Showing papers by "Peter Wipf published in 2019"

Ferroptosis as a Novel Therapeutic Target for Friedreich's Ataxia.

Abstract: Friedreich ataxia (FRDA) is a progressive neuro- and cardio-degenerative disorder characterized by ataxia, sensory loss, and hypertrophic cardiomyopathy. In most cases, the disorder is caused by GAA repeat expansions in the first introns of both alleles of the FXN gene, resulting in decreased expression of the encoded protein, frataxin. Frataxin localizes to the mitochondrial matrix and is required for iron-sulfur-cluster biosynthesis. Decreased expression of frataxin is associated with mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. Ferropotosis is a recently identified pathway of regulated, iron-dependent cell death, which is biochemically distinct from apoptosis. We evaluated whether there is evidence for ferroptotic pathway activation in cellular models of FRDA. We found that primary patient-derived fibroblasts, murine fibroblasts with FRDA-associated mutations, and murine fibroblasts in which a repeat expansion had been introduced (knockin/knockout) were more sensitive than normal control cells to erastin, a known ferroptosis inducer. We also found that the ferroptosis inhibitors ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS11-92) and ethyl 3-amino-4-(cyclohexylamino)benzoate, used at 500 nM, were efficacious in protecting human and mouse cellular models of FRDA treated with ferric ammonium citrate (FAC) and an inhibitor of glutathione synthesis [L-buthionine (S,R)-sulfoximine (BSO)], whereas caspase-3 inhibitors failed to show significant biologic activity. Cells treated with FAC and BSO consistently showed decreased glutathione-dependent peroxidase activity and increased lipid peroxidation, both hallmarks of ferroptosis. Finally, the ferroptosis inhibitor SRS11-92 decreased the cell death associated with frataxin knockdown in healthy human fibroblasts. Taken together, these data suggest that ferroptosis inhibitors may have therapeutic potential in FRDA.

Mitochondrial energetics is impaired in very long-chain acyl-CoA dehydrogenase deficiency and can be rescued by treatment with mitochondria-targeted electron scavengers.

Abstract: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common defect of mitochondrial long-chain fatty acid β-oxidation. Patients present with heterogeneous clinical phenotypes affecting heart, liver and skeletal muscle predominantly. The full pathophysiology of the disease is unclear and patient response to current therapeutic regimens is incomplete. To identify additional cellular alterations and explore more effective therapies, mitochondrial bioenergetics and redox homeostasis were assessed in VLCAD-deficient fibroblasts, and several protective compounds were evaluated. The results revealed cellular and tissue changes, including decreased respiratory chain (RC) function, increased reactive oxygen species (ROS) production and altered mitochondrial function and signaling pathways in a variety of VLCAD-deficient fibroblasts. The mitochondrially enriched electron and free radical scavengers JP4-039 and XJB-5-131 improved RC function and decreased ROS production significantly, suggesting that they are viable candidate compounds to further develop to treat VLCAD-deficient patients.

Hsp70 Binds to the Androgen Receptor N-terminal Domain and Modulates the Receptor Function in Prostate Cancer Cells.

Abstract: The androgen receptor (AR) is a key driver and therapeutic target in androgen-sensitive prostate cancer, castration-resistant prostate cancer (CRPC), and CRPC resistant to abiraterone and enzalutamide, two second-generation inhibitors of AR signaling. Because current AR inhibitors target a functioning C-terminal ligand-binding domain (LBD), the identification and characterization of cofactors interacting with the N-terminal domain (NTD) of AR may lead to new approaches to target AR signaling in CRPC. Using a pull-down approach coupled with proteomics, we have identified Hsp70 as a cofactor for the NTD of AR in prostate cancer cells. Hsp70 inhibition using siRNA or small molecules indicated that Hsp70 played an important role in the expression and transactivation of endogenous AR. Prostate-specific antigen (PSA) promoter/enhancer-driven luciferase assays showed that Hsp70 was also required for transactivation of AR mutant lacking LBD. Furthermore, clonogenic assays showed that an Hsp70 inhibitor, either alone or in synergy with enzalutamide, can inhibit the proliferation of 22Rv1, a widely used enzalutamide-resistant CRPC prostate cancer cell line. These findings suggest that Hsp70 is a potential therapeutic target for the treatment of enzalutamide-resistant CRPC.

ETHE1 and MOCS1 deficiencies: Disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts

Abstract: Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Asymmetric Total Synthesis and Biological Evaluation of (+)-Cycloclavine

Abstract: The first total synthesis of natural (+)-cycloclavine uses a catalytic­ asymmetric cyclopropanation of allene, a regiospecific Pd-catalyzed­ enone formation, and two intramolecular Diels–Alder reactions for indole/indoline annulations. The binding properties of natural (+)- and unnatural (–)-cycloclavine on 16 CNS receptors revealed significant stereospecificity and unique binding profiles in comparison to LSD, psilocin, and DMT. Differential 5-HT affinities, as well as novel sigma­-1 receptor properties bode well for potential therapeutic developments of clavine alkaloid scaffolds.

Synthesis and Optimization of Kv7 (KCNQ) Potassium Channel Agonists: The Role of Fluorines in Potency and Selectivity

Abstract: Based on the potent Kv7 agonist RL-81, we prepared new lead structures with greatly improved selectivity for Kv7.2/Kv7.3 over related potassium channels, i.e., Kv7.3/Kv7.5, Kv7.4, and Kv7.4/7.5. RL-36 and RL-12 maintain an agonist EC2x of ca. 1 μM on Kv7.2/Kv7.3 in a high-throughput assay on an automated electrophysiology platform in HEK293 cells but lack activity on Kv7.3/Kv7.5, Kv7.4, and Kv7.4/7.5, resulting in a selectivity index SI > 10. RL-56 is remarkably potent, EC2x 0.11 ± 0.02 μM, and still shows an SI = 2.5. We also identified analogues with significant selectivity for Kv7.4/Kv7.5 over Kv7.2/Kv7.3. The extensive use of fluorine in iterative core structure modifications highlights the versatility of these substituents, including F, CF3, and SF5, to span orders of magnitude of potency and selectivity in medicinal chemistry lead optimizations.

In-flow photooxygenation of aminothienopyridinones generates iminopyridinedione PTP4A3 phosphatase inhibitors.

Abstract: A continuous flow photooxygenation of 7-aminothieno[3,2-c]pyridin-4(5H)-ones to produce 7-iminothieno[3,2-c]pyridine-4,6(5H,7H)-diones has been developed, utilizing ambient air as the sole reactant. N-H Imines are formed as the major products, and excellent functional group tolerance and conversion on gram-scale without the need for chromatographic purification allow for facile late-stage diversification of the aminothienopyridinone scaffold. Several analogs exhibit potent in vitro inhibition of the cancer-associated protein tyrosine phosphatase PTP4A3, and the SAR supports an exploratory docking model.

Next-Generation Cell-Active Inhibitors of the Undrugged Oncogenic PTP4A3 Phosphatase

Abstract: Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.

Specific RITA modification produces hyperselective cytotoxicity while maintaining in vivo antitumor efficacy

Abstract: The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA9s heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these “hyperselective” agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA9s SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA9s broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P

A New Synthesis of Gefitinib

Abstract: A four-step synthesis of the FDA-approved anticancer agent gefitinib was developed starting from 2,4-dichloro-6,7-dimethoxyquinazoline. Reaction temperatures were highly practical (0–55 °C), and chromatographic purifications were avoided. The ionic liquid trimethylammonium heptachlorodialuminate was used to monodemethylate the dimethoxyquinazoline core. In the final step, a selective dehalogenation was employed to provide gefitinib in 14% overall yield on a gram scale.

Amelioration of Mucositis in Proton Therapy of Fanconi Anemia Fanca-/- Mice by JP4-039.

Abstract: Background/aim We tested JP4-039, a GS-nitroxide radiation damage mitigator in proton therapy of Fanconi anemia (FA) mice. Materials and methods Fanca-/- and Fanca+/+ bone marrow stromal cells were pre-treated with JP4-039 and irradiated with either protons or photons (0-10 GyRBE) followed by clonogenic survival and β-Galactosidase senescence analysis. Fanca-/- and Fanca+/+ mice were pretreated with JP4-039 for 10 min prior to oropharyngeal irradiation with either protons or photons (0 or 30 GyRBE) followed by sacrifice and measurement of oral cavity ulceration, distant hematopoietic suppression, and real-time polymerase chain reaction analysis. Results JP4-039 reduced oral cavity ulceration in Fanca-/- mice, transcripts Nfkb, Ap1, Sp1, and Nrf2, and proton therapy induced distant marrow suppression. Conclusion JP4-039 protected Fanca-/- and Fanca+/+ cells and mouse oral cavity from both proton and photon radiation.

Amelioration of Amyotrophic Lateral Sclerosis in SOD1 G93A Mice by M 2 Microglia from Transplanted Marrow.

Abstract: Background/Aim: The cause of fatal neuromuscular amyotrophic lateral sclerosis (ALS) is not known. Materials and Methods: Ninety-day-old superoxide-dismutase-1 G93A (SOD1 G93A ) mice demonstrating level 1 paralysis, received 9.0 Gy total body irradiation (TBI) from a cesium source at 340 cGy per minute, and intravenous transplantation with 1×10 6 C57BL/6 green fluorescent protein (GFP)+ donor bone marrow cells. Results: Paralysis-free survival was prolonged in TBI and bone marrow-transplanted SOD1 G93A mice from 100 to over 250 days (p=0.0018). Other mice transplanted with SOD1 G93A marrow or marrow treated with the free-radical scavenger MMS350 showed no therapeutic effect. GFP+ macrophage-2 (M2) microglial cells of bone marrow origin, were seen at sites of degenerating anterior horn motor neurons. SOD1 G93A mice had a disruption in the blood-brain barrier permeability which was reversed by marrow transplant from C57BL/6 mice. SOD1 G93A marrow showed unexpected robust hematopoiesis in vitro, and radioresistance. Conclusion: After TBI, M2 microglial cells from transplanted donor marrow extended the paralysis-free interval in SOD1 G93A mice.

Substituted β-lapachones for treating cancer

Abstract: Compounds, compositions and methods useful for treating cancer and neurodegeneration are provided. The compounds comprise a mitochondria-targeting moiety linked to β-lapachone or a β-lapachone derivative.

1,2,3-triazole inhibitors of p97 aaa atpase activity

Abstract: The present technology is directed to methods of inhibiting or modulating p97 and compounds and compositions useful in such methods. Diseases and conditions that can be treated with the compounds and compositions of the present technology include, but are not limited to, antibacterial infection, antiviral infection, cancer and neurodegenerative disorders susceptible to treatment by modulation of p97.