In silico drug repositioning on F508del-CFTR: A proof-of-concept study on the AIFA library.

Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings.

Inhibition of Fibrillar Assemblies of l-Phenylalanine by Crown Ethers: A Potential Approach toward Phenylketonuria.

In this article, our aim is to investigate the interaction of l-phenylalanine (l-Phe) fibrils with crown ethers (CEs). For this purpose, two different CEs (15-Crown-5 (15C5) and 18-Crown-6 (18C6)) were used. Interestingly, we have observed that both CEs have the ability to arrest fibril formation. However, 18C6 was found to be a better candidate compared to 15C5. Field emission scanning electron microscopy and fluorescence lifetime imaging microscopy were used to monitor the fibril-arresting kinetics of CEs. The arresting process was further confirmed by fluorescence correlation spectroscopy and nuclear magnetic resonance studies.

L-Phenylalanine preloading reduces the (10)B(n, α)(7)Li dose to the normal brain by inhibiting the uptake of boronophenylalanine in boron neutron capture therapy for brain tumours.

Boron neutron capture therapy (BNCT) is a cellular-level particle radiation therapy that combines the selective delivery of boron compounds to tumour tissue with neutron irradiation. Previously, high doses of one of the boron compounds used for BNCT, L-BPA, were found to reduce the boron-derived irradiation dose to the central nervous system. However, injection with a high dose of L-BPA is not feasible in clinical settings. We aimed to find an alternative method to improve the therapeutic efficacy of this therapy. We examined the effects of oral preloading with various analogues of L-BPA in a xenograft tumour model and found that high-dose L-phenylalanine reduced the accumulation of L-BPA in the normal brain relative to tumour tissue. As a result, the maximum irradiation dose in the normal brain was 19.2% lower in the L-phenylalanine group relative to the control group. This study provides a simple strategy to improve the therapeutic efficacy of conventional boron compounds for BNCT for brain tumours and the possibility to widen the indication of BNCT to various kinds of other tumours.

Brain-derived neurotrophic factor inhibits phenylalanine-induced neuronal apoptosis by preventing RhoA pathway activation.

Phenylketonuria (PKU) is neuropathologically characterized by neuronal cell loss, white matter abnormalities, dendritic simplification, and synaptic density reduction. The neuropathological effect may be due to the 'toxicity' of the high concentration of phenylalanine, while little is known about the related treatments to block this effect. In this study, we reported that brain-derived growth factor (BDNF) protected neurons from phenylalanine-induced apoptosis and inhibition of Trk receptor by K252a or downregulation of TrkB abrogated the effect of BDNF. We further demonstrated that phenylalanine-induced RhoA activation and myosin light chain phosphorylation were inhibited by pretreatment with BDNF, while phenylalanine activates the mitochondria-mediated apoptosis through the RhoA/Rho-associated kinase pathway. Thus our studies indicate that the protective effect of BDNF against phenylalanine-induced neuronal apoptosis is probably mediated by suppression of RhoA signaling pathway via TrkB receptor. Taken together, these findings suggest a potential neuroprotective action of BDNF in prevention and treatment of PKU brain injury.

The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose.

A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.

Alanine prevents the in vitro inhibition of glycolysis caused by phenylalanine in brain cortex of rats.

Glycolysis is the main route that provides energy to brain functioning. In this study we investigated the in vitro effects of phenylalanine, the main metabolite known to accumulate in phenylketonuria, and/or alanine, on pyruvate kinase activity, glucose utilization, lactate release, and ADP concentration in brain cortex homogenates from 30-day-old Wistar rats. We found that phenylalanine decreased PK activity, glucose utilization, and lactate release, and increased ADP brain levels. We also verified that alanine per se did not modify these parameters, but prevented the effects of phenylalanine. Our data suggest that the inhibition of pyruvate kinase by phenylalanine decreases glycolysis and energy production, and that alanine, a known competitor of phenylalanine on the enzyme activity, prevents the reduction of glycolysis and energy production caused by phenylalanine, probably by preventing the enzyme inhibition provoked by the amino acid. These results suggest that inhibition of brain PK activity by phenylalanine may be related to the diminution of glucose metabolism observed in the brain of phenylketonuric patients and may be one of the mechanisms responsible for the neurological dysfunction found in these patients.

Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity.

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.

Beneficial effects of Batimastat (BB-94), a matrix metalloproteinase inhibitor, in rat experimental colitis.

BACKGROUND AND AIMS: Matrix metalloproteinases (MMPs) represent a group of enzymes that regulate cell-matrix composition playing a major role in the inflammatory response. In the present study we evaluated the ability of the MMP inhibitor Batimastat (BB-94) to modify the course of experimental colitis induced in the rat by trinitrobenzensulfonic acid (TNB). METHODS: Colitis was induced in 40 rats by intracolonic administration of TNB. Animals were divided into four groups of ten rats each: group 1 received only intracolonic TNB, group 2 received TNB+5 mg/kg intraperitoneal BB-94, group 3 TNB+10 mg/kg BB-94 and group 4 TNB+20 mg/kg BB-94. The MMP inhibitor was administered 30 min before induction of colitis and twice daily until death. Ten rats receiving only intracolonic 0.9% saline served as controls. Animals were killed after seven days; segments of colon were removed and used for histological score of inflammation and myeloperoxidase (MPO) activity. RESULTS: Rats receiving only intracolonic 0.9% saline showed no evidence of colitis. The inflammation score was 0.9, MPO activity 0.235 U/mg. Group 1 (TNB-treated rats) exhibited a high inflammation score (12.4) and MPO activity (0.715 U/mg). Conversely, BB-94-treated rats showed, compared to the TNB group, a significantly lower inflammation score and MPO activity in a dose-dependent fashion. Group 2: inflammatory score 10.1, MPO activity 0.474 (p < 0.05 vs. TNB); group 3: inflammatory score 8.3, MPO activity 0.287 (p < 0.01 vs. TNB); group 4: inflammatory score 5.0, MPO activity 0.256 (p < 0.01 vs. TNB). CONCLUSIONS: Treatment with BB-94 has dose-dependent beneficial effects on the inflammatory alterations in rat experimental colitis. Thus, the inhibition of MMPs may represent a novel therapeutic approach for treatment of intestinal inflammation.

Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.

The protein-retaining effects of growth hormone during fasting involve inhibition of muscle-protein breakdown.

The metabolic response to fasting involves a series of hormonal and metabolic adaptations leading to protein conservation. An increase in the serum level of growth hormone (GH) during fasting has been well substantiated. The present study was designed to test the hypothesis that GH may be a principal mediator of protein conservation during fasting and to assess the underlying mechanisms. Eight normal subjects were examined on four occasions: 1) in the basal postabsorptive state (basal), 2) after 40 h of fasting (fast), 3) after 40 h of fasting with somatostatin suppression of GH (fast-GH), and 4) after 40 h of fasting with suppression of GH and exogenous GH replacement (fast+GH). The two somatostatin experiments were identical in terms of hormone replacement (except for GH), meaning that somatostatin, insulin, glucagon and GH were administered for 28 h; during the last 4 h, substrate metabolism was investigated. Compared with the GH administration protocol, IGF-I and free IGF-I decreased 35 and 70%, respectively, during fasting without GH. Urinary urea excretion and serum urea increased when participants fasted without GH (urea excretion: basal 392 +/- 44, fast 440 +/- 32, fast-GH 609 +/- 76, and fast+GH 408 +/- 36 mmol/24 h, P < 0.05; serum urea: basal 4.6 +/- 0.1, fast 6.2 +/- 0.1, fast-GH 7.0 +/- 0.2, and fast+GH 4.3 +/- 0.2 mmol/1, P < 0.01). There was a net release of phenylalanine across the forearm, and the negative phenylalanine balance was higher during fasting with GH suppression (balance: basal 9 +/- 3, fast 15 +/- 6, fast-GH 17 +/- 4, and fast+GH 11 +/- 5 nmol/min, P < 0.05). Muscle-protein breakdown was increased among participants who fasted without GH (phenylalanine rate of appearance: basal 17 +/- 4, fast 26 +/- 9, fast-GH 33 +/- 7, fast+GH 25 +/- 6 nmol/min, P < 0.05). Levels of free fatty acids and oxidation of lipid decreased during fasting without GH (P < 0.01). In summary, we find that suppression of GH during fasting leads to a 50% increase in urea-nitrogen excretion, together with an increased net release and appearance rate of phenylalanine across the forearm. These results demonstrate that GH-possibly by maintenance of circulating concentrations of free IGF-I--is a decisive component of protein conservation during fasting and provide evidence that the underlying mechanism involves a decrease in muscle protein breakdown.

L-2,5-dihydrophenylalanine, an inducer of cathepsin-dependent apoptosis in human promyelocytic leukemia cells (HL-60).

L-2,5-Dihydrophenylalanine (DHPA), a phenylalanine analogue, induced apoptosis in human promyelocytic leukemia cells (HL-60). This apoptosis was demonstrated by morphological changes of the cells, such as fragmentation of nuclei and chromatin condensation, and by some evidence found in biochemical analysis, such as DNA ladder and activation of caspase 3. The DHPA-induced apoptosis was prevented by a pan-caspase inhibitor, Z-VAD-fmk, and a cysteine protease inhibitor, E-64d, which inhibits calpains and cathepsin B and L. A calpain inhibitor, Z-LL-H, did not affect this apoptosis. A cathepsin B specific inhibitor, CA074-Me, prevented only chromatin condensation. However, E-64d and a cathepsin L specific inhibitor, Z-FY(t-Bu)-dmk, protected the cells from both chromatin condensation and oligonucleosomal DNA fragmentation. As proceeding to the apoptotic process, the activities of both cathepsin B and L increased gradually. These results indicated that DHPA was an inducer of cathepsin-dependent apoptosis in HL-60 cells.

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).

The effects of RB101, a mixed inhibitor of enkephalin-catabolizing enzymes, on carrageenin-induced spinal c-Fos expression are completely blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist.

We have demonstrated that pre-administered RB101 (40 mg/kg, i.v.), a mixed inhibitor of enkephalin-catabolizing enzymes, decreased spinal c-Fos expression induced 1 h and 30 min after intraplantar (i.pl.) carrageenin (41% reduction, p<0.01). These effects were completely blocked by pre-administered beta-funaltrexamine (10 mg/kg, i.v., 24 h prior to stimulation), a selective long-lasting mu-opioid receptor antagonist. In conclusion, these results clearly demonstrate that the effects of endogenous enkephalins on noxiously evoked spinal c-Fos expression are essentially mediated via mu-opioid receptors.

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex.

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (alpha-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation.

RB101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl)-L-phenylalanine benzyl ester) is a recently developed full inhibitor of the enkephalin-catabolizing enzymes able to cross the blood-brain barrier, whereas RB38A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freund's complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg, i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non-inflamed paws. RB101 (10 and 20 mg/kg, i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg, i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 micrograms) blocked the effect of morphine only in non-inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues.

Effect of tyrosine on the potentiation by aspartame and phenylalanine of metrazol-induced convulsions in rats.

Male rats were treated by oral intubation with tyrosine (Tyr), at doses of 0.5 and 1.0 g/kg body weight, alone or together with 1 g aspartame (APM)/kg body weight, or an equivalent dose of phenylalanine (Phe; 0.5 g/kg body weight); the effects on seizures induced by an effective dose of metrazol (ED50) were observed. Tyr (0.5 g/kg body weight) had a protective effect against the Phe-potentiation of metrazol-induced clonic-tonic convulsions. At the same dose Tyr had no effect on the seizure-promoting activity of APM, but at 1 g/kg it reduced the proconvulsant potential of the sweetener. Analysis of the brain and plasma amino acid concentrations indicated that the Tyr to Phe ratio tended to be enhanced in Tyr-Phe treated rats compared with those treated with Phe alone. This ratio remained essentially constant in the brain of APM-treated rats, compared with those treated with APM plus 1 g Tyr/kg body weight, whereas an increase in this ratio in the plasma was observed. These results confirm that Tyr antagonizes the proconvulsant effect of Phe and APM and they further suggest that no simple relationship exists between the relative brain concentrations of the two amino acids and the response to metrazol convulsions.

Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogues.

Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, utilizing a tetrahydropterin and molecular oxygen as cosubstrates. Several approaches were taken to examining the identity of the rate-limiting step in catalysis. Steady-state kinetic parameters were determined with a series of ring-substituted phenylalanines. The Vmax value was unchanged with substrates ranging in reactivity from tyrosine to 4-fluorophenylalanine. Neither 4-pyridylalanine N-oxide, a model of tyrosine phenoxide, nor 4-hydroxy-3-pyridylalanine N-oxide or alpha-amino-3-hydroxy-4-pyridone-1- propionic acid, models of a hydroxycyclohexadienone intermediate, was an effective inhibitor. There was no solvent isotope effect on either the Vmax or the V/KTyr value. These results establish that no chemistry occurs at the amino acid in the rate-limiting step and no exchangeable proton is in flight in the rate-limiting step. The results are consistent with a model in which the slow step in catalysis is formation of the hydroxylating intermediate.

Analgesic responses elicited by endogenous enkephalins (protected by mixed peptidase inhibitors) in a variety of morphine-sensitive noxious tests.

It has been suggested that the endogenous opioid peptides, methionine and leucine enkephalin, participate only in naloxone-facilitated antinociceptive responses. To reassess this proposal, analgesic effects resulting from complete inhibition of enkephalin metabolism by intracerebroventricular (i.c.v.) administration of the mixed inhibitor RB 38A (R,S)HONHCOCH2CH(CH2 phi)CONHCH(CH2 phi)COOH) were compared to the effects of morphine (i.c.v.) in various assays commonly used to select analgesics: mouse hot plate-test, tail flick test with mice and rats, electrical stimulation of the tail (TES), paw pressure test with rats, and phenylbenzoquinone-induced writhing test with mice. The ED50s of morphine vs. ED50s of RB 38A in the writhing, hot plate (jumping) and tail flick tests with mice were 0.24 nmol vs. 38 nmol, 1 nmol vs. 36 nmol and 3.2 nmol vs. 285 nmol, respectively. RB 38A (ED30 153 nmol) was only 15 times less active in the tail flick test with rats than morphine and only halve as active in the paw pressure test. Noxious TES in rat was very sensitive to the inhibitory action of endogenous opioids protected by RB 38A, particularly the post-vocalization response which was also shown to be alleviated by antidepressants. All the analgesic effects observed were reversed by naloxone. This first direct evidence of analgesia resulting from peptidase inhibition, in the tail flick test with mice and rats, hot plate (paw lick) and TES shows that the pain suppressive effects of endogenous opioid peptides are not restricted to naloxone-facilitated noxious stimuli but occur more generally, in all morphine-sensitive tests. The differential effects of RB 38A in the various assays is likely to be related to the amount of enkephalins released and to the efficiency of peptidase inactivation in particular brain regions implicated in the control of a given nociceptive input. This mechanism could account for the reduction in side-effects compared to those of morphine following chronic administration of RB 38A.