Biochem. Biophys. Rep. 2025
Human citrate synthase (hCS) was kinetically characterized through full progress curve kinetic modelling using kinetic simulation, and global fitting of the direct AcCoA to CoA transition along with a coupled thiol probe reaction to better determine the kinetics with low substrate concentration. Our analysis provides one of the most rigorous kinetic analyses of any citrate synthase ruling out the need to invoke complex cooperative mechanisms to explain progress curve data. Furthermore, we collected and modeled stopped-flow pH-dependent kinetic data with CoA and popular thiol probes such as Ellman’s reagent (DTNB) and 4,4′-Dithiodipyridine (DPS), providing the opportunity for detailed kinetic simulations using these thiol probes with CoA-producing enzymes. Global fitting suggests that the DPS/CoA bimolecular rate constant increased 100-fold via protonation of the pyridine ring (pKa = 5.2), quantifying its kinetic advantage relative to DTNB. To explore the kinetic effects of polar substituents on the pyridine ring, we synthesized three different DPS analogs by adding either an alcohol, amine, or carboxylic acid moiety to the pyridine ring. Of these, the alcohol group provided the most similar kinetic characteristics to DPS but greatly increases thiol probe polarity offering an alternative to the original DPS.
Bioorg. Med. Chem. Lett. 2024
Arylalkylamine N-acetyltransferase (AANAT) catalyzes the rate-limiting step in melatonin synthesis and is a potential target for disorders involving melatonin overproduction, such as seasonal affective disorder. Previously described AANAT inhibitor bromoacetyltryptamine (BAT) and benzothiophenes analogs were reported to react with CoASH to form potent bisubstrate inhibitors through AANAT’s alkyltransferase function, which is secondary to its role as an acetyltransferase. We replaced the bromoacetyl group in BAT with various Michael acceptors to mitigate possible off-target activity of its bromoacetyl group. Additionally, we modified the length of the carbon linker between the Michael acceptor and indole bicycle of tryptamine to determine its effect on potency. An AANAT enzymatic assay showed a two-carbon linker present in BAT was optimal, while none of the new warheads had activity. Kinetic analysis indicated that these Michael acceptors reacted with CoASH much slower than BAT and not within the timeframe of our enzymatic assay. Additionally, we confirmed earlier reports that the acetyltransferase function of AANAT follows an ordered bi bi mechanism in which AcCoA binds before serotonin. In contrast, BAT’s alkyltransferase kinetics revealed a bi uni mechanism in which BAT binds to AANAT before CoASH. Our model combines both functions of AANAT into one kinetic mechanism.
ChemMedChem, 2023
Circadian rhythm (CR) dysregulation negatively impacts health and contributes to mental disorders. The role of melatonin, a hormone intricately linked to CR, is still a subject of active study. The enzyme arylalkylamine N-acetyltransferase (AANAT) is responsible for melatonin synthesis, and it is a potential target for disorders that involve abnormally high melatonin levels, such as seasonal affective disorder (SAD). Current AANAT inhibitors suffer from poor cell permeability, selectivity, and/or potency. To address the latter, we have employed an X-ray crystal-based model to guide the modification of a previously described AANAT inhibitor, containing a rhodanine-indolinone core. We made various structural modifications to the core structure, including testing the importance of a carboxylic acid group thought to bind in the CoA site, and we evaluated these changes using MD simulations in conjunction with enzymatic assay data. Additionally, we tested three AANAT inhibitors in a zebrafish locomotion model to determine their effects in vivo. Key discoveries were that potency could be modestly improved by replacing a 5-carbon alkyl chain with rings and that the central rhodanine ring could be replaced by other heterocycles and maintain potency.
Biophysical Journal, 2022
The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinson’s disease drug levodopa (L-Dopa). LAT1 is found in the blood-brain barrier, testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases, such as autism and epilepsy, as well as cancer. In this study, we combine metainference molecular dynamics simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1’s substrate-binding site and ligands, including both inhibitors and substrates. We then performed metainference molecular dynamics simulations using cryoelectron microscopy structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate-binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target.
Nucleosides, Nucleotides & Nucleic Acids, 2022
We report the synthesis and cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells of novel 1,2,3- and 1,2,4-triazolyl analogs of ribavirin. We modified ribavirin’s carboxamide moiety to test the effects of lipophilic groups. 1-β-D-Ribofuranosyl-1H-1,2,3-triazoles were prepared using Click Chemistry, whereas an unprecedented application of a prior 1,2,4-triazole ring synthesis was used for 1-β-D-ribofuranosyl-1H-1,2,4-triazole analogs. Though cytotoxicity was mediocre and there was no correlation with lipophilicity, we discovered that a structurally similar concentrative nucleoside transporter 2 (CNT2) inhibitor was modestly cytotoxic (MCF-7 IC50 of 42 µM). These syntheses could be used to efficiently investigate variation in the nucleobase.
The L‐type amino acid transporter 1 (LAT1, SLC7A5) imports dietary amino acids and amino acid drugs (e.g. L‐DOPA) into the brain, and it plays a role in cancer metabolism. Though there have been numerous reports of LAT1‐targeted amino acid‐drug conjugates (prodrugs), identifying the structural determinants to enhance substrate activity has been challenging. In this work, we investigated the position and orientation of a carbonyl group in linking hydrophobic moieties including the anti‐inflammatory drug ketoprofen to L‐tyrosine and L‐phenylalanine. We found that esters of meta‐carboxyl L‐phenylalanine had better LAT1 transport rates than the corresponding acylated L‐tyrosine analogs. However, as the size of the hydrophobic moiety increased, we observed a decrease in LAT1 transport rate with a concomitant increase in potency of inhibition. Our results have important implications for designing amino acid prodrugs that target LAT1 at the blood‐brain barrier (BBB) or on cancer cells.