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Activation of pyruvate dehydrogenase (PDH) by inhibition of pyruvate dehydrogenase kinase (PDHK) has the potential for the treatment of diabetes mellitus and its complications, caused by the malfunction of the glycolytic system and glucose oxidation. In this paper, we describe the identification of novel PDHK inhibitors with a fluorene structure. High-throughput screening using our in-house library provided compound 6 as a weak inhibitor that occupied the allosteric lipoyl group binding site in PDHK2. Structure-based drug design (SBDD) while addressing physicochemical properties succeeded in boosting inhibitory activity approximately 700-fold. Thus obtained compound 32 showed favorable pharmacokinetics profiles supported by high membrane permeability and metabolic stability, and exhibited activation of PDH in rat livers and a glucose lowering effect in Zucker fatty rats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondrial dysfunction is considered one of the major pathogenic mechanisms of sepsis-induced cardiomyopathy (SIC). Pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of mitochondrial metabolism, is essential for maintaining mitochondrial function. However, its specific role in SIC remains unclear. To investigate this, we established an in vitro model of septic cardiomyopathy using lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Our study revealed a significant increase in PDK4 expression in LPS-treated H9C2 cardiomyocytes. Inhibiting PDK4 with dichloroacetic acid (DCA) improved cell survival, reduced intracellular lipid accumulation and calcium overload, and restored mitochondrial structure and respiratory capacity while decreasing lactate accumulation. Similarly, Oxamate, a lactate dehydrogenase inhibitor, exhibited similar effects to DCA in LPS-treated H9C2 cardiomyocytes. To further validate whether PDK4 causes cardiomyocyte and mitochondrial damage in SIC by promoting lactate production, we upregulated PDK4 expression using PDK4-overexpressing lentivirus in H9C2 cardiomyocytes. This resulted in elevated lactate levels, impaired mitochondrial structure, and reduced mitochondrial respiratory capacity. However, inhibiting lactate production reversed the mitochondrial dysfunction caused by PDK4 upregulation. In conclusion, our study highlights the pathogenic role of PDK4 in LPS-induced cardiomyocyte and mitochondrial damage by promoting lactate production. Therefore, targeting PDK4 and its downstream product lactate may serve as promising therapeutic approaches for treating SIC., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Camptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest., Aim of the Study: This study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs., Methods: The cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2's antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model., Results: FLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects., Conclusion: The study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Backgruound: Reactive oxygen species (ROS) and inflammation are reported to have a fundamental role in the pathogenesis of ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury. The present study investigated the role of pyruvate dehydrogenase kinase 4 (PDK4) in ROS production and inflammation following IR injury., Methods: We used a streptozotocin-induced diabetic C57BL6/J mouse model, which was subjected to IR by clamping both renal pedicles. Cellular apoptosis and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia work station., Results: Following IR injury in diabetic mice, the expression of PDK4, rather than the other PDK isoforms, was induced with a marked increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This was accompanied by a pronounced ROS activation, as well as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) production. Notably, sodium dichloroacetate (DCA) attenuated renal IR injury-induced apoptosis which can be attributed to reducing PDK4 expression and PDHE1α phosphorylation levels. DCA or shPdk4 treatment reduced oxidative stress and decreased TNF-α, IL-6, IL-1β, and MCP-1 production after IR or hypoxia-reoxygenation injury., Conclusion: PDK4 inhibition alleviated renal injury with decreased ROS production and inflammation, supporting a critical role for PDK4 in IR mediated damage. This result indicates another potential target for reno-protection during IR injury; accordingly, the role of PDK4 inhibition needs to be comprehensively elucidated in terms of mitochondrial function during renal IR injury.

Many cancer cells exhibit enhanced glycolysis, which is seen as one of the hallmark metabolic alterations, known as Warburg effect. Substantial evidence shows that upregulated glycolytic enzymes are often linked to malignant growth. Using glycolytic inhibitors for anticancer treatment has become appealing in recent years for therapeutic intervention in cancers with highly glycolytic characteristic, including non-small cell lung cancer (NSCLC). In this work, we studied the anticancer effects and the underlying mechanisms of combination of benzerazide hydrocholoride (Benz), a hexokinase 2 (HK2) inhibitor and 64, a pyruvate dehydrogenase kinase 1 (PDK1) inhibitor, in several NSCLC cell lines. We found that combination of Benz and 64 exhibited strong synergistic anticancer effects in NCI-H1975, HCC827, NCI-H1299 and SK-LU-1 cell lines. With this combination treatment, we observed changes of certain mechanistic determinants associated with metabolic stress caused by glycolysis restriction, such as mitochondrial membrane potential depolarization, overproduction of reactive oxygen species [1], activation of AMPK and down-regulation of mTOR, which contributed to enhanced apoptosis. Moreover, Benz and 64 together significantly suppressed the tumor growth in HCC827 cell mouse xenograft model. Taken together, our study may suggest that combined inhibition of HK2 and PDK1 using Benz and 64 could be a viable anticancer strategy for NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Lung adenocarcinoma (LUAD) is one of the most prevalent and aggressive types of lung cancer. Metabolic reprogramming plays a critical role in the development and progression of LUAD. Pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA) are two key enzymes involved in glucose metabolism, whilst their aberrant expressions are often associated with tumorigenesis. Herein, we investigated the anticancer effects of combined inhibition of PDK1 and LDHA in LUAD in vitro and in vivo and its underlying mechanisms of action. The combination of a PDK1 inhibitor, 64, and a LDHA inhibitor, NHI-Glc-2, led to a synergistic growth inhibition in 3 different LUAD cell lines and more than additively suppressed tumor growth in the LUAD xenograft H1975 model. This combination also inhibited cellular migration and colony formation, while it induced a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) resulting in mitochondrial depolarization and apoptosis in LUAD cells. These effects were related to modulation of multiple cell signaling pathways, including AMPK, RAS/ERK, and AKT/mTOR. Our findings demonstrate that simultaneous inhibition of multiple glycolytic enzymes (PDK1 and LDHA) is a promising novel therapeutic approach for LUAD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas.

We have previously demonstrated a high antitumor potential of NOS inhibitor T1023 (1-isobutanoyl-2-isopropylisothiourea hydrobromide): antitumor antiangiogenic activity in several animal tumor models and its ability to synergistically enhance the antitumor effects of bevacizumab, cyclophosphamide and γ-radiation. At the same time, rather rapid adaptation of experimental neoplasias to T1023 treatment was often observed. We attempted to enhance the antitumor activity of this NOS inhibitor by supplementing its molecular structure with a PDK-inhibiting fragment, dichloroacetate (DCA), which is capable of hypoxia-oriented toxic effects. We synthesized compound T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). Its toxic properties, NOS-inhibiting and PDK-inhibiting activity in vivo, and antitumor activity on the mouse Ehrlich carcinoma model (SEC) were investigated in compare with T1023 and Na-DCA. We found that the change of the salt-forming acid from HBr to DCA does not increase the toxicity of 1-isobutanoyl-2-isopropylisothiourea salts, but significantly expands the biochemical and anti-tumor activity. New compound T1084 realizes in vivo NOS-inhibiting and PDK-inhibiting activity, quantitatively, at the level of the previous compounds, T1023 and Na-DCA. In two independent experiments on SEC model, a pronounced synergistic antitumor effect of T1084 was observed in compare with T1023 and Na-DCA at equimolar doses. There were no signs of SEC adaptation to T1084 treatment, while experimental neoplasia rapidly desensitized to the separate treatment of both T1023 and Na-DCA. The totality of the data obtained indicates that the combination of antiangiogenic and hypoxia-oriented toxic effects (in this case, within the molecular structure of the active substance) can increase the antitumor effect and suppress the development of hypoxic resistance of neoplasias. In general, the proposed approach can be used for the design of new anticancer agents.

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 μM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM., Competing Interests: Declaration of competing interest Yawara Kawano reports a relationship with Janssen Pharmaceuticals Inc that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Takeda Pharmaceutical Co Ltd that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Sanofi that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Bristol Myers Squibb Co that includes: speaking and lecture fees. Yawara Kawano reports a relationship with Ono Pharmaceutical Co Ltd that includes: speaking and lecture fees., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel strategy to treat Ewing sarcoma. The most potent compound 12 is suitable for progression to in vivo studies. The specific attributes of 12 included nanomolar inhibitory potency against both phosphoinositide-dependent kinase-1 (PDK1) and Aurora A (AurA) kinase, with acceptable in vitro ADME-Tox properties (cytotoxicity in 2 healthy and 14 hematological and solid cancer cell-lines; inhibition of PDE4C1, SIRT7, HDAC4, HDAC6, HDAC8, HDAC9, AurB, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and hERG). X-ray crystallography and docking studies led to the identification of the key AurA and PDK1/12 interactions. Finally, in vitro drug-intake kinetics and in vivo PK appear to indicate that these compounds are attractive lead-structures for the design and synthesis of PDK1/AurA dual-target molecules to further investigate the in vivo efficacy against Ewing Sarcoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Objective: To study the effect of miR-138 on the function of osteosarcoma (OS) T follicular helper cells (Tfh cells) and its mechanism., Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with osteosarcoma (OS group) and healthy volunteers (control group). CD4+CXCR5+ Tfh cells and CD9+ B cells were sorted by flow cytometry. qRT-PCR was used to detect the expression of miR-138 and PDK1 in the peripheral blood and CD4+CXCR5+ Tfh cells. Flow cytometry was employed to detect the proportion of CD4+CXCR5+ Tfh cells in CD4+ T cells, the level of CD40L in CD4+CXCR5+ Tfh cells, and the expression of CD27 and CD38 in B cells. Western blot was used to determine the protein expression of PDK1, PI3K, p-Akt, Akt, p-mTOR, and mTOR. In addition, dual-luciferase reporter assay was performed to verify the relationship between miR-138 and PDK1. ELISA method was used to determine the levels of IgM, IgG, IL-10, and IL-21., Results: Compared with that of the control group, the expression of miR-138 in PBMC and CD4+CXCR5+ Tfh cells of the OS group was lower; overexpression of miR-138 could promote the maturation of Tfh cells and immature B cells. The results of the dual-luciferase report experiment showed that miR-138 can target and negatively regulate PDK1, and PDK1 can reverse the effect of miR-138 on the function of Tfh cells and immature B cells., Conclusion: miR-138 inhibits the PI3K/Akt/mTOR pathway by targeting and negatively regulating PDK1 to alleviate the dysfunction of T follicular helper cells in OS., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2021 Baoen Jiang et al.)

Metabolic reprogramming has been recognized as an essential emerging cancer hallmark. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been reported to have anti-cancer effects by reversing tumor-associated glycolysis. This study was performed to explore the anti-cancer potential of DCA in lung cancer alone and in combination with chemo- and targeted therapies using two non-small cell lung cancer (NSCLC) cell lines, namely, A549 and LNM35. DCA markedly caused a concentration- and time-dependent decrease in the viability and colony growth of A549 and LNM35 cells in vitro. DCA also reduced the growth of tumor xenografts in both a chick embryo chorioallantoic membrane and nude mice models in vivo. Furthermore, DCA decreased the angiogenic capacity of human umbilical vein endothelial cells in vitro. On the other hand, DCA did not inhibit the in vitro cellular migration and invasion and the in vivo incidence and growth of axillary lymph nodes metastases in nude mice. Treatment with DCA did not show any toxicity in chick embryos and nude mice. Finally, we demonstrated that DCA significantly enhanced the anti-cancer effect of cisplatin in LNM35. In addition, the combination of DCA with gefitinib or erlotinib leads to additive effects on the inhibition of LNM35 colony growth after seven days of treatment and to synergistic effects on the inhibition of A549 colony growth after 14 days of treatment. Collectively, this study demonstrates that DCA is a safe and promising therapeutic agent for lung cancer.

Ovarian clear cell carcinoma (CCC) exhibits an association with endometriosis, resistance to oxidative stress, and poor prognosis owing to its resistance to conventional platinum-based chemotherapy. A greater understanding of the molecular characteristics and pathogenesis of ovarian cancer subtypes may facilitate the development of targeted therapeutic strategies, although the mechanism of drug resistance in ovarian CCC has yet to be determined. In this study, we assessed exome sequencing data to identify new therapeutic targets of mitochondrial function in ovarian CCC because of the central role of mitochondria in redox homeostasis. Copy number analyses revealed that chromosome 17q21-24 (chr.17q21-24) amplification was associated with recurrence in ovarian CCC. Cell viability assays identified an association between cisplatin resistance and chr.17q21-24 amplification, and mitochondrion-related genes were enriched in patients with chr.17q21-24 amplification. Patients with high expression of pyruvate dehydrogenase kinase 2 (PDK2) had a worse prognosis than those with low PDK2 expression. Furthermore, inhibition of PDK2 synergistically enhanced cisplatin sensitivity by activating the electron transport chain and by increasing the production of mitochondrial reactive oxygen species. Mouse xenograft models showed that inhibition of PDK2 with cisplatin inhibited tumor growth. This evidence suggests that targeting mitochondrial metabolism and redox homeostasis is an attractive therapeutic strategy for improving drug sensitivity in ovarian CCC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background : Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods : Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results : Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell's epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions : Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1β and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications., (© 2021 Wiley-VCH GmbH.)

Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium ( Stm ) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) - which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) - on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm , but interestingly not Mycobacterium tuberculosis , in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Doorn, Schouten, van Veen, Walburg, Esselink, Heemskerk, Vrieling and Ottenhoff.)

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.

Pyruvate is irreversibly decarboxylated to acetyl coenzyme A by mitochondrial pyruvate dehydrogenase complex (PDC). Decarboxylation of pyruvate is considered a crucial step in cell metabolism and energetics. The cancer cells prefer aerobic glycolysis rather than mitochondrial oxidation of pyruvate. This attribute of cancer cells allows them to sustain under indefinite proliferation and growth. Pyruvate dehydrogenase kinases (PDKs) play critical roles in many diseases because they regulate PDC activity. Recent findings suggest an altered metabolism of cancer cells is associated with impaired mitochondrial function due to PDC inhibition. PDKs inhibit the PDC activity via phosphorylation of the E1a subunit and subsequently cause a glycolytic shift. Thus, inhibition of PDK is an attractive strategy in anticancer therapy. This review highlights that PDC/PDK axis could be implicated in cancer's therapeutic management by developing potential small-molecule PDK inhibitors. In recent years, a dramatic increase in the targeting of the PDC/PDK axis for cancer treatment gained an attention from the scientific community. We further discuss breakthrough findings in the PDC-PDK axis. In addition, structural features, functional significance, mechanism of activation, involvement in various human pathologies, and expression of different forms of PDKs (PDK1-4) in different types of cancers are discussed in detail. We further emphasized the gene expression profiling of PDKs in cancer patients to prognosis and therapeutic manifestations. Additionally, inhibition of the PDK/PDC axis by small molecule inhibitors and natural compounds at different clinical evaluation stages has also been discussed comprehensively., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Danhong injection (DHI) is a compound Chinese medicine widely used in China for treatment of ischemic cardio-cerebrovascular diseases. However, limited data are available regarding the protective effect of DHI on the ischemic penumbra in ischemic stroke. This study aimed to investigate the effect of intravenous DHI on neuronal injure in the ischemic penumbra after cerebral ischemia/reperfusion (CI/R), focusing especially on the involvement of intracellular energy metabolism coupling. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion for 60 min followed by reperfusion with or without intravenous DHI (0.5, 1.0, or 2.0 mL/kg) once daily for 7 days. Post-treatment with DHI ameliorated neurological defects, diminished cerebral infarction, alleviated cerebral edema, improved microcirculatory perfusion after 7days of reperfusion, and inhibited apoptosis and enhanced neuronal survival in the ischemic penumbra. In addition, DHI significantly ameliorated oxidative stress, reduced DNA damage, and inhibited the activation of PARP1/AIF pathway, thereby restoring cytoplasmic glycolytic activity. Furthermore, this drug increased PDH activity by inhibiting the HIF1α/PDK1 signaling pathway, thus eliminating the inhibitory effect of CI/R on mitochondrial metabolism. The results of this study suggest that DHI can alleviate cerebral edema after CI/R and rescue the ischemic penumbra, and these protective effects are due to the regulation of intracellular energy metabolic coupling., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The phosphorylation of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase (PDK) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is upregulated in various types of human cancer. Because PDK4 regulation is critical for metabolic changes in cancer cells, it is an attractive target for cancer therapy given its ability to shift glucose metabolism. It was previously shown that a novel PDK4 inhibitor, cryptotanshinone (CPT), suppressed the three‑dimensional (3D)‑spheroid formation of pancreatic and colorectal cancer cells. In the present study, the effects of CPT on the invasiveness of bladder cancer cells were investigated. CPT significantly suppressed the invasiveness and 3D‑spheroid formation of T24 and J82 bladder cancer cells. CPT also suppressed the phosphorylation of PDH and β‑catenin, as well as the expression of N‑cadherin, which are all critical for inducing epithelial‑mesenchymal transition (EMT). The knockdown of β‑catenin or PDK4 using specific small interfering RNAs suppressed N‑cadherin expression and invasiveness in T24 cells. An mTOR inhibitor also suppressed the phosphorylation of β‑catenin and N‑cadherin expression. Furthermore, CPT injection significantly suppressed pancreatic tumor growth and peritoneal dissemination of highly metastatic SUIT‑2 pancreatic cancer cells in a mouse orthotopic pancreatic cancer model, without evident toxicity. Moreover, immunohistochemistry analyses demonstrated decreased β‑catenin expression in CPT‑treated pancreatic tumors compared with control tumors. Taken together, these results indicate that CPT reduced the invasiveness and metastasis of bladder cancer cells by suppressing EMT via the mTOR/β‑catenin/N‑cadherin pathway.

Influenza A virus (IAV) infection alters lung epithelial cell metabolism in vitro by promoting a glycolytic shift. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. A/WSN/33 IAV-inoculated C57BL/6 mice were treated daily from 1 day post-inoculation (d.p.i.) with 2-deoxy-d-glucose (2-DG) to inhibit glycolysis and with the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) to promote flux through the TCA cycle. To block OXPHOS, mice were treated every other day from 1 d.p.i. with the Complex I inhibitor rotenone (ROT). 2-DG significantly decreased nocturnal activity, reduced respiratory exchange ratios, worsened hypoxemia, exacerbated lung dysfunction, and increased humoral inflammation at 6 d.p.i. DCA and ROT treatment normalized oxygenation and airway resistance and attenuated IAV-induced pulmonary edema, histopathology, and nitrotyrosine formation. None of the treatments altered viral replication. These data suggest that a shift to glycolysis is host-protective in influenza., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis., (© 2021 Wiley Periodicals LLC.)

Due to the lack of early diagnostic and effective treatment modalities, hepatocellular carcinoma (HCC) is still the most lethal cancer with a high mortality on a global scale. Recent studies have highlighted the key roles of microRNAs (miRs) in HCC development. In the study, we attempted to investigate the potential role of miR-9-5p in the progression of HCC. Expression of pyruvate dehydrogenase kinase 4 (PDK4) and miR-9-5p was examined in HCC tissues collected from HCC patients and cell lines. The proliferation, migration, invasion, and apoptosis of HCC cells, and levels of oxygen consumption rate, extracellular acidification rate and reactive oxygen species (ROS) as well as the tumorigenicity of transfected cells in vivo were measured after gain- and loss-of-function experiments in HCC cells. It was revealed that miR-9-5p was upregulated, while PDK4 was poorly expressed in HCC tissues and cells, associating with a poor prognosis of HCC patients. miR-9-5p directly targeted PDK4 and could downregulate its expression, thus leading to promoted cell proliferation, invasion and migration, enhanced mitochondrial activity and energy metabolism, and suppressed apoptosis in HCC cells, along with increased tumorigenicity in mouse xenograft models. Altogether, miR-9-5p facilitated mitochondrial energy metabolism of HCC cells by downregulating PDK4, promoting the development of HCC. miR-9-5p and PDK4 may serve as potential therapeutic targets for preventing recurrence and metastasis of HCC.

Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis enabled us to identify a potent compound 6u, which inhibited PDKs with an EC 50 value of 0.09 μM, and reduced various cancer cells proliferation with IC 50 values ranging from 1.1 to 3.8 μM, while show weak effect against non-cancerous L02 cell (IC 50  > 10 μM). In the A375 xenograft model, 6u displayed an obvious antitumor activity at a dose of 5 mg/kg, but with no negative effect to the mice weight. Molecular docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375 cell apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose metabolic pathway in A375 cell by decreasing lactate formation and increasing ROS production and OCR consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Metabolic reprogramming between resistance and tolerance occurs within the immune system in response to sepsis. While metabolic tissues such as the liver are subjected to damage during sepsis, how their metabolic and energy reprogramming ensures survival is unclear. Employing comprehensive metabolomic, lipidomic, and transcriptional profiling in a mouse model of sepsis, we show that hepatocyte lipid metabolism, mitochondrial tricarboxylic acid (TCA) energetics, and redox balance are significantly reprogrammed after cecal ligation and puncture (CLP). We identify increases in TCA cycle metabolites citrate, cis-aconitate, and itaconate with reduced fumarate and triglyceride accumulation in septic hepatocytes. Transcriptomic analysis of liver tissue supports and extends the hepatocyte findings. Strikingly, the administration of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate reverses dysregulated hepatocyte metabolism and mitochondrial dysfunction. In summary, our data indicate that sepsis promotes hepatic metabolic dysfunction and that targeting the mitochondrial PDC/PDK energy homeostat rebalances transcriptional and metabolic manifestations of sepsis within the liver., Competing Interests: RM, MZ, DL, NB, EQ, CK, XZ, JP, CF, PS, JM, CM, MQ No competing interests declared

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo . Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δ ψ ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δ ψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii , is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii 's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases ( T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA. IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii , which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii , followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies., (Copyright © 2021 Ferrarini et al.)

PDK1 (3-Phosphoinositide dependent protein kinase-1) is a member in the PI3K (phosphatidylinositol 3 kinase) pathway and is implicated in neurodevelopmental disease with microcephaly. Although the role of PDK1 in neurogenesis has been broadly studied, it remains unknown how PDK1 may regulate oligogenesis in the central nervous system (CNS). To address this question, we generated oligodendrocyte (OL) lineage cells specific PDK1 conditional knockout (cKO) mice. We find that PDK1 cKOs display abnormal white matter (WM), massive loss of mature OLs and severe defect in myelination in the CNS. In contrast, these mutants exhibit normal neuronal development and unchanged apoptosis in the CNS. We demonstrate that deletion of PDK1 severely impairs OL differentiation. We show that genetic or pharmacological inhibition of PDK1 causes deficit in the mammalian target of rapamycin (mTor) signaling and down-regulation of Sox10. Together, these results highlight a critical role of PDK1 in OL differentiation during postnatal CNS development., (Copyright © 2020. Published by Elsevier Inc.)

Most cancer cells primarily produce their energy through a high rate of glycolysis followed by lactic acid fermentation even in the presence of abundant oxygen. Pyruvate dehydrogenase kinase (PDK) 1, an enzyme responsible for aerobic glycolysis via phosphorylating and inactivating pyruvate dehydrogenase (PDH) complex, is commonly overexpressed in tumors and recognized as a therapeutic target in colorectal cancer. Hemistepsin A (HsA) is a sesquiterpene lactone isolated from Hemistepta lyrata Bunge (Compositae). Here, we report that HsA is a PDK1 inhibitor can reduce the growth of colorectal cancer and consequent activation of mitochondrial ROS-dependent apoptotic pathway both in vivo and in vitro. Computational simulation and biochemical assays showed that HsA directly binds to the lipoamide-binding site of PDK1, and subsequently inhibits the interaction of PDK1 with the E2 subunit of PDH complex. As a result of PDK1 inhibition, lactate production was decreased, but oxygen consumption was increased. Mitochondrial ROS levels and mitochondrial damage were also increased. Consistent with these observations, the apoptosis of colorectal cancer cells was promoted by HsA with enhanced activation of caspase-3 and -9. These results suggested that HsA might be a potential candidate for developing a novel anti-cancer drug through suppressing cancer metabolism.

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in aging populations. Here, we applied metabolomics to human sera of patients with nAMD during an active (exudative) phase of the pathology and found higher lactate levels and a shift in the lipoprotein profile (increased VLDL-LDL/HDL ratio). Similar metabolomics changes were detected in the sera of mice subjected to laser-induced choroidal neovascularization (CNV). In this experimental model, we provide evidence for two sites of lactate production: first, a local one in the injured eye, and second a systemic site associated with the recruitment of bone marrow-derived inflammatory cells. Mechanistically, lactate promotes the angiogenic response and M2-like macrophage accumulation in the eyes. The therapeutic potential of our findings is demonstrated by the pharmacological control of lactate levels through pyruvate dehydrogenase kinase (PDK) inhibition by dichloroacetic acid (DCA). Mice treated with DCA exhibited normalized lactate levels and lipoprotein profiles, and inhibited CNV formation. Collectively, our findings implicate the key role of the PDK/lactate axis in AMD pathogenesis and reveal that the regulation of PDK activity has potential therapeutic value in this ocular disease. The results indicate that the lipoprotein profile is a traceable pattern that is worth considering for patient follow-up. KEY MESSAGES: Lactate and lipoprotein profile are associated with the active phase of AMD and CNV development. Lactate is a relevant and functional metabolite correlated with AMD progression. Modulating lactate through pyruvate dehydrogenase kinase led to a decrease of CNV progression. Pyruvate dehydrogenase kinase is a new therapeutic target for neovascular AMD.

Objective: Despite recent advances in treatment options, pancreatic cancer remains the most deadly major cancer. Targeting metabolism represents an emerging anti-cancer strategy., Results: Metagenomic 16S analysis was employed to explore the effect of Dichloroacetate (DCA) on the composition of the fecal microbiota and metabolomic profile was assessed on in vivo pancreatic cancer mouse xenograft model. Pancreatic cancer xenograft mice displayed a shift of microbiota' profile as compared to control mice without DCA treatment and a significant decrease of the purine bases inosine xanthine together with their metabolically-related compound hypoxanthine were observed in the DCA treated group as compared to the control group. Two aminoacids methionine and aspartic acid resulted decreased and increased respectively. DCA affects tumor environment and studies are needed in order to understand whether DCA supplementation could be supportive as synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

Most cancer cells feature an altered glucose metabolism from oxidative phosphorylation to cytoplasmic glycolysis. Pyruvate dehydrogenase kinases (PDKs) and lactate dehydrogenase A (LDHA) play crucial roles in promotion of glycolysis, thus the inhibition of both enzymes is considered a promising strategy for developing of anticancer therapeutics. Herein, we describe the first discovery of series novel dual inhibitors targeting PDKs and LDHA. We identified 6 hits from a library database containing 485465 compounds through a high-throughput virtual screening assay. Hit-to-lead optimization enabled us to discover two compounds, namely 20e and 20k, which inhibited PDKs with IC 50 values of 0.8, and 1.6 μM, respectively, and inhibited LDHA with IC 50 values of 0.15 and 0.7 μM, respectively. Meanwhile, the two compounds reduced A549 cell proliferation with EC 50 values of 13.2, and 15.7 μM. Furthermore, 20e and 20k decreased the lactate formation, and increased oxygen consumption, suggesting the two compounds modulated the glucose metabolic pathways in cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Budding at the tumor invasive front has been correlated with the malignant properties of many cancers. Malic enzyme 1 (ME1) promotes the Warburg effect in cancer cells and induces epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC). Therefore, we investigated the role of ME1 in tumor budding in OSCC. Tumor budding was measured in 96 human OSCCs by immunostaining for an epithelial marker (AE1/AE3), and its expression was compared with that of ME1. A significant correlation was observed between tumor budding and ME1 expression. The correlation increased with the progression of cancer. In human OSCC cells, lactate secretion decreased when lactate fermentation was suppressed by knockdown of ME1 and lactate dehydrogenase A or inhibition of pyruvate dehydrogenase (PDH) kinase. Furthermore, the extracellular pH increased, and the EMT phenotype was suppressed. In contrast, when oxidative phosphorylation was suppressed by PDH knockdown, lactate secretion increased, extracellular pH decreased, and the EMT phenotype was promoted. Induction of chemical hypoxia in OSCC cells by CoCl 2 treatment resulted in increased ME1 expression along with HIF1α expression and promotion of the EMT phenotype. Hypoxic conditions also increased matrix metalloproteinases expression and decreased mitochondrial membrane potential, mitochondrial oxidative stress, and extracellular pH. Furthermore, the hypoxic treatment resulted in the activation of Yes-associated protein (YAP), which was abolished by ME1 knockdown. These findings suggest that cancer cells at the tumor front in hypoxic environments increase their lactate secretion by switching their energy metabolism from oxidative phosphorylation to glycolysis owing to ME1 overexpression, decrease in extracellular pH, and YAP activation. These alterations enhance EMT and the subsequent tumor budding. Tumor budding and ME1 expression are thus considered useful markers of OSCC malignancy, and ME1 is expected to be a relevant target for molecular therapy.

Th2 cytokine such as IL-4, IL -5 and IL-13 are important therapeutic targets for Th2-type chronic inflammation. Several biologics targeting Th2 cytokine and its receptors are effective in clinical practice; however, the development of small-molecule compounds that inhibit Th2 cytokine productions is awaited. We found that an inhibitor for pyruvate dehydrogenase kinase (PDHK) suppresses the differentiation of IL-5/IL-13-producing Th2 cells. The expression of the Th2-related transcriptional factors Pparγ was decreased by treatment with inhibitor, whereas Gata3, a master regulator of Th2 cell differentiation, remained unchanged. The oxygen consumption rate was unaffected, whereas the level of farnesylated proteins was decreased by the PDHK inhibitor. Furthermore, the inhibitors for farnesyltransferase and hydroxymethylglutaryl-CoA reductase showed an inhibitory effect similar to that of the PDHK inhibitor. These results suggest that the mevalonate biosynthesis and subsequent protein prenylation may be novel therapeutic target for Th2 cell-dependent immune dysregulation, such as in allergic diseases., Competing Interests: Declaration of competing interest All authors declare no financial relationships with other people or organizations that could inappropriately influence our work., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The Warburg effect is a unique metabolic feature of the majority of tumor cells and is closely related to chemotherapeutic resistance. Pyruvate dehydrogenase kinase 1 (PDK1) is considered a 'switch' that controls the fate of pyruvate in glucose metabolism. However, to date, to the best of our knowledge, there are only a few studies to available which had studied the reduction of chemotherapeutic resistance via the metabolic reprogramming of tumor cells with PDK1 as a target. In the present study, it was found dicoumarol (DIC) reduced the phosphorylation of pyruvate dehydrogenase (PDH) by inhibiting the activity of PDK1, which converted the metabolism of human hepatocellular carcinoma (HCC) cells to oxidative phosphorylation, leading to an increase in mitochondrial reactive oxygen species ROS (mtROS) and a decrease in mitochondrial membrane potential (MMP), thereby increasing the apoptosis induced by oxaliplatin (OXA). Furthermore, the present study elucidated that the targeting of PDK1 may be a potential strategy for targeting metabolism in the chemotherapy of HCC. In addition, DIC as an 'old drug' exhibits novel efficacy, bringing new hope for antitumor therapy.

In cancer cells, aerobic glycolysis rather than oxidative phosphorylation (OxPhos) is generally preferred for the production of ATP. In many cancers, highly expressed pyruvate dehydrogenase kinase 1 (PDK1) reduces the activity of pyruvate dehydrogenase (PDH) by inducing the phosphorylation of its E1α subunit (PDHA1) and subsequently, shifts the energy metabolism from OxPhos to aerobic glycolysis. Thus, PDK1 has been regarded as a target for anticancer treatment. Here, we report that ilimaquinone (IQ), a sesquiterpene quinone isolated from the marine sponge Smenospongia cerebriformis , might be a novel PDK1 inhibitor. IQ decreased the cell viability of human and murine cancer cells, such as A549, DLD-1, RKO, and LLC cells. The phosphorylation of PDHA1, the substrate of PDK1, was reduced by IQ in the A549 cells. IQ decreased the levels of secretory lactate and increased oxygen consumption. The anticancer effect of IQ was markedly reduced in PDHA1-knockout cells. Computational simulation and biochemical assay revealed that IQ interfered with the ATP binding pocket of PDK1 without affecting the interaction of PDK1 and the E2 subunit of the PDH complex. In addition, similar to other pyruvate dehydrogenase kinase inhibitors, IQ induced the generation of mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in the A549 cells. The apoptotic cell death induced by IQ treatment was rescued in the presence of MitoTEMPO, a mitochondrial ROS inhibitor. In conclusion, we suggest that IQ might be a novel candidate for anticancer therapeutics that act via the inhibition of PDK1 activity.

Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteosarcoma is a common malignant bone cancer easily to metastasize. Much safer and more efficient strategies are still needed to suppress osteosarcoma growth and lung metastasis. We recently presented a pure physical method to fabricate Ångstrom-scale silver particles (AgÅPs) and determined the anti-tumor efficacy of fructose-coated AgÅPs (F-AgÅPs) against lung and pancreatic cancer. Our study utilized an optimized method to obtain smaller F-AgÅPs and aimed to assess whether F-AgÅPs can be used as an efficient and safe agent for osteosarcoma therapy. We also investigated whether the induction of apoptosis by altering glucose metabolic phenotype contributes to the F-AgÅPs-induced anti-osteosarcoma effects. Methods: A modified method was developed to prepare smaller F-AgÅPs. The anti-tumor, anti-metastatic and pro-survival efficacy of F-AgÅPs and their toxicities on healthy tissues were compared with that of cisplatin (a first-line chemotherapeutic drug for osteosarcoma therapy) in subcutaneous or orthotopic osteosarcoma-bearing nude mice. The pharmacokinetics, biodistribution and excretion of F-AgÅPs were evaluated by testing the levels of silver in serum, tissues, urine and feces of mice. A series of assays in vitro were conducted to assess whether the induction of apoptosis mediates the killing effects of F-AgÅPs on osteosarcoma cells and whether the alteration of glucose metabolic phenotype contributes to F-AgÅPs-induced apoptosis. Results: The newly obtained F-AgÅPs (9.38 ± 4.11 nm) had good stability in different biological media or aqueous solutions and were more effective than cisplatin in inhibiting tumor growth, improving survival, attenuating osteolysis and preventing lung metastasis in osteosarcoma-bearing nude mice after intravenous injection, but were well tolerated in normal tissues. One week after injection, about 68% of F-AgÅPs were excreted through feces. F-AgÅPs induced reactive oxygen species (ROS)-dependent apoptosis of osteosarcoma cells but not normal cells, owing to their ability to selectively shift glucose metabolism of osteosarcoma cells from glycolysis to mitochondrial oxidation by inhibiting pyruvate dehydrogenase kinase (PDK). Conclusion: Our study suggests the promising prospect of F-AgÅPs as a powerful selective anticancer agent for osteosarcoma therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The mechanistic contributions of cancer-associated fibroblasts (CAFs) in breast cancer progression remain to be fully understood. While altered glucose metabolism in CAFs could fuel cancer cells, how such metabolic reprogramming emerges and is sustained needs further investigation. Studying fibroblasts isolated from patients with benign breast tissues and breast cancer, in conjunction with multiple animal models, we demonstrate that CAFs exhibit a metabolic shift toward lactate and pyruvate production and fuel biosynthetic pathways of cancer cells. The depletion or suppression of the lactate production of CAFs alter the tumor metabolic profile and impede tumor growth. The glycolytic phenotype of the CAFs is in part sustained through epigenetic reprogramming of HIF-1α and glycolytic enzymes. Hypoxia induces epigenetic reprogramming of normal fibroblasts, resulting in a pro-glycolytic, CAF-like transcriptome. Our findings suggest that the glucose metabolism of CAFs evolves during tumor progression, and their breast cancer-promoting phenotype is partly mediated by oxygen-dependent epigenetic modifications., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells., Methods: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-Kras G12D , LSL-Kras G12D , and LSL-Hras G12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition., Results: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30 + chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl + clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion., Conclusions: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

We aimed to assess the neuroprotective effect of a pyruvate dehydrogenase kinase (PDK) inhibitor, Nov3r after ischemia/reperfusion (IR) injury in rats. IR injury was induced by applying 150 mmHg of intraocular pressure for 50 min. Nov3r was orally administered (100 mg/kg) 3 h before and 24 h after IR injury. TUNEL-positive cells increased and immunoreactive RBPMS-positive cells decreased in the rat retinas after IR injury. Administration of Nov3r significantly ameliorated the increase in TUNEL-positive cells and prevented the RBPMS-positive cell decrease. Similarly, the number of IR-induced Iba1-positive microglial cells was significantly reduced with Nov3r treatment. Among metabolic parameters, IR damage induced the elevation of lactate and pyruvate, and the reduction of ATP. Oral administration of Nov3r ameliorated these changes. Our data suggest that the Nov3r had a retinal neuroprotective effect in IR injury in rats. This finding suggests that the regulation of pyruvate dehydrogenase (PDH) activity has potential therapeutic value by enabling metabolic reprograming in diseases associated with ischemic retinal damage, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ischemic optic neuropathy and glaucoma., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice., Methods: We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1 Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1 Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay., Results: Some intestinal crypts from Lkb1 Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1 Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile., Conclusions: LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The majority of cancers detected every year are treated with anti-cancer compounds. Unfortunately, many tumors become resistant to antineoplastic drugs. One option is to use cocktails of compounds acting on different targets to try to overcome the resistant cells. This type of approach can produce good results, but is often accompanied by a sharp increase of associated side effects. The strategy presented herein focuses on the use of a single compound acting on two different biological targets enhancing potency and lowering the toxicity of the chemotherapy. In this light, the approach presented in the current study involves the dual inhibition of human pyruvate dehydrogenase kinase-1 (PDHK1) and tubulin polymerization using mono-, di- and tri-chloroacetate-loaded benzophenones and benzothiophenones. Synthesized molecules were evaluated in vitro on tubulin polymerization and on pyruvate dehydrogenase kinase 1. The cell cycle distribution after treatment of DA1-3b leukemic cells with active compounds was tested. Twenty-two benzo(thio)phenones have been selected by the National Cancer Institute (USA) for evaluation of their anti-proliferative potential against NCI-60 cancer cell lines including multidrug-resistant tumor cell lines. Seventeen molecules proved to be very effective in combating the growth of tumor cells exhibiting inhibitory activities up to nanomolar range. The molecular docking of best antitumor molecules in the study was realized with GOLD in the tubulin and PDHK1 binding sites, and allowed to understand the positioning of active molecules. Chloroacetate-loaded benzo(thio)phenones are dual targeted tubulin- and pyruvate dehydrogenase kinase 1 (PDHK1)-binding antitumor agents and exhibited superior antitumor activity compared to non-chlorinated congeners particularly on leukemia, colon, melanoma and breast cancer cell lines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dichloroacetate (DCA) is a metabolic modulator that inhibits pyruvate dehydrogenase activity and promotes the influx of pyruvate into the tricarboxylic acid cycle for complete oxidation of glucose. DCA stimulates oxidative phosphorylation (OXPHOS) more than glycolysis by altering the morphology of the mitochondria and supports mitochondrial apoptosis. As a consequence, DCA induces apoptosis in cancer cells and inhibits the proliferation of cancer cells. Recently, the role of miRNAs has been reported in regulating gene expression at the transcriptional level and also in reprogramming energy metabolism. In this article, we indicate that DCA treatment leads to the upregulation of let-7a expression, but DCA-induced cancer cell death is independent of let-7a. We observed that the combined effect of DCA and let-7a induces apoptosis, reduces reactive oxygen species generation and autophagy, and stimulates mitochondrial biogenesis. This was later accompanied by stimulation of OXPHOS in combined treatment and was thus involved in metabolic reprogramming of MDA-MB-231 cells.

Background: 3-Phosphoinositide Dependent Protein Kinase-1 (PDK1) is being lately considered as an attractive and forthcoming anticancer target. A Protein Data Bank (PDB) cocrystallized crystal provides not only rigid theoretical data but also a realistic molecular recognition data that can be explored and used to discover new hits., Objective: This incited us to investigate the co-crystallized ligands' contacts inside the PDK1 binding pocket via a structure-based receptor-ligand pharmacophore generation technique in Discovery Studio 4.5 (DS 4.5)., Methods: Accordingly, 35 crystals for PDK1 were collected and studied. Every single receptorligand interaction was validated and the significant ones were converted into their corresponding pharmacophoric features. The generated pharmacophores were scored by the Receiver Operating Characteristic (ROC) curve analysis., Results: Consequently, 169 pharmacophores were generated and sorted, 11 pharmacophores acquired good ROC-AUC results of 0.8 and a selectivity value above 8. Pharmacophore 1UU3_2_01 was used in particular as a searching filter to screen NCI database because of its acceptable validity criteria and its distinctive positive ionizable feature. Several low micromolar PDK1 inhibitors were revealed. The most potent hit illustrated anti-PDK1 IC50 values of 200 nM with 70% inhibition against SW480 cell lines., Conclusion: Eventually, the active hits were docked inside the PDK1 binding pocket and the recognition points between the active hits and the receptor were analyzed that led to the discovery of new scaffolds as potential PDK1 inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Phosphoinositide-Dependent Kinase 1 (PDK1) is now widely studied in malignant solid tumors. Researchers have previously revealed that targeting PDK1 is thought of as a promising anticancer treatment strategy. The aim of this study was designed to evaluate the anticancer activity of GSK-470, a novel and highly specific inhibitor of PDK1, in Pheochromocytoma (PCC) tumor model., Methods: PC12 cells were xenografted into nude mice to build PCC tumor model. Animals were treated with GSK-470 vs vehicle. Mean tumor volume was calculated and compared across groups. TUNEL was used to detect apoptosis. The effects of PDK1 inhibitor GSK-470 on activation of the Akt signaling and its downstream Akt/mTOR pathway in xenotransplant tumor tissues were examined by western bolt., Results: The mean tumor volume in GSK-470 group was significantly less than that in control group. TUNEL results found that cell apoptosis was markedly increased in GSK-470 group compared with the control group. The western bolt analysis showed that the phosphorylation of Akt at threonine 308 was significantly reduced in GSK-470 group. Also, GSK-470 strongly inhibited phosphorylation of mTOR on Ser2448, a marker for mTORC1 activity, as well as phosphorylation of p70S6K, best characterized targets of mTOR., Conclusion: Our results showed that GSK-470 exhibited potent anticancer activity in PC12 tumor-bearing mice. Also, we found that this effect appeared to be mediated by the inhibition of the Akt/mTOR pathway. The present study once again provides new insights into the therapeutic effects of inhibiting PDK1 in the treatment of malignant PCC. Therefore, we propose that GSK-470 might be an effective therapeutic agent for the treatment of malignant PCC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Combined chronic treatment of Ehrlich solid carcinoma (EC) with an NOS inhibitor 1-isobutanoyl-2-isopropylisothiourea hydrobromide (T1023) and a PDK1 inhibitor dichloroacetate was accompanied by statistically significant synergetic antitumor effects manifested in a significant and stable suppression of neoplasm growth (by 55-65%). Separate treatment with T1023 and dichloroacetate induced moderate short-term inhibition of tumor growth (by 30-35%) followed by weakening of tumor sensitivity to these substances. These results attest to synergetic antitumor effects NOS inhibitor T1023 and PDK1 inhibitor dichloroacetate producing antiangiogenic and hypoxia-targeted cytotoxic effects, during their combined administration, which allows overcoming the adaptive potential of the tumors.