Urea-based methionyl-tRNA synthetase inhibitors were designed, synthesized, and evaluated for their potential toward treating human African trypanosomiasis (HAT). With the aid of a homology model and ...a structure–activity-relationship approach, low nM inhibitors were discovered that show high selectivity toward the parasite enzyme over the closest human homologue. These compounds inhibit parasite growth with EC50 values as low as 0.15 μM while having low toxicity to mammalian cells. Two compounds (2 and 26) showed excellent membrane permeation in the MDR1-MDCKII model and encouraging oral pharmacokinetic properties in mice. Compound 2 was confirmed to enter the CNS in mice. Compound 26 had modest suppressive activity against Trpanosoma brucei rhodesiense in the mouse model, suggesting that more potent analogues or compounds with higher exposures need to be developed. The urea-based inhibitors are thus a promising starting point for further optimization toward the discovery of orally available and CNS active drugs to treat HAT.
The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A ...panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs.
The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are ...the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNATyr.
An “extra pocket” (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases.
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•Leishmania donovani is a parasite causing visceral leishmaniasis.•The structure of tyrosyl-tRNA synthetase from Leishmania donovani is described.•Substantial differences between parasite enzyme and both human homologs exist.•Selective inhibition of tyrosyl-tRNA synthetases from diverse parasites is possible.
Better therapeutics are greatly needed to treat patients infected with trypanosomatid parasites such as Trypanosoma cruzi or Trypanosoma brucei. This report describes 28 new imidazopyridines and ...triazolopyrimidines with potent and selective antitrypanosomal activity. Drug-like properties were demonstrated in a number of in vitro assays. In vivo efficacy was observed for 19 and 20 in acute mouse models of T. cruzi infection. Compounds 19 and 20 represent potential leads for new anti-Chagas disease drugs.
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A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), led to the identification of ...N-(2-aminoethyl)-N-phenyl benzamides as a starting point for hit-to-lead medicinal chemistry. Eighty two analogues were prepared, which led to the identification of a set of highly potent N-(2-aminoethyl)-N-benzyloxyphenyl benzamides with the most potent compound 73 having an in vitro EC50=0.001μM. The compounds displayed drug-like properties when tested in a number of in vitro assays. Compound 73 was orally bioavailable and displayed good plasma and brain exposure in mice, cured 2 out of 3 mice infected with Trypanosoma brucei in acute model when dosed orally at 50mg/kg once per day for 4days. Given its potent antiparasitic properties and its ease of synthesis, compound 73 represents a potential lead for the development of drug to treat Human African Trypanosomiasis.
American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes ...debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl‐tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment‐based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment‐binding hotspot adjacent to the ATP‐binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.
The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and 12 compounds exhibited an in ...vitro EC50 ≤ 1 μM against Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) parasites, respectively. Based on promising results of in vitro activity (EC50 < 100 nM), cytotoxicity, metabolic stability, protein binding, and pharmacokinetics (PK) properties, compound 20 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi (Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 20 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 20 represents a potential lead for the development of drugs to treat trypanosomiasis.
Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, ...dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.
In the 100 years since the discovery of Chagas disease, only two drugs have been developed and introduced into clinical practice, and these drugs were introduced over 40 years ago. The tools of drug ...discovery have improved dramatically in the interim; however, this has not translated into new drugs for Chagas disease. This has been largely because the main practitioners of drug discovery are pharmaceutical companies who are not financially motivated to invest in Chagas disease and other "orphan" diseases. As a result, it has largely been up to academic groups to bring drug candidates through the discovery pipeline and to clinical trials. The difficulty with drug discovery in academia has been the challenge of bringing together the diverse expertise in biology, chemistry, and pharmacology in concerted efforts towards a common goal of developing therapeutics. Funding is often inadequate, but lack of coordination amongst academic investigators with different expertise has also contributed to the slow progress. The purpose of this chapter is to provide an overview of approaches that can be accomplished in academic settings for preclinical drug discovery for Chagas disease. The chapter addresses methods of drug screening against Trypanosoma cruzi cultures and in animal models and includes general topics on compound selection, testing for drug-like properties (including oral bioavailability), investigating the pharmacokinetics and toxicity of compounds, and finally providing parameters to help with triaging compounds.
Kinesins are a family of motor proteins conserved throughout eukaryotes. In our present study we characterize a novel kinesin, Kinesin(CaaX), orthologs of which are only found in the kinetoplastids ...and not other eukaryotes. Kinesin(CaaX) has the CVIM amino acids at the C-terminus, and CVIM was previously shown to be an ideal signal for protein farnesylation in T. brucei. In this study we show Kinesin(CaaX) is farnesylated using radiolabeling studies and that farnesylation is dependent on the CVIM motif. Using RNA interference, we show Kinesin(CaaX) is essential for T. brucei proliferation. Additionally RNAi Kinesin(CaaX) depleted T. brucei are 4 fold more sensitive to the protein farneysltransferase (PFT) inhibitor LN-59, suggesting that Kinesin(CaaX) is a target of PFT inhibitors' action to block proliferation of T. brucei. Using tetracycline-induced exogenous tagged Kinesin(CaaX) and Kinesin(CVIMdeletion) (non-farnesylated Kinesin) expression lines in T. brucei, we demonstrate Kinesin(CaaX) is farnesylated in T. brucei cells and this farnesylation has functional effects. In cells expressing a CaaX-deleted version of Kinesin, the localization is more diffuse which suggests correct localization depends on farnesylation. Through our investigation of cell cycle, nucleus and kinetoplast quantitation and immunofluorescence assays an important role is suggested for Kinesin(CaaX) in the separation of nuclei and kinetoplasts during and after they have been replicated. Taken together, our work suggests Kinesin(CaaX) is a target of PFT inhibition of T. brucei cell proliferation and Kinesin(CaaX) functions through both the motor and farnesyl groups.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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