Targeting myeloperoxidase limits myeloid cell immunosuppression enhancing immune checkpoint therapy for pancreatic cancer.

Pancreatic ductal adenocarcinoma is a devastating disease characterized by an extreme resistance to current therapies, including immune checkpoint therapy. The limited success of immunotherapies can be attributed to a highly immunosuppressive pancreatic cancer microenvironment characterized by an extensive infiltration of immune suppressing myeloid cells. While there are several pathways through which myeloid cells contribute to immunosuppression, one important mechanism is the increased production of reactive oxygen species. Here, we evaluated the contribution of myeloperoxidase, a myeloid-lineage restricted enzyme and primary source of reactive oxygen species, to regulate immune checkpoint therapy response in preclinical pancreatic cancer models. We compared treatment outcome, immune composition and characterized myeloid cells using wild-type, myeloperoxidase-deficient, and myeloperoxidase inhibitor treated wild-type mice using established subcutaneous pancreatic cancer models. Loss of host myeloperoxidase and pharmacological inhibition of myeloperoxidase in combination with immune checkpoint therapy significantly delayed tumor growth. The tumor microenvironment and systemic immune landscape demonstrated significant decreases in myeloid cells, exhausted T cells and T regulatory cell subsets when myeloperoxidase was deficient. Loss of myeloperoxidase in isolated myeloid cell subsets from tumor-bearing mice resulted in decreased reactive oxygen species production and T cell suppression. These data suggest that myeloperoxidase contributes to an immunosuppressive microenvironment and immune checkpoint therapy resistance where myeloperoxidase inhibitors have the potential to enhance immunotherapy response. Repurposing myeloperoxidase specific inhibitors may provide a promising therapeutic strategy to expand therapeutic options for pancreatic cancer patients to include immunotherapies.

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic 'gatekeeper' enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Šresolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Clinically relevant mitochondrial-targeted therapy improves chronic outcomes after traumatic brain injury.

Pioglitazone, an FDA-approved compound, has been shown to target the novel mitochondrial protein mitoNEET and produce short-term neuroprotection and functional benefits following traumatic brain injury. To expand on these findings, we now investigate the dose- and time-dependent effects of pioglitazone administration on mitochondrial function after experimental traumatic brain injury. We then hypothesize that optimal pioglitazone dosing will lead to ongoing neuroprotection and cognitive benefits that are dependent on pioglitazone-mitoNEET signalling pathways. We show that delayed intervention is significantly more effective than early intervention at improving acute mitochondrial bioenergetics in the brain after traumatic brain injury. In corroboration, we demonstrate that mitoNEET is more heavily expressed, especially near the cortical contusion, in the 18 h following traumatic brain injury. To explore whether these findings relate to ongoing pathological and behavioural outcomes, mice received controlled cortical impact followed by initiation of pioglitazone treatment at either 3 or 18 h post-injury. Mice with treatment initiation at 18 h post-injury exhibited significantly improved behaviour and tissue sparing compared to mice with pioglitazone initiated at 3 h post-injury. Further using mitoNEET knockout mice, we show that this therapeutic effect is dependent on mitoNEET. Finally, we demonstrate that delayed pioglitazone treatment improves serial motor and cognitive performance in conjunction with attenuated brain atrophy after traumatic brain injury. This study illustrates that mitoNEET is the critical target for delayed pioglitazone intervention after traumatic brain injury, mitochondrial-targeting is highly time-dependent after injury and there is an extended therapeutic window to effectively treat mitochondrial dysfunction after brain injury.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase.

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2).

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Development of an a priori computational approach for brain uptake of compounds in an insect model system.

Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure-property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r2 > 0.5, with the descriptors log(P*MW-0.5) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW-0.5) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines.

Dual monoamine oxidase B and acetylcholine esterase inhibitors for treating movement and cognition deficits in a C. elegans model of Parkinson's disease.

Parkinson's disease (PD) is an age-associated neurodegenerative movement disorder that leads to loss of dopaminergic neurons and motor deficits. Approaches to neuroprotection and symptom management in PD include use of monoamine oxidase B (MAO-B) inhibitors. Many patients with PD also exhibit memory loss in the later stages of disease progression, which is treated with acetylcholine esterase (AChE) inhibitors. We sought to identify a dual-mechanism compound that would inhibit both MAO-B and AChE enzymes. Our screen identified a promising compound (7) with balanced MAO-B (IC50 of 16.83 µM) and AChE inhibition activity (AChE IC50 of 22.04 µM). Application of this compound 7 increased short-term associative memory and significantly prevented 6-hydroxy-dopamine toxicity in dopaminergic neurons in the Caenorhabditis elegans nematode. These findings present a platform for future development of dual-mechanism drugs to treat neurodegenerative diseases such as PD.

Synthesis of Illudinine from Dimedone and Identification of Activity as a Monoamine Oxidase Inhibitor.

The fungal metabolite illudinine is prepared in seven steps and ca. 55% overall yield from dimedone using an "open and shut" (ring-opening and ring-closing) strategy. Tandem ring-opening fragmentation and olefination of dimedone establishes alkyne and vinylarene functionality linked by a neopentylene tether. Oxidative cycloisomerization then provides the illudinine framework. The key innovation in this second-generation synthesis of illudinine is the use of the nitrile functional group, rather than an ester, as the functional precursor to the carboxylic acid of illudinine. The small, linear nitrile (C≡N) is associated with improved selectivity, π-conjugation, and reactivity at multiple points in the synthetic sequence relative to the carboxylic acid ester. Preliminary assays indicate that illudinine and several related synthetic analogues are monoamine oxidase inhibitors, which is the first reported indication of biological activity associated with this natural product. Illudinine was found to inhibit monoamine oxidase B (MAO-B) with an IC50 of 18 ± 7.1 µM in preliminary assays.

Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

PURPOSE: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia. METHODS: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness. RESULTS: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay. CONCLUSION: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.

Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.

CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism. Dynamic regulation of CoA in different tissues and organs supports metabolic flexibility. Two mammalian Nudix hydrolases, Nudt19 and Nudt7, degrade CoA in vitro Nudt19 and Nudt7 possess conserved Nudix and CoA signature sequences and specifically hydrolyze the diphosphate bond of free CoA and acyl-CoAs to form 3',5'-ADP and 4'-(acyl)phosphopantetheine. Limited information is available on these enzymes, but the relatively high abundance of Nudt19 and Nudt7 mRNA in the kidney and liver, respectively, suggests that they play specific roles in the regulation of CoA levels in these organs. Here, we analyzed Nudt19-/- mice and found that deletion of Nudt19 elevates kidney CoA levels in mice fed ad libitum, indicating that Nudt19 contributes to the regulation of CoA in vivo Unlike what was observed for the regulation of Nudt7 in the liver, Nudt19 transcript and protein levels in the kidney did not differ between fed and fasted states. Instead, we identified chenodeoxycholic acid as a specific Nudt19 inhibitor that competed with CoA for Nudt19 binding but did not bind to Nudt7. Exchange of the Nudix and CoA signature motifs between the two isoforms dramatically decreased their kcat Furthermore, substitutions of conserved residues within these motifs identified amino acids playing different roles in CoA binding and hydrolysis in Nudt19 and Nudt7. Our results reveal that the kidney and liver each possesses a distinct peroxisomal CoA diphosphohydrolase.

The MitoNEET Ligand NL-1 Mediates Antileukemic Activity in Drug-Resistant B-Cell Acute Lymphoblastic Leukemia.

Disease relapse in B-cell acute lymphoblastic leukemia (ALL), either due to development of acquired resistance after therapy or because of de novo resistance, remains a therapeutic challenge. In the present study, we have developed a cytarabine (Ara-C)-resistant REH cell line (REH/Ara-C) as a chemoresistance model. REH/Ara-C 1) was not crossresistant to vincristine or methotrexate; 2) showed a similar proliferation rate and cell surface marker expression as parental REH; 3) demonstrated decreased chemotaxis toward bone marrow stromal cells; and 4) expressed higher transcript levels of cytidine deaminase (CDA) and mitoNEET (CISD1) than the parental REH cell line. Based on these findings, we tested NL-1, a mitoNEET inhibitor, which induced a concentration-dependent decrease in cell viability with a comparable IC50 value in REH and REH/Ara-C. Furthermore, NL-1 decreased cell viability in six different ALL cell lines and showed inhibitory activity in a hemosphere assay. NL-1 also impaired the migratory ability of leukemic cells, irrespective of the chemoattractant used, in a chemotaxis assay. More importantly, NL-1 showed specific activity in inducing death in a drug-resistant population of leukemic cells within a coculture model that mimicked the acquired resistance and de novo resistance observed in the bone marrow of relapsed patients. Subsequent studies indicated that NL-1 mediates autophagy, and inhibition of autophagy partially decreased NL-1-induced tumor cell death. Finally, NL-1 showed antileukemic activity in an in vivo mouse ALL model. Taken together, our study demonstrates that mitoNEET has potential as a novel antileukemic drug target in treatment refractory or relapsed ALL.

4-Hydroxynonenal and 4-Oxononenal Differentially Bind to the Redox Sensor MitoNEET.

MitoNEET is a CDGSH iron-sulfur protein that has been a target for drug development for diseases such as type-2 diabetes, cancer, and Parkinson's disease. Functions proposed for mitoNEET are as a redox sensor and regulator of free iron in the mitochondria. We have investigated the reactivity of mitoNEET toward the reactive electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) that are produced from the oxidation of polyunsaturated fatty acid during oxidative stress. Proteomic, electrophoretic, and spectroscopic analysis has shown that HNE and ONE react in a sequence selective manner that was unexpected considering the structure similarity of these two reactive electrophiles.

Binding of thiazolidinediones to the endoplasmic reticulum protein nutrient-deprivation autophagy factor-1.

Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]-containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.

Inhibition of the FAD containing ER oxidoreductin 1 (Ero1) protein by EN-460 as a strategy for treatment of multiple myeloma.

Multiple myeloma (MM) cells demonstrate high basal endoplasmic reticulum (ER) stress and are typically exquisitely sensitive to agents such as proteasome inhibitors that activate the unfolded protein response. The flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum oxidoreductin enzyme (Ero1L) catalyzes de-novo disulfide bridge formation of ER resident proteins and contributes to proper protein folding. Here we show that increased Ero1L expression is prognostic of poor outcomes for MM patients relapsing on therapy. We propose that targeting protein folding via inhibition of Ero1L may represent a novel therapeutic strategy for the treatment of MM. In this report we show that treatment of MM cells with EN-460, a known inhibitor of ERO1L, was sufficient to inhibit cell proliferation and induce apoptosis. Furthermore, we show that cell death correlated in part with induction of ER stress. We also show that EN460 inhibited the enzyme activity of Ero1L, with an IC50 of 22.13 µM, consistent with previous reports. However, EN-460 was also found to inhibit other FAD-containing enzymes including MAO-A (IC50 = 7.91 µM), MAO-B (IC50 = 30.59 µM) and LSD1 (IC50 = 4.16 µM), suggesting overlap in inhibitor activity and the potential need to develop more specific inhibitors to enable pharmacological validation of ERO1L as a target for the treatment of MM. We additionally prepared and characterized azide-tagged derivatives of EN-460 as possible functional probe compounds (e.g., for photo-affinity labeling) for future target-engagement studies and further development of structure-activity relationships.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Disulfiram-based disulfides as narrow-spectrum antibacterial agents.

Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator.

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.