMET and physicochemical investigations of poly-N-methylated peptides designed to inhibit Abeta aggregation.

N-Methylation is a common strategy for improving oral bioavailability of peptide-based lead structures. Herein, we present a detailed study on how the degree of N-methylation affects the absorption-distribution-metabolism-excretion-toxicity (ADMET) properties such as solubility, membrane transport, proteolytic stability, and general cell toxicity of the investigated peptides. As representative structures we chose hexapeptides 1-8. These peptides, corresponding to N-methylated analogues of residues 16-21 and 32-37 of the Abeta-peptide, pathological hallmark of Alzheimer's disease (AD), have previously been shown to inhibit aggregation of Abeta fibrils in vitro. This study suggests that poly-N-methylated peptides are non-toxic and have enhanced proteolytic stability over their non-methylated analogues. Furthermore, solubility in aqueous solution is seen to increase with increased degree of N-methylation, while membrane transport was found to be low for all investigated hexapeptides. The present results, together with those reported in the literature, suggest that poly-N-methylated peptides, especially shorter or equal to six residues, can be suitable candidates for drug design.

Poorly soluble marketed drugs display solvation limited solubility.

We determined the intrinsic aqueous solubility of 15 poorly soluble drugs with solubilities ranging from 2.9 nM to 1.1 microM. We then analyzed the data from a physicochemical perspective, using experimentally determined solid-state properties and easily interpretable two-dimensional molecular descriptors, to better understand the factors underlying poor solubility. The analysis shows that poorly soluble drugs that have reached the market are solubility limited by solvation rather than by their solid state.

Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers.

Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.

Beta- and gamma-di- and tripeptides as potential substrates for the oligopeptide transporter hPepT1.

The hPepT1-mediated transport properties of a series of 11 synthesized beta- and gamma-peptides have been studied in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but only two beta-dipeptides act as substrates and are transported across the cell monolayers. These two are less-efficient substrates than alpha-peptides. Larger derivatives than beta-dipeptides do not act as hPepT1 substrates, but instead, they appear to be substrates for P-glycoprotein efflux.

Orally active antiviral tripeptide glycyl-prolyl-glycinamide is activated by CD26 (dipeptidyl peptidase IV) before transport across the intestinal epithelium.

The tripeptide amide glycyl-prolyl-glycinamide (GPG-amide) is a new antiretroviral drug candidate, but its absorption mechanism is unknown. In this investigation, the transport and metabolism of GPG-amide were studied in a model of the human intestinal epithelium, Caco-2 cell monolayers. The results show that when the tripeptide amide came into contact with the apical enterocyte membrane, it was degraded by CD26 (dipeptidyl peptidase IV) to glycylproline and the antiretrovirally active metabolite glycinamide. Glycinamide retained antiretroviral activity in vitro after transport through the Caco-2 cell monolayers. The transport of glycinamide across Caco-2 cell monolayers occurred via passive diffusion with an apparent permeability coefficient of about 2 x 10(-6) cm s(-1), which suggests that it is absorbed by the oral route in sufficient amounts to be considered for oral administration. In conclusion, the tripeptide GPG-amide acts as a prodrug that is activated by CD26 to release the orally active antiretroviral compound glycinamide.

Mechanism of the glutathione transferase-catalyzed conversion of antitumor 2-crotonyloxymethyl-2-cycloalkenones to GSH adducts.

Human glutathione (GSH) transferase (hGSTP1-1) processes with similar kinetic efficiencies the antitumor agents 2-crotonyloxymethyl-2-cyclohexenone (COMC-6), 2-crotonyloxymethyl-2-cycloheptenone (COMC-7), and 2-crotonyloxymethyl-2-cyclopentenone (COMC-5) to 2-glutathionylmethyl-2-cyclohexenone, 2-glutathionylmethyl-3-glutathionyl-2-cycloheptenone, and 2-glutathionylmethyl-2-cyclopentenone, respectively. This process likely involves initial enzyme-catalyzed Michael addition of GSH to the COMC derivative to give a glutathionylated enol(ate), which undergoes nonstereospecific ketonization, either while bound to the active site or free in solution, to a glutathionylated exocyclic enone. Free in solution, GSH reacts at the exomethylene carbon of the exocyclic enone, displacing the first GSH to give the final product. This mechanism is supported by the observation of multiphasic kinetics in the presence of high concentrations of hGSTP1-1 and the ability to trap kinetically competent exocyclic enones in aqueous acid using COMC-6 and COMC-7 as substrates. That the exocyclic enone is formed by nonstereospecific ketonization of an enol(ate) species is indicated by the observation that COMC-6 (chirally labeled with deuterium at the exomethylene carbon) gives stereorandomly labeled exocyclic enone. The isozymes hGSTP1-1, hGSTA1-1, hGSTA4-4, and hGSTM2-2 catalyze the conversion of COMC-6 to final product with similar efficiencies (K(m) = 0.08-0.34 mM, k(cat) = 1.5-6.1 s(-)(1)); no activity was detected with the rat rGSTT2-2 isozyme. Molecular docking studies indicate that in hGSTP1-1, the hydroxyl group of Tyr108 might serve as a general acid catalyst during substrate turnover. The possible significance of these observations with respect to the metabolism of COMC derivatives in multidrug resistant tumors is discussed.

The cyclopentenone product of lipid peroxidation, 15-A(2t)-isoprostane (8-isoprostaglandin A(2)), is efficiently conjugated with glutathione by human and rat glutathione transferase A4-4.

Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain alpha,beta-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A(2)/J(2)-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain alpha,beta-unsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA(2) and PGJ(2). Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A(2t)-IsoP (8-iso-PGA(2)) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a k(cat)/K(m) value of >200 s(-)(1) mM(-)(1). The k(cat)/K(m) value for conjugation of 15-A(2t)-IsoP by the homologous rat GST A4-4 is >2000 s(-)(1) mM(-)(1). Similar high enzyme activities were observed when PGA(2) was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A(2t)-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.

Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus.

Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.