Diastereoselective synthesis of 2,3,5-trisubstituted tetrahydrofurans via cyclofunctionalization reactions. Evidence of stereoelectronic effects.

The work described herein considers the impact of stereoelectronic effects and allylic 1,3-strain in controlling the cyclofunctionalization reaction when a hydroxyl group is at the allylic position. The stereoelectronic arguments are supported by independent iodocyclization reactions performed using two secondary alcohols. The transition-state pathways involved in these reactions are established through a comparison of relative reaction rates. A bi-directional approach is used to demonstrate the potential of the iodocyclization reaction to differentiate a terminus in molecules with a pseudo C(2) axis of symmetry, showing that two-directional synthesis can be used to differentiate between alternative transition-state pathways.

2',6'-Dimethylphenoxyacetyl: a new achiral high affinity P(3)-P(2) ligand for peptidomimetic-based HIV protease inhibitors.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.

Kinetic studies of the branched chain amino acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin beta-lactone.

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.

Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids.

Potent and selective dipeptidyl boronic acid proteasome inhibitors are described. As compared to peptidyl aldehyde compounds, boronic acids in this series display dramatically enhanced potency. Compounds such as 15 are promising new therapeutics for treatment of cancer and inflammatory diseases.

Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism.

We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site-directed small molecule inhibitors. Clasto-lactacystin beta-lactone is a time-dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc-Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a kinact/[I] of 1,700 M-1 s-1. Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Ogamma of the hydroxyl group on the N-terminal threonine of the beta-subunit is the sole modification made by the beta-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit. Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Ogamma moiety on Thr1. High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids. This modification is also blocked by prior treatment with beta-lactone.

Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells.

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde.

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.

Ureido-based peptidomimetic inhibitor of herpes simplex virus ribonucleotide reductase: an investigation of inhibitor bioactive conformation.

We have been investigating peptidomimetic inhibitors of herpes simplex virus (HSV) ribonucleotide reductase (RR). These inhibitors bind to the HSV RR large subunit and consequently prevent subunit association and subsequent enzymatic activity. This report introduces a new series of compounds that contain an extra nitrogen (a ureido function) at the inhibitor N-terminus. This nitrogen improves inhibitor binding potency 50-fold over our first published inhibitor series. Evidence supports that this improvement in potency results from a new hydrogen-bonding contact between the inhibitor and the RR large subunit. This report also provides evidence for the bioactive conformation around two important amino acid residues contained in our inhibitors. A tert-butyl group, which contributes 100-fold to inhibitor potency but does not directly bind to the large subunit, favors an extended beta-strand conformation that is prevalent in solution and in the bound state. More significantly, the bioactive conformation around a pyrrolidine-modified asparagine residue, which contributes over 30 000-fold to inhibitor potency, is elucidated through a series of conformationally restricted analogues.

Middle ear pressure variations during nitrous oxide and oxygen anaesthesia.

Many authors have reported middle ear pressure variations during inhalation of high concentrations of nitrous oxide. An on-going study on subjects anaesthetized with nitrous oxide and oxygen supplemented with halogens or narcotics and excluding operations on the ear enables us to register three typical curves of middle ear pressure according to the patency of the Eustachian tube. We recorded significant negative middle ear pressures during the recovery period when there was important obstruction of the Eustachian tube. The presence of a tympanic "neomembrane" due to an old perforation associated with important obstruction of the Eustachian tube could lead to a tympanic perforation that may be unnoticed by the anaesthetist if it is not specifically investigated. In our series, we report one case of tympanic perforation and one case of haemotympanum as examples of such consequences.

[Educational activity and psychosocial prevention : a model of intervention.]. / Activité éducative et prévention psychosociale : un modèle d'intervention.

The authors describe the socio-economic conditions of the retired in 1980 (poverty, poor health, lack of adequate services, etc.) and explain how these conditions are related to capitalist social organization. Their analysis leads them to set down the principal goal of the community workers in regard to the elderly population as follows : contribute to the organization and to the development of conditions favorable to the mobilization of the retired and elderly workers who, as a group, have the greatest potential for changing their conditions. To attain this long-term objective, the pre-retirement clientele possess the most favorable potential, and this is why the retirement preparation intervention is defined in an educationel orientation of psycho-social prevention. Seconding to the authors, the apprenticeships showed permit the elderly to assume both personal and collective control - thereby making it possible to transform the existing conditions.

[On the problem of retirement crisis.]. / Pour une problématique de la crise de retraite.

Retirement is first defined as a transitory period from a working situation to a situation of definite cessation of work. In order to understand this transitory process, the author looks into the importance of work as a determinant of life conditions and as a structuring influence in life conducts. The change brought about by retirement involves many upheavals on a social level (relationship, loss of income, economical dependency) as well as on a personal level (return to a home life, modification of relationships within the couple and the family life). The importance of these upheavals is such and their effects so acute that retirement is defined as a crisis situation.