Mitochondrial assay of platelets exposed to thrombin.

<p>Platelets were plated on XF96 plates in MAS buffer, and first a basal rate of oxygen consumption was measured, followed by injection of thrombin (0.5 U/ml). This was followed by injection of (A) saponin (60 μg/ml), pyruvate (5 mM), malate (2.5 mM) and ADP (1 mM) for complex I substrates (D) or saponin (60 μg/ml), succinate (10 mM) and ADP (1 mM) for complex II substrates. Then oligomycin (1 μg/ml) and antimycin A (10 μM) were injected sequentially. From this different parameters of respiration were calculated—(B, E) state 3 (substrate sensitive—oligomycin sensitive OCR) and state 4 (oligomycin sensitive—AA sensitive OCR). (C, F) Respiratory control ratio (RCR) was calculated using the formulae—state3/state4. Data expressed as mean±SEM from one representative donor, n = 3–5 replicates per sample. *p<0.01, different from control.</p

Changes in nucleotides after exposure of platelets to thrombin.

<p>Platelets (1 x 10⁸) were plated onto Cell-Tak coated 48 well plates, and exposed to either media or thrombin (0.5 U/ml) for 30 min and the samples were extracted to measure (A) NAD, AMP, ADP and ADP by HPLC. (B) The ATP/ADP ratio and (C) energy charge (ATP + 1/2ADP)/(ATP+ADP+AMP) was calculated. Data expressed as mean±SEM from one representative donor, n = 3 replicates per sample. *p<0.05, **p<0.01, different from control.</p

Effect of inhibiting mitochondrial fatty acid oxidation on thrombin stimulated platelets.

<p>Platelets were plated on Cell-Tak coated XF96 plates, and pre-treated with etomoxir (25 μM) for 1h prior to bioenergetic measurements. (A) Basal OCR of platelets were measured prior to injection of thrombin (0.5 U/ml), followed by 1 μg/ml oligomycin (O), 0.6 μM FCCP (F) and 10 μM antimycin A (AA). (B) Indices of mitochondrial function, basal, thrombin responsive, ATP-linked, proton leak, maximal, reserve capacity and non-mitochondrial OCR were calculated. (C) Basal and thrombin responsive ECAR were calculated from parallel ECAR measurements. Platelets were pre-treated with TMZ (0–1000 μM) for 3h before bioenergetic assay. (D) Change in OCR after thrombin injection presented as a percentage of control. Data expressed as mean±SEM from one representative donor, n = 3–5 replicates per sample. *p<0.01, different from control. #p<0.01, different from thrombin. $p<0.01 different from etomoxir.</p

Effect of Gln on thrombin stimulated platelets.

<p>Platelets were plated on XF96 plates Cell-Tak coated, and incubated in either regular XF DMEM media or media without Gln for 1h, before bioenergetic measurements. (A) Basal OCR of platelets were measured ahead of thrombin injection (0.5 U/ml), followed by 1 μg/ml oligomycin (O), 0.6 μM FCCP (F) and 10 μM antimycin A (AA). (B) Indices of mitochondrial function, basal, thrombin responsive, ATP-linked, proton leak, maximal, reserve capacity and non-mitochondrial OCR were calculated. (C) Basal and thrombin responsive ECAR were calculated from parallel ECAR measurements. Bioenergetic assays were performed in XF DMEM media containing different concentrations of Gln (0–4 mM). (D) Basal OCR prior to thrombin injection, thrombin linked, ATP linked and maximal OCR after thrombin injection presented as a percentage of highest concentration of Gln (4 mM) after subtraction of no Gln OCR. Platelets were pre-treated with Aza (0–50 μM) for 3h before bioenergetic assay. (E) Change in OCR after thrombin injection presented as a percentage of control. Data expressed as mean±SEM from one representative one donor, n = 3–5 replicates per sample. *p<0.01, different from control. #p<0.01, different from thrombin. $p<0.01 different from Ctrl-no glut. @p<0.01, thrombin linked OCR different from Gln (500 μM). %p<0.01, ATP-linked OCR different from Gln (500 μM). +p<0.01, maximal OCR different from Gln (500 μM).</p

Bioenergetic profile of platelets exposed to thrombin.

<p>(A) OCR and (B) ECAR were measured in platelets by establishing a basal rate then injecting thrombin (0.5 U/ml) and following over 64 min. (C) either media or thrombin were injected (0.5 U/ml) and then sequential injections of 1 μg/ml oligomycin (O), 0.6 μM FCCP (F) and 10 μM Antimycin A (AA). (D) Different parameters of mitochondrial function were calculated—basal (basal OCR—AA sensitive OCR), thrombin (thrombin response—basal OCR), ATP-linked (thrombin response—oligomycin sensitive OCR), proton leak (oligomycin sensitive—AA sensitive OCR), maximal (FCCP sensitive—AA sensitive OCR), reserve capacity (FCCP sensitive—thrombin responsive OCR) and non-mitochondrial (AA sensitive OCR). (E) Simultaneously ECAR measured by first establishing a basal rate followed by injection of thrombin (0.5 U/ml). All the bioenergetic measurements were normalized to protein content per well. Data expressed as mean±SEM from one representative donor, n = 3–5 replicates per sample. *p<0.01, different from control.</p

Effect of inhibiting glycolysis on thrombin stimulated platelets.

<p>Platelets were plated on Cell-Tak coated XF96 plates, and pre-treated with 2DG (120 mM) for 1h before the bioenergetic assay. (A) Basal OCR of platelets were measured prior to thrombin injection (0.5 U/ml), followed by 1 μg/ml oligomycin (O), 0.6 μM FCCP (F) and 10 μM antimycin A (AA). (B) Indices of mitochondrial function, basal, thrombin responsive, ATP-linked, proton leak, maximal, reserve capacity and non-mitochondrial OCR were calculated. (C) Basal and thrombin responsive ECAR were calculated from parallel ECAR measurements. Platelets were pre-treated with koningic acid (10 μM) for 1h prior to the bioenergetic assay. (E) Change in OCR after thrombin injection presented as a percentage of control. Data expressed as mean±SEM from one representative one donor, n = 3–5 replicates per sample. *p<0.01, different from control. %p<0.05, different from control. #p<0.01, different from thrombin. &p<0.05, different from thrombin. $p<0.01 different from 2DG.</p

Effect of supplementation of BSA-palmitate on thrombin stimulated platelets.

<p>Platelets were plated on Cell-Tak coated XF96 plates, and pre-incubated with either BSA or BSA-palmitate (palmitate 200 μM) for 1h prior to bioenergetic measurements. (A) Basal OCR of platelets were measured prior to injection of thrombin (0.5 U/ml), followed by 1 μg/ml oligomycin (O), 0.6 μM FCCP (F) and 10 μM Antimycin A (AA). (B) Indices of mitochondrial function, basal, thrombin responsive, ATP-linked, proton leak, maximal, reserve capacity and non-mitochondrial OCR were calculated. (C) Basal and thrombin responsive ECAR were calculated from parallel ECAR measurements. Data expressed as mean±SEM from one representative donor, n = 3–5 replicates per sample. *p<0.01, different from control. #p<0.01, different from thrombin. %p<0.05 different from BSA-palmitate, $p<0.01 different from BSA-palmitate.</p

Regulation of autophagy, mitochondrial dynamics, and cellular bioenergetics by 4-hydroxynonenal in primary neurons

<p>The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5–10 μM), 4-HNE activates autophagy, whereas at higher concentrations (15 μM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.</p

Identification of Compounds That Decrease Glioblastoma Growth and Glucose Uptake <i>in Vitro</i>

Tumor heterogeneity has hampered the development of novel effective therapeutic options for aggressive cancers, including the deadly primary adult brain tumor glioblastoma (GBM). Intratumoral heterogeneity is partially attributed to the tumor initiating cell (TIC) subset that contains highly tumorigenic, stem-like cells. TICs display metabolic plasticity but can have a reliance on aerobic glycolysis. Elevated expression of GLUT1 and GLUT3 is present in many cancer types, with GLUT3 being preferentially expressed in brain TICs (BTICs) to increase survival in low nutrient tumor microenvironments, leading to tumor maintenance. Through structure-based virtual screening (SBVS), we identified potential novel GLUT inhibitors. The screening of 13 compounds identified two that preferentially inhibit the growth of GBM cells with minimal toxicity to non-neoplastic astrocytes and neurons. These compounds, SRI-37683 and SRI-37684, also inhibit glucose uptake and decrease the glycolytic capacity and glycolytic reserve capacity of GBM patient-derived xenograft (PDX) cells in glycolytic stress test assays. Our results suggest a potential new therapeutic avenue to target metabolic reprogramming for the treatment of GBM, as well as other tumor types, and the identified novel inhibitors provide an excellent starting point for further lead development

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as <b>63</b> exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug–target residence time was determined via SPR. Analysis of these data suggests that drug–target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target