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The intestinal protozoan Cryptosporidiumis 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.

ABSTRACTNew classes of antiparasitic drugs active against Trypanosoma bruceiare 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. bruceicultures 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.

ABSTRACTChagas disease, caused by the protozoan pathogen Trypanosoma cruzi, remains a challenging infection due to the unavailability of safe and efficacious drugs. Inhibitors of the trypanosome sterol 14α-demethylase enzyme (CYP51), including azole antifungal drugs, are promising candidates for development as anti-Chagas disease drugs. Posaconazole is under clinical investigation for Chagas disease, although the high cost of this drug may limit its widespread use. We have previously reported that the human protein farnesyltransferase (PFT) inhibitor tipifarnib has potent anti-T. cruziactivity by inhibiting the CYP51 enzyme. Furthermore, we have developed analogs that minimize the PFT-inhibitory activity and enhance the CYP51 inhibition. In this paper, we describe the efficacy of the lead tipifarnib analog compared to that of posaconazole in a murine model of T. cruziinfection. The plasma exposure profiles for each compound following a single oral dose in mice and estimated exposure parameters after repeated twice-daily dosing for 20 days are also presented. The lead tipifarnib analog had potent suppressive activity on parasitemia in mice but was unsuccessful at curing mice, whereas posaconazole as well as benznidazole cured 3 of 5 and 4 of 6 mice, respectively. The efficacy results are consistent with posaconazole having substantially higher predicted exposure than that of the tipifarnib analog after repeat twice-daily administration. Further changes to the tipifarnib analogs to reduce plasma clearance are therefore likely to be important. A crystal structure of a trypanosomal CYP51 bound to a tipifarnib analog is reported here and provides new insights to guide structure-based drug design for further optimized compounds.

ABSTRACTHuman 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 bruceidemonstrated 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. bruceiMetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. bruceicultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. bruceigrowth 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. bruceiMetRS 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.

ABSTRACTNew antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparumPFT and are effective at killing P. falciparumin vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.

ABSTRACTTrypanosoma cruziis the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy againstT. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound,N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruziactivity associated with inhibition of oxidosqualene cyclase.

ABSTRACTTrypanosoma cruziis the protozoan parasite that causes Chagas’ disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruziinfections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133–138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruziinfections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance inT. cruziin vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruziinfection.