Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection.

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.

Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model.

BACKGROUND: Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. OBJECTIVES: To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. METHODS: Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. RESULTS: Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. CONCLUSIONS: The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 µg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC50 of 4nM.

Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides: New potent Trypanosoma brucei inhibitors.

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.

A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

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.

Inhibitors of methionyl-tRNA synthetase have potent activity against Giardia intestinalis trophozoites.

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.

Induced resistance to methionyl-tRNA synthetase inhibitors in Trypanosoma brucei is due to overexpression of the target.

New classes of antiparasitic drugs active against Trypanosoma brucei are needed to combat human African trypanosomiasis. Inhibitors of methionyl-tRNA synthetase (MetRS) have excellent potential to be developed for this purpose (S. Shibata, J. R. Gillespie, A. M. Kelley, A. J. Napuli, Z. Zhang, K. V. Kovzun, R. M. Pefley, J. Lam, F. H. Zucker, W. C. Van Voorhis, E. A. Merritt, W. G. Hol, C. L. Verlinde, E. Fan, and F. S. Buckner, Antimicrob. Agents Chemother. 55:1982-1989, 2011). In order to assess the potential for resistance to develop against this new class of inhibitors, T. brucei cultures were grown in the presence of MetRS inhibitors or comparison drugs. Resistance up to ∼50 times the baseline 50% inhibitory concentration (IC50) was induced against a MetRS inhibitor after ∼120 days. A similar level of resistance to the clinical drug eflornithine was induced after ∼50 days and for pentamidine after ∼80 days. Thus, resistance was induced more slowly against MetRS inhibitors than against clinically used drugs. The parasites resistant to the MetRS inhibitor were shown to overexpress MetRS mRNA by a factor of 35 over the parental strain. Southern analysis indicated that the MetRS gene was amplified in the genome by nearly 8-fold. When injected into mice, the MetRS inhibitor-resistant parasites caused a reduced level of infection, indicating that the changes associated with resistance attenuated their virulence. This finding and the fact that resistance to MetRS inhibitors developed relatively slowly are encouraging for further development of this class of compounds. Published studies on other antitrypanosomal drugs have primarily shown that alterations in membrane transporters were the mechanisms responsible for resistance. This is the first published report of induced drug resistance in the African trypanosome due to overexpression of the target enzyme.

Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.

Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 µM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

Synthesis and Structure-Activity Relationships of Imidazopyridine/Pyrimidine- and Furopyridine-Based Anti-infective Agents against Trypanosomiases.

Neglected tropical diseases remain among the most critical public health concerns in Africa and South America. The drug treatments for these diseases are limited, which invariably leads to fatal cases. Hence, there is an urgent need for new antitrypanosomal drugs. To address this issue, a large number of diverse heterocyclic compounds were prepared. Straightforward synthetic approaches tolerated pre-functionalized structures, giving rise to a structurally diverse set of analogs. We report on a set of 57 heterocyclic compounds with selective activity potential against kinetoplastid parasites. In general, 29 and 19 compounds of the total set could be defined as active against Trypanosoma cruzi and T. brucei brucei, respectively (antitrypanosomal activities <10 µM). The present work discusses the structure-activity relationships of new fused-ring scaffolds based on imidazopyridine/pyrimidine and furopyridine cores. This library of compounds shows significant potential for anti-trypanosomiases drug discovery.

Phenotypic Drug Discovery for Human African Trypanosomiasis: A Powerful Approach.

The work began with the screening of a library of 700,000 small molecules for inhibitors of Trypanosoma brucei growth (a phenotypic screen). The resulting set of 1035 hit compounds was reviewed by a team of medicinal chemists, leading to the nomination of 17 chemically distinct scaffolds for further investigation. The first triage step was the assessment for brain permeability (looking for brain levels at least 20% of plasma levels) in order to optimize the chances of developing candidates for treating late-stage human African trypanosomiasis. Eleven scaffolds subsequently underwent hit-to-lead optimization using standard medicinal chemistry approaches. Over a period of six years in an academic setting, 1539 analogs to the 11 scaffolds were synthesized. Eight scaffolds were discontinued either due to insufficient improvement in antiparasitic activity (5), poor pharmacokinetic properties (2), or a slow (static) antiparasitic activity (1). Three scaffolds were optimized to the point of curing the acute and/or chronic T. brucei infection model in mice. The progress was accomplished without knowledge of the mechanism of action (MOA) for the compounds, although the MOA has been discovered in the interim for one compound series. Studies on the safety and toxicity of the compounds are planned to help select candidates for potential clinical development. This research demonstrates the power of the phenotypic drug discovery approach for neglected tropical diseases.

Structure-guided discovery of selective methionyl-tRNA synthetase inhibitors with potent activity against Trypanosoma brucei.

Based on crystal structures of Trypanosoma brucei methionyl-tRNA synthetase (TbMetRS) bound to inhibitors, we designed, synthesized, and evaluated two series of novel TbMetRS inhibitors targeting this parasite enzyme. One series has a 1,3-dihydro-imidazol-2-one containing linker, the other has a rigid fused aromatic ring in the linker. For both series of compounds, potent inhibition of parasite growth was achieved with EC50 < 10 nM and most compounds exhibited low general toxicity to mammalian cells with CC50s > 20 000 nM. Selectivity over human mitochondrial methionyl tRNA synthetase was also evaluated, using a cell-based mitochondrial protein synthesis assay, and selectivity in a range of 20-200-fold was achieved. The inhibitors exhibited poor permeability across the blood brain barrier, necessitating future efforts to optimize the compounds for use in late stage human African trypanosomiasis.

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

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 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.

Triazolopyrimidines and Imidazopyridines: Structure-Activity Relationships and in Vivo Efficacy for Trypanosomiasis.

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.

Glycogen synthase kinase 3 is a potential drug target for African trypanosomiasis therapy.

Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 "short" (Tb10.161.3140) than for T. brucei GSK-3 "long" (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found.

The crystal structure of the drug target Mycobacterium tuberculosis methionyl-tRNA synthetase in complex with a catalytic intermediate.

Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.

1-Benzyl-3-aryl-2-thiohydantoin Derivatives as New Anti-Trypanosoma brucei Agents: SAR and in Vivo Efficacy.

A high throughput screening and subsequent hit validation identified compound 1 as an inhibitor of Trypanosoma brucei parasite growth. Extensive structure-activity relationship optimization based on antiparasitic activity led to the highly potent compounds, 1-(4-fluorobenzyl)-3-(4-dimethylamino-3-chlorophenyl)-2-thiohydantoin (68) and 1-(2-chloro-4-fluorobenzyl)-3-(4-dimethylamino-3-methoxyphenyl)-2-thiohydantoin (76), with a T. brucei EC50 of 3 and 2 nM, respectively. This represents >100-fold improvement in potency compared to compound 1. In vivo efficacy experiments of 68 and 76 in an acute mouse model of Human African Trypanosomiasis showed a 100% cure rate after 4 days of oral treatment at 50 mg/kg twice per day.

New Class of Antitrypanosomal Agents Based on Imidazopyridines.

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.