Describing inhibitor specificity for the amino acid transporter LAT1 from metainference simulations.

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.

l-Type amino acid transporter 1 activity of 1,2,3-triazolyl analogs of l-histidine and l-tryptophan.

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.

Cellular fatty acid level regulates the effect of tolylfluanid on mitochondrial dysfunction and insulin sensitivity in C2C12 skeletal myotubes.

Previous research suggests that the endocrine disrupting chemical tolylfluanid (TF) may promote metabolic dysfunction and insulin resistance in humans. The potential impact of TF on skeletal muscle metabolism has yet to be fully investigated. The purpose of this study was to determine whether TF can promote insulin resistance and metabolic dysfunction in mammalian skeletal muscle cells. C2C12 murine skeletal myotubes were exposed to 1 ppm TF for 24 h. To examine the potential effect of cellular fatty acid levels on TF-dependent regulation of mitochondrial metabolism and insulin signaling, we treated skeletal myotubes with 0.25 mM or 1.0 mM oleic acid (OA) during TF exposure trials. Tolylfluanid (1-10 ppm) reduced lipid accumulation by approximately 20% in 0.25 and 1.0 mM OA treated cells. The addition of 0.25 mM OA completely inhibited the TF-dependent reduction in maximal mitochondrial oxygen consumption rate (OCR) while 1.0 mM OA exacerbated the TF-dependent reduction in mitochondrial OCR. Exposing skeletal myotubes to 1 ppm TF promoted an 80% reduction in mitochondrial membrane potential, which was completely inhibited by 0.25 mM OA and partially inhibited by1.0 mM OA. The addition of 0.25 mM OA promoted a TF-dependent increase in insulin-dependent P-Akt (Ser473). In contrast, the addition of 1.0 mM OA promoted a significant reduction in insulin-dependent P-Akt (Ser473). Further, the addition of 1 ppm TF significantly reduced insulin-dependent mTORC1 activity regardless of OA concentration. Finally, TF significantly reduced insulin-dependent protein synthesis in the 1 mM OA treated cells only. Our results demonstrate that the effect of 1 ppm TF on mitochondrial function and insulin-dependent protein synthesis in skeletal myotubes was largely dependent upon cellular fatty acid levels.

LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

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.

LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates.

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.

Synthesis, characterization, and PK/PD studies of a series of spirocyclic pyranochromene BACE1 inhibitors.

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 Aß in wild type rats after a single dose.

Evaluation of Rhodanine Indolinones as AANAT Inhibitors.

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.

Mechanistic pharmacokinetic-pharmacodynamic modeling of BACE1 inhibition in monkeys: development of a predictive model for amyloid precursor protein processing.

This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of ß-site amyloid precursor protein (APP)-cleaving enzyme (BACE1), GNE-629 [(4S,4a'S,10a'S)-2-amino-8'-(2-fluoropyridin-3-yl)-1-methyl-3',4',4a',10a'-tetrahydro-1'H-spiro[imidazole-4,10'-pyrano[4,3-b]chromen]-5(1H)-one] and GNE-892 [(R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one], and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid ß (Aß) in the plasma and cerebrospinal fluid (CSF), and secreted APPß (sAPPß) and secreted APPα (sAPPα) in the CSF were measured after a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aß and CSF Aß, sAPPα, and sAPPß using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 after adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC50 of 0.0033 µM and 0.065 µM, respectively. These differences in CSF and free plasma IC50 suggest that different mechanisms are involved in Aß formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aß, sAPPß, and sAPPα in the CSF, and Aß in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.

Synthesis of ribavirin 1,2,3- and 1,2,4-triazolyl analogs with changes at the amide and cytotoxicity in breast cancer cell lines.

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-ß-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-ß-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 Effects of Prodrug Size and a Carbonyl Linker on l-Type Amino Acid Transporter 1-Targeted Cellular and Brain Uptake.

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.

Prodrug thiamine analogs as inhibitors of the enzyme transketolase.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

Non-charged thiamine analogs as inhibitors of enzyme transketolase.

Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Reevaluating the Substrate Specificity of the L-Type Amino Acid Transporter (LAT1).

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.

8-Tetrahydropyran-2-yl chromans: highly selective beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors.

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 Aß1-40 at 60 mg/kg (PO).

Discovery of 7-tetrahydropyran-2-yl chromans: ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors that reduce amyloid ß-protein (Aß) in the central nervous system.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aß1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aß1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

Spirocyclic ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: from hit to lowering of cerebrospinal fluid (CSF) amyloid ß in a higher species.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.