Small Molecular Weight Compounds Research Articles

BDNF mimetic alleviates body weight gain in obese mice by enhancing mitochondrial biogenesis in skeletal muscle

Background7,8-Dihydroxyflavone (7,8-DHF) is a small molecular weight compound that mimics the functions of brain-derived neurotrophic factor (BDNF). The current study aims to elucidate the molecular mechanism of 7,8-DHF-induced body weight regulation. MethodsObese female C57/BL6 (20-week-old) mice that have been fed with high-fat diet for 13 weeks were treated with 7,8-DHF for 9 weeks. Various biochemical and molecular analyses were performed to examine the signal transduction pathway, metabolite content, and mitochondrial mass in the animals. Moreover, systemic energy metabolism and insulin sensitivity were determined by indirect calorimetry and insulin/glucose-tolerance tests. We have also determined the metabolic actions of 7,8-DHF on cultured myotubes. Results7,8-DHF treatment increased cellular respiration by promoting mitochondrial biogenesis in cultured skeletal muscle cells. In diet-induced obese mice, subsequent 7,8-DHF consumption triggered the AMPK/CREB/PGC-1α pathways to increase the muscular mitochondrial content. Systemic energy metabolism was thus elevated, which reduced the body weight gain in obese animals. Consequently, hyperlipidemia, hyperglycemia hyperinsulinemia, and ectopic lipid accumulation in skeletal muscle and liver of the obese animals were alleviated after 7,8-DHF treatment. Moreover, insulin sensitivity of the obese muscle was improved after 7,8-DHF consumption. Conclusion7,8-DHF treatment increases muscular mitochondrial respiration and systemic energy expenditure, which alleviates the body weight gain and partially reverse the metabolic abnormalities induced by obesity.

Multicomponent Reactions for Multitargeted Compounds for Alzheimer`s Disease.

Alzheimer's Disease (AD) is a multifactorial and fatal neurodegenerative disorder affecting around 35 million people worldwide, which is characterized by decline of cholinergic function, deregulation of amyloid beta (Aβ) oligomers formation and Aβ fibril deposition. Multi-Target- Directed Ligands (MTDLs) have emerged as an original strategy for developing new therapeutic agents on AD. Multicomponent Reactions (MCRs) are a useful alternative to sequential multistep syntheses, allowing scaffold diversity and a rapid and easy access to biologically relevant compounds. The biological diversity of MCRs is very rich providing great possibilities for researchers interested in bioactive small molecular weight compounds. Since the MTDL strategy has been used to develop compounds endowed with the capacity to interact with different targets, versatile compound libraries may be obtained by MCRs according to the well established features of each target. Thus, either MTDLs or monotarget compounds have been developed by MCRs to address different factors implicated in AD. This work focuses on antioxidants, calcium channel modulators, both AChE and BuChE inhibitors, BACE1 inhibitors, and modulators of the nuclear factor (erythroid-derived 2)-like 2. First, we discuss the Biginelli reaction and its use for developing new interesting compounds for AD, followed by the contribution of Ugi reaction and, finally, the interest of other MCRs in the same topic.

Temperature profiling to maximize energy yield with reduced water input in a lignocellulosic ethanol biorefinery

Softwood biomass is an attractive renewable feedstock for bioethanol production. The net energy yield of the related biorefinery processes has been limited, however, by its high lignin content, which is recalcitrant to hydrolysis and fermentation. New understanding of the causes of the inhibiting effects is critical to approach the optimal energy/water nexus in a biorefinery. This paper introduces a new prehydrolysis simultaneous saccharification and fermentation process to convert sulfite pretreated Monterey pine into bioethanol, resulting in an extremely high titer of 82.6 g/L or 10 vol%. The new process was carried out at a solid content of 25% by using a commercial enzyme and Saccharomyces cerevisiae. Sugars in the pretreatment spent liquor were concentrated and mixed into the fermentation broth for complete utilization of the sugars, but it was found that this liquor can also affect the ethanol titer during fermentation. Control experiments suggested that sugar-based pretreatment by-products were not the major contributors to the inhibition, while small molecular weight compounds played a major role in affecting the fermentability of the slurry under high temperature. Cell viability tests showed that reducing fermentation temperature from 35 °C to 28 °C can overcome the impacts of pretreatment by-products on cell growth and ethanol production. Without the need of detoxification, the resulting ethanol titer is approaching the theoretical yield and is currently among the highest in softwood conversion. The net energy yield of the new process was 2410 MJ per ton oven-dried wood, which is approximately 730–1690 MJ higher than that of the other biorefinery processes. The water input before reclamation was 3.65 tons per ton dried wood, which is 25.8–51.2% lower than most of the other processes.

General Anesthesia Inhibits the Activity of the "Glymphatic System".

INTRODUCTION: According to the “glymphatic system” hypothesis, brain waste clearance is mediated by a continuous replacement of the interstitial milieu by a bulk flow of cerebrospinal fluid (CSF). Previous reports suggested that this cerebral CSF circulation is only active during general anesthesia or sleep, an effect mediated by the dilatation of the extracellular space. Given the controversies regarding the plausibility of this phenomenon and the limitations of currently available methods to image the glymphatic system, we developed original whole-brain in vivo imaging methods to investigate the effects of general anesthesia on the brain CSF circulation.METHODS: We used magnetic resonance imaging (MRI) and near-infrared fluorescence imaging (NIRF) after injection of a paramagnetic contrast agent or a fluorescent dye in the cisterna magna, in order to investigate the impact of general anesthesia (isoflurane, ketamine or ketamine/xylazine) on the intracranial CSF circulation in mice.RESULTS: In vivo imaging allowed us to image CSF flow in awake and anesthetized mice and confirmed the existence of a brain-wide CSF circulation. Contrary to what was initially thought, we demonstrated that the parenchymal CSF circulation is mainly active during wakefulness and significantly impaired during general anesthesia. This effect was especially significant when high doses of anesthetic agent were used (3% isoflurane). These results were consistent across the different anesthesia regimens and imaging modalities. Moreover, we failed to detect a significant change in the brain extracellular water volume using diffusion weighted imaging in awake and anesthetized mice.CONCLUSION: The parenchymal diffusion of small molecular weight compounds from the CSF is active during wakefulness. General anesthesia has a negative impact on the intracranial CSF circulation, especially when using a high dose of anesthetic agent.

Magnolol protects Ctenopharyngodon idella kidney cells from apoptosis induced by grass carp reovirus

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

Bacteriocins of lactic acid bacteria and their application on food as biopreservatives. (II)

Lactic acid bacteria produce a variety of small molecular weight compounds, which have antimicrobial properties. Such substances are: organic acids, alcohols, carbon dioxide, diacetyl, hydrogen peroxide and bacteriocins. Many of these compounds have a wide inhibitory spectrum but the bacteriocins are able to inhibit species, namely, related with the bacteriocin-producing strain. In the last years bacteriocins have gained a lot of concern because some of them are able to inhibit the growth of pathogenic bacteria, like Listeria monocytogenes. The term "biopreservation" refers to the extension of storage life, as well as to the enhancement of the food safety, using the bacteriocin-producing lactic acid strains or their metabolic antibacterial products. In this review will be reported bacteriocins, which are produced by the lactic acid bacteria and will be discussed the potential application of the bacteriocinogenic strains or their bacteriocins on the foods, as protective cultures or as protective compounds, respectively.

An in silico approach in predicting the possible mechanism involving restoration of wild-type p53 functions by small molecular weight compounds in tumor cells expressing R273H mutant p53.

R273H mutant p53 is a DNA-contact mutant that renders p53 dysfunctional due to a single substitution of Arg273 for His273. Rescuing R273 mutant p53 implies that a competent molecule would have to bind to the site of DNA-contact hot spots to complement the loss of contact with the DNA-binding domain. Here, curcumin, flavokawain B, and alpinetin were docked against the crystal structure of R273H mutant p53 in silico. Consequently, all the compounds bind to the cavity of R273H mutant p53 with a dissociation constant estimated to have 36.57, 70.77, and 75.11 µM for curcumin, flavokawain B, and alpinetin, respectively. Subsequently, each molecule was able to bind to the R273H mutant p53 by interacting with the DNA-contact hot spot Arg248 and mutant R273H, thereby compensating for the loss of direct contact with the DNA-binding domain. Furthermore, all the molecules were able to induce a direct contact with the consensus site of the DNA binding domain, thus maintaining DNA-contact residues with the DNA. The present findings offer preliminary indirect supporting evidence that small molecular weight compounds may certainly rescue DNA-contact mutant p53, which may lay a foundation for designing a competent and effective molecule capable of rescuing mutant p53 in tumor cells expressing R273H mutant p53.

The molecular weight distribution of dissolved organic carbon after application off different sludge disintegration techniques

Abstract The effect of sludge disintegration on molecular weight distribution of dissolved organic carbon (DOC) is demonstrated by size exclusion chromatography with online organic carbon detection (SEC-OCD). Ozone, ultrasound and sodium hydroxide were used to disintegrate excess sludge. Significant differences on the molecular weight distribution of the released DOC were found for the different treatments. Ozonation did not change the DOC distribution based on the molecular size, compared to untreated sludge. Small molecular weight compounds were the dominant constituents solubilized by sodium hydroxide treatment. Ultrasound disintegrated the sludge flocs and dissolved large molecules into the sludge liquid phase. Especially for sodium hydroxide and ultrasound treatment the analysis with SEC-OCD clearly shows the preferential fractions of organic carbon, which are more quickly transferred to methane in batch anaerobic experiments. For sodium hydroxide, it is the small molecular weight fraction as for ultrasound treatment the degradation is evenly distributed throughout all weight fractions.

Abstract IA19: Targeting the PRC2 complex through EED for anti-cancer therapy

Abstract The Polycomb Repressive Complex-2 (PRC2) plays important roles in regulating gene expression through its histone H3 lysine methyl transferase activity. Dysregulation of PRC2 is observed in multiple cancers. For example, gain of function mutations of the EZH2 catalytic subunit of PRC2 have been reported in B-cell lymphomas, melanoma and parathyroid carcinoma. Components of the complex are also overexpressed in a number of cancers. The PRC2-complex have therefore become a major target for anti-cancer drug discovery. We and others, have reported anti-tumor activity of small molecular weight agents targeting the SAM-binding pocket of EZH2 in preclinical models and a number of EZH2 inhibitors ore showing promising activity in clinical trials. EED and SUZ12 are two additional components of the PRC2 core complex required for regulation of its catalytic function. High resolution structures and other reports have showed that binding of EED to tri-methylated histone H3 Lysine 27 (H3K27Me3) further stimulates the catalytic effect of EZh2. This suggests that small molecular weight compounds binding to and interfering with the EED stimulatory effect on Ezh2 may be able to modulate PRC2 activity. Following this possibility, we launched a comprehensive effort to discover non-catalytic site PRC2 binders. The discovery and characterization of EED226, a highly potent and selective EED-binding inhibitor of PRC2 will be presented. Data will be presented to demonstrate that EED226 inhibits PRC2 function via a distinct allosteric mechanism by binding to the H3K27Me3 binding pocket of EED. Furthermore, EED226 potently inhibits cellular H3K27Me3 levels, modulates target gene expression similar to the SAM competitive Ezh2 inhibitors and inhibits the proliferation of cell lines carrying mutant Ezh2. Oral administration of EED226 to EZH2-mutant tumor bearing mice leads to profound tumor regression. Our data therefore demonstrates that the multi-subunit PRC2 complex can be targeted at non-catalytic binding pockets to achieve anti-tumor activity. Some unique features of the anti-tumor activity achieved through targeting PRC2 will be discussed. Citation Format: Peter W. Atadja. Targeting the PRC2 complex through EED for anti-cancer therapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA19.

A metabolomics-based approach identifies changes in the small molecular weight compound composition of the peanut as a result of dry-roasting

Raw peanuts in the USA are subjected to thermal processing, such as dry-roasting, prior to consumption. A multi-instrument metabolomics-based platform along with targeted analyses was used to determine changes in the low-molecular-weight compound composition of peanuts due to dry-roasting. Runner and virginia-type peanut seeds were characterized using several analytical platforms including (RP)/UPLC-MS/MS (positive and negative ion mode ESI) and HILIC/UPLC-MS/MS with negative ion mode ESI. Of the 383 compounds identified, 16 compounds were unique to the roasted peanuts. Using pathway analysis, compounds associated with arginine and proline metabolism were found to be the most changed. Products of chemical degradation and compounds contained within the vesicular bodies of the peanut increased after roasting. Dry-roasting had a significant impact on the levels and types of low-molecular-weight compounds present. These findings provide useful information about composition changes due to roasting.

Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts

Melanin pigment is a negatively charged polymer found in pigmented human tissues. In the eye, iris, ciliary body, choroid and retinal pigment epithelium (RPE) are heavily pigmented. Several drug molecules are known to bind to melanin, but larger sets of drugs have not been compared often in similar test conditions. In this study, we introduce a powerful tool for screening of melanin binding. The binding of a set of 34 compounds to isolated porcine RPE melanin was determined by cassette (n-in-one) dosing in rapid equilibrium dialysis inserts and the binding was quantitated with LC-MS/MS analytics. The compounds represented large variety in melanin binding (from 8.6%, ganciclovir) to over 95% bound (ampicillin and ciprofloxacin). The data provides information on melanin binding of small molecular weight compounds that are used for ocular (e.g. brinzolamide, ganciclovir) and systemic (e.g. tizanidine, indomethacin) therapy. Interestingly, competition among compounds was seen for melanin binding and the binding did not show any correlation with plasma protein binding. These results increase the understanding of melanin binding of ocular drugs and can be further exploited to predict pharmacokinetics in the eye. Pigment binding provides an interesting option for improved drug distribution to retina and choroid that are difficult target tissues in drug delivery.

Pharmaceutical Chaperones and Proteostasis Regulators in the Therapy of Lysosomal Storage Disorders: Current Perspective and Future Promises

Different approaches have been utilized or proposed for the treatment of lysosomal storage disorders (LSDs) including enzyme replacement and hematopoietic stem cell transplant therapies, both aiming to compensate for the enzymatic loss of the underlying mutated lysosomal enzymes. However, these approaches have their own limitations and therefore the vast majority of LSDs are either still untreatable or their treatments are inadequate. Missense mutations affecting enzyme stability, folding and cellular trafficking are common in LSDs resulting often in low protein half-life, premature degradation, aggregation and retention of the mutant proteins in the endoplasmic reticulum. Small molecular weight compounds such as pharmaceutical chaperones (PCs) and proteostasis regulators have been in recent years to be promising approaches for overcoming some of these protein processing defects. These compounds are thought to enhance lysosomal enzyme activity by specific binding to the mutated enzyme or by manipulating components of the proteostasis pathways promoting protein stability, folding and trafficking and thus enhancing and restoring some of the enzymatic activity of the mutated protein in lysosomes. Multiple compounds have already been approved for clinical use to treat multiple LSDs like migalastat in the treatment of Fabry disease and others are currently under research or in clinical trials such as Ambroxol hydrochloride and Pyrimethamine. In this review, we are presenting a general overview of LSDs, their molecular and cellular bases, and focusing on recent advances on targeting and manipulation proteostasis, including the use of PCs and proteostasis regulators, as therapeutic targets for some LSDs. In addition, we present the successes, limitations and future perspectives in this field.

Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation.

Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

Identification of novel small molecules that bind to the loop2 region of sclerostin - an in silico computational analysis.

The goal of this study was to identify small molecular weight compounds that bind to sclerostin using in-silico methods because of the established importance of sclerostin-based therapies for the treatment of disease characterized by low bone mass. The zinc database (Zdb) revealed that nine potential molecules bind to the loop2 region (functional site) of sclerostin with ADME/T properties that are within an acceptable range defined for human use. Compounds 30160056 and 56871042 showed the highest docking score. Density functional theory (by HOMO, LUMO and MESP analysis) and MM/GBSA analysis showed that four compounds 30160056, 56871042, 72112226 and 43920281 exhibit high stability among the nine small molecules identified. Induced Docking Fit and Pymol software analyses revealed that the identified compounds differ in the interaction with amino acids in the loop2 region of sclerostin. Six compound exhibited interaction with Ile95 and 2 compounds with Asn93, an amino acid in the loop2 region known to be involved in sclerostin's inhibitory effect, suggesting that the identified compounds have the potential to bind and neutralize sclerostin function. Furthermore, compound 43920281 showed a low risk of toxicity and drug-like characteristic features compared to all nine identified compounds. In conclusion, in silico analysis identified a novel compound 43920281 as a potent anti-sclerostin therapeutic for drug development for the treatment of osteoporosis.

Abstract 3776: Mechanistic evaluation of AG311 - an OXPHOS inhibitor - as a potential treatment for breast cancer

Abstract About fifty percent of breast cancer patients receiving a combination of surgery, radiation and systemic therapy will not remain cancer-free. Most solid tumors, including breast tumors, have hypoxic regions that can contribute to chemoresistance, radioresistance, and poor differentiation in tumors resulting in a poor clinical outcome in patients with advanced disease. It has been reported that mitochondrial respiration remains active in these hypoxic microenvironments. Complex I of the mitochondrial electron transport chain (ETC) has been shown to switch to a de-active catalytic state under hypoxic conditions. The purpose of this study was to investigate the potential anticancer action of a novel compound with antimitochondrial activity under hypoxic conditions. AG311 (5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine) is a small molecular weight compound shown to inhibit complex I activity in vitro and to drastically reduce mitochondrial oxygen consumption rate by 62.9% at 7.5 μM in breast cancer cells (p<0.001). In two triple negative breast cancer mouse models (MDA-MB-435 and 4T1), AG311 significantly reduced tumor volume by 85% and 81%, respectively (p<0.001 for both). In this current study, the effect of the microenvironment on AG311 mitochondrial inhibition was examined. First, it was shown that AG311 inhibited complex I activity not only in vitro, but also in breast cancer cells (54% inhibition, p<0.001) and tumor homogenate (50% inhibition, p = 0.01). AG311 induced greater cytotoxicity in cells (MDA-MB-435) cultured in glucose-depleted media (IC50 15.5 μM vs 20.0 μM, p<0.01), a condition favoring mitochondrial ATP production, as compared to normal glucose concentrations. Further, co-treatment of AG311 with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor and stimulator of oxidative phosphorylation, showed a synergistic effect on cell kill (CI = 0.7 at 20 μM). Importantly, hypoxic conditions (1% O2) significantly sensitized cancer cells to AG311-induced cell death (from 43.3% to 30.1%, p = 0.019). Further, the effect of AG311 on the deactive form of complex I, which is promoted under hypoxic conditions was assessed by measuring NADH oxidation rate. The switch to the de-active state was thermally-induced in mitochondrial homogenates and once in this state, complex I activity was sensitized to AG311 inhibition (from 45.4% to 65.0% inhibition, p = 0.04). Thus, a mitochondrial inhibitor that preferentially inhibits the de-active state of complex I in hypoxic tumor regions could potentially provide a therapeutic benefit. Citation Format: Anja Bastian, Satoshi Matsuzaki, Kenneth M. Humphries, Lora C. Bailey-Downs, Aleem Gangjee, Michael A. Ihnat. Mechanistic evaluation of AG311 - an OXPHOS inhibitor - as a potential treatment for breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3776.

Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish.

Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish with another PPARα agonist, gemfibrozil, also increased expression of the mbp promoter-driven fluorescent reporter in an SREBF-dependent manner. These results suggest that activation of SREBFs by small molecular weight compounds may be a feasible therapeutic approach to stimulate myelination.

ApoE: In Vitro Studies of a Small Molecule Effector.

Apolipoprotein E4 (apoE4), one of three isoforms of apoE, is the major risk factor for developing late onset Alzheimer's disease. The only differences among these isoforms (apoE2, apoE3, and apoE4) are single amino acid changes. Yet these proteins are functionally very different. One approach to ameliorating the effect of apoE4 with respect to Alzheimer's disease would be to find small molecular weight compounds that affect the behavior of apoE4. Few studies of this approach have been carried out in part because there was no complete structure of any full-length apoE isoform until 2011. Here, we focus on one small molecular weight compound, EZ-482, and explore the effects of its binding to apoE. Using hydrogen-deuterium exchange, we determined that EZ-482 binds to the C-terminal domains of both apoE3 and apoE4. The binding to apoE4, however, is accompanied by a unique N-terminal allosteric effect. Using fluorescence methods, we determined an apparent dissociation constant of approximately 8 μM. Although EZ-482 binds to the C-terminal domain, it blocks heparin binding to the N-terminal domain. The residues of apoE that bind heparin are the same as those involved in apoE binding to LDL and LRP-1 receptors. The methods and the data presented here may serve as a template for future studies using small molecular weight compounds to modulate the behavior of apoE.

Targeting cancer stem cells with p53 modulators.

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest.

Direct Label-Free Measurement of the Distribution of Small Molecular Weight Compound Inside Thick Biological Tissue using Coherent Raman Microspectroscopy

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 /cm inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.Sci. Rep. 5, 13868; http://dx.doi.org/10.1038/srep13868 (2015).

Microdosing of protein drugs.

Poor pharmacokinetics (PK) can seriously limit clinical utility. Knowing early whether a new compound is likely to have the desired PK profile at therapeutic doses is therefore important. One approach, microdosing, has shown high success with small molecular weight compounds, despite early skepticism. Vlaming et al. report the first, and successful, clinical application of a microdose of a humanized recombinant protein. But what is the likely success for this class of drugs more generally?