Transdural retrieval of retropulsed transforaminal lumbar interbody fusion cages.

Transforaminal lumbar interbody fusions (TLIFs) are performed for various lumbar spine pathologies. Posterior migration of an interbody cage is a complication that may result in neurologic injury and require reoperation. Sparse information exists regarding the safety and efficacy of a transdural approach for cage retrieval. We describe a surgical technique, in which centrally retropulsed cages were safely retrieved transdurally. A patient with prior L3-S1 posterior lumbar fusion and L4-S1 TLIFs presented with radiculopathy and weakness in dorsiflexion. Imaging revealed posterior central migration of TLIF cages causing compression of the traversing L5 nerve root. Cages were removed transdurally; the correction was performed with an all-posterior T10-pelvis fusion. Aside from temporary weakness in right-sided dorsiflexion, the patient experienced complete resolution in their radiculopathy and strength returned to its presurgical state by 3 months. The transdural approach for interbody removal can be safely performed and should be a tool in the spine surgeon's armamentarium.

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.

Physics-Informed Neural Networks with Group Contribution Methods.

Thermophysical properties of organic compounds are used in countless scientific, engineering, and industrial settings in developing theories, designing new systems and devices, analyzing costs and risks, and improving existing infrastructure. Often, due to costs, safety, prior interest, or procedural difficulties, experimental values for desired properties are not available and must be predicted. The literature is filled with prediction techniques, but even the best traditional methods have significant errors compared to what is possible considering experimental uncertainty. Recently, machine learning and artificial intelligence techniques have been applied to the property prediction problem, but the examples to date do not extrapolate well outside the data set used for training the model. This work demonstrates a solution to this problem by combining chemistry and physics when training the model and builds upon prior traditional and machine learning methods. Two case studies are presented. The first is for parachor which is used for surface tension prediction. Surface tensions are needed to design distillation columns, adsorption processes, gas-liquid reactors, liquid-liquid extractors, improve oil reservoir recovery, and undertake environmental impact studies or remediation actions. A set of 277 compounds is divided into training, validation, and test sets, and a multilayered physics-informed neural network (PINN) is developed. The results demonstrate that better extrapolation by deep learning models can be developed by adding in physics-based constraints. Second, a set of 1600 compounds is utilized for training, validating, and testing a PINN to improve normal boiling point predictions based on group contribution methods and physics-based constraints. The results show that the PINN performs better than any other method with a normal boiling point mean absolute error of 6.95 °C on training and 11.2 °C on test data. Key observations are that (1) a balanced split by compound type is important to have representative compound families in each of the train, validation, and test sets and (2) constraining group contributions being positive improves predictions on the test set. While this work demonstrates improvements for only surface tension and normal boiling point, the results offer significant hope that PINNs can improve prediction of other relevant thermophysical properties over existing approaches.

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.

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.

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.

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.

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.

Trace element concentrations, risks and their correlation with metallothionein genes polymorphism: A case study of narrow-ridged finless porpoises (Neophocaena asiaeorientalis) in the East China Sea.

The concentration of trace elements (TEs) and their risk to narrow-ridged finless porpoises (Neophocaena asiaeorientalis) are still unclear. The present study determined the concentration of typical TEs in liver, kidney, and muscle tissues from porpoises in the East China Sea, assessed potential health risk of TEs to porpoises, and explored the relationship between TE concentration and metallothionein genes (MTs) polymorphism. It was found that Zn, Cu, Mn, Cd and Hg were highly accumulated in liver, and Cd was highly accumulated in kidney. The concentrations of Cr, As, Pb and Ni were very low in all three tissues. TE concentrations showed significant positive correlation with body length, and sexual variation. The levels of most TEs were higher in tissues of porpoises in Ningbo and Nantong than in Pingtan, which is likely related to the local environment pollution level. The risk assessment showed that porpoises from Nantong and Ningbo could face health risks due to Hg, As, Cd, Pb, and Cr exposure. Moreover, two polymorphic sites on the MT4 gene were found to be significantly associated with increased levels of Hg, Cd, Zn and Mn. Whether these two polymorphic sites are involved in expression of MTs, or other functional processes, needs further research.

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.

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.

Mitigation of opioid off-target effects and identification of structural drivers of opioid receptor engagement for BACE-1 small molecule inhibitors.

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.

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 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

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.

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.

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.

The effect of changing the sequence of cuff inflation and device fixation with the LMA-Supreme® on device position, ventilatory complications, and airway morbidity: a clinical and fiberscopic study.

BACKGROUND: The conventional sequence when using supraglottic airway devices is insertion, cuff inflation and fixation. Our hypothesis was that a tighter fit of the cuff and tip could be achieved with a consequently lower incidence of air leak, better separation of gastrointestinal and respiratory tracts and less airway morbidity if the device were first affixed and the cuff then inflated. METHODS: Our clinical review board approved the study (public registry number DRKS00003174). An LMA Supreme® was inserted into 184 patients undergoing lower limb arthroscopy in propofol-remifentanil anaesthesia who were randomly assigned to either the control (inflation then fixation; n = 92) or study group (fixation then inflation; n = 92). The cuff was inflated to 60 cmH2O. The patients' lungs were ventilated in pressure-controlled mode with 5 cmH2O PEEP, Pmax to give 6 ml kg-1 tidal volume, and respiratory rate adjusted to end-tidal CO2 of 4.8 and 5.6 kPa. Correct cuff and tip position were determined by leak detection, capnometry trace, oropharyngeal leak pressure, suprasternal notch test, and lube-tube test. Bowl and cuff position and the presence of glottic narrowing were assessed by fiberscopic examination. Postoperative dysphagia, hoarseness and sore throat were assessed with a questionnaire. Ventilatory impairment was defined as a tidal volume < 6 ml kg-1 with Pmax at oropharyngeal leak pressure, glottic narrowing was defined as an angle between the vocal cords under 16 degrees. RESULTS: The incidence of incorrect device position (18% vs. 21%), failed ventilation (10% vs. 9%), leak pressure (24.8 vs. 25.2 cmH2O, p = 0.63), failed lube-tube test (16.3% vs. 17.6%) and glottic narrowing (19.3% vs. 14.1%, p = 0.35) was similar in both groups (control vs. study, resp.). When glottic narrowing occurred, it was more frequently associated with ventilatory impairment in the control group (77% vs. 39%; p = 0.04). Airway morbidity was more common in the control group (33% vs. 19%; p < 0.05). CONCLUSIONS: Altering the sequence of cuff inflation and device fixation does not affect device position, oropharyngeal leak pressures or separation of gastrointestinal and respiratory tracts. It reduces the incidence of glottic narrowing with impaired ventilation and also perioperative airway morbidity.