New prolyl endopeptidase inhibitors: in vitro and in vivo activities of azabicyclo[2.2.2]octane, azabicyclo[2.2.1]heptane, and perhydroindole derivatives.

A series of potent and selective prolylendopeptidase (PEP) inhibitors of the alpha-keto heterocyclic type has been obtained by replacing the classical central proline of 1-[1-(4-phenylbutanoyl)-L-prolyl]pyrrolidine (SUAM 1221,3) by non-natural amino acids PHI, ABO, and ABH. These 4-phenylbutanoyl side-chain-containing inhibitors exhibited potent in vitro inhibitory potencies with IC50 around 30 nM (compounds 24 and 25). Modulation of the side chain by replacement of the terminal phenyl ring by the dicyclopropyl moiety afforded derivatives 30 and 32 with improved potencies (IC50 between 10 and 20 nM). Furthermore, replacing the linear 4-phenylbutanoyl side chain by the (2-phenylcyclopropyl)carbonyl entity provided potent inhibitors with IC50 culminating at 0.9 nM on a rat cortex enzymatic preparation (compound 70). The configuration of the cyclopropyl ring had to be R,R in order to obtain not only a strong PEP inhibition in vitro but also a good activity in vivo, exemplified by inhibitor 68, which gave ID50 ip and po of 0.3 and 1 mg/kg, respectively. Finally, demonstration of the cognition-enhancing properties of compound 54 was given in the passive avoidance test using scopolamine-induced amnesia in the rat, where it dose dependently inhibited the scopolamine-induced decrease in avoidance response.

Myristoyl-CoA:protein N-myristoyltransferase activity in cancer cells. Purification and characterization of a cytosolic isoform from the murine leukemia cell line L1210.

Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the N-terminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) Eur. J. Biochem. 201, 257-263], a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with [14C]myristoyl-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC50 of about 0.1 microM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.

In vivo immunopharmacological properties of tuftsin and four analogs.

Four analogs of the natural macrophage-activator peptide tuftsin (T-K-P-R) were synthesized with the aim of obtaining compounds more effective in the stimulation of the immune system than tuftsin. Modifications to the parent tuftsin molecule were (i) substitution of the proline (P) residue, and/or (ii) replacement of the N-terminal residue threonine (T). The study presented here shows that the integrity of the NH2 terminus is not mandatory for a full biological tuftsin-like activity. Our data also suggest that the analogue F-(psi)-K-ABO-R, where ABO is a non-natural amino acid, is a promising agent for immunotherapy of infectious and neoplasic diseases for which tuftsin has already demonstrated some efficacy.

Synthesis and ACE inhibitory activity of the stereoisomers of perindopril (S 9490) and perindoprilate (S 9780).

Perindopril, a powerful ACE inhibitor contains 5 chiral carbons, thus there is the possibility of 2(5) = 32 stereoisomers for the general structure 1. These 32 stereoisomers were synthesized by cross-coupling the 8 stereoisomers of perhydroindole 2-carboxylic acid benzylester with the 4 stereoisomers of 2-(1-carbethoxybutylamino) propionic acid 4, then hydrogenating the resulting benzylesters. Each stereoisomer of perindopril furnished by saponification the corresponding diacid stereoisomer 2 of perindoprilate which is the active form of perindopril. For each of the 32 stereoisomers 2 the in vitro ACE inhibitory potency (IC50) was determined. Four of them, including perindoprilate, had activities in the nanomolar range, and four more were ca. 10 x less active. The four acid esters 1 corresponding respectively to the four most active diacids 2 in vitro were studied (1 mg/kg via the oral route) for their in vivo activity in dogs. It could be concluded that p.o. absorption of the active acid esters 1 and their activation to the active diacid 2 depended only on the chiralities of the two ring junction carbons of the perhydroindole ring.

N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes.

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.

Approaches to some biochemical mechanisms of action of tuftsin and analogues.

Tuftsin, T-K-P-R, is a phagocytosis-stimulating peptide described as a natural immunostimulant. Four analogues of this peptide were synthesized. These compounds were assayed for their ability to compete with [3H]tuftsin for its specific receptor from thioglycollate-elicited mouse peritoneal macrophages. They were also tested for their ability to change level in intracellular cGMP and to stimulate phagocytosis through the nitroblue tetrazolium reduction measurement. Surprisingly, all the analogues were poor competitors of [3H]tuftsin binding but possess potent tuftsin-like activities.

Configuration and preferential solid-state conformations of perindoprilat (S-9780). Comparison with the crystal structures of other ACE inhibitors and conclusions related to structure-activity relationships.

The conformation of perindoprilat, an antihypertensive drug, is studied in the solid state by X-ray analysis. The resolution of its structure reveals important analogies between its observed conformation and that of several ACE inhibitors of the same family. This comparison points out a constant relative orientation of the functional groups, regardless of the molecular environment. This angular constancy appears to us as not being accidental and is a good argument for the spatial design of the ACE binding site. Although ACE is a carboxydipeptidase, the binding site may not contain two but one unique hydrophobic pocket receiving the C-terminal end of the inhibitors.

Central cardiovascular effects of morphinomimetic peptides in dogs.

Morphinomimetic peptides were injected into the cisterna magna of chloralosed dogs. Methionine enkepalin (500 microgram/kg i.c.) was found ineffective but beta-endorphin (50 microgram/kg i.c.) induced an initial and transient increase in blood pressure and heart rate followed by a delayed hypotension and bradycardia. The synthetic pentapeptides, [d-ala2]met-enkephalin (500 microgram/kg i.c.), [d-ala2]met-enkephalinamide 500 microgram/kg i.c.) also induced a marked hypotensive, bradycardic and sympatho-inhibitory effect. High doses of naloxone (100 microgram/kg i.v.) were required to antagonize these effects transiently.