Genotoxic effects induced by iprodione and tebuconazole in meristematic cells of Allium cepa: responses dependent on concentration and exposure time.

The present work explores the genotoxicity of the fungicides iprodione (IP) and tebuconazole (TB) using the Allium cepa assay as an in vivo biological model. Both short-term and long-term exposures were studied, revealing concentration- and time-dependent cytological and genotoxic effects. IP exhibited genotoxicity over a wider concentration range (5-50 µg/ml) and required 30 h of exposure, while TB showed genotoxicity at higher concentrations (10 and 30 µg/ml) within a 4-h exposure period. The study highlights the importance of assessing potential risks associated with fungicide exposure, including handling, disposal practices, and concerns regarding food residue. Moreover, the research underscores the genotoxic effects of IP and TB on plant cells and provides valuable insights into their concentration and time-response patterns.

Cytogenotoxic activity of the pesticides imidacloprid and iprodione on Allium cepa root meristem.

Effects of imidacloprid and iprodione, isolated and in mixture, were assessed by using seed germination and root growth test, flow cytometry, and chromosomal aberrations test on Allium cepa root meristem. The highest concentrations of imidacloprid, including field concentration, increased the frequency of sub-G1 particles, decreased the frequency of nuclei in G2/M, increased the coefficient of variation of G1 (CVG1) and the frequency of aberrant cells, and inhibited the mitotic index culminating in the reduction in root length. All doses of iprodione also presented cytogenotoxic action. The highest concentration of the fungicide affected the growth of A. cepa roots. In response to exposure to pesticide mixtures, the cell cycle of A. cepa was blocked in the G1 phase. The mixtures with low doses of the pesticides significantly decreased the mitotic index, and as a consequence, the genotoxicity was reduced. In the mixtures with the highest doses of the agrochemicals, the blockage of the cell cycle was insufficient for damage repair, resulting in a significant increase of chromosomal aberrations. The results suggest caution in the use of pesticides doses that induce cytological abnormalities in non-target organisms.

Residue behavior and removal of iprodione in garlic, green garlic, and garlic shoot.

BACKGROUND: Iprodione is considered to be an endocrine-disturbing pesticide, which could harm consumers. The garlic crop has three edible parts: the garlic, the green garlic, and the garlic shoot, which correspond to different stages of its growth. In this study, iprodione residue dissipation and distribution in these three edible parts were investigated, and dietary risk was evaluated. RESULTS: Iprodione residues were present in these samples in the following order: green garlic > garlic shoot > > garlic. The dissipation of iprodione in green garlic was slow with a half-life of 5.82-19.25 days. A very high RQchronic value of 207.35-407.30% suggested that the residual iprodione in green garlic had an unacceptable level of risk. Iprodione residue was significantly eliminated (59-90%) by an alkaline solution. The order for removing iprodione by soaking was the alkaline solutions (0.5% and 2% NaHCO3 ) > the acidic solutions (5% and 10% of vinegar) ≈ the neutral solutions (the 1% and 2% of table salt) > tap water. Processing factors (PFs) were <1, indicating that processing could decrease the iprodione residue level. CONCLUSION: This work could contribute to establishing maximum residue limits (MRLs) for iprodione in garlic, green garlic, and garlic shoots, and could provide guidance on the safe and appropriate use of iprodione in the garlic crop. © 2020 Society of Chemical Industry.

Discovery and Development of S6821 and S7958 as Potent TAS2R8 Antagonists.

In humans, bitter taste is mediated by 25 TAS2Rs. Many compounds, including certain active pharmaceutical ingredients, excipients, and nutraceuticals, impart their bitter taste (or in part) through TAS2R8 activation. However, effective TAS2R8 blockers that can either suppress or reduce the bitterness of these compounds have not been described. We are hereby reporting a series of novel 3-(pyrazol-4-yl) imidazolidine-2,4-diones as potent and selective TAS2R8 antagonists. In human sensory tests, S6821 and S7958, two of the most potent analogues from the series, demonstrated efficacy in blocking TAS2R8-mediated bitterness and were selected for development. Following data evaluation by expert panels of a number of national and multinational regulatory bodies, including the US, the EU, and Japan, S6821 and S7958 were approved as safe under conditions of intended use as bitter taste blockers.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry.

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

The fungicide iprodione affects midgut cells of non-target honey bee Apis mellifera workers.

The honey bee Apis mellifera is an important pollinator of agricultural crops and natural forests. Honey bee populations have declined over the years, as a result of diseases, pesticides, and management problems. Fungicides are the main pesticides found in pollen grains, which are the major source of protein for bees. The objective of this study was to evaluate the cytotoxic effects of the fungicide iprodione on midgut cells of adult A. mellifera workers. Bees were fed on iprodione (LD50, determined by the manufacturer) for 12 or 24 h, and the midgut was examined using light and transmission electron microscopies. The expression level of the autophagy gene atg1 was assessed in midgut digestive cells. Cells of treated bees had signs of apoptosis: cytoplasmic vacuolization, apical cell protrusions, nuclear fragmentation, and chromatin condensation. Ultrastructural analysis revealed some cells undergoing autophagy and necrosis. Expression of atg1 was similar between treated and control bees, which can be explained by the facts that digestive cells had autolysosomes, whereas ATG-1 is found in the initial phases of autophagy. Iprodione acts by inhibiting the synthesis of glutathione, leading to the generation of reactive oxygen species, which in turn can induce different types of cell death. The results indicate that iprodione must be used with caution because it has side effects on non-target organisms, such as pollinator bees.

Antitussive and Anti-inflammatory Dual-active Agents Developed from Natural Product Lead Compound 1-Methylhydantoin.

Natural products play an important role in drug discovery. This work employed a natural product 1-methylhydantoin as the lead compound to develop novel dual-active drugs. 1-Methylhydantoin was isolated from Oviductus Ranae, which is a traditional Chinese medicine that has been used for tussive and inflammation treatment for a long time. An in silico study screened the more active 1-methylhydantoin derivatives. Antitussive assessment indicated that the newly synthesized agent had similar bioactivity with the natural product. An anti-inflammatory model used xylene induced ear edema model. At the same dosage (100 mg/Kg), the newly prepared agent had an inhibition rate 53.18% which was much higher than that of the lead compound (22.69%). The results might be ascribed to the cyclooxygenases-1 (COX-1) and cyclooxygenases-2 (COX-2) selectivity, and the fitness of the compound, and the binding pocket. The anti-particulate matter (PM 2.5) acute pneumonia was evaluated through an in vivo model constructed by nasal instillation with PM 2.5 suspension. The results of the above models suggested that this novel agent had remarkable antitussive, anti-inflammatory, and anti-PM 2.5 acute pneumonia activities.

Toxicological effects of comercial formulations of fungicides based on procymidone and iprodione in seedlings and root tip cells of Allium cepa.

In this study the phytotoxic, cytotoxic, genotoxic and mutagenic effects of two commercial fungicide-active compounds, procymidone (PR) and iprodione (IP), were determined. The parameters evaluated were germination and root growth, mitotic index, chromosomal and nuclear aberrations, and molecular analyses were also performed in the model plant Allium cepa L. The results demonstrated that the active compounds PR and IP were phytotoxic, delaying germination and slowing the development of A. cepa seedlings. Moreover, PR and IP showed cytogenotoxicity towards A. cepa meristematic cells, inducing chromosomal changes and cell death. The mutagenic activity of the active compounds was demonstrated by the detection of DNA changes in simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers in the treated cells compared to the negative control. Together, these results contribute to a better understanding of the damage caused by these substances in living organisms and reveal a promising strategy for prospective studies of the toxic effects of environmental pollutants.

Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay.

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency. In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10-6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492. In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

Recent applications of hydantoin and thiohydantoin in medicinal chemistry.

Hydantoin, imidazolidine-2,4-dione, is a non-aromatic five-membered heterocycle, which is considered a valuable, privileged scaffold in medicinal chemistry. The importance of the hydantoin scaffold in drug discovery has been reinforced by several medicines in clinical use, such as phenytoin, nitrofurantoin, and enzalutamide. Hydantoin has five potential substituent sites, including two hydrogen bond acceptors and two hydrogen bond donors. Two additional attractive features of hydantoin scaffolds are their synthetic feasibility for core scaffolds via established cyclization reactions and their ease of accepting various substituents. Because of these characteristics, many hydantoin derivatives with different substituents have been designed and synthesized and exhibit a broad spectrum of biological and pharmacological activities against, for example, cancers, microbial infections, metabolic diseases, and epilepsy. In this review, recent contributions of hydantoin, thiohydantoin, and selenohydantoin scaffolds to medicinal chemistry are described; some major compounds are presented to emphasize their importance, and their structure-activity relationships (SARs) are briefly addressed. Major discussions are devoted to the structural features or novelty of each scaffold and its SAR. The publications in this review encompass those from 2012 to 2018.

The effects of iprodione fungicide on survival, behavior, and brood development of honeybees (Apis mellifera L.) after one foliar application during flowering on mustard.

A semifield study to assess the effects of iprodione on honeybees at label use rates was conducted on a bloom mustard crop. The present study followed the Organisation for Economic Co-operation and Development guideline 75 tunnel test and consisted of 3 groups: the iprodione-treated group, the untreated control group, and the toxic reference item group. In addition to the tunnels used for biological assessments, a tunnel was set up in the treatment and control groups to determine the level of residues in flowers, nectar, and pollen. The major endpoints to assess the effects of the application of iprodione were mortality, flight intensity, behavior, condition of the colonies, and development of the brood. Residue analysis showed that honeybees were exposed to significant residues of iprodione. However, no adverse effects were observed on overall mortality, flight intensity, behavior, or brood development of honeybees compared to control. It is concluded that iprodione does not adversely affect the health of honeybees when applied in agriculture at commercially relevant rates in a worst-case exposure scenario. Environ Toxicol Chem 2018;37:3086-3094. © 2018 SETAC.

Variation in Fungicide Sensitivity Among Rhizoctonia Isolates Recovered from Potatoes in South Africa.

Rhizoctonia is a major pathogen of potato causing substantial yield losses worldwide. Control of Rhizoctonia diseases is based predominantly on the application of fungicides. However, little is known about the fungicide response variability of different Rhizoctonia anastomosis groups associated with potato diseases in South Africa. A total of 131 Rhizoctonia isolates were obtained from potato growing regions of South Africa from 2012 to 2014 and evaluated for sensitivity to fungicides in vitro and in vivo. The fungicides comprised six chemical formulations and one bio-fungicide representing seven Fungicide Resistance Action Committee groups. All Rhizoctonia anastomosis groups were sensitive to tolclofos-methyl (EC50: 0.001 to 0.098 µg a.i. ml-1) and fludioxonil (EC50: 0.06 to 0.09 µg a.i. ml-1) and showed variation in sensitivity to pencycuron, iprodione, benomyl, and Bacillus subtilis QST 713. However, for azoxystrobin, Rhizoctonia isolates exhibited variable sensitivity ranging from sensitivity (EC50: <0.09 µg a.i. ml-1) to insensitivity with EC50 values exceeding 5 µg a.i. ml-1. In greenhouse and field trials, tolclofos-methyl and fludioxonil exhibited significantly greater control of stem and black scurf whereas azoxystrobin was the least effective. This work demonstrated variable sensitivity within and among anastomosis groups of R. solani and binucleate Rhizoctonia to different fungicides. Information on fungicide sensitivity of Rhizoctonia isolates is crucial in the development of effective Rhizoctonia control strategies and facilitates monitoring of fungicide insensitive isolates in the pathogen population.

Synthesis and Evaluation of Novel Diazaspiro Hydantoins as Potential Anticonvulsants.

BACKGROUND: Epilepsy, one of the most frequent neurological afflictions in man characterized by excessive temporary neuronal discharges resulting in uncontrolled convulsion, requires special medical attention. Though several new anticonvulsants are introduced, some types of seizures are still not adequately treated with current therapy. Toxicity, intolerance, and lack of efficacy for certain types of seizure are some of the limitations of the current medications. METHODS: Maximal electroshock (MES) seizure model was used in the present study to evaluate the anticonvulsant activity of the drugs. Seizures were induced in ten weeks old male Wistar rats (200-220 g) by delivering electro shock of 150 mA for 0.2 sec by means of a convulsiometer through a pair of ear clip electrodes. The test compounds (1-10, 100 mg/kg) were administered by oral route 30 mins before the maximal electroshock seizure test by suspending in carboxymethylcellulose (1%). The animals were observed closely for 2 mins. The percentage of inhibition of seizure relative to control was recorded and calculated. Phenytoin (100 mg/kg, p.o) was used as a standard drug. The data was analysed by using one way ANOVA followed by dunnett's test. RESULTS: In our present series of compounds the active compounds possess all the requirements essential for anticonvulsant activity as proposed by Dimmock and others. In this study, it reveals that, compounds showing anticonvulsant activity with more lipophilic N-substitution group are more active than hydrophobic substitution in the hydantoin ring. The rapid onset of action is believed to be due to the substitution of more lipophilic propyl group in the N-substitution in the hydantoin moiety. Evidently, this distal hydrophobic centre alters the bioavailability of the molecules. CONCLUSION: The results are encouraging and show that, the hydantoins are more potential molecules for the treatment of anticonvulsant. Anticonvulsants have greatly improved the lives of people with epilepsy. Approximately 70% of patients can achieve complete freedom from seizures with appropriate treatment. Lipophilicity appears to govern the MES activity. If there is lipophilic moiety, then MES activity is favoured. All the compounds have shown promising and significant protective effect on maximal electroshock induced seizures when compared to vehicle treated control rats.

Novel seleno-hydantoin palladium(II) complex - antimigratory, cytotoxic and prooxidative potential on human colon HCT-116 and breast MDA-MB-231 cancer cells.

Selenium and palladium containing compounds separately exert multifunctional effects on cells. While selenium containing compounds usually exert antioxidative properties, palladium(II) containing compounds are cytotoxic and prooxidative. Here we investigated biological effects of bicyclic seleno-hydantoin cis-7a-ethyl-5-methyl-5-phenylselanylmethyl-tetrahydro-pyrrolo[1,2-c]imidazole-1,3-dione (Hid-Se), and its palladium(II) complex, trans-bis-(cis-7a-ethyl-5-methyl-5-phenylselanylmethyl-tetrahydro-pyrrolo[1,2-c]imidazole-1,3-dionato) palladium(II) chloride ((Hid-Se)2Pd) on human colon HCT-116 and breast MDA-MB-231 cancer cell lines. Hid-Se and (Hid-Se)2Pd showed prooxidative and cytotoxic character. In all performed experiments (Hid-Se)2Pd proved to be more active, i.e. this substance exerted greater prooxidative effect, cytotoxicity and influence on cell migration potential. Even though Hid-Se and (Hid-Se)2Pd enhanced migration of HCT-116 cells, very important feature of these substances is the strong antimigratory potential on metastatic MDA-MB-231 cells.

Effects of Fungicide and Adjuvant Sprays on Nesting Behavior in Two Managed Solitary Bees, Osmia lignaria and Megachile rotundata.

There is a growing body of empirical evidence showing that wild and managed bees are negatively impacted by various pesticides that are applied in agroecosystems around the world. The lethal and sublethal effects of two widely used fungicides and one adjuvant were assessed in cage studies in California on blue orchard bees, Osmia lignaria, and in cage studies in Utah on alfalfa leafcutting bees, Megachile rotundata. The fungicides tested were Rovral 4F (iprodione) and Pristine (mixture of pyraclostrobin + boscalid), and the adjuvant tested was N-90, a non-ionic wetting agent (90% polyethoxylated nonylphenol) added to certain tank mixtures of fungicides to improve the distribution and contact of sprays to plants. In separate trials, we erected screened cages and released 20 paint-marked females plus 30-50 males per cage to document the behavior of nesting bees under treated and control conditions. For all females in each cage, we recorded pollen-collecting trip times, nest substrate-collecting trip times (i.e., mud for O. lignaria and cut leaf pieces for M. rotundata), cell production rate, and the number of attempts each female made to enter her own or to enter other nest entrances upon returning from a foraging trip. No lethal effects of treatments were observed on adults, nor were there effects on time spent foraging for pollen and nest substrates and on cell production rate. However, Rovral 4F, Pristine, and N-90 disrupted the nest recognition abilities of O. lignaria females. Pristine, N-90, and Pristine + N-90 disrupted nest recognition ability of M. rotundata females. Electroantennogram responses of antennae of O. lignaria females maintained in the laboratory did not differ significantly between the fungicide-exposed and control bees. Our results provide the first empirical evidence that two commonly used fungicides and a non-ionic adjuvant can disrupt nest recognition in two managed solitary bee species.

Adjuvant antiarrhythmic therapy in patients with implantable cardioverter defibrillators.

The risk of sudden cardiac death from ventricular fibrillation or ventricular tachycardia in patients with cardiomyopathy related to structural heart disease has been favorably impacted by the wide adaptation of implantable cardioverter defibrillators (ICDs) for both primary and secondary prevention. Unfortunately, after ICD implantation both appropriate and inappropriate ICD therapies are common. ICD shocks in particular can have significant effects on quality of life and disease-related morbidity and mortality. While not indicated for primary prevention of ICD therapies, beta-blockers and antiarrhythmic drugs are a cornerstone for secondary prevention of them. This review will summarize our current understanding of adjuvant antiarrhythmic drug therapy in ICD patients. The review will also discuss the roles of nonantiarrhythmic drug approaches that are used in isolation and in combination with antiarrhythmic drugs to reduce subsequent risk of ICD shocks.

Structural destabilization of DNA duplexes containing single-base lesions investigated by nanopore measurements.

The influence of DNA duplex structural destabilization introduced by a single base-pair modification was investigated by nanopore measurements. A series of 11 modified base pairs were introduced into the context of an otherwise complementary DNA duplex formed by a 17-mer and a 65-mer such that the overhanging ends comprised poly(dT)23 tails, generating a representative set of duplexes that display a range of unzipping mechanistic behaviors and kinetic stabilities. The guanine oxidation products 8-oxo-7,8-dihydroguanine (OG), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) were paired with either cytosine (C), adenine (A), or 2,6-diaminopurine (D) to form modified base pairs. The mechanism and kinetic rate constants of duplex dissociation were determined by threading either the 3' or 5' overhangs into an α-hemolysin (α-HL) channel under an electrical field and measuring the distributions of unzipping times at constant force. In order of decreasing thermodynamic stability (as measured by duplex melting points), the rate of duplex dissociation increases, and the mechanism evolves from a first-order reaction to two sequential first-order reactions. These measurements allow us to rank the kinetic stability of lesion-containing duplexes relative to the canonical G:C base pair in which the OG:C, Gh:C, and Sp:C base pairs are, respectively, 3-200 times less stable. The rate constants also depend on whether unzipping was initiated from the 3' versus 5' side of the duplex. The kinetic stability of these duplexes was interpreted in terms of the structural destabilization introduced by the single base-pair modification. Specifically, a large distortion of the duplex backbone introduced by the presence of the highly oxidized guanine products Sp and Gh leads to a rapid two-step unzipping. The number of hydrogen bonds in the modified base pair plays a lesser role in determining the kinetics of duplex dissociation.

The activity of 16 new hydantoin compounds on the intrinsic and overexpressed efflux pump system of Staphylococcus aureus.

AIM: To evaluate a new series of 16 hydantoin derivatives for activity against the intrinsic and overexpressed efflux pumps of the ATTC 25923 Staphylococcus aureus and the clinical Staphylococcus aureus HPV-107 strain, respectively. MATERIALS AND METHODS: The hydantoin compounds were evaluated for activity against the efflux pumps of the ATTC 25923 S. aureus and the clinical S. aureus HPV-107 strains by the aid of the automated ethidium bromide method. Compounds that inhibited the efflux pumps of either strain were evaluated for ability to reduce or reverse resistance of these strains to oxacillin. RESULTS: Although most of the hydantoins inhibited the efflux pumps of the ATTC strain, none reduced the resistance of this strain to oxacillin. In contrast, the inhibition of the Qac efflux pump present in HPV-107 was inhibited to some degree, by much higher concentrations of the hydantoin compounds than that needed for similar activity against the ATTC strain; only hydantoin PI8a significantly reduced the minimum inhibitory concentration of oxacillin against the HPV-107 strain. CONCLUSION: Hydantoin compound PI8a may have potential for therapy of a methicillin-resistant S. aureus infection whose multidrug-resistant phenotype is due to overexpression of an efflux pump.

Activity of fourteen new hydantoin compounds on the human ABCB1 efflux pump.

BACKGROUND: Multidrug resistance (MDR) is one of the major concerns in the treatment of cancer and one of the major causes of therapy failure. The overexpression of an ABC transporter, the ABCB1, is often associated with MDR in cancer. Previously it was observed that hydantoin compounds can modulate the activity of the ABCB1 pump. MATERIALS AND METHODS: Fourteen hydantoin derivatives were synthesized and studied for their capacity to increase accumulation of ethidium bromide (EB) by mouse lymphoma cancer cells that were transfected with the human ABCB1 gene and overexpress the human ABCB1 pump. RESULTS: It was observed that the accumulation of EB by the cells in the presence of four of the newly synthesized hydantoins was strongly increased. Similar but milder effects were also observed for the other seven hydantoins; the remaining three had no activity. CONCLUSION: The 14 hydantoin compounds studied belong to three different structural groups. Structure-activity relationships were studied and important molecular substituents that were possibly responsible for increased the activity of the molecules were identified. This important information may lead to the continuation of our work and to the future synthesis of more active compounds.

Novel pharmacological chaperones that correct phenylketonuria in mice.

Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.