Andrew J. Kennedy

Development of amidine-based sphingosine kinase 1 nanomolar inhibitors and reduction of sphingosine 1-phosphate in human leukemia cells.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has been identified as an accelerant of cancer progression. The sphingosine kinases (SphKs) are the sole producers of S1P, and thus, SphK inhibitors may prove effective in cancer mitigation and chemosensitization. Of the two SphKs, SphK1 overexpression has been observed in a myriad of cancer cell lines and tissues and has been recognized as the presumptive target over that of the poorly characterized SphK2. Herein, we present the design and synthesis of amidine-based nanomolar SphK1 subtype-selective inhibitors. A homology model of SphK1, trained with this library of amidine inhibitors, was then used to predict the activity of additional, more potent, inhibitors. Lastly, select amidine inhibitors were validated in human leukemia U937 cells, where they significantly reduced endogenous S1P levels at nanomolar concentrations.

Discovery, biological evaluation, and structure-activity relationship of amidine based sphingosine kinase inhibitors.

Sphingosine 1-phosphate (S1P), a potent phospholipid growth and trophic factor, is synthesized in vivo by two sphingosine kinases. Thus these kinases have been proposed as important drug targets for treatment of hyperproliferative diseases and inflammation. We report here a new class of amidine-based sphingosine analogues that are competitive inhibitors of sphingosine kinases exhibiting varying degrees of enzyme selectivity. These inhibitors display K(I) values in the submicromolar range for both sphingosine kinases and, in cultured vascular smooth muscle cells, decrease S1P levels and initiate growth arrest.

Amixicile, a Novel Inhibitor of Pyruvate:Ferredoxin Oxidoreductase, Shows Efficacy against Clostridium difficile in a Mouse Infection Model

ABSTRACT Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

A rapid assay for assessment of sphingosine kinase inhibitors and substrates.

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-(32)P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K(m), and the K(i) values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.

Raising the Roof: The Preferential Pharmacological Stimulation of Th1 and Th2 Responses Mediated by NKT Cells

Natural killer T (NKT) cells serve as a bridge between the innate and adaptive immune systems, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease, and cancer. NKT cells are a subset of T cells that express cell‐surface markers characteristic of both natural killer cells and T cells. These unique immunologic cells have been demonstrated to serve as a link between the innate and adaptive immune systems through their potent cytokine production following the recognition of a range of lipid antigens, mediated through presentation of the major histocompatibility complex (MHC) class I like CD1d molecule, in addition to the NKT cells cytotoxic capabilities upon activation. Although a number of glycolipid antigens have been shown to complex with CD1d molecules, most notably the marine sponge derived glycolipid alpha‐galactosylceramide (α‐GalCer), there has been debate as to the identity of the endogenous activating lipid presented to the T‐cell receptor (TCR) via the CD1d molecule on antigen‐presenting cells (APCs). This review aims to survey the use of pharmacological agents and subsequent structure–activity relationships (SAR) that have given insight into the binding interaction of glycolipids with both the CD1d molecules as well as the TCR and the subsequent immunologic response of NKT cells. These studies not only elucidate basic binding interactions but also pave the way for future pharmacological modulation of NKT cell responses.

Synthesis and structure-activity relationships of tyrosine-based inhibitors of autotaxin (ATX).

Autotaxin (ATX) is a secreted soluble enzyme that generates lysophosphatidic acid (LPA) through its lysophospholipase D activity. Because of LPAs role in neoplastic diseases, ATX is an attractive therapeutic target due to its involvement in LPA biosynthesis. Here we describe the SAR of ATX inhibitor, VPC8a202, and apply this SAR knowledge towards developing a high potency inhibitor. We found that electron density in the pyridine region greatly influences activity of our inhibitors at ATX.

Pitt-Hopkins Mouse Model has Altered Particular Gastrointestinal Transits In Vivo.

Pitt–Hopkins syndrome (PTHS) is a neurodevelopmental disorder, classified as an autism spectrum disorder that is caused by the haploinsufficiency of Transcription Factor 4 (TCF4). The most common non‐neurological symptoms in PTHS patients are gastrointestinal (GI) disturbances, mainly gastroesophageal reflux and severe constipation (in about 30 and 75% of PTHS patients, respectively). We hypothesized that the recently recognized mouse model of PTHS will exhibit problems with their gut function. We conducted series of in vivo tests on 15‐ to 19‐ week old male mice, heterozygous for the TCF4 functional deletion, mimicking the TCF4 haploinsufficiency in PTHS patients, and their wild type littermates. Data collection and initial analysis were performed blindly, that is, the genotyping key was received after the mean values were calculated for each individual animal, and then mean/median of each group was subsequently calculated. Body weight, fecal pellet output, and fluid content were similar between the groups, indicating normal gross growth of PTHS mice and their overall physiological GI motility and intestinal secretion/absorption. There were no significant differences in gut length and gross appearance pointing out that PTHS mice have normal gut in gross anatomical terms. However, the assessment of gut transit indicates that, while whole‐gut transit velocity was similar between the groups, the upper GI and distal colon transit velocities were significantly reduced in the PTHS mice. This is the first evidence of specific gut related problems in the PTHS mice. Our study also validates the TCF4 functional knockout mice as an animal model to study PTHS‐associated GI disturbances. Autism Res 2015, 8: 629–633.

Tet1 oxidase regulates neuronal gene transcription, active DNA hydroxymethylation, object location memory, and threat recognition memory

A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the CNS. In this study, we used Tet1 gene knockout (Tet1KO) mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to: altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning) and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

Drugging the methylome: DNA methylation and memory.

Abstract Over the past decade, since epigenetic mechanisms were first implicated in memory formation and synaptic plasticity, dynamic DNA methylation reactions have been identified as integral to long-term memory formation, maintenance, and recall. This review incorporates various new findings that DNA methylation mechanisms are important regulators of non-Hebbian plasticity mechanisms, suggesting that these epigenetic mechanisms are a fundamental link between synaptic plasticity and metaplasticity. Because the field of neuroepigenetics is so young and the biochemical tools necessary to probe gene-specific questions are just now being developed and used, this review also speculates about the direction and potential of therapeutics that target epigenetic mechanisms in the central nervous system and the unique pharmacokinetic and pharmacodynamic properties that epigenetic therapies may possess. Mapping the dynamics of the epigenome in response to experiential learning, even a single epigenetic mark in isolation, remains a significant technical and bioinformatic hurdle facing the field, but will be necessary to identify changes to the methylome that govern memory-associated gene expression and effectively drug the epigenome.

Experience-dependent epigenomic reorganization in the hippocampus

Using a hippocampus-dependent contextual threat learning and memory task, we report widespread, coordinated DNA methylation changes in CA1 hippocampus of Sprague-Dawley rats specific to threat learning at genes involved in synaptic transmission. Experience-dependent alternations in gene expression and DNA methylation were observed as early as 1 h following memory acquisition and became more pronounced after 24 h. Gene ontology analysis revealed significant enrichment of functional categories related to synaptic transmission in genes that were hypomethylated at 24 h following threat learning. Integration of these data sets with previously characterized epigenetic and transcriptional changes in brain disease states suggested significant overlap between genes regulated by memory formation and genes altered in memory-related neurological and neuropsychiatric diseases. These findings provide a comprehensive resource to aid in the identification of memory-relevant therapeutic targets. Our results shed new light on the gene expression and DNA methylation changes involved in memory formation, confirming that these processes are dynamic and experience-dependent. Finally, this work provides a roadmap for future studies to identify linkage of memory-associated genes to altered disease states.