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Trypanosoma brucei sub-species are vector borne kinetoplastid parasites that cause the potentially lethal disease Human African trypanosomiasis. The target-based therapy for curing this parasitic disease relies on one drug, Eflornithine. The roles of mitogen-activated protein kinases in regulating key cellular processes in eukaryotic cells such as proliferation, stress response and differentiation plus their druggability make them attractive targets for therapeutic exploitation. The extracellular-regulated kinase 8 homolog in T. brucei (TbERK8) is a MAPK that is required for the parasite to proliferate normally in culture. We examined the importance of TbERK8 for permitting T. brucei to thrive in mice. Here we show that depleting TbERK8 in vivo negatively affected the virulence of T. brucei reducing its ability to progress to lethal infections or cause significant pathology in mice, which validates it as an attractive target.

Human African trypanosomiasis (HAT) is a lethal, vector-borne disease caused by the parasite Trypanosoma brucei. Therapeutic strategies for this neglected tropical disease suffer from disadvantages such as toxicity, high cost, and emerging resistance. Therefore, new drugs with novel modes of action are needed. We screened cultured T. brucei against a focused kinase inhibitor library to identify promising bioactive compounds. Among the ten hits identified from the phenotypic screen, AZ960 emerged as the most promising compound with potent antiparasitic activity (IC50=120nM) and was shown to be a selective inhibitor of an essential gene product, T. brucei extracellular signal-regulated kinase 8 (TbERK8). We report that AZ960 has a Ki of 1.25μM for TbERK8 and demonstrate its utility in establishing TbERK8 as a potentially druggable target in T. brucei.

The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.

The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC(50) of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Previous studies on DNA polymerase epsilon indicate that this enzyme is involved in replication of chromosomal DNA. In this study, we examined the expression of DNA polymerases alpha, delta, and epsilon during mouse testis development and germ cell differentiation. The steady-state levels of mRNAs encoding DNA polymerase epsilon and the recombination enzyme Rad51 remained constant during testis development, whereas the mRNA levels of DNA polymerases alpha and delta declined from birth until sexual maturity. Immunohistochemical staining methods, using a stage-specific model of the seminiferous epithelium, revealed dramatic differences between DNA polymerase alpha and epsilon distribution. As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication. The distribution of Rad51 was similar, but there was a dramatic peak in late pachytene cells. In contrast, DNA polymerase epsilon was detectable in mitotically proliferating spermatogonia but not in the early stages of meiotic prophase. However, DNA polymerase epsilon reappeared in late pachytene cells and remained through the two meiotic divisions, and was present in haploid spermatids up to the stage at which the flagellum starts developing. Overall, the results suggest that DNA polymerase epsilon functions in mitotic replication, in the completion of recombination in late pachytene cells, and in repair of DNA damage in round spermatids. In contrast, DNA polymerases alpha and delta appear to be involved in meiotic DNA synthesis, which occurs early in meiotic prophase, in addition to functioning in DNA replication in proliferating spermatogonia.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas’ disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3′ mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.

We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood–brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood–brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain., Author Summary African trypanosomiasis, or sleeping sickness, is caused by the single-cell parasite Trypanosoma brucei (T. brucei). Two parasite-derived enzyme proteins have been hypothesized to play an important role in the viability of the parasite or its ability to produce disease in the human host. Utilizing RNA interference that blocks the production of these proteins in the parasite, we show that elimination of parasite cathepsin B cures infection in mice. RNAi of the second enzyme protein, brucipain, results in the prolongation of life of half the infected mice, but does not cure. Further experiments carried out in a culture system show that brucipain facilitates the migration of parasites across a model of the blood–brain barrier. This suggests that while brucipain is not necessary for the viability of the organisms, it may play a role in infection by allowing parasites to reach the central nervous system and produce the severe second stage of sleeping sickness.

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN(2) in T. brucei.

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.

The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the six T. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei.

Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.

DNA joining events are required for the completion of DNA replication, DNA excision repair and genetic recombination. Five DNA ligase activities, I–V, have been purified from mammalian cell extracts and three mammalian LIG genes, LIG1 , LIG3 and LIG4 , have been cloned. During DNA replication, the joining of Okazaki fragments by the LIG1 gene product appears to be mediated by an interaction with proliferating cell nuclear antigen (PCNA). This interaction may also occur during the completion of mismatch, nucleotide excision and base excision repair (BER). In addition, DNA ligase I participates in a second BER pathway that is carried out by a multiprotein complex in which DNA ligase I interacts directly with DNA polymerase β. DNA ligase IIIα and DNA ligase IIIβ, which are generated by alternative splicing of the LIG3 gene, can be distinguished by their ability to bind to the DNA repair protein, XRCC1. The interaction between DNA ligase IIIα and XRCC1, which occurs through BRCT motifs in the C-termini of these polypeptides, implicates this isoform of DNA ligase III in the repair of DNA single-strand breaks and BER. DNA ligase II appears to be a proteolytic fragment of DNA ligase IIIα. The restricted expression of DNA ligase IIIβ suggests that this enzyme may function in the completion of meiotic recombination or in a postmeiosis DNA repair pathway. Complex formation between DNA ligase IV and the DNA repair protein XRCC4 involves the C-terminal region of DNA ligase IV, which contains two BRCT motifs. This interaction, which stimulates DNA joining activity, implies that DNA ligase IV functions in V(D)J recombination and non-homologous end-joining of DNA double-strand breaks. At the present time, it is not known whether DNA ligase V is derived from one of the known mammalian LIG genes or is the product of a novel gene.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.