BACKGROUND Myocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4‐hydroxy trans ‐2‐nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury . METHODS AND RESULTS We report here that β‐alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD 1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD 1‐transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD 1 prevented the accumulation of acrolein and 4‐hydroxy trans ‐2‐nonenal–protein adducts in ischemic hearts and delayed acrolein or 4‐hydroxy trans ‐2‐nonenal–induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP , high‐energy phosphates, intracellular pH, and glycolysis during low‐flow ischemia in the wild‐type mice hearts were attenuated in the ATPGD 1‐transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH , but not both, failed to protect against I/R injury. CONCLUSIONS Either exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

System L amino acid transporters are a member of the Solute Carrier transporter Family (SLC). L-Amino acid transporter 1 (LAT1) belong to SLC7 and requires the heavy chained 4F2hc chaperone protein for amino acid transport. LAT1 is expressed in tumor cells and cancerous cells in vivo are strongly linked to LAT1 expression. Herein, we provide historical aspects regarding initial Structure Activity Relationship (SAR) findings reported in 2002 with the oocyte model and in 2008 with S2-LAT1 and S2-LAT2 cell lines. We summarize a series of dichloro- dibromo- and diiodo- tyrosine analogs that were prepared, and tested their potential to inhibit leucine transport in vitro to afford IC50 values with a few being low microM LAT1 inhibitors. We then summarize our efforts regarding novel LAT1 inhibitors with sub-nanomolar (nM) IC50s to produce JPH203 which has completed Phase I clinical trial and is currently in Phase II trial in Japan. We describe differences observed between LAT1 and LAT2. Overall, we provide an expanded SAR model regarding potent and selective LAT1 inhibitors.

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of (1)-piperideine-6-carboxylate and -aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques.

Endogenous histidyl dipeptides such as carnosine (β-alanine-l-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (β-alanine-Nτ-methylhistidine) and dimethyl balenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of Nτ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at Nτ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe2+ chelating ability of balenine was higher than that of carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of β-alanyl residue and the imidazole residue of l-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.

High (millimolar) concentrations of the histidine containing dipeptide - carnosine (beta-alanine-L-histidine) are present in the skeletal muscle. The dipeptide has been shown to buffer intracellular pH, chelate transition metals, and scavenge lipid peroxidation products; however, its role in protecting against tissue injury remains unclear. In this study, we tested the hypothesis that carnosine protects against post ischemia by augmenting HIF-1a angiogenic signaling by Fe2+ chelation. We found that wild type (WT) C57BL/6 mice, subjected to hind limb ischemia (HLI) and supplemented with carnosine (1g/L) in drinking water, had improved blood flow recovery and limb function, enhanced revascularization and regeneration of myocytes compared with HLI mice placed on water alone. Carnosine supplementation enhanced the bioavailability of carnosine in the ischemic limb, which was accompanied by increased expression of proton-coupled oligopeptide transporters. Consistent with our hypothesis, carnosine supplementation augmented HIF-1 alpha and VEGF expression in the ischemic limb and the mobilization of proangiogenic Flk-1(+)/Sca-1(+) cells into circulation. Pretreatment of murine myoblast (C2C12) cells with octyl-D-carnosine or carnosine enhanced HIF-1 alpha protein expression, VEGF mRNA levels and VEGF release under hypoxic conditions. Similarly pretreatment of WT C57/B16 mice with carnosine showed enhanced blood flow in the ischemic limb following HLI surgery. In contrast, pretreatment of hypoxic C2C12 cells with methylcarcinine, a carnosine analog, lacking Fe2+ chelating capacity, had no effect on HIF-1a levels and VEGF release. Collectively, these data suggest that carnosine promotes post ischemic revascularization via augmentation of pro-angiogenic HIF-1 alpha/VEGF signaling, possibly by Fe2+ chelation.

Neoplastic cells exhibit higher oxidative stress compared to normal cells; however, antioxidants based clinical trials have mostly failed. Another attractive therapeutic approach is to further increase the oxidative stress in cancer cells leading to cell death. Herein, we show that Procyanidin B2 3,3″‐di‐O‐gallate (B2G2), the most active constituent of grape seed extract, treatment causes cell death in human prostate cancer (PCa) cells (LNCaP and 22Rv1) via increasing the reactive oxygen species (ROS) generation. Mechanistically, B2G2 treatment decreased the mitochondrial electron transport chain complex III activity leading to enhanced mitochondrial superoxide generation and decreased ATP production in LNCaP cells. Additional molecular studies revealed that B2G2‐induced cell death was mediated mainly through ROS‐induced sustained activation of ERK1/2, which was due to inhibition of MAP kinase phosphatase (MKP) activity as over‐expression of MKP3 in LNCaP cells conferred significant protection against B2G2‐induced cell death. Along with ERK1/2, AMP‐activated protein kinase α (AMPKα) was also activated by B2G2 treatment, and pre‐treatment with AMPKα inhibitor compound C significantly reversed the cytotoxic effects of B2G2 in LNCaP cells. Furthermore, pre‐treatment of MKP3 over‐expressing LNCaP cells with compound C further reduced the B2G2‐induced cell death, suggesting the involvement of AMPKα along with MKP3 and ERK1/2 in the biological effects of B2G2. Together, these results for the first time identified that oxidative stress and MKP3 inhibition play a critical role in B2G2‐induced cell death in PCa cells through sustained activation of both ERK1/2 and AMPKα. These results offer a unique opportunity to control this deadly malignancy through B2G2 use.

Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14–57) which were characterized by 1H NMR, 13C NMR and LC/MS–MS. These compounds were assessed for their effect on the in vitro anchorage independent growth of human lung cancer cell line H2122 and IC50 values calculated. Two of the more potent compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0h post ip (50mg/Kg) dose.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Histone lysine methyl transferase 2 (EZH2) inhibitor GSK126 and a novel deuterated internal standard GSK126-d7 were chemically prepared. We performed in vitro experiments using the prepared GSK126 to: i) confirm in vitro EZH2 inhibitory activity; ii) conduct Sprague-Dawley (SD) rat liver microsomal incubations and identified Phase I metabolites; iii) determine whether or not GSK126 was an Organic Anion Transporter (OAT) substrate; and, iv) determine oral bioavailability by conducting oral and orbital sinus dosing (OSD) experiments and determining blood concentration versus time profiles. GSK126 was shown to decrease the expression of H3K27Me3 protein in medulloblastoma D283 cells and was able to decrease cell viability in KO99L cells, a novel T cell lymphoma cell line. Three in vitro hepatic Phase I mono-oxidative metabolites (L-M1, L-M2 and L-M3) were observed and also detected in rat liver and urine samples from the in vivo studies. GSK126 was found to be an OAT1 and OAT2 substrate, but not an OAT3 or OAT4 substrate. Our Pharmacokinetic (PK) results indicate: 1) GSK126 has very poor oral bioavailability (< 2%); 2) co-administration of probenecid, a prototypical OAT inhibitor, did not significantly alter observed PK; and 3) tissue distribution studies demonstrate that GSK126 predominately distributes to the liver and kidneys after an OSD.

Abstract Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition. Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.

Presently the relationship between CD28, biological marker of senescence, and ovariectomy is not well understood. We show that ovariectomy leads to CD28 loss on T cells and estrogen (E2) repletion and medicarpin (Med) inhibits this effect. We thus propose that Med/E2 prevents bone loss by delaying premature T cell senescence.

Medicarpin, a pterocarpan class of naturally occurring benzopyran furanobenzene compound was synthesized in gram scale to investigate its effects on murine bone cells and in ovariectomized (OVx) mice. Medicarpin, at as low as 10−10 M suppressed osteoclastogenesis in bone marrow cells (BMCs). Medicarpin-induced apoptosis of mature osteoclasts isolated from long bones. Effects of medicarpin in osteoclasts appear to be independent of estrogen receptor (ER) activation as ICI 180,782 failed to abrogate its effects on osteoclasts. In calvarial osteoblasts, medicarpin (10−10 M) blocked nuclear factor kappaB (NF-κB) signaling assessed by tumor necrosis factor alpha (TNFα)-stimulated nuclear translocation of p65 subunit of NF-κB. Medicarpin also inhibited the expression of TNFα in mouse calvarial osteoblasts. This effect was ER dependent as ICI 180,782 reversed the suppressive effect of medicarpin on TNFα mRNA levels in osteoblasts. In addition, like 17β-estradiol, presence of medicarpin inhibited TNFα-induced upregulation of interleukin-1, and -6 mRNA levels in osteoblasts. In co-cultures consisting of calvarial osteoblasts and BMCs, presence of medicarpin increased osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) ratio and reduced mRNA levels of osteoclast markers including tartrate-resistant acid phosphatase and RANK. OVx mice administered medicarpin (10.0 mg kg−1 day−1) orally for 30 days had reduced formation of osteoclasts but increased formation of osteoprogenitor cells in BMCs compared with OVx + vehicle group. Medicarpin treatment to OVx mice maintained parameters of trabecular microarchitecure. Medicarpin exhibited no uterine estrogenicity. Our findings point towards direct and indirect inhibitory effects of medicarpin on osteoclastogenesis in vitro that contribute to its bone sparing effect in OVx mice.