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

Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.JAGS was funded by the European Union, grant FP7-HEALTH-2007-B-2.3.4-1.223048, NANOTRYP and Ministerio de Economía y Competitividad, Spain Plan Nacional de Investigación grant SAF2011- 30528. JLA was funded by Instituto de Salud Carlos III, Spain, grant FIS. 11/02571. HPdK was supported by a grant from the Medical Research Council (84733)

Instability of aquaglyceroporin (Aqp) 2 contributes to drug resistance in trypanosoma brucei

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance

New Approaches to Overcome Transport Related Drug Resistance in Trypanosomatid Parasites.

Leishmania and Trypanosoma are members of the Trypanosomatidae family that cause severe human infections such as leishmaniasis, Chagas disease, and sleeping sickness affecting millions of people worldwide. Despite efforts to eradicate them, migrations are expanding these infections to developing countries. There are no vaccines available and current treatments depend only on chemotherapy. Drug resistance is a major obstacle for the treatment of these diseases given that existing drugs are old and limited, with some having severe side effects. Most resistance mechanisms developed by these parasites are related with a decreased uptake or increased efflux of the drug due to mutations or altered expression of membrane transporters. Different new approaches have been elaborated that can overcome these mechanisms of resistance including the use of inhibitors of efflux pumps and drug carriers for both active and passive targeting. Here we review new formulations that have been successfully applied to circumvent resistance related to drug transporters, opening alternative ways to solve drug resistance in protozoan parasitic diseases.JAGS was supported by “Programa Estatal de Investigación,Desarrollo e Innovación Orientada a los Retos de la Sociedad” (grant SAF- SAF2015-71714-R MINECO/FEDER) and JVP was supported by “Programa de Promoción de Empleo Joven e Implantación de la Garantía Juvenil en I+D+i”, MIMECO, Spain.Ye

Agathadiol, a labdane diterpenoid from juniper berries, is a positive allosteric modulator of CB1R

The neutral fraction of a juniper (Juniperus communis L.) berries acetone extract could positively modulate the activity of type 1 - cannabinoid receptor (CB1R). Bioactivity-directed fractionation identified the labdane diterpenoid agathadiol (4) as a positive allosteric modulator of CB1R, while closely related analogues were inactive. Agathadiol (4) is a minor constituent of juniper, but could be more conveniently obtained by semisynthesis from agathic acid (8), a major constituent of Manila copal

Progress towards new treatments for human African trypanosomiasis

The treatment of African trypanosomiasis has essentially remained unchanged for decades. A mountain of excellent work has been produced on many aspects of trypanosome biochemistry, biology, genetics, etc., but this has not translated into new therapies, although the disease burden has steadily increased through the latter half of the twentieth century. The only new drug to be introduced in the last 50 years or so is eflornithine, in the late 1970s, for the treatment of late-stage gambiense sleeping sickness only. However, this was in many ways unsatisfactory and melarsoprol remained the first-line treatment for late-stage sleeping sickness until an alarming increase in treatment failures necessitated change. Since the emerging sleeping sickness epidemic became widely recognised, around the year 2000, needs-driven development of new drugs, and the preservation of the production of old drugs, has been the result of dedicated work by organisations such as the World Health Organisation, the Drugs for Neglected Diseases initiative (DNDi), the Access to Essential Medicines campaign, and the Consortium for Parasitic Drug Development (CPDD) among others, much of it in partnership with academia and the pharmaceutical industry. This has already resulted in milestones such as the donations of free treatments by producers; improved drug distribution, case finding and clinical care; an improved 10-day melarsoprol treatment; the first clinical trial for an oral sleeping sickness drug—pafuramidine and the introduction of eflornithine–nifurtimox combination therapy to begin replacing melarsoprol. While these efforts have undoubtedly contributed to reducing the disease burden in central Africa, newer treatments are still very necessary, especially as most current treatments are threatened by drug resistance. Here, we review recent advances in understanding drug resistance mechanisms, progress towards new drugs, and new delivery systems to improve efficacy

The dimerization of Delta(9)-tetrahydrocannabinolic acid A (THCA-A)

The renewed interest in dimeric salicylates as broad-spectrum anti-inflammatory and antidiabetic agents provided a rationale to investigate the dimerization of the substituted salicylate Delta(9)-tetrahydrocannabinolic acid (THCA-A, 3a) as a strategy to solve its instability to decarboxylation and to generate analogues and/or pro-drugs of this native pre-cannabinoid. Activation of the carboxylic group with the DCC-HOBt-DMAP protocol afforded a high yield of the OBt ester 4, that was next converted into the highly crystalline di-depsidic dimer 5 upon treatment with DMAP. The mono-depsidic dimer 6 was also formed when the reaction was carried out with partially decarboxylated THCA-A samples. The structure of the depsidic dimers was established by spectroscopic methods and by aminolysis of 5 into the pre-cannabinoid amide 7. Both dimers showed excellent shelf stability and did not generate significant amounts of Delta(9)-THC upon heating. However, only the didepsidic dimer 5 activated PPAR-gamma, the major target of pre-cannabinoids, but strong binding to serum proteins abolished this activity, also shielding it from the action of esterases. (C) 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection