Human citrate synthase (hCS) was kinetically characterized through full progress curve kinetic modelling using kinetic simulation, global fitting of the direct AcCoA to CoA transition, and 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 DPS.

Arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in melatonin biosynthesis and a regulator of circadian rhythm, with potential relevance to mood disorders such as seasonal affective disorder (SAD). We report a series of hydantoin indolinone-based AANAT inhibitors, developed as more stable alternatives to a previously reported rhodanine scaffold. Guided by docking studies and prior structure-activity data, we modified four regions of the molecule to improve potency. Substitution at the 5-position of the indolinone ring led to marked increases in activity, with compound 5g (bearing a CH3CO2CH2- substituent) resulting in an IC₅₀ of 1.1 μM-representing a 19-fold improvement over the parent compound. Kinetic mechanism studies were also conducted with respect to acetyl-CoA and serotonin to explore inhibitor binding. These findings establish a promising starting point for the development of more potent AANAT inhibitors as chemical probes for studying melatonin's function.

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.

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. Starting from a known AANAT inhibitor containing a rhodanine-indolinone scaffold, we made various structural modifications and evaluated compound potency using an enzymatic assay in conjunction with MD simulations to predict binding mode (depicted). We found that potency was enhanced by using rings to join a carboxylic acid to the central rhodanine ring and that the rhodanine ring could be replaced with alternative heterocycles.image

The pyruvate dehydrogenase complex (PDC) is at the interface of glycolysis and the TCA cycle and is one of the major regulators of fuel selection and metabolic remodeling. Downregulation of PDC in cancer, type 2 diabetes, and heart disease has made this enzymatic complex a valuable drug target. In mammals, the PDC specific kinase inhibits PDC activity by phosphorylating the E1 subunit and blocking the pyruvate binding site. PDK inhibition is reversed by a pyruvate dehydrogenase specific phosphatase (PDP). Here we look to further activate PDC by inhibiting PDK with reversible synthetic inhibitors. The ZINC compound database currently contains over a billion compounds, where we have found about 10.5 million of these are characterized as drug-like, commercially available, and potentially in-stock. We prepared and docked this virtual library to known structures of PDK isozymes 1-4 to the lipoamide, ATP, and pzf3 binding sites. In the top 15K docked compounds of each PDK isozyme, we found only 8 compounds that were found to be in all four. These compounds have a common biphenyl structure that has not been identified in the literature. We synthesized six of these and tested them using a plate reader-based absorbance assay. We found two of these compounds to be active with IC50 values of approximately 30 μM. The project described was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Grant # 5P20GM103427 and R15GM15295-01.

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-beta-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-beta-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 mu M). These syntheses could be used to efficiently investigate variation in the nucleobase.

The human Large neutral amino acids transporter small subunit 1 (LAT1 or SLC7A5), mediates the import of large neutral amino acids, thyroid hormones, and pharmaceutical drugs in a 1:1 exchange for intracellular amino acids (e.g., glutamine). LAT1 is found in the blood-brain-barrier (BBB), testis, bone marrow and placenta, where multiple genetic variations of LAT1 have been associated with numerous diseases and disorders. For example, LAT1 is overexpressed in numerous cancer types, where LAT1’s ability to transport amino acids in particular leucine, is hijacked to activate the mechanistic target of rapamycin (mTOR) pathway, promoting cancer cell growth and the Warburg Effect.

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.

We present a highly reproducible method for investigating the startle flight responses of wild type Drosophila melanogaster to light-off stimuli, using the automated Zantiks MWP unit. The built-in, live video-tracking of the Zantiks unit measured distance travelled between frames for 24 flies after light-off stimuli, whilst providing video-recordings of each startle. Using light-off stimuli which elicited peak startling, we found evidence for habituation of the startle response after only a few consecutive trials. Distance travelled on startle trials was reduced when a prepulse stimulus of shorter duration was introduced before the light-off stimulus, providing behavioural evidence for prepulse inhibition (PPI). Deficits in habituation and PPI are linked to various psychiatric disorders and our method holds great potential for use alongside genetic and pharmacological manipulations. Here, we demonstrate the capability of this highly automated, high throughput technology to streamline behavioural research on Drosophila, using a replicable, controlled environment.

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 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 analogues. 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 or on cancer cells.

A series of 1,2,3-triazole analogs of the amino acids l-histidine and l-tryptophan were modeled, synthesized and tested for l-type amino acid transporter 1 (LAT1; SLC7A5) activity to guide the design of amino acid-drug conjugates (prodrugs). These triazoles were conveniently prepared by the highly convergent Huisgen 1,3-dipolar cycloaddition (Click Chemistry). Despite comparable predicted binding modes, triazoles generally demonstrated reduced cell uptake and LAT1 binding potency relative to their natural amino acid counterparts. The structure-activity relationship (SAR) data for these triazoles has important ramifications for treating cancer and brain disorders using amino acid prodrugs or LAT1 inhibitors.

The L-type amino acid transporter 1 (LAT1, SLC7A5) transports essential amino acids across the blood-brain barrier (BBB) and into cancer cells. To utilize LAT1 for drug delivery, potent amino acid promoieties are desired, as prodrugs must compete with millimolar concentrations of endogenous amino acids. To better understand ligand-transporter interactions that could improve potency, we developed structural LAT1 models to guide the design of substituted analogues of phenylalanine and histidine. Furthermore, we evaluated the structure-activity relationship (SAR) for both enantiomers of naturally occurring LAT1 substrates. Analogues were tested in cis-inhibition and trans-stimulation cell assays to determine potency and uptake rate. Surprisingly, LAT1 can transport amino acid-like substrates with wide-ranging polarities including those containing ionizable substituents. Additionally, the rate of LAT1 transport was generally nonstereoselective even though enantiomers likely exhibit different binding modes. Our findings have broad implications to the development of new treatments for brain disorders and cancer.

The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described.

Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood–brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer cells, as part of a tumor’s increased metabolic demands. Previously, amino acid prodrugs have been shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine’s carboxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined. Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stimulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure–activity relationship (SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates (i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by parent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the corresponding methyl ester derivative. We concluded that HA’s are transported by LAT1. In addition, our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen bonding may be as important as charge for interaction with the transporter binding site.

Application of safety lead optimization screening strategies during the early stage of drug discovery led to the identification of a series of CNS-active small molecule inhibitors with opioid off-target effects, as evidenced by potent agonistic activity in functional cell-based assays for mu (MOP), kappa (KOP) and delta (DOP) opioid receptors. The translation of these effects was confirmed in vivo with the following observations: hypoactivity and decreased fecal production in rats (characteristic of MOP agonism); increased urine production in rats (characteristic of KOP agonism); and decreased intestinal transit time in mice, which was partially blocked by the MOP antagonist naloxone, demonstrating that the in vivo effects were specific for MOP. Based on the confirmation of in vitro-in vivo translatability, an in vitro screening strategy was implemented that resulted in the identification of an optimized backup molecule, devoid of in vivo off-target opioid effects. In addition, in silico modeling by docking of the various molecules to the opioid receptors allowed the identification of the structural drivers of these off-target effects, which can be applied to future chemical-design criteria.Thus, implementation of the safety lead optimization strategy described in this article demonstrates the utility and impact of such approaches on risk mitigation and identification of lead small molecules with improved safety profiles.

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50=110 nM) demonstrated a reduction in CSF Aβ in wild type rats after a single dose.

A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2' and P3 moieties were explored. A conformationally restricted P2' methyl group provided inhibitors with excellent cell potency (37-137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aβ1-40 at 60 mg/kg (PO).

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.