Quality assessment of combinatorial pooled libraries using mass spectrometry.

Parallel synthesis techniques aim to prepare collections of single compounds which, once tested, can easily be identified by their sole location in the synthesic array. On the other hand, true combinatorial chemistry produces libraries of compounds as mixtures of variable size which require a deconvolution procedure for identification of the active hits or leads. In the latter case, analytical methods are crucial for the success of the strategy and mass spectrometry plays a major role. If the goal is to identify all the library components, including expected products as well as by-products, various mass spectrometric techniques may be necessary. Library components can be separated according to their mass by increasing mass resolution or by their elution time by coupling liquid chromatography and mass spectrometry. The efficiency of such separation techniques are discussed as a function of the size and the degeneracy of the library. Library members possess common structural features which impart similar fragmentation patterns after ionization in the gas phase. This feature can be exploited by tandem mass spectrometry to specifically detect subfamilies of products. Examples of precursor ion scans, product ion scans and constant neutral loss scans will be shown that facilitate partial characterization of libraries. To solve the difficult problem of the quantitative analysis of libraries, i.e., to evaluate their equimolarity, the use of an evaporative light scattering detector (ELSD) or a chemiluminescent nitrogen detector (CLND) is suggested as more appropriate.

Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.

Physico-chemical and biological analysis of true combinatorial libraries.

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.

Limitations of the coupling of amino acid mixtures for the preparation of equimolar peptide libraries.

The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.

Solution conformation by NMR and molecular modeling of three sulfide-free somatostatin octapeptide analogs compared to angiopeptin.

The conformation in dimethylsulfoxide of the somatostatin derivative angiopeptin and of three disulfide-free analogs was estimated by two-dimensional nuclear magnetic resonance spectroscopy at room temperature. The resulting 3D molecular graphics were compared and shown to reflect the observed differences in the inhibition of restenosis after rat aorta balloon injury by these octapeptide inhibitors. Angiopeptin and its active analog 2 displayed a relatively rigid conformation of the cyclic hexapeptide backbone due to the presence of two well-defined hydrogen bonds, further stabilized by a third hydrogen bond outside the ring. No such constraints were detected for the two biologically inactive analogs, which, compared to 2, had a two-atom longer or shorter hexapeptide ring. The well-defined structure of compound 2 may serve as an improved pharmacophore for this new class of drugs.

Combinatorial peptide libraries: robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high-performance capillary electrophoresis techniques.

Combinatorial peptide libraries are a new source of compounds from which a large number of pharmacological leads will emerge in the next few years. A large body of literature shows that this approach is of considerable interest in many areas of biological sciences for the search of enzyme substrates and inhibitors, of receptor agonists or antagonists, or of antigen sequences. Nevertheless, the analytical investigation of such complex mixtures as libraries which contain up to millions of individual molecules is still poorly documented. In this work, we present analytical solutions for their characterization. NMR and tandem mass spectrometry (MS/MS) can provide an in deep description of any type of combinatorial libraries, while MS and high-performance capillary electrophoresis can bring a rapid and overall information at the routine level, sufficient to ensure a first assessment of their composition. MS in the fast atom bombardment mode was used to describe the libraries O1X2X3X4X5 or O1X2X3X4 (Oi and Xi are fixed and random residue in position i, respectively). Advantage was taken of the high proton affinity of arginine and of its induction of charge remote fragmentation to interpret the MS spectra of whole libraries and neutral losses (MS/MS) in the model sublibraries ArgGlyX3X4 and NipValX3X4 (Nip,4-nitrophenylalanine). Two-dimensional NMR allowed the incorporation of the individual residues during synthesis to be tested in 24 sublibraries O1X2X3X4. While from the pharmacological point of view, impressive discoveries made with combinatorial peptide libraries have already been reported, our results show that they should be complemented by appropriate analytical tools, crucial for the proper characterization and exploitation of these libraries.

Synthesis of 1,2-diacyl-3-nicotinoyl glycerol derivatives and evaluation of their acute effects on plasma lipids in the rat.

Nicotinic acid (CAS 59-67-6) is the only hypolipidemic agent whose activity has been shown both on atherosclerotic lesions and on long term mortality. Unfortunately, its use is hindered by the frequent occurrence ( > 70%) of adverse reactions (i.e. cutaneous rash, pruritus and, most significantly, flush). New prodrugs of nicotinic acid have been prepared by the use of diacylglycerol esters. In the rat, after acute oral administration of these products, a significant decrease of the free fatty acid plasma levels was obtained without the dramatic increase in nicotinic acid plasma levels observed after the oral administration of an equimolecular dose of nicotinic acid. The most interesting ester, S 16961 ((d,l)-1,2-dipalmitoyl-3-nicotinoyl glycerol, CAS 160555-46-4) is undergoing clinical trials.

S 16118 (p-guanidobenzoyl-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin) is a potent and long-acting bradykinin B2 receptor antagonist, in vitro and in vivo.

The in vitro and in vivo effects of S 16118 [p-guanidobenzoyl-[Hyp3, Thi5,D-Tic7,Oic8]bradykinin (BK)], a new BK receptor antagonist, were studied. S 16118 inhibited the contraction produced by BK in the rabbit jugular vein, but was ineffective in the rabbit aorta, indicating the BK B2 receptor specificity of the compound. In isolated organs from various species including humans, S 16118 was a potent antagonist (Ki, pA2 or pKB value from 9.58-7.37). The effect of S 16118 was specific as it did not show any affinity for a number of other receptors or channels and did not possess residual agonistic properties in most of the tissues studied. Furthermore, S 16118 is a poor secretagogue agent either in the rat or human mast cells and is resistant to degradation with an in vitro half-life in blood from different species, including humans, of more than 24 hr. In vivo, in the rabbit, i.v. injection of S 16118 inhibited the hypotension induced by BK up to 4 hr after administration. In the guinea pig, it was also effective in inhibiting the bronchoconstriction induced by BK, although when administered i.v. it had a shorter duration than in the rabbit. However, in the same species, when aerosolized, S 16118 was effective and long-acting against BK-induced bronchoconstriction. Changes in permeability induced by BK injection in the guinea pig trachea and bronchus, and by BK superfusion in the hamster cheek pouch, were abolished by i.v. pretreatment with S 16118.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of blood levels of endothelin and neurokinin receptor antagonists at the rat portal and jugular veins after oral administration as a tool in peptide drug design.

The blood concentration of three representative endothelin and four neurokinin receptor antagonists were monitored both at the portal and jugular vein of rats 30, 60 and 90 min after oral administration. Peptide-derived structures, in the size range tetra-pentapeptides, were shown to be absorbed in the reverse order of their log P values, to be weakly metabolized in the first hepatic transit and to maintain high blood levels during the observation time. These interesting results obtained by a simple and convenient UV assay, stress once again the importance of monitoring oral absorption early in the process of peptide drug design.

Evaluation and comparison of urinary pyridinium crosslinks in two rat models of bone loss--ovariectomy and adjuvant polyarthritis--using a new automated HPLC method.

A specific HPLC system was developed to assess urinary excretion of collagen crosslinks (pyridinoline (Pyr) and deoxypyridinoline (D.Pyr)) in two models of osteopenia in rats, ovariectomy and adjuvant polyarthritis. The sensitivity of this method was in the picomolar range. In ovariectomized rats, a specific model of bone resorption, Pyr and D.Pyr levels rose early, reaching a peak 2 weeks after surgery. Both levels remained raised during the whole observation period (6 weeks) with no change in the Pyr/D.Pyr ratio. So, in this high bone turnover model, hyperresorption is reflected by the parallel increase of both crosslinks resulting in a significant decrease of bone mineral density (BMD) at 6 weeks (-7.3% vs. control). In polyarthritic rats, in the 2 post-adjuvant weeks, Pyr levels increased in parallel with inflammatory parameters, whereas D.Pyr levels remained unchanged. This is in agreement with our previous report that at the end of the 2nd week after adjuvant there is no change in bone resorption. From the 3rd week, both Pyr and D.Pyr increased. The Pyr/D.Pyr ratio was always significantly higher in polyarthritic rats. These results suggest that the early increase of Pyr level reflects non-osseous collagen breakdown and that bone resorption occurs at a later stage when D.Pyr rises, leading to a dramatic decrease of BMD at 4 weeks (-17.7% vs. control). Taken together, our results suggest that in rat as in human, urinary Pyr is a marker of bone and cartilage breakdown, whereas D.Pyr is a specific marker of bone loss. This automated method described may constitute a very useful tool to evaluate bone and/or cartilage breakdown in rats and for the assessment of protective treatments.

In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats.

Flavonoids form an important family of compounds widely present in plants and therefore in food, sometimes as substitutes for synthetic antioxidants. Although the excretion routes of flavonoids in animals has been explored, little is known about the details of their conjugation in the xenobiotic metabolism pathway. In this study, we investigate the metabolism of diosmetin as a model compound in the rat, particularly its level in blood after treatment (100 mg/kg, po) and the presence of its glucuronide(s) in both blood and urine. We demonstrate that after po treatment of the rat, a rapid glucuronidation takes place and that diosmetin circulates as glucuronides, whereas no free diosmetin is present in blood and urine. The glucuronides formed are present in the blood plasma at a high level (approximately 10 micrograms/ml), for at least 6 hr after the treatment and the conjugates are excreted in urine. We have detected four different glucuronides in blood and characterized the two major ones after purification by a combination of MS, NMR, and UV spectroscopy: diosmetin-7,3'-diglucuronide and diosmetin-3'-glucuronide. A brief characterization of the in vitro glucuronidation of some flavonoid compounds using liver microsomal preparations suggests that this important class of natural compounds might be conjugated by a specific isoform of UDP-glucuronosyltransferase. Thus, this work brings evidence that diosmetin is rapidly glucuronoconjugated in the rat and provides an explanation for the low po bioavailability of the unchanged compound that can be generalized to this important class of natural compounds.

Kinetics of photoperoxidation of arachidonic acid: molecular mechanisms and effects of antioxidants.

The kinetics of photoperoxidation of [1-14C]arachidonic acid (20:4n-6) at 1.32 mM was studied either with the unsaturated fatty acid alone or in the presence of 10 microM of antioxidants and/or inhibitors of eicosanoid metabolism. The photosensitizer used was meso-tetraphenylporphine. The time-course of the reactions was followed by ultraviolet spectral analysis, thiobarbituric acid reactivity and high-performance liquid chromatographic analysis of aliquots sampled every 15 min during the 4 h of irradiation. The kinetics of photoperoxidation of 20:4n-6 can be divided into three main successive steps: (i) monohydroperoxidation, characterized by the appearance of conjugated diene patterns and monohydroperoxidized 20:4n-6; (ii) secondary oxidation characterized by polyoxygenated products such as dihydroperoxidized 20:4n-6 possessing conjugated triene patterns; and (iii) the disappearance of conjugated patterns and the oxidative cleavage of the products of the two first steps into aldehydic molecules reacting with thiobarbituric acid. During the first 90 min of irradiation, the mechanism of monohydroperoxidation (step one) is purely or predominantly type II photoperoxidation involving only singlet oxygen. This step was inhibited by beta-carotene and by BW755C (3-amino-1-[3-trifluoromethylphenyl]2-pyrazoline). In contrast, the reactions involved in the second and third steps were predominantly type I photoperoxidation involving radical mechanisms. These latter steps were inhibited by beta-carotene, BW755C, vitamin E and probucol. Indomethacin and 5,8,11,14-eicosatetraynoic acid did not alter 20:4n-6 photoperoxidation. This in vitro model of lipid photoperoxidation allows the screening of antioxidants in accordance with their singlet oxygen quenching and/or free radical scavenging properties.

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.

Metabolism of amineptine in rat, dog and man.

After oral administration of amineptine (7-[(10-11)-dihydro-5H-dibenzo(a,d)cycloheptane-5yl] amino heptanoic acid), an original tricyclic antidepressant, seven metabolites were isolated from urine and plasma of rat, dog and man. The metabolic pathways were similar for the three species studied. The two major pathways consisted of the beta-oxidation of the heptanoic side chain leading to pentanoic (first step) and propanoic (second step) side chain metabolites and the hydroxylation of the dibenzocycloheptyl ring on carbon atom 10 (C10) causing the formation of two diastereoisomers. Lactamization by internal dehydration of beta-oxidized metabolites appeared to be a minor route of biotransformation. Conjugation reactions were of minor importance in the rat, in contrast to findings for dog and man. Urinary elimination was the major route of excretion in man while in dog and in rat faecal excretion was predominant.