New small molecules, ISA27 and SM13, inhibit tumour growth inducing mitochondrial effects of p53.

BACKGROUND: p53 is a transcription factor with tumour suppressor properties, which is able to induce mitochondrial apoptosis independently of its transcriptional activity. We recently synthesised two new compounds (ISA27 and SM13), which block p53-MDM2 interaction and induce apoptosis in p53 wild-type (WT) tumour cells. The aim of this study was to verify the effectiveness of these compounds in tumours carrying a mutated form of p53 gene with no transcriptional activity. METHODS: In vitro we evaluated the effectiveness of our compounds in cancer cell lines carrying WT, mutated and null p53 gene. In vivo study was performed in Balb/c nude mice and the mitochondrial-dependent apoptotic signalling was evaluated by western blot. RESULTS: Both ISA27 and SM13 reduced cell proliferation and induced apoptosis in vitro in cells carrying either p53 WT or mutated gene, suggesting that its effect is independent from p53 transcriptional activity. On the contrary, SM13 had no effect in a p53 null cell line. In vivo, ISA27 and SM13 induced cancer cell death in a dose-dependent manner through the activation of the mitochondrial-dependent death signalling in p53-mutated cells. In vivo, SM13 reduced tumour growth. CONCLUSIONS: Our study proposes SM13 as anticancer compound to use for the treatment of p53-dependent tumours, even in the absence of p53 transcriptional activity.

Mitochondrial control of nuclear apoptosis.

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.

Cell penetrating peptides in ocular drug delivery: State of the art.

Despite the increasing number of effective therapeutics for eye diseases, their treatment is still challenging due to the presence of effective barriers protecting eye tissues. Cell Penetrating Peptides (CPPs), synthetic and natural short amino acid sequences able to cross cellular membrane thanks to a transduction domain, have been proposed as possible enhancing strategies for ophthalmic delivery. In this review, a general description of CPPs classes, design approaches and proposed cellular uptake mechanisms will be provided to the reader as an introduction to ocular CPPs application, together with an overview of the main problems related to ocular administration. The results obtained with CPPs for the treatment of anterior and posterior segment eye diseases will be then introduced, with a focus on non-invasive or minimally invasive administration, shifting from CPPs capability to obtain intracellular delivery to their ability to cross biological barriers. The problems related to in vitro, ex vivo and in vivo models used to investigate CPPs mediated ocular delivery will be also addressed together with potential ocular toxicity issues.

New thiol inhibitors of neutral endopeptidase EC 3.4.24.11: synthesis and enzyme active-site recognition.

Selective, as well as mixed, inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE), are of major clinical interest in the treatment of hypertension and cardiac failure. New thiol inhibitors, corresponding to the general formula HS-CH(R1)-CH2-CH(R2)-CONH-CH(R3)-COOH, were designed in order to explore the putative S1 subsite of the active site of NEP. The inhibitors were also tested on ACE and the most representative on thermolysin (TLN) for comparison. The relatively low inhibitory potencies exhibited by these compounds (IC50S in the 10(-7) M range for NEP and in the 10(-6) M range for ACE) as compared to that of thiorphan (IC50S 2.10 x 10(-9) M on NEP and 1.40 x 10(-7) M on ACE) clearly indicate the absence of the expected energetically favorable interactions with the active site of both peptidases. A 100-fold loss of potency for these inhibitors was also observed for thermolysin as compared to thiorphan. Using the mutated Glu102-NEP, it was possible to demonstrate that the inhibitors do not fit the S1 subsite of NEP but interact with the S'1 and S'2 subsites through binding of their R1 and R2 residues and that the C-terminal amino acid is located outside the active site. These results seem to indicate that these thiol inhibitors are not well adapted for optimal recognition of the S1 subsite of NEP, and probably ACE, and that other zinc-chelating moieties such as carboxylate or phosphonate groups may be preferred for this purpose.

Exploration of neutral endopeptidase active site by a series of new thiol-containing inhibitors.

With the aim of characterizing the active site of the neutral endopeptidase [EC 3.4.24.11 (NEP)] and especially its putative S1 subsite, two series of new thiol inhibitors designed to interact with the S1, S'1, and S'2 subsites of the enzyme have been synthesized. These molecules correspond to the general formula HSCH(R1)CH(R2)CONHCH(R3)COOH (series I) and HSCH(R1)CH(R2)CONHCH(R3)CONHCH(R4)COOH (series II). Due to the synthetic pathway used, these inhibitors were obtained as mixtures of four stereoisomers. HPLC separation of the stereoisomers of 17 HSCH[CH2CH(CH3)2]CH(CH2Ph)CONHCH(CH3)COOH allowed the stereochemical dependence of the inhibitory potency to be determined. The most active isomer 17b (IC50 = 3.6 nM) is assumed to have the S,S,S stereochemistry, as deduced from both NMR and HPLC data. Although none of the inhibitors obtained were significantly more active than thiorphan, HSCH2CH(CH2Ph)CONHCH2COOH (IC50 = 4 nM), which interacts only with the S'1 and S'2 subsites of NEP, their enhanced hydrophobicity is expected to improve their pharmacokinetic properties. All these compounds displayed low affinities for ACE (IC50s > 1 microM). The determination of the IC50s of two inhibitors of series II for NEP and for a mutated enzyme in which Arg102 was replaced by Glu102 allowed their mode of binding to the active site of NEP to be characterized. The R2 and R3 chains fit the S'1-S'2 subsites, while the R4 group is probably located outside the active site. Taken together these results indicate that the R1 chain of these inhibitors creates no additional stabilizing interactions with the active site of NEP. Two hypotheses may account for this: there is no hydrophobic S1 subsite in NEP or the inhibitors have structures which are too constrained for optimized interactions with the active site.

Ketomethylene and (cyanomethylene)amino pseudopeptide analogues of the C-terminal hexapeptide of neurotensin.

A series of pseudopeptide analogues of the C-terminal hexapeptide of neurotensin (NT8-13), namely [Tyr11 psi[COCH2]Phe12]-, [Ile12 psi[COCH2]Phe13]-, and [Tyr11 psi[CH(CN)NH]Ile12]NT8-13 with different stereochemistries, has been synthesized and evaluated for its potency in displacing labeled NT from rat cortex membranes. Ketomethylene pseudohexapeptides were prepared from the corresponding Boc-protected ketomethylene dipeptide derivatives, previously formed, using different solid phase synthesis (SPS) conditions, while (cyanomethylene)amino analogues were directly prepared by SPS using Fmoc strategy. H-Arg-Arg-Pro-Tyr psi[COCH2]-Phe-Leu-OH was nearly as potent as NT8-13 and [Phe12]NT8-13 in binding to the receptor. Comparison of the affinities for the pseudohexapeptides, here reported, with those of the psi-[CH2NH] analogues indicates the importance of the CO group in the amide or surrogate linkage at 11-12 and 12-13 positions in the receptor binding process.

Synthesis and stereochemical structure-activity relationships of 1,3-dioxoperhydropyrido[1,2-c]pyrimidine derivatives: potent and selective cholecystokinin-A receptor antagonists.

The synthesis and stereochemical structure--activity relationships of a new class of potent and selective non-peptide cholecystokinin-A (CCK-A) receptor antagonists based on the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine skeleton are described. The most potent member of this series of eight diastereoisomers, (4aS,5R)-2-benzyl-5-[N-[(tert-butoxycarbonyl)-L-tryptophyl]-amino] - 1,3-dioxoperhydropyrido[1,2-c]pyrimidine, displays nanomolar CCK-A receptor affinity and higher than 8000-fold potency at the CCK-A than at the CCK-B receptor. As CCK-A antagonist, this compound inhibits the CCK-8-evoked amylase release from pancreatic acinar cells at a low concentration, similar to that of the typical antagonist Devazepide. Highly strict stereochemical requirements for CCK-A receptor binding and selectivity have been found. The L-Trp and the 4a,5-trans disposition of the bicyclic perhydropyrido[1,2-c]pyrimidine are essential for binding potency and selectivity.

5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK(1) receptor antagonists: structural modifications at the tryptophan domain.

Analogues of the previously reported potent and highly selective CCK(1) receptor antagonist (4aS, 5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido-[1, 2-c]pyrimidine (2a) were prepared to explore the structural requirements at the Boc-tryptophan domain for CCK(1) receptor affinity. Structural modifications of 2a involved the Trp side chain, its conformational freedom, the Boc group, and the carboxamide bond. Results of the CCK binding and in vitro functional activity evaluation showed three highly strict structural requirements: the type and orientation of the Trp side chain, the H-bonding acceptor carbonyl group of the carboxamide bond, and the presence of the Trp amino protection Boc. Replacement of this acid-labile group with 3, 3-dimethylbutyryl or tert-butylaminocarbonyl conferred acid stability to analogues 14a and 15a, which retained a high potency and selectivity in binding to CCK(1) receptors, as well as an in vivo antagonist activity against the acute pancreatitis induced by caerulein in rats. Oral administration of compounds 14a and 15a also produced a lasting antagonism to the hypomotility induced by CCK-8 in mice, suggesting a good bioavailability and metabolic stability.

5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK(1)receptor antagonists: structure-activity relationship studies on the substituent at N2-position.

To establish structure-activity relationships a new series of analogues of the highly potent and selective CCK(1) receptor antagonist (4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]-pyrimidine (1a) modified at N2-position of the central scaffold has been prepared and evaluated as CCK receptor ligands. With this aim the N2-benzyl group has been replaced by methyl, cyclohexyl, aromatic groups, 1-phenylethyl, and 1-carboxy-2-phenylethyl group. Then, substituents with different electronic and steric properties were introduced into different positions of the phenyl group of analogues 19a and 19b. The results of the CCK receptor binding and in vitro functional activity evaluation suggest the importance of the lipophilic character and an appropriate spatial orientation of the moiety linked at the N2-position of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine template for potent and selective binding and antagonist activity at CCK(1) receptor subtype. The 2-cyclohexyl and (2S)-1-naphthyl derivatives 18a and (2S)-20a have emerged as more potent and selective CCK(1) receptor antagonists than the lead compound 1a. Additionally, the results confirm the (4aS,5R)-stereochemistry at the central bicyclic skeleton as an essential structural requirement for potent binding to this receptor subtype.

Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models.

1. The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[N alpha-tert-butoxicarbonyl)L-tryptophyl] amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158. 2. IQM-95,333 displaced [3H]-CCK-8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50 > 10 microM). IQM-95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. 3. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCKA receptors. 4. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCKA receptor-mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. 5. Low doses (50-100 micrograms kg-1, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCKA receptors. 6. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10-5,000 micrograms kg-1). Typical CCKA and CCKB antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range. 7. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. 8. In conclusion, IQM-95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic-like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

Prolonged antinociceptive activity of pseudodipeptide analogues of Lys-Trp(Nps) and Trp(Nps)-Lys.

Peptide bond substitution in the molecules of Lys-Trp(Nps) (LTN) and Trp(Nps)-Lys (TNL) by an aminomethylene and ketomethylene bond, respectively, afforded pseudodipeptides with analgesic activity. The new compounds Lys psi(CH2NH)-Trp(Nps)-OMe (LTNAM) and Trp(Nps)psi(COCH2)(R,S)-Lys (TNLKM) induced a dose-dependent and naloxone-reversible analgesia following intracerebroventricular (ICV) administration to mice. The antinociceptive effects were longer lasting compared to those induced by the parent compounds. The pseudodipeptides protected Met-enkephalin degradation by rat striatal slices and, combined with an ineffective dose of the opioid peptide, induced analgesia. LTNAM and TNLKM were as potent as LTN to inhibit brain aminopeptidase in vitro and ex vivo. An increased resistance to proteolysis of the pseudodipeptides may explain their prolonged analgesic activity.

Intramolecular C-H--O interaction between lactam oxygen and N-alkyl protons.

We report evidence of an unusual C-H--O interaction between an alpha-methylene hydrogen of the alkylamine chain of substituted (N,N-dimethylamino)propyl-azetidinones, substituted (N,N-dimethylamino)propyl-thiazolidinones and substituted (N,N-dimethylamino)propyl-thiazinone and the lactam carbonyl oxygen. NMR analysis results, supported by molecular mechanic predictions, were in agreement with ab initio calculations. The observed interaction shorting the nitrogen-nitrogen distance in the H1-histamine antagonist, 2-(4-methylphenyl)-3-[3-(N,N-dimethylamino)propyl]-1,3-thiazolidin-4-one (1) could explain its fitting with the H1-antihistaminic pharmacophoric model and the high antihistaminic activity.

Synthesis and structure-activity relationships of 2-(substituted phenyl)-3-[3-(N,N-dimethylamino)propyl]-1,3-thiazolidin-4-ones acting as H1-histamine antagonists.

2-(Substituted-phenyl)-3-[3-(N,N-dimethylamino)propyl]-1,3-thiazolidi n-4- ones (1-15) showed dependence of the potency of the H1-histamine antagonism on the m- and p-substituents suggesting that the aromatic moiety binds the receptor by a strong pi-interaction. Electron-withdrawing substituents decrease the potency while the electron-donating alkyl substituents, enhancing the aryl HOMO energy, increase the antihistamine activity. The m-substituents with the capability to form hydrogen bonds, seems to share an extra-interaction with hydrogen accepting or donating groups of the histamine receptor and exhibits very high potency.

A novel route to synthesize Freidinger lactams by micowave irradiation.

The incorporation of a Freidinger-like lactam structure into the backbone of peptides has been proven to be an useful strategy in the design of a variety of conformationally restricted targets. Several different strategies have been developed toward Freidinger lactams but no one resulted to be completely facile. Here, we report an efficient strategy that involves the iodo-derivatives in side chain of an appropriate amino acid used as electrophilic agent, and the standard solid phase peptide synthesis assisted by microwave irradiation. The methodology developed could be useful to perform Freidinger-like lactams with defined stereochemistry for routine use in solid phase peptide chemistry.

Ketomethylenebestatin: synthesis and aminopeptidase inhibition.

The synthesis of (6R,5S,2RS)-6-amino-5-hydroxy-2-isobutyl-4-oxo-7- phenylheptanoic acid (9), a carbaanalogue of the aminopeptidase (AP) inhibitor bestatin (1) is described. This synthesis was carried out by a malonic ester alkylation with the suitably protected halomethyl ketone of (2S,3R)-AHPBA*), followed by a second alkylation with isobutyl bromide of the resulting 4-ketodiester, and subsequent decarboxylation and deprotection. The inhibitory potencies of the 1:1 diastereomeric mixture 9 against AP-B, AP-M and Leu-AP were approximately 10-fold lower than those of bestatin.

Ketomethylene analogues of phosphoryl dipeptides related to phosphoramidon: synthesis and inhibition of proteases.

Non-rhamnose-containing phosphoramidon analogues, in which the amide bond was replaced by the isosteric ketomethylene group, have been synthesized in order to stabilize these compounds to peptidase degradation. The key step in this synthesis was suitable alkylation of a 4-ketodiester, prepared from Z-Leu chloromethyl ketone and dimethyl malonate. The ketomethylene dipeptide derivatives P-Leu psi (COCH2)(RS)Xaa-OMe (Xaa = Trp, Phe) are good inhibitors of thermolysin, ACE and specially enkephalinase.

Synthesis and inhibitory activities against aminopeptidase B and enkephalin-degrading enzymes of ketomethylene dipeptide analogues of arphamenines.

Three ketomethylene pseudodideptide analogues [(S)Lys psi(COCH2)(R and S)Phe (14 or 15 and 15 or 14) and (S)Lys psi(COCH2)(xi Trp (19)] of natural arphamenine A [(S)Arg psi(COCH2(R,S)Phe (1)] were easily prepared by a route involving two successive main reactions: a malonic ester alkylation with Z-protected lysine iodomethyl ketone and the introduction of a benzyl or (indol-3-yl)methyl moiety in position 2 of the resulting 4-ketodiester. The isomer of 1 with reversed sequence, (S)Phe psi(COCH2)(R,S)Arg (22) was synthesized by guanidylation and subsequent deprotection of Z-(S)Phe psi(COCH2)(R,S)Orn. The inhibitory effects of compounds 14, 15, 19, and 22, and the related ketomethylene dipeptides (S)Ala psi(COCH2)(R,S)Phe (3), (S)Phe psi(COCH2)(R,S)X [X = Ala (4), Orn (5)] and (S)Trp psi(COCH2)(R,S)Y [Y = Orn (6), Lys (7), Arg (8)] on aminopeptidase B (AP-B), and enkephalin-degrading enzymes [aminopeptidase N (APN) and neutral endopeptidase (NEP)] were compared with that of the model compound 1.