The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition

Signed zeros of Gaussian vector fields-density, correlation functions and curvature

We calculate correlation functions of the (signed) density of zeros of Gaussian distributed vector fields. We are able to express correlation functions of arbitrary order through the curvature tensor of a certain abstract Riemann-Cartan or Riemannian manifold. As an application, we discuss one- and two-point functions. The zeros of a two-dimensional Gaussian vector field model the distribution of topological defects in the high-temperature phase of two-dimensional systems with orientational degrees of freedom, such as superfluid films, thin superconductors and liquid crystals.Comment: 14 pages, 1 figure, uses iopart.cls, improved presentation, to appear in J. Phys.

Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation.

New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18-118 nM to 180-890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3.IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs

Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal – adult worm killing – drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin that were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 is represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.  AUTHOR SUMMARY Onchocerciasis and lymphatic filariasis are human filarial tropical diseases, which can cause blindness and severe dermatitis (onchocerciasis) or lymphedema and hydrocele (lymphatic filariasis). Current strategies to eliminate these diseases include the mass drug administration (MDA) of drugs that target the progeny of the filariae, the microfilariae, and temporarily inhibit filarial embryogenesis and, therefore, the transmission of the disease. However, MDA has several limitations that delay the goal of elimination including the lack of a drug with a short term regimen and a potent macrofilaricidal effect. As an alternative approach, the antibiotic doxycycline has been proven to be effective in depleting Wolbachia endosymbionts from adult filariae, which then leads to permanent sterilization and death of the adult worms. Due to contraindications for doxycycline and prolonged treatment regimen of at least 4 weeks, there is an urgent need for new anti-filarial drugs with an improved safety profile and shorter regimens. The current study demonstrates that the boron-pleuromutilin derivative AN11251 provides an excellent in vivo anti-Wolbachia depletion in the Litomosoides sigmodontis filarial mouse model that is superior to doxycycline and comparable to rifampicin, allowing for regimens as short as 10-14 days. Combination with doxycycline for 7 days had no significant beneficial effect on efficacy, achieving Wolbachia reductions of more than 97%. Therefore, AN11251 shows potent anti-Wolbachia activity in the L. sigmodontis mouse model and may also present an alternative pre-clinical candidate for filariasis treatment

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.</p