Betulinic Acid Hydroxamate is Neuroprotective and Induces Protein Phosphatase 2A-Dependent HIF-1α Stabilization and Post-transcriptional Dephosphorylation of Prolyl Hydrolase 2

Huntington’s disease (HD) is a neurodegenerative disorder characterized by unwanted choreatic movements, behavioral and psychiatric disturbances, and dementia. The activation of the hypoxic response pathway through the pharmacological inhibition of hypoxia-inducing factor (HIF) prolyl-hydroxylases (PHDs) is a promising approach for neurodegenerative diseases, including HD. Herein, we have studied the mechanism of action of the compound Betulinic acid hydroxamate (BAH), a hypoximimetic derivative of betulinic acid, and its efficacy against striatal neurodegeneration using complementary approaches. Firstly, we showed the molecular mechanisms through which BAH modifies the activity of the PHD2 prolyl hydroxylase, thus directly affecting HIF-1α stability. BAH treatment reduces PHD2 phosphorylation on Ser-125 residue, responsible for the control of its hydrolase activity. HIF activation by BAH is inhibited by okadaic acid and LB-100 indicating that a protein phosphatase 2A (PP2A) is implicated in the mechanism of action of BAH. Furthermore, in striatal cells bearing a mutated form of the huntingtin protein, BAH stabilized HIF-1α protein, induced Vegf and Bnip3 gene expression and protected against mitochondrial toxin-induced cytotoxicity. Pharmacokinetic analyses showed that BAH has a good brain penetrability and experiments performed in a mouse model of striatal neurodegeneration induced by 3-nitropropionic acid showed that BAH improved the clinical symptoms. In addition, BAH also prevented neuronal loss, decreased reactive astrogliosis and microglial activation, inhibited the upregulation of proinflammatory markers, and improved antioxidant defenses in the brain. Taken together, our results show BAH’s ability to activate the PP2A/PHD2/HIF pathway, which may have important implications in the treatment of HD and perhaps other neurodegenerative diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01089-4.     

Falcarindiol allosterically modulates GABAergic currents in cultured rat hippocampal neurons

Falcarindiol (1), a C-17 polyacetylenic diol, shows a pleiotropic profile of bioactivity, but the mechanism(s) underlying its actions are largely unknown. Large amounts of 1 co-occur in water hemlock (Oenanthe crocata) along with the convulsant polyacetylenic toxin oenanthotoxin (2), a potent GABA(A) receptor (GABA(A)R) inhibitor. Since these compounds are structurally and biogenetically related, it was considered of interest to evaluate whether 1 could affect GABAergic activity, and for this purpose a model of hippocampal cultured neurons was used. Compound 1 significantly increased the amplitude of miniature inhibitory postsynaptic currents, accelerated their onset, and prolonged the decay kinetics. This compound enhanced also the amplitude of currents elicited by 3 μM GABA and accelerated their fading, reducing, however, currents evoked by a saturating (10 mM) GABA concentration. Moreover, kinetic analysis of responses to 10 mM GABA revealed that 1 upregulated the rate and extent of desensitization and slowed the current onset and deactivation. Taken together, these data show that 1 exerts a potent modulatory action on GABA(A)Rs, possibly by modulating agonist binding and desensitization, overall potentially decreasing the toxicity of co-occurring GABA-inhibiting convulsant toxins.     

Effect of chirality and lipophilicity in the functional activity of evodiamine and its analogues at TRPV1 channels

Background and Purpose

Evodiamine, a racemic quinazolinocarboline alkaloid isolated from the traditional Chinese medicine Evodiae fructus, has been reported to act as an agonist of the transient receptor potential vanilloid type-1 (TRPV1) cation channel both in vitro and in vivo. Evodiamine is structurally different from all known TRPV1 activators, and has significant clinical potential as a thermogenic agent. Nevertheless, the molecular bases for its actions are still poorly understood.

Experimental Approach

To investigate the structure-activity relationships of evodiamine, the natural racemate was resolved, and a series of 23 synthetic analogues was prepared, using as the end point the intracellular Ca²⁺ elevation in HEK-293 cells stably overexpressing either the human or the rat recombinant TRPV1.

Key Results

S-(+) evodiamine was more efficacious and potent than R-(−) evodiamine, and a new potent lead (Evo30) was identified, more potent than the reference TRPV1 agonist, capsaicin. In general, potency and efficacy correlated with the lipophilicity of the analogues. Like other TRPV1 agonists, several synthetic analogues could efficiently desensitize TRPV1 to activation by capsaicin.

Conclusions and Implications

Evodiamine qualifies as structurally unique lead structure to develop new potent TRPV1 agonists/desensitizers.

Linked Articles

This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10     

Oxyhomologation of the amide bond potentiates neuroprotective effects of the endolipid N-palmitoylethanolamine

The endolipid N-palmitoylethanolamine (PEA) shows a pleiotropic pattern of bioactivities, whose mechanistic characterization is still unclear and whose pharmacological potential is substantially limited by rapid metabolization by the amido hydrolyzing enzymes fatty acid amide hydrolases and N-acylethanolamine-hydrolyzing acid amidase. To overcome this problem, we have synthesized a new series of PEA homologs and characterized their activity on two in vitro models of neurodegeneration (oxidative stress, excitotoxicity). PEA partially prevented tert-butylhydroperoxide (t-BOOH; 100 microM; 3 h)-induced cell death (maximal effect, 26.3 +/- 7.5% in comparison with t-BOOH-untreated cells at 30 microM), whereas it was ineffective against the L-glutamate (1 mM; 24 h)-induced excitotoxicity at all concentrations tested (0.01-30 microM). Oxyhomologation of the amide bond, although leading to an increased enzymatic stability, also potentiated neuroprotective activity, especially for N-palmitoyl-N-(2-hydroxyethyl)hydroxylamine (EC(50) = 2.1 microM). These effects were not mediated by cannabinoid/vanilloid-dependent mechanisms but rather linked to a decreased t-BOOH-induced lipoperoxidation and reactive oxygen species formation and L-glutamate-induced intracellular Ca(2+) overload. The presence of the hydroxamic group and the absence of either redox active or radical scavenger moieties suggest that the improved neuroprotection is the result of increased metal-chelating properties that boost the antioxidant activity of these compounds.     

Antiproliferative effects of daucane esters from Ferula communis and F. arrigonii on human colon cancer cell lines

Certain jaesekanadiol p-hydroxy- and p-methoxybenzoates - typical of Ferula communis and Ferula arrigonii sardinian plants - show antiproliferative activity on human colon cancer less. The inhibitory doses 50%, calculated after 72 h of treatment, revealed that the antiproliferative capacity of the compounds was in the following descending order: ferutinin > 2alpha-OH-ferutidin > ferutidin > siol anisate > lapiferin > jaeskeanadiol. Evidence is presented that interaction with type II estrogen-binding sites (EBS) underlies this activity.     

Biological properties of jatrophane polyesters,new microtubule-interacting agents

PURPOSE. The biological activities of macrocyclic jatrophane polyesters 1-3 from the Sardinian endemism Euphorbia semiperfoliata Viv. have not been evaluated in depth. We investigated the microtubule-interacting and antiproliferative activities of these drugs and the molecular mechanisms underlying their effects. METHODS. We tested jatrophanes for their interaction with purified bovine brain tubulin by an in vitro polymerization assay and by electron microscopy. At a cellular level, the effects of jatrophanes on microtubular architecture, nuclear morphology, cell viability, cell cycle perturbations, and p53 and Raf-1/Bcl-2 involvement were investigated. RESULTS. Jatrophanes exhibited microtubule-interacting activity. They stimulated purified tubulin assembly in vitro, and induced paclitaxel-like microtubules, as revealed by electron microscopy. In the cells, rearrangement of microtubule architecture was in contrast to the bundling produced by paclitaxel. Jatrophanes inhibited the growth of some human cancer cell lines without inducing cell cycle arrest in the G2/M phase. Moreover, they influenced p53 expression and Raf-1/Bcl-2 activation. CONCLUSIONS. Despite their structural difference from paclitaxel and other microtubule-interacting agents, jatrophanes may represent a new type of tubulin binder.