Replacement of glycine with dicarbonyl and related moieties in analogues of the C-terminal pentapeptide of cholecystokinin: CCK(2) agonists displaying a novel binding mode.

Recent advances in the field of cholecystokinin have indicated the possible occurrence of multiple affinity states of the CCK(2) receptor. Besides, numerous pharmacological experiments performed "in vitro" and "in vivo" support the eventuality of different pharmacological profiles associated to CCK(2) ligands. Indeed, some agonists are essentially anxiogenic and uneffective in memory tests, whereas others are not anxiogenic and appear as able to reinforce memory. The reference compound for the latter profile is the CCK-8 analogue BC 264 (Boc-Tyr(SO(3)H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH(2)). However, although tetrapeptide ligands based on CCK-4 (Trp-Met-Asp-Phe-NH(2)) are known to possess sufficient structural features for CCK(2) recognition, none shares the properties of BC 264. Hence we have developed new short peptidic or pseudo-peptidic derivatives containing the C-terminal tetrapeptide of BC 264. Our results indicate that some compounds characterized by the presence of two carbonyl groups at the N-terminus, as in 2b (HO(2)C-CH(2)-CONH-Trp-(NMe)Nle-Asp-Phe-NH(2)), are likely to show a BC 264-like profile, bind to the CCK(2) receptor in a specific way, and display remarkable affinities (2b: 0.28 nM on guinea-pig cortex membrane preparations). This original binding mode is discussed and further enlightened by NMR and molecular modeling studies.

Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors.

An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournié-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R1)CONHCH(R1')CON(R)CH(R2')COOH have been synthesized. The introduction of well-selected beta-branched chains in positions R1 and R1', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapto-3-phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S1' and S2' subsites, whereas additional interactions with the S1 binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10(-5) mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.

Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach.

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln76 of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 microM range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain and natural amino acids in P1' and P2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P1' and P2' components, respectively. This led to new inhibitors of TeNt with Ki values in the 3-4 microM range. These molecules are the most potent inhibitors of TeNt described so far.

Synthesis and biological properties of new constrained CCK-B antagonists: discrimination of two affinity states of the CCK-B receptor on transfected CHO cells.

To improve our knowledge of the bioactive conformation of CCK-B antagonists, we have developed a new series of constrained dipeptoids whose synthesis and biochemical properties are reported here. These compounds, of general structure N alpha-[(2-adamantyloxy)carbonyl]-alpha-methyltryptophanyl-(4 -X)-proline, were designed by introducing a cyclization in the structure of the previously described CCK-B/peptoid antagonist RB 210, N-[N-[(2-adamantyloxy)carbonyl]-DL-alpha-methyltryptophanyl] -N-(2-phenylethyl)glycine (Blommaert et al. J. Med. Chem. 1993, 36, 2868-2877), by means of a five-membered ring. Structure-affinity relationship studies showed that an R configuration of Trp-C alpha and a cis configuration of the pyrrolidine substituents were favorable for receptor recognition. The most potent compounds of this new series had similar affinities for the CCK-B receptor as RB 210 and proved to be far more efficient in inhibiting inositol phosphate production in CHO cells stably transfected with rat brain CCK-B receptor, with IC50 values approaching those of the commonly used antagonists L-365,260 and PD-134,308. Moreover, binding studies performed using transfected CHO cells showed that two affinity states of the CCK-B receptor can be discriminated by some of these compounds which also have different biological profiles and are therefore highly interesting tools for the biochemical and pharmacological characterization of CCK-B receptor heterogeneity.

Design of orally active dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme with long duration of action.

Mercaptoacyl dipeptides, containing a glycine linked to a C-terminal 5-phenylproline, have been synthesized in order to obtain new highly efficient dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), which are involved in the control of blood pressure and fluid homeostasis. These compounds have been designed (i) to fit optimally the ACE pharmacophore previously described (Fournié-Zaluski, M. C.; et al. J. Med. Chem. 1994, 37, 1070-1083), through interaction with the S1, S1', and S2' subsites of this enzyme, (ii) and to interact with the S1' and S2' subsites of NEP with the 5-phenylproline moiety outside the catalytic domain (Coric, P.; et al. J. Med. Chem. 1996, 39, 1210-1219). Replacement of Gly by Ala in these mercaptoacyl dipeptides induced an about 100-fold decrease in ACE inhibition. This shows that, in agreement with molecular modeling studies, a steric constraint as weak as a methyl group hinders optimal ACE active site recognition. Among these compounds, the dual inhibitor 26 (RB 106) (Ki, ACE = 0.35 nM; NEP = 1.6 nM) showed excellent pharmacokinetic properties with an almost complete in vivo inhibition of NEP and ACE for more than 4 h after oral administration in mice of a low dose (2.6 x 10(-5) mol/kg) of the inhibitor. Moreover, RB 106 remained active 12 h after oral administration. In spontaneous hypertensive rats, a chronic treatment of orally administered RB 106 (25 mg/kg/day) induced a prolonged hypotensive effect (-28 mmHg) still significant 2 days after the end of the treatment. In DOCA salt rats, a hypotensive response and a significant natriuresis were observed after i.v. administration. RB 106, which is one of the most potent dual inhibitors described to date, could have interesting clinical applications in long term treatment of congestive heart failure and myocardial ischemia.

Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: synthesis, biological properties, and antinociceptive activities.

The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H(3)N(+)-CH(R(1))-P(O)(OH)-CH(2)-CH(R(2))-CONH-CH(R(3))-COO(-), able to act as transition-state analogues and to fit the S(1), S(1)', and S(2)' subsites of both enzymes were designed. Selection of the R(1), R(2), and R(3) residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K(i) values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors.

Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme.

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to fit its S(1), S(1)', and S(2)' subsites. This analysis confirmed that the S(1) subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S(1)' subsite is hydrophobic whereas the S(2)' subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H(3)N(+)CH(CH(2)CH(2)SO(3)(-))CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K(i) of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II and cholecystokinin CCK(8) in the central nervous system.

Novel constrained CCK-B dipeptoid antagonists derived from pipecolic acid.

A new series of 4-substituted pipecolic acid derivatives was prepared and incorporated into dipeptoids. The resulting products behave as moderately potent CCK-B antagonists but their constrained structure and its comparison with structurally related compounds yield valuable information about the conformational requirements for optimal recognition of the CCK-B receptor by antagonists.

Investigation of the structural parameters involved in the delta-opioid selectivity of several families of opioid peptides.

Three series of highly delta-opioid selective peptides are now available, and each family is used as template to investigate the structural parameters involved in delta-receptor recognition and in the modulation of the selectivity of the parent peptide. The first series includes cyclic derivatives such as Tyr-D-Pen-Gly-Phe-D-Pen(DPDPE) and Tyr-D-Pen-Gly-Phe-Pen(DPLPE); the second are the synthetic linear constrained peptides [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(DSTBULET), Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr (OtBu)(BUBU) and especially Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC)] and the last one the natural peptides [Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (deltorphin or dermenkephalin) and Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH2 ([D-Ala2] deltorphin I)]. In the present study, the possibly of transposing some of the decisive factors of delta-selectivity evidenced in the two other families, to the linear constrained peptides series was examined. With this aim in view, residues such as Phe3, pClPhe4 or Asp were introduced in the sequence of DSTBULET, BUBU or BUBUC. Direct comparison between the biochemical profiles of the [pClPhe4] analogs of the linear constrained peptides and their parent compounds shows that the addition of an electronegative atom on the Phe4 residue of enkephalin sequences is not an absolute parameter for delta-selectivity improvement. The hydrophobic delta-receptor subsite seems able to receive a range of molecular volumes and electronegativities. By contrast, this subsite cannot interact with a Phe3 aromatic ring introduced in this series of peptides. Moreover, the results obtained with linear peptides including additional negatively charged residues demonstrate that the proposed location of the delta-receptors in a cationic membrane environment is not adequate to explain the selectivity profile of a number of compounds.

Nucleomimetic strategy for the inhibition of HIV-1 nucleocapsid protein NCp7 activities.

We report the synthesis and biological properties of three modified dinucleotides T*G, G*T and T*T in which the natural phosphodiester linkage has been replaced by a methylene carboxamide unit. They have been designed to act as nucleomimetics of a sequence recognized by the HIV-1 nucleocapsid protein NCp7 and to inhibit this interaction.