Phosphorus amino acid analogues as inhibitors of leucine aminopeptidase

A variety of phosphorus amino acid and dipeptide analogues have been synthesized and evaluated as inhibitors of the metalloenzyme leucine aminopeptidase from porcine kidney. Two phosphonate dipeptides were found to be modest inhibitors of the enzyme (8e, Ki = 58 microM; 8h, Ki = 340 microM). The phosphinic acid (17-OH) and phosphinamide (17-NH2) analogues related to bestatin were prepared by condensation of the phosphinate amino acid derivative 11, via a trivalent phosphonite ester 12, with leucine isocyanate derivatives 13. These compounds also proved to be unexceptional in their inhibition of LAP (17-O-, Ki = 56 microM; 17-NH2, Ki = 40 microM). A series of simple (alpha-aminoalkyl)phosphonic acid and -phosphinic acids were also evaluated, and the most potent inhibitors were found to be the phosphonic acid analogues of L-Leu and L-Phe [R)-3e, Ki = 0.23 microM; (R)-3h, Ki = 0.42 microM). Slow-binding behavior was observed for (R)-3e (kon = 400 +/- 55 M-1 s-1) and (R)-3h (kon = 445 +/- 50 M-1 s-1). The phosphinic acid analogues of Leu and Phe are 100-fold less potent than the phosphonate derivatives. The fact that tetrahedral phosphorus analogues are less potent inhibitors of LAP than they are of other zinc peptidases suggests that the mechanism of LAP may be fundamentally different than that of the latter enzymes.     

Identification of phosphinate dipeptide analog inhibitors directed against the Plasmodium falciparum M17 leucine aminopeptidase as lead antimalarial compounds

Previous studies have pinpointed the M17 leucyl aminopeptidase of Plasmodium falciparum (PfLAP) as a target for the development of new antimalarials. This metallo-exopeptidase functions in the terminal stages of hemoglobin digestion and is inhibited by bestatin, a natural analog of Phe-Leu. By screening novel phosphinate dipeptide analogues for inhibitory activity against recombinant PfLAP, we have discovered two compounds, 4 (hPheP[CH2]Phe) and 5 (hPheP[CH2]Tyr), with inhibitory constants better than bestatin. These compounds are fast, tight-binding inhibitors that make improved contacts within the active site of PfLAP. Both compounds inhibit the growth of P. falciparum in vitro, exhibiting IC50 values against the chloroquine-resistant clone Dd2 of 20-40 and 12-23 muM, respectively. While bestatin exhibited some in vivo activity against Plasmodium chabaudi chabaudi, compound 4 reduced parasite burden by 92%. These studies establish the PfLAP as a prime target for the development of antimalarial drugs and provide important new lead compounds.     

Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes

Amastatin [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-L-valyl-L-valyl-L- aspartic acid] and bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] are slow-binding, competitive inhibitors of aminopeptidase M (AP-M) with net inhibition constants (Ki) of 1.9 X 10(-8) and 4.1 X 10(-6) M, respectively. The effect of inhibitor structure on net Ki and on slow-binding inhibition was evaluated for analogues of both inhibitors on AP-M and leucine aminopeptidase (LAP). The (2S)-hydroxyl group contributes to the stabilization of a collision complex [EI], which is formed rapidly. In contrast, increasing the peptide chain length of the inhibitor produces more potent inhibitors as a consequence of a slower binding process. A statine analogue of amastatin [(3S,4S)-Sta-Val-Val-Asp] stimulated rather than inhibited LAP. AP-M binds tri- and tetrapeptide inhibitors more strongly than dipeptide inhibitors, whereas LAP binds dipeptide inhibitors more strongly. The difference in binding can be used to distinguish cytosolic from membrane-bound aminopeptidases.     

Inhibition of aminopeptidases by peptides containing ketomethylene and hydroxyethylene amide bond replacements

Inhibitors of aminopeptidase enzymes have been prepared by the synthesis of peptide substrate analogues in which the scissile amide bond has been replaced with the hydrolytically stable ketomethylene (-COCH2-) and hydroxyethylene [-CH(OH)CH2-] functionalities. Two synthetic strategies were used to prepare the inhibitors, and the advantages and disadvantages of each are discussed. The synthesis of peptides that contain the hydroxyethylene isostere was complicated by competing lactone and lactam formation, and attempts to prepare free N-terminal dipeptide hydroxyethylene isostere derivatives were unsuccessful. All ketomethylene isosteres examined were weak inhibitors of both leucine aminopeptidase and aminopeptidase M. However, the ketomethylene inhibitor LysK(RS)Phe (58) (Ki = 4 nM) is a potent inhibitor comparable to the natural product, arphamenine A (ArgKPhe; Ki = 2.5 nM). Normal Michaelis-Menten kinetics for inhibition of membrane leucine aminopeptidase are observed in the absence of magnesium ion, but nonlinear kinetics were obtained in the presence of Mg2+.     

Synthesis of sulfur-containing analogues of bestatin. Inhibition of aminopeptidases by alpha-thiolbestatin analogues

Sulfur-containing amino acid and peptide analogues of bestatin [((2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] (1) have been synthesized and evaluated as inhibitors of aminopeptidase M (AP-M), leucine aminopeptidase (LAP), and aminopeptidase B (AP-B). The 2-thiolbestatin analogue (6) was found to be a potent inhibitor of all three aminopeptidases (AP-M, Ki = 4.4 microM; LAP, Ki = 0.55 microM; AP-B, Ki = 4.6 nM) but only a slightly better inhibitor of these aminopeptidases than the parent hydroxy-containing compound 1. Synthetic analogues of L-leucinethiol(4), a strong inhibitor of aminopeptidases, were prepared in which the carbon alpha to the thiol groups was substituted with methyl, methyl carboxylate, and carboxamide derivatives and found to be much weaker inhibitors of all aminopeptidases. A thioamide analogue of bestatin (49) is a modest inhibitor of AP-M (Ki = 40 microM), LAP (Ki = 0.33 microM), and AP-B (Ki = 2.4 microM). These results suggest that the sulfur atoms in 2-thiolbestatin and bestatin thioamide do not interact strongly with the active-site zinc atom of these aminopeptidases when the inhibitors are bound to the enzyme. These results are not consistent with proposed models for the inhibition of aminopeptidases by bestatin and related analogues.     

Retro-inverso concept applied to the complete inhibitors of enkephalin-degrading enzymes

Peptide retro-inverso modification was applied to the complete hydroxamate inhibitors of the three zinc metallopeptidases (neutral endopeptidase 24-11 (NEP, EC 3.4.24.11), aminopeptidase N (APN, EC 3.4.11.2), and a dipeptidylaminopeptidase (DAP) involved in the in vitro enkephalin degradation by brain tissues. Compounds corresponding to the general formula RN(OH)CO(CH2)nCH(CH2Ph)NHCOCH(R')COOH (n = 0, 1) were synthesized. In the first series of inhibitors (n = 0), the "retro-inverso" modification induced a large decrease in inhibitory potency for NEP as compared to that of the parent compounds. In contrast, the presence of a methylene group between the hydroxamate and CH alpha in the second series (n = 1) led to derivatives with inhibitory potencies in the nanomolar range, similar to their analogues with a natural amide bond. On the other hand, the retro-inverso modification led to a slight improvement in the inhibition of DAP and APN, in the first series of inhibitors, while the inverse result occurred in the second series. Thus, compounds containing an alpha-amino acid moiety in P'1 position behave as weak inhibitors of the three enzymes, with IC50 values in the micromolar range, and compounds bearing a beta-amino acid moiety in the same position are more specific than the parent compounds for NEP inhibition.     

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.     

Design of novel inhibitors of aminopeptidases. Synthesis of peptide-derived diamino thiols and sulfur replacement analogues of bestatin

Investigations were directed toward inhibition of an aminopeptidase, isolated from rat brain, which has been implicated in the metabolic inactivation of enkephalins. The design rationale and synthesis of novel peptidyl diamino thiol inhibitors of rat brain aminopeptidase are presented, along with accompanying structure-activity analysis. Some of the reported compounds are highly active aminopeptidase inhibitors and possess enzyme inhibitory potency in the nanomolar range (62; I50 = 1 nM). Analysis of the data permits speculations on possible modes of binding of diamino thiols to aminopeptidase. Other investigations were directed toward understanding the mode of enzyme binding of the naturally occurring aminopeptidase inhibitor bestatin. On the basis of published models of enzyme binding, replacement of the C-2 hydroxyl group of bestatin by a sulfhydryl group was anticipated to lead to enhanced inhibition due to a strengthened interaction of this group with enzymic zinc. Contrary to expectations, "thiobestatin" inhibited rat brain aminopeptidase with only the same degree of effectiveness as the corresponding alcohol. Speculations on the possible mode of enzyme-inhibitor binding of bestatin are offered.     

Design and synthesis of HIV-1 protease inhibitors incorporating oxazolidinones as P2/P2' ligands in pseudosymmetric dipeptide isosteres

A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type HIV-1 protease have been determined. A comparison of the inhibitor-protease structures with the LPV-protease structure provides valuable insight into the binding mode of the new inhibitors to the protease enzyme. Based on the crystal structures and knowledge of structure-activity relationships, new inhibitors can be designed with enhanced enzyme inhibitory and antiviral potencies.     

Synthesis and biological evaluation of phosphonamidate peptide inhibitors of enkephalinase and angiotensin-converting enzyme

The effectiveness of phosphonamidate peptide analogues as inhibitors of rat kidney or human brain metalloendopeptidase (enkephalinase, E.C. 3.4.24.11) and angiotensin-converting enzyme (ACE, 3.4.14.1) has been explored with a series of enkephalin analogues in which the scissile Gly3-Phe4 amide bond has been replaced with a phosphonamidate moiety. These compounds exhibited good inhibitory potency against enkephalinase with several of the analogues having Ki values in the submicromolar range as contrasted to micromolar or higher toward ACE. Within a series of [(N-acylamino)methyl] phosphonamidates there was a dramatic decrease in inhibitory activity against enkephalinase as the N-acyl moiety was substituted with larger, more hydrophobic acyl groups. Likewise, the inhibitory activity of the [(N-acylamino)methyl] phosphonamidates against ACE was attenuated by larger phenylalkyl acyl functionalities, although not to the same degree as against enkephalinase. However, phosphonamidate pentapeptide analogues of (Leu)enkephalin and (D-Ala2,D-Leu5)enkephalin showed good inhibitory potency against both enzymes. Interestingly, these two (Leu)enkephalin phosphonamidate analogues were completely inactive in the electrically stimulated guinea pig ileum and mouse vas deferens preparations. Conformational factors that may be involved in this inactivity are discussed.     

Potent and systemically active aminopeptidase N inhibitors designed from active-site investigation.

Derivatives of amino acids bearing various zinc-coordinating moieties (SH, COOH, CONHOH, and PO3H2) were synthesized and tested for their ability to inhibit aminopeptidase N (APN). Among them, beta-amino thiols were found to be the most efficient with IC50's in the 11-50 nM range. These results suggest that the S1 subsite of APN is a deep but not very large hydrophobic pocket, optimally fitting side chains of moderate bulk endowed with some degree of freedom. The iv administration of the inhibitors, alone, did not induce antinociceptive responses on the hot plate test in mice. However, in presence of 10 mg/kg acetorphan, a prodrug of the neutral endopeptidase inhibitor thiorphan, these compounds gave a large increase in the jump latency time with ED50's of 2 and 2.4 mg/kg for the disulfides of methioninethiol H2NCH(CH2CH2SCH3)CH2S]2 and S-oxomethioninethiol [H2NCH(CH2CH2S(O)CH3)CH2S]2, respectively. These results show that the disulfide forms of beta-amino thiols are efficient prodrugs of aminopeptidase N inhibitors capable of crossing the blood-brain barrier.     

以氨肽酶 N为靶点的N-取代-2，5-吡咯烷二酮类肽的设计合成及活性研究

目的 寻找具有氨肽酶N(APN,CD13)抑制活性的新型化合物并测定其抑制氨肽酶N的活性;考察目标化合物与APN活性位点的结合,研究目标化合物与酶的相互作用关系。方法 以光学纯的天冬酰胺为原料,经Boc保护、环合、酯化、脱Boc保护、酰化、氢化还原、羟肟酸化等反应合成目标化合物。借助FlexX对接软件,研究目标化合物与APN活性位点的结合情况;采用体外抑酶试验测定目标化合物抑制APN的活性。结果 合成了14个未见文献报道的 N-取代-2,5-吡咯烷二酮类肽类APN抑制剂,其结构经¹H-NMR、MS谱确证。结论 目标化合物均对APN/CD13具有一定的抑制活性,其中,化合物7h的活性较好,与计算机对接结果一致。     

Potential latentiation forms of biologically active compounds based on action of leucine aminopeptidase. Dipeptide derivatives of the tricycloaliphatic alpha-amino acid, adamantanine.

Some glycine, leucine and phenylalanine dipeptide derivatives of the transport inhibitory, tricycloaliphatic alpha-amino acid, adamantanine (1), have been synthesized using classical methods of peptide synthesis with the aim of improving the latter's bioavailability. Although test doses of glycyladamantanine and L-leucyladamantanine appeared to be absorbed in vivo as evidenced by its appearance in the uring following intraperitoneal administration, they were not hydrolyzed by a purified preparation of leucine aminopeptidase in vitro. Indeed, they were inhibitors of this enzyme. Adamantanylglycine, adamantanyl-L-leucine, and adamantanyl-L-phenylalanine were also not hydrolyzed by leucine aminopeptidase.     

Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs

Prodrugs of phosphinic dual inhibitors of the enkephalin degrading enzymes, neutral endopeptidase (NEP) and aminopeptidase N (APN), corresponding to the formula H(3)N(+)CH(R(1))P(O)(OR)CH(2)CH(CH(2)Bip)CONHCH(CH(3))COOCH(2)Ph, with R(1) = CH(3) or Ph and R being a benzyl ester, a S-acyl-2-thioethyl derivative, or an acyloxyalkyl group, were synthesized to improve the poor central bioavailability of their precursors. As expected, these compounds (50 mg/kg, iv or ip) induced long lasting ( approximately 2 h) antinociceptive responses in the hot plate test in mice with a ceiling effect varying between 25 and 42% of analgesia. A very rapid hydrolysis of the carboxylate ester contrasting with a slow deprotection of the phosphinate group (t(1/2) approximately 1 h) was observed in serum while 80% of free drug was obtained after 1 h incubation with brain membranes. These results account for the long duration of action observed with these prodrugs.     

alpha-Keto amide inhibitors of aminopeptidases.

The design and synthesis of 3-amino-2-oxo-4-phenylbutanoic acid amides (alpha-keto amides), a new class of aminopeptidase inhibitor, are described. These compounds, illustrated by the Phe-Leu analogue 2, are effective inhibitors of arginyl aminopeptidase (Ki = 1.5 microM), cytosol aminopeptidase (Ki = 1.0 microM), and microsomal aminopeptidase (Ki = 2.5 microM). The ketone carbonyl of the alpha-keto amide was found to hydrate readily in an aqueous DMSO solution, due to the electron-withdrawing effect of the neighboring amide group. A mechanism of inhibition is proposed for the alpha-keto amides that is similar to that proposed for the structurally related aminopeptidase inhibitor bestatin and its analogues, wherein the inhibitor may interact with the S1'-S2' subsite of the enzyme rather than the S1-S1' subsite. Like bestatin, the alpha-keto amides are slow-binding inhibitors of all three enzymes.     

Novel pyrazolopyrimidopyridazinones with potent and selective phosphodiesterase 5 (PDE5) inhibitory activity as potential agents for treatment of erectile dysfunction

Pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones and their analogues, potentially useful for the treatment of erectile dysfunction, were synthesized and evaluated as inhibitors of phosphodiesterase 5 (PDE5). Several compounds showed IC50 values in the low nanomolar range, and in particular, compound 5r, displaying high potency toward PDE5 (IC50 = 8.3 nM) and high selectivity versus PDE6 (240-fold) appeared to be a very promising new lead both in comparison with the potent but not selective sildenafil and in comparison with some analogues previously reported by us. SAR studies in this triheterocyclic scaffold led us to conclude that the best arranged groups are a methyl in position 1, a benzyl in position 3, a phenyl in position 9, and a linear four-carbon chain in position 6.     

Aminopeptidase N (APN/CD13) as a target for anti-cancer agent design

APN is an important zinc dependent metallo-exopeptidase; it has been considered as a suitable target for anti-cancer drug design. In this review we focus on the most effective and the most promising inhibitors of aminopeptidase N. Their binding modes to the enzyme, the attempt to explain the origin of the inhibitory activity, as well as the structure-activity relationship for some of these compounds are discussed. Besides, the structural and electronic requirements of the enzyme active site and the binding pockets, together with the specificity towards the ligands are presented.     

Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among these compounds is L-ArgNO2-L-Dbu-NH2 (1) (Ki = 130 nM), which also exhibits the highest selectivity over eNOS (>1,500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than 1. Both of the results demonstrate the importance of the alpha-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.     

Synthesis and inhibitory activities against enkephalin degrading aminopeptidase of H-Trp(Nps)-Lys-OMe analogues bearing chelating groups.

With the aim of increasing the inhibitory potency of the analgesic dipeptide H-Trp(Nps)-Lys-OMe against enkephalin-degrading aminopeptidases, the following derivatives bearing chelating groups at the N-terminus have been synthesized: Ac-Trp(Nps)-Lys-OMe (3), HS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (4), n = 2 (5)], MeOCO(CH2)n-Trp(Nps)-Lys-OMe [n = 1 (6), n = 2 (7)] and analogues in which the N alpha-amino group has been replaced by a methoxycarbonyl group (8) and a bidentate hydroxamate function (9), respectively. The inhibitory activities of all these compounds and the S-protected derivatives EtNHCOS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (16), n = (17)] against the mentioned enzyme, isolated from rat striatum, are compared with those of the parent dipeptide 2 and bestatin. All the new derivatives showed, in general, inhibitory potencies of the same order of magnitude as compound 2.     

Inhibition of metallopeptidases by flavonoids and related compounds

To elucidate possible mechanisms of activity in medicinal plants containing flavonoids, the inhibitory potency of twenty flavones, flavonols, flavanones, phenylacrylic acids and various hydroxylated phenylacetic acids on the activity of neutral endopeptidase (NEP; EC 3.4.24.11), angiotensin-converting enzyme (ACE; EC 3.4.15.1) and aminopeptidase N (APN; EC 3.4.11.2) was investigated in vitro. The screening generally resulted that inhibition of these enzymes requires free hydroxyl groups at the flavone molecule. Flavone and methoxylated compounds (sinensetin) were without effects. Flavonoids with free hydroxyl functions in position 3',4' and 5,7 inhibited the activity of NEP (quercetin, luteolin, fisetin), with myricetin (IC50 = 42 microM) as strongest inhibitor. Inhibition of ACE and APN did not depend on this class of compounds and substitution pattern. E.g. 3,4-dihydroxyphenylacetic acid and 4-methylcatechol (urinary metabolites of flavonoids) also inhibited both APN and ACE activity, but not NEP activity. The results demonstrate that some of the pharmacological activities of flavonoids might be related to the inhibition of metallopeptidases responsible for the splitting of regulatory neuropeptides.