JNT-517 for Phenylketonuria
Recruiting in Palo Alto (17 mi)
+14 other locations
Age: 18+
Sex: Any
Travel: May Be Covered
Time Reimbursement: Varies
Trial Phase: Phase 1
Recruiting
Sponsor: Jnana Therapeutics
No Placebo Group
Trial Summary
What is the purpose of this trial?This trial tests a new oral medication, JNT-517, for safety and tolerance. It involves healthy participants and people with phenylketonuria (PKU). Researchers aim to see if JNT-517 can safely reduce certain amino acids in people with PKU.
Do I have to stop taking my current medications for this trial?
The trial protocol does not specify whether you need to stop taking your current medications. However, you should discuss your current medications with the study team to ensure they don't interfere with the trial.
What data supports the idea that JNT-517 for Phenylketonuria is an effective drug?
The available research does not provide any data on JNT-517 for Phenylketonuria or any other condition. Therefore, there is no information to support the idea that JNT-517 is an effective drug for treating Phenylketonuria.
12345What safety data exists for JNT-517 treatment for Phenylketonuria?
The provided research does not contain any safety data for JNT-517 or its synonyms related to Phenylketonuria. The articles focus on unrelated topics such as enzyme specificity, metabolism of other drugs, and analytical methods for different compounds. Therefore, no relevant safety data for JNT-517 is available in the provided research.
678910Eligibility Criteria
This trial is for healthy adults aged 18-55, and those with phenylketonuria (PKU) aged 18-65. Healthy participants must not smoke and have a BMI of 18-40 kg/m2. Women should agree to use two forms of contraception. PKU patients need a confirmed diagnosis and at least two plasma Phe levels >600 μM in the past year.Inclusion Criteria
Your body mass index (BMI) is between 18 and 40.
I am between 18 and 55 years old.
I agree to use two effective birth control methods if I can have children.
+7 more
Exclusion Criteria
I don't have conditions that affect how drugs work in my body.
I have not had any cancer except for non-melanoma skin cancer in the last 5 years.
I have not received a COVID-19 vaccine in the last 14 days.
+9 more
Participant Groups
The study tests JNT-517's safety, tolerability, dosage effects, and impact on urinary Phe in healthy individuals and those with PKU across four parts: single & multiple dose studies, bioavailability comparison between tablet/suspension forms with food effect analysis in healthy volunteers, and a placebo-controlled test in PKU patients.
6Treatment groups
Experimental Treatment
Group I: JNT-517 Tablet Fed Then Suspension Then Tablet Fasted (Part C)Experimental Treatment2 Interventions
Single dose of JNT-517 tablet in a fed state, JNT-517 suspension, and JNT-517 tablet in a fasted state in a sequential, open-label manner. Each treatment is separated by a minimum of 5 half-lives.
Group II: JNT-517 Tablet Fasted Then Tablet Fed Then Suspension (Part C)Experimental Treatment2 Interventions
Single dose of JNT-517 tablet in a fasted state, JNT-517 tablet in a fed state, and JNT-517 suspension in a sequential, open-label manner. Each treatment is separated by a minimum of 5 half-lives.
Group III: JNT-517 Suspension Then Tablet Fasted Then Tablet Fed (Part C)Experimental Treatment2 Interventions
Single dose of JNT-517 suspension, JNT-517 tablet in a fasted state, and JNT-517 tablet in a fed state in a sequential, open-label manner. Each treatment is separated by a minimum of 5 half-lives.
Group IV: JNT-517 SAD (Part A)Experimental Treatment2 Interventions
Single dose of JNT-517 or placebo in fasted state.
Group V: JNT-517 PKU (Part D)Experimental Treatment2 Interventions
JNT-517 or placebo daily for 4 weeks. Dose is based on data from Parts A, B, and C.
Group VI: JNT-517 MAD (Part B)Experimental Treatment2 Interventions
JNT-517 or placebo once or twice daily for 14 days, with first daily dose given after an overnight fast.
Find a Clinic Near You
Research Locations NearbySelect from list below to view details:
Rare Disease ResearchAtlanta, GA
Children's Hospital of PhiladelphiaPhiladelphia, PA
University of Texas Health Science Center at HoustonHouston, TX
UT Southwestern Medical CenterDallas, TX
More Trial Locations
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Who Is Running the Clinical Trial?
Jnana TherapeuticsLead Sponsor
References
International consensus on the use of tau PET imaging agent 18F-flortaucipir in Alzheimer's disease. [2022]Label="PURPOSE">Positron emission tomography (PET) with the first and only tau targeting radiotracer of 18F-flortaucipir approved by FDA has been increasingly used in depicting tau pathology deposition and distribution in patients with cognitive impairment. The goal of this international consensus is to help nuclear medicine practitioners procedurally perform 18F-flortaucipir PET imaging.
Evaluation of Tau Radiotracers in Chronic Traumatic Encephalopathy. [2023]Chronic traumatic encephalopathy (CTE) is a neurologic disorder associated with head injuries, diagnosed by the perivascular accumulation of hyperphosphorylated tau protein (phospho-tau) identified at autopsy. Tau PET radiopharmaceuticals developed for imaging Alzheimer disease are under evaluation for brain injuries. The goal of this study was to conduct a head-to-head in vitro evaluation of 5 tau PET radiotracers in subjects pathologically diagnosed with CTE. Methods: Autoradiography was used to assess the specific binding and distribution of 3H-flortaucipir (also known as Tauvid, AV-1451, and T807), 3H-MK-6240 (also known as florquinitau), 3H-PI-2620, 3H-APN-1607 (also known as PM-PBB3 and florzolotau), and 3H-CBD-2115 (also known as 3H-OXD-2115) in fresh-frozen human postmortem CTE brain tissue (stages I-IV). Immunohistochemistry was performed for phospho-tau with AT8, 3R tau with RD3, 4R tau with RD4 and amyloid-β with 6F/3D antibodies. Tau target density (maximum specific binding) was quantified by saturation analysis with 3H-flortaucipir in tissue sections. Results: 3H-flortaucipir demonstrated a positive signal in all CTE cases examined, with varying degrees of specific binding (68.7% ± 10.5%; n = 12) defined by homologous blockade and to a lesser extent by heterologous blockade with MK-6240 (27.3% ± 13.6%; n = 12). The 3H-flortaucipir signal was also displaced by the monoamine oxidase (MAO)-A inhibitor clorgyline (43.9% ± 4.6%; n = 3), indicating off-target binding to MAO-A. 3H-APN-1607 was moderately displaced in homologous blocking studies and was not displaced by 3H-flortaucipir; however, substantial displacement was observed when blocking with the β-amyloid-targeting compound NAV-4694. 3H-MK-6240 and 3H-PI-2620 had negligible binding in all but 2 CTE IV cases, and binding may be attributed to pathology severity or mixed Alzheimer disease/CTE pathology. 3H-CBD-2115 showed moderate binding, displaced under homologous blockade, and aligned with 4R-tau immunostaining. Conclusion: In human CTE tissues, 3H-flortaucipir and 3H-APN-1607 revealed off-target binding to MAO-A and amyloid-β, respectively, and should be considered if these radiotracers are used in PET imaging studies of patients with brain injuries. 3H-MK-6240 and 3H-PI-2620 bind to CTE tau in severe- or mixed-pathology cases, and their respective 18F PET radiotracers warrant further evaluation in patients with severe suspected CTE.
Tau PET Imaging with [18F]PM-PBB3 in Frontotemporal Dementia with MAPT Mutation. [2021]Flortaucipir (AV-1451) and pyridinyl-butadienyl-benzothiazole 3 (PBB3) are newly developed and commonly used positron emission tomography (PET) tracers to detect tau deposition in tauopathies, including frontotemporal dementia (FTD). [18F]PM-PBB3, as a second-generation compound, has not been described in FTD so far.
N-[18F]-FluoropropylJDTic for κ-opioid receptor PET imaging: Radiosynthesis, pre-clinical evaluation, and metabolic investigation in comparison with parent JDTic. [2018]To image kappa opioid receptor (KOR) for preclinical studies, N-fluoropropylJDTic 9 derived from the best-established KOR antagonist JDTic, was labeled with fluorine-18.
Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters. [2022]Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as in vitro models. The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced V max of [3H]uridine uptake in ENT1 and ENT2 without affecting K m. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.
Key amino acid residues responsible for the differences in substrate specificity of human UDP-glucuronosyltransferase (UGT)1A9 and UGT1A8. [2021]Human UDP-glucuronosyltransferase (UGT)1A9 is one of the major isoforms in liver and extrahepatic tissues, catalyzing the glucuronidation of a variety of drugs, dietary constituents, steroids, fatty acids, and bile acids. UGT1A9 shows high amino acid homology with UGT1A7, UGT1A8, and UGT1A10 with overlapping substrate specificity. However, the affinities for substrates are different among them. Amino acid alignment analysis revealed that 14 amino acids, Cys3, Arg42, Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Asn152, Leu173, Leu219, His221, Arg222, and Glu241, are unique to UGT1A9 compared with UGT1A7, UGT1A8, and UGT1A10. In this study, we constructed expression systems in human embryonic kidney 293 cells for seven mutants (Mut) UGT1A9, Mut 1 (R42Q), Mut 2 (K91M, A92D), Mut 3 (Y106F, G111S, D115G), Mut 4 (N152A), Mut 5 (L173A), Mut 6 (L219F, H221Q, R222Y), and Mut 7 (E241A), in which the amino acids were substituted to those of UGT1A8. Using these mutants, the effects of the amino acid changes on the activities of 4-methylumbelliferone (4-MU), p-nitrophenol (p-NP), and 3-hydroxydesloratadine glucuronidations were investigated. For 4-MU and p-NP O-glucuronidations, Mut 1 and Mut 4 exhibited higher K(m) values and Mut 3 and Mut 4 exhibited higher V(max) values compared with wild-type UGT1A9. It is interesting to note that only Mut 4 was active toward 3-hydroxydesloratadine O-glucuronidation that is specific for UGT1A8. The findings reveal that the residues Arg42 and Asn152 may have a large contribution to the difference in the substrate specificity with that of UGT1A8, although all of the unique amino acids of UGT1A9 would be collectively involved in the catalytic property.
Retigabine N-glucuronidation and its potential role in enterohepatic circulation. [2013]The metabolism of retigabine in humans and dogs is dominated by N-glucuronidation (), whereas in rats, a multitude of metabolites of this new anticonvulsant is observed (). The comparison of the in vivo and in vitro kinetics of retigabine N-glucuronidation in these species identified a constant ratio between retigabine and retigabine N-glucuronide in vivo in humans and dog. An enterohepatic circulation of retigabine in these species is likely to be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine N-glucuronide does not occur in this species. In the rat, 90% of retigabine N-glucuronidation is catalyzed by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas family 2 UGT enzymes contribute also. Of ten recombinant human UGTs, only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and our activity and inhibition data, we conclude that UGT1A4 is a major retigabine N-glucuronosyl transferase in vivo and significantly contributes to the enterohepatic cycling of the drug.
Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography-ionspray mass spectrometry. [2019]An LC-MS-MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS-MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5-10000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.
In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism. [2017]1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with 75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.
N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II. [2013]Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.
Characterisation of a myristoyl CoA:glycylpeptide N-myristoyl transferase activity in rat brain: subcellular and regional distribution. [2019]An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60src. This N-myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Michaelis-Menten kinetics yielding Km values of 100 microM and 60 microM, and Vmax values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and src-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N-myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord.
560G>A (rs4986782) (R187Q) Single Nucleotide Polymorphism in Arylamine N-Acetyltransferase 1 Increases Affinity for the Aromatic Amine Carcinogens 4-Aminobiphenyl and N-Hydroxy-4-Aminobiphenyl: Implications for Cancer Risk Assessment. [2022]Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.
Human liver nicotinamide N-methyltransferase: ion-pairing radiochemical assay, biochemical properties and individual variation. [2019]Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridine, and structurally related compounds. The products of this reaction are positively charged pyridinium ions that cannot be removed from aqueous solution by simple organic solvent extraction. We developed an assay for human liver NNMT based on the extraction of the positively charged reaction product into 60% isoamyl alcohol in toluene in the presence of the ion-pairing reagent 1-heptanesulfonic acid. Nicotinamide was the methyl acceptor for the reaction, and [14C-methyl]-S-adenosyl-L-methionine (Ado-Met) served as the methyl donor. Apparent Km values of human liver NNMT for nicotinamide and Ado-Met were 347 and 1.76 mumol/l, respectively. The product of the reaction was identified as N1-methylnicotinamide (NMN) by high performance liquid chromatography. NNMT activity was inhibited by the reaction products, NMN and S-adenosyl-L-homocysteine. NNMT activity was not affected by inhibitors of other methyltransferases including Ca2+, SKF 525A and 3,4-dimethoxy-5-hydroxybenzoic acid. Individual variation in NNMT activity was studied by measuring hepatic enzyme activities in 163 human liver biopsy samples obtained during clinically-indicated surgery. The average NNMT activity in these samples was 51.5 +/- 32.5 U per mg protein (mean +/- SD), and there was not a significant correlation of enzyme activity with patient age or with the time of storage of the biopsy samples at -80 degrees C. The distribution of activities was bimodal, and approximately 26% of the samples were included in a subgroup with high NNMT activity. It will now be possible to test the hypothesis that individual differences in hepatic NNMT activity might be related to variation in the N-methylation of pyridine compounds and to individual differences in either toxicity or the therapeutic efficacy of such compounds.
Substrate specificity and inhibition studies of human serotonin N-acetyltransferase. [2021]Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.
A new, robust, and nonradioactive approach for exploring N-myristoylation. [2021]Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.