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AbstractProfiling approaches have been increasingly employed for the characterization of disease‐relevant phenotypes or compound perturbation as they provide a broad, unbiased view on impaired cellular states. We report that morphological profiling using the cell painting assay (CPA) can detect modulators of de novo pyrimidine biosynthesis and of dihydroorotate dehydrogenase (DHODH) in particular. The CPA can differentiate between impairment of pyrimidine and folate metabolism, which both affect cellular nucleotide pools. The identified morphological signature is shared by inhibitors of DHODH and the functionally tightly coupled complex III of the mitochondrial respiratory chain as well as by UMP synthase, which is downstream of DHODH. The CPA appears to be particularly suited for the detection of DHODH inhibitors at the site of their action in cells. As DHODH is a validated therapeutic target, the CPA will enable unbiased identification of DHODH inhibitors and inhibitors of de novo pyrimidine biosynthesis for biological research and drug discovery. Show less

Computational modeling of inhibitors for metalloenzymes in virtual drug development campaigns has proven challenging. To overcome this limitation, a technique for predicting the binding pose of metal-binding pharmacophores (MBPs) is presented. Using a combination of density functional theory (DFT) calculations and docking using a genetic algorithm, inhibitor binding was evaluated in silico and compared with inhibitor-enzyme cocrystal structures. The predicted binding poses were found to be consistent with the cocrystal structures. The computational strategy presented represents a useful tool for predicting metalloenzyme-MBP interactions. Show less

Bioisosteres are a useful approach to address pharmacokinetic liabilities and improve drug-like properties. Specific to developing metalloenzyme inhibitors, metal-binding pharmacophores (MBPs) have been combined with bioisosteres, to produce metal-binding isosteres (MBIs) as alternative scaffolds for use in fragment-based drug discovery (FBDD). Picolinic acid MBIs have been reported and evaluated for their metal-binding ability, pharmacokinetic properties, and enzyme inhibitory activity. However, their structural, electronic, and spectroscopic properties with metal ions other than Zn(II) have not been reported, which might reveal similarities and differences between MBIs and the parent MBPs. To this end, [M(TPA)(MBI)]⁺ (M = Ni(II) and Co(II), TPA = tris(2-pyridylmethyl)amine) is presented as a bioinorganic model system for investigating picolinic acid, four heterocyclic MBIs, and 2,2'-bipyridine. These complexes were characterized by X-ray crystallography as well as NMR, IR, and UV-vis spectroscopies, and their magnetic moments were accessed. In addition, [(Tp^Ph,Me)Co(MBI)] (Tp^Ph,Me = hydrotris(3,5-phenylmethylpyrazolyl)borate) was used as a second model compound, and the limitations and attributes of the two model systems are discussed. These results demonstrate that bioinorganic model complexes are versatile tools for metalloenzyme inhibitor design and can provide insights into the broader use of MBIs. Show less

Advanced stages of cancer are highly associated with short overall survival in patients due to the lack of long-term treatment options following the standard form of care. New options for cancer therapy are needed to improve the survival of cancer patients without disease recurrence. Auranofin is a clinically approved agent against rheumatoid arthritis that is currently enrolled in clinical trials for potential repurposing against cancer. Auranofin mainly targets the anti-oxidative system catalyzed by thioredoxin reductase (TrxR), which protects the cell from oxidative stress and death in the cytoplasm Show less

Abstract It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the “slow” form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-β–D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2 by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand. Show less

Aberrant activation of the PI3K pathway is one of the commonest oncogenic events in human cancer. AKT is a key mediator of PI3K oncogenic function, and thus has been intensely pursued as a therapeutic target. Multiple AKT inhibitors, broadly classified as either ATP-competitive or allosteric, are currently in various stages of clinical development. Herein, we review the evidence for AKT dependence in human tumours and focus on its therapeutic targeting by the two drug classes. We highlight the future prospects for the development and implementation of more effective context-specific AKT inhibitors aided by our increasing knowledge of both its regulation and some previously unrecognised non-canonical functions. Show less

Entinostat is a synthetic benzamide derivative histone deacetylase (HDAC) inhibitor, which potently and selectively inhibits class I and IV HDAC enzymes. This action promotes histone hyperacetylation and transcriptional activation of specific genes, with subsequent inhibition of cell proliferation, terminal differentiation and apoptosis. This oral HDAC inhibitor has been evaluated in Phase I and II trials in patients with advanced malignancies, and is in general well tolerated. Entinostat does not currently have regulatory approval for clinical use; however promising preclinical and clinical data exist in hormone-resistant breast cancer. An ECOG-ACRIN Phase III registration study is ongoing in advanced breast cancer (E2112, NCT02115282) and aims to confirm the overall survival advantage observed with the combination of exemestane and entinostat/placebo in the Phase II setting (ENCORE301 trial). This article provides an overview of the chemistry, pharmacokinetics/pharmacodynamics and available clinical data for entinostat with a focus on advanced breast cancer. Show less

The enzyme cytochrome c oxidase (CcO) or complex IV (EC 1.9.3.1) is a large transmembrane protein complex that serves as the last enzyme in the respiratory electron transport chain of eukaryotic mitochondria. CcO promotes the switch from glycolytic to oxidative phosphorylation (OXPHOS) metabolism and has been associated with increased self-renewal characteristics in gliomas. Increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure, and patients with primary glioblastoma multiforme and high tumor CcO activity have worse clinical outcomes than those with low tumor CcO activity. Therefore, CcO is an attractive target for cancer therapy. We report here the characterization of a CcO inhibitor (ADDA 5) that was identified using a high throughput screening paradigm. ADDA 5 demonstrated specificity for CcO, with no inhibition of other mitochondrial complexes or other relevant enzymes, and biochemical characterization showed that this compound is a non-competitive inhibitor of cytochrome c When tested in cellular assays, ADDA 5 dose-dependently inhibited the proliferation of chemosensitive and chemoresistant glioma cells but did not display toxicity against non-cancer cells. Furthermore, treatment with ADDA 5 led to significant inhibition of tumor growth in flank xenograft mouse models. Importantly, ADDA 5 inhibited CcO activity and blocked cell proliferation and neurosphere formation in cultures of glioma stem cells, the cells implicated in tumor recurrence and resistance to therapy in patients with glioblastoma. In summary, we have identified ADDA 5 as a lead CcO inhibitor for further optimization as a novel approach for the treatment of glioblastoma and related cancers. Show less

Glucose metabolism has long been thought to operate with exquisite specificity and near-optimal efficiency. New findings show, however, that two glycolytic enzymes produce minor products that inhibit other enzymes involved in central carbon metabolism unless they are further metabolized by a novel enzyme. Show less

Aminotriazole (ATZ) is commonly used as a catalase (CAT) inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP) overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H2O2) levels, malondialdehyde (MDA) contents, myeloperoxidase (MPO) levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1) expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD50 of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation. Show less