Keeping Balance Between Genetic Stability and Plasticity at the Telomere and Subtelomere of Trypanosoma brucei

Telomeres, the nucleoprotein complexes at chromosome ends, are well-known for their essential roles in genome integrity and chromosome stability. Yet, telomeres and subtelomeres are frequently less stable than chromosome internal regions. Many subtelomeric genes are important for responding to environmental cues, and subtelomeric instability can facilitate organismal adaptation to extracellular changes, which is a common theme in a number of microbial pathogens. In this review, I will focus on the delicate and important balance between stability and plasticity at telomeres and subtelomeres of a kinetoplastid parasite, Trypanosoma brucei, which causes human African trypanosomiasis and undergoes antigenic variation to evade the host immune response. I will summarize the current understanding about T. brucei telomere protein complex, the telomeric transcript, and telomeric R-loops, focusing on their roles in maintaining telomere and subtelomere stability and integrity. The similarities and differences in functions and underlying mechanisms of T. brucei telomere factors will be compared with those in human and yeast cells

Regulation of Antigenic Variation by Trypanosoma brucei Telomere Proteins Depends on Their Unique DNA Binding Activities

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoprotein (VSG), to evade the host immune response. Such antigenic variation is a key pathogenesis mechanism that enables T. brucei to establish long-term infections. VSG is expressed exclusively from subtelomere loci in a strictly monoallelic manner, and DNA recombination is an important VSG switching pathway. The integrity of telomere and subtelomere structure, maintained by multiple telomere proteins, is essential for T. brucei viability and for regulating the monoallelic VSG expression and VSG switching. Here we will focus on T. brucei TRF and RAP1, two telomere proteins with unique nucleic acid binding activities, and summarize their functions in telomere integrity and stability, VSG switching, and monoallelic VSG expression. Targeting the unique features of TbTRF and TbRAP10 s nucleic acid binding activities to perturb the integrity of telomere structure and disrupt VSG monoallelic expression may serve as potential therapeutic strategy against T. brucei

Trypanosoma brucei TIF2 and TRF Suppress VSG Switching Using Overlapping and Independent Mechanisms

Trypanosoma brucei causes debilitating human African trypanosomiasis and evades the host\u27s immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. We previously showed that two interacting telomere proteins, TbTRF and TbTIF2, are essential for cell proliferation and suppress VSG switching by inhibiting DNA recombination events involving the whole active VSG expression site. We now find that TbTIF2 stabilizes TbTRF protein levels by inhibiting their degradation by the 26S proteasome, indicating that decreased TbTRF protein levels in TbTIF2-depleted cells contribute to more frequent VSG switching and eventual cell growth arrest. Surprisingly, although TbTIF2 depletion leads to more subtelomeric DNA double strand breaks (DSBs) that are both potent VSG switching inducers and detrimental to cell viability, TbTRF depletion does not increase the amount of DSBs inside subtelomeric VSG expression sites. Furthermore, expressing an ectopic allele of F2H-TbTRF in TbTIF2 RNAi cells allowed cells to maintain normal TbTRF protein levels for a longer frame of time. This resulted in a mildly better cell growth and partially suppressed the phenotype of increased VSG switching frequency but did not suppress the phenotype of more subtelomeric DSBs in TbTIF2-depleted cells. Therefore, TbTIF2 depletion has two parallel effects: decreased TbTRF protein levels and increased subtelomeric DSBs, both resulting in an acute increased VSG switching frequency and eventual cell growth arrest

Lead Optimization of Dual Tubulin and Hsp27 Inhibitors

Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These compounds exhibited extensive anti-cancer activities against the proliferation of various human cancer cell lines. In the current study, a systematic ligand based structural optimization led to new analogs that significantly inhibited the growth of a panel of breast cancer cell lines. Furthermore, the most potent compounds were examined with tubulin polymerization assay and Hsp27 chaperone activity assay. The compounds showed potent tubulin polymerization inhibition but no Hsp27 inhibitory effect. The structural optimization dissected the dual activity and improved the selectivity of the compounds for tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27 inhibition. The modification of these structural fragments eliminated Hsp27 inhibition, but did not harm tubulin-targeting effects of the compounds. This result further defined the structureeactivity relationship between the tubulin and Hsp27 effects of these compounds

Lead Optimization of Dual Tubulin and Hsp27 Inhibitors

Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These compounds exhibited extensive anti-cancer activities against the proliferation of various human cancer cell lines. In the current study, a systematic ligand based structural optimization led to new analogs that significantly inhibited the growth of a panel of breast cancer cell lines. Furthermore, the most potent compounds were examined with tubulin polymerization assay and Hsp27 chaperone activity assay. The compounds showed potent tubulin polymerization inhibition but no Hsp27 inhibitory effect. The structural optimization dissected the dual activity and improved the selectivity of the compounds for tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27 inhibition. The modification of these structural fragments eliminated Hsp27 inhibition, but did not harm tubulin-targeting effects of the compounds. This result further defined the structureeactivity relationship between the tubulin and Hsp27 effects of these compounds

Validating TbRAP1-Interacting Candidates

Telomeres are specialized protein-DNA complexes located at the ends of eukaryotic chromosomes. They act as caps on the ends of chromosomes to preserve DNA from degradation and rearrangements and are therefore essential for genome stability. Trypanosoma brucei, a protozoan parasite that causes sleeping sickness in humans and nagana in cattle, evades the host immune responses by regularly switching its surface antigen, Variant Surface Glycoproteins (VSG). VSGs are expressed exclusively from regions adjacent to telomeres. Hence, understanding the VSG regulation by the telomere complex would help in developing means to eliminate this parasite. Our recent studies indicate that TbRAP1 is a protein associated with the telomere complex and is important for VSG silencing and VSG switching. In order to better understand the underlying mechanisms of TbRAP1-mediated VSG regulation, we attempted to identify proteins that interact with TbRAP1. By using TbRAP1 as bait, we identified a number of TbRAP1-interacting candidates in a yeast-2-hybrid screen. We have now subcloned these candidates into yeast expression vectors and tested their interactions with TbRAP1 directly by the liquid yeast 2-hybrid assay using Ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate of the reporter gene product -glactosidase

Validating TbRAP1-Interacting Candidates

Telomeres are specialized protein-DNA complexes located at the ends of eukaryotic chromosomes. They act as caps on the ends of chromosomes to preserve DNA from degradation and rearrangements and are therefore essential for genome stability. Trypanosoma brucei, a protozoan parasite that causes sleeping sickness in humans and nagana in cattle, evades the host immune responses by regularly switching its surface antigen, Variant Surface Glycoproteins (VSG). VSGs are expressed exclusively from regions adjacent to telomeres. Hence, understanding the VSG regulation by the telomere complex would help in developing means to eliminate this parasite. Our recent studies indicate that TbRAP1 is a protein associated with the telomere complex and is important for VSG silencing and VSG switching. In order to better understand the underlying mechanisms of TbRAP1-mediated VSG regulation, we attempted to identify proteins that interact with TbRAP1. By using TbRAP1 as bait, we identified a number of TbRAP1-interacting candidates in a yeast-2-hybrid screen. We have now subcloned these candidates into yeast expression vectors and tested their interactions with TbRAP1 directly by the liquid yeast 2-hybrid assay using Ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate of the reporter gene product -glactosidase

Pharmacokinetic Study of an Anti-trypanosome Agent with Different Formulations and Administration Routes in Mice by HPLC-MS/MS

Previously compound 12 showed great anti-trypanosome activity without toxicity in an in vivo study. In the current study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to investigate its pharmacokinetics in mouse plasma. A protein precipitation method was applied to extract the compound, and it was then separated using a Kinetex C-18 column with mobile phase consisting of acetonitrile-0.1% formic acid water (50:50, v/v) at a flow rate of 300 mu l/min. The analytes were detected with the multiple reaction monitoring in negative electrospray ionization source for quantitative response of the compounds. Compound 12 was detected at m/z 477.0 -\u3e 367.2, while the internal standard compound 14 was detected at m/z 499.2 -\u3e 268.2. Inter- and intra-day precision wa

Synthesis and Biological Evaluation of Imidamide Analogs as Selective Anti-trypanosomal Agents

Human African trypanosomiasis is caused by a protozoan parasite Trypanosoma brucei majorly infecting people living in sub-Saharan Africa. Current limited available treatments suffer from drug resistance, severe adverse effects, low efficacy, and costly administrative procedures in African countries with limited medical resources. Therefore, there is always a perpetual demand for advanced drug development and invention of new strategies to combat the disease. Previous work in our lab generated a library of sulfonamide analogs as selective tubulin inhibitors, based on the structural difference between mammalian and trypanosome tubulin proteins. Further lead derivatization was performed in the current study and generated 25 potential drug candidates to improve the drug efficacy and uptake by selectively targeting the parasite\u27s P2 membrane transporter protein with imidamide moiety. One of the newly synthesized analogs, compound 25 with a di-imidamide moiety, has shown greater potency with an IC50 of 1 nM to selectively inhibit the growth of trypanosome cells without affecting the viability of mammalian cells. Western blot analyses reveal that the compound suppressed tubulin polymerization in T. brucei cells. A detailed structure-activity relationship (SAR) was summarized that will be used to guide future lead optimization

Lead Optimization of Selective Tubulin Inhibitors As Anti-Trypanosomal Agents

Previously synthesized tubulin inhibitors showed promising in vitro selectivity and activity against Human African Trypanosomiasis. Current aim is to improve the ligand efficiency and reduce overall hydrophobicity of the compounds, by lead optimization. Via combinatorial chemistry, 60 new analogs were synthesized. For biological assay Trypanosoma brucei brucei Lister 427 cell line were used as the parasite model and for the host model human embryonic kidney cell line HEK-293 and mouse macrophage cell line RAW 264.7 were used to test efficacy. Of the newly synthesized compounds 5, 39, 40, and 57 exhibited IC s below 5 µM inhibiting the growth of trypanosome cells and not harming the mammalian cells at equipotent concentration. Comparably, the newly synthesized compounds have a reduced amount of aromatic moieties resulting in a decrease in molecular weight. Due to importance of tubulin polymerization during protozoan life cycle its activity was assessed by western blot analyses. Our results indicated that compound 5 had a profound effect on tubulin function. A detailed structure activity relationship (SAR) was summarized that will be used to guide future lead optimization. 5