Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways

Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.This work is funded by grants from the Wellcome Trust (101050/Z/13/Z) and the MRC (MR/K011022/1) and supported by the Gurdon Institute core grant from Cancer Research UK (C6946/A14492) and the Wellcome Trust (092096/Z/10/Z). This research was supported in part by the Intramural Research Program of NIAMS at the NIH (1Z01AR041126-17). M.V. was supported by a Svenska Sällskapet för Medicinsk Forskning (SSMF) postdoctoral fellowship. S.W. was supported in part by fellowships from the Gates Cambridge Trust and NIH-Cambridge MD/PhD Program (T32GM007367). M.O. was supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science (JSPS). K.M. is supported by Human Frontier Science Program (RGP0021/2016), JSPS KAKENHI grants JP16H01321 and JP16H01222, and by a Grant for Basic Science Research Projects from The Sumitomo Foundation (150810). V.P. was supported by the Wellcome Trust (081277), the Wallonia-Brussels International Excellence Grant, The Research Foundation – Flanders (FWO) (Odysseus Return Grant G0F7716N), the KU Leuven Research Fund (BOFZAP starting grant StG/15/021BF, C1 grant C14/16/077, and project financing)

Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Human African trypanosomiasis (HAT), a devastating and fatal parasitic disease endemic in sub-Saharan Africa, urgently needs novel targets and efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine exhibits specific antitrypanosomal activity toward T. b. rhodesiense, the causative agent of the acute form of HAT. Here we applied a chemical proteomics approach to find the cellular target of this compound. Adenosine kinase, a key enzyme of the parasite purine salvage pathway, was isolated and identified as compound binding partner. Direct binding assays using recombinant protein, and tests on an adenosine kinase knock-down mutant of the parasite produced by RNA interference confirmed TbrAK as the putative target. Kinetic analyses showed that the title compound is an activator of adenosine kinase and that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition. Whereas hyperactivation as a mechanism of action is well known from drugs targeting cell signaling, this is a novel and hitherto unexplored concept for compounds targeting metabolic enzymes, suggesting that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides

Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs

Diverse Effects on Mitochondrial and Nuclear Functions Elicited by Drugs and Genetic Knockdowns in Bloodstream Stage Trypanosoma brucei

The parasite Trypanosoma brucei causes human African trypanosomiasis, which is fatal unless treated. Currently used drugs are toxic, difficult to administer, and often are no longer effective due to drug resistance. The search for new drugs is long and expensive, and determining which compounds are worth pursuing is a key challenge in that process. In this study we sought to determine whether different compounds elicited different responses in the mammalian-infective stage of the parasite. We also examined whether genetic knockdown of parasite molecules led to similar responses. Our results show that, depending on the treatment, the replication of the parasite genomes, proper division of the cell, and mitochondrial function can be affected. Surprisingly, these different responses were not able to predict which compounds affected the long term proliferative potential of T. brucei. We found that some of the compounds had irreversible effects on the parasites within one day, so that even cells that appeared healthy could not proliferate. We suggest that determining which compounds set the parasites on a one-way journey to death may provide a means of identifying those that could lead to drugs with high efficacy

Crystal Structures of T. b. rhodesiense Adenosine Kinase Complexed with Inhibitor and Activator: Implications for Catalysis and Hyperactivation

Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) and its derivatives exhibit specific antitrypanosomal activity toward T. b. rhodesiense, the causative agent of the acute form of HAT. We found that compound 1 would target the parasite adenosine kinase (TbrAK), an important enzyme of the purine salvage pathway, by acting via hyperactivation of the enzyme. This represents a novel and hitherto unexplored strategy for the development of trypanocides. These findings prompted us to investigate the mechanism of action at the molecular level. The present study reports the first three-dimensional crystal structures of TbrAK in complex with the bisubstrate inhibitor AP5A, and in complex with the activator (compound 1). The subsequent structural analysis sheds light on substrate and activator binding, and gives insight into the possible mechanism leading to hyperactivation. Further structure-activity relationships in terms of TbrAK activation properties support the observed binding mode of compound 1 in the crystal structure and may open the field for subsequent optimization of this compound series

Ribosylurea accumulates in yeast urc4 mutants.

Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms

NOVEL 2-SUBSTITUTED 2’/3’-C-METHYL-ADENOSINE DERIVATIVES: SYNTHESIS AND BIOLOGICAL EVALUATION AGAINST TRYPANOSOMA BRUCEI

Human African trypanosomiasis (HAT), which is also known as sleeping sickness, is a devastating parasitic disease that affects more than 300000 people of sub-Saharan Africa each year. The causative agent of this affliction is the protozoan Trypanosoma brucei, which is introduced in the mammalian host by the tsetse fly. T. brucei attacks the central nervous system leading to dementia, epileptic attacks, coma, and, if left untreated, death. Current treatment for this disease, including suramin, pentamidine, melarsoprol, and difluoromethylornithine (DFMO), is often antiquated, highly toxic and frequently ineffective. Therefore, new highly effective and not toxic drugs are needed. Like most obligate intracellular parasites, T. brucei has lost the capacity to synthesize purines de novo and depends on the salvage pathway of nucleosides from the body fluids of the host. Bloodstream T. brucei can take up different types of purines and interconverts them into essential cellular nucleotides. Cordycepin (3’-deoxyadenosine) is an adenosine derivatives able to cure mice inoculated with the human pathogenic T. brucei even after parasites have penetrated into the brain, but requires co-administration with the adenosine deaminase (ADA) inhibitor coformycin to prevent deamination. However, the toxicity of coformycin has stimulated the search of adenosine analogues active against the parasite, but resistant to ADA. In our previous work, we found that the introduction of a methyl group in position 2’- or 3’- of the sugar moiety of adenosine (2’-C-methyladenosine and 3’-C-methyladenosine, respectively) confers a certain grade of resistance to ADA.(1) In fact, 3’-MeAdo is resistant to ADA, while 2’-MeAdo is deaminated by ADA even though the rate of deamination was 1/25 that observed with adenosine. Moreover, some 2, N6-disubstituted adenosine analogs have been reported to show antitrypanosomal activity.(2) Based on these findings a new series of 2-substituted-2’-C-methyl-, and 3’-C-methyl-adenosine derivatives were synthesized and tested for their antiprotozoal activity. The results of this study will be discussed. (1) Franchetti, P, et al. J Med Chem 1998, 41, 1708-1715; Franchetti, P, et al. J Med Chem 2005, 48, 4983-4989; Cappellacci, L, et al. J Med Chem 2008, 51, 4260-4269. (2) Rodenko, B, et al. Antimicrob. Agents Chemother. 2007, 51, 3796-3802