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Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.

Objectives Drugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis. Methods The in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50, Graphical abstract Image 1

Aim: To develop a bioanalytical method to support pharmacokinetic evaluation of DNDI-VL-2098 in mouse, rat, dog and hamster following oral administration. Results & methodology: A robust LC–MS/MS bioanalytical method was developed to quantify DNDI-VL-2098. DNDI-VL-2098 showed time-dependent recovery loss in acetonitrile precipitated plasma in all species. Acid-lysed whole blood was identified as a matrix in which recovery was stable over time. A two-step extraction procedure was used, with protein precipitation followed by liquid–liquid extraction with methyl tert-butyl ether. The assay was validated in the dynamic range of 5–5000 ng/ml for mouse, rat and dog blood, and a fit-for-purpose method was developed for hamster. Conclusion: A specific LC–MS/MS assay for DNDI-VL-2098 was developed and validated in hemolyzed blood.

[Image: see text] Antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines were previously shown to exhibit potent antileishmanial and antitrypanosomal activities, culminating in a new clinical investigational drug for visceral leishmaniasis (DNDI-0690). To offset development risks, we continued to seek further leads with divergent candidate profiles, especially analogues possessing greater aqueous solubility. Starting from an efficacious monoaryl derivative, replacement of the side chain ether linkage by novel amine, amide, and urea functionality was first explored; the former substitution was well-tolerated in vitro and in vivo but elicited marginal alterations to solubility (except through a less stable benzylamine), whereas the latter groups resulted in significant solubility improvements (up to 53-fold) but an antileishmanial potency reduction of at least 10-fold. Ultimately, we discovered that O-carbamate 66 offered a more optimal balance of increased solubility, suitable metabolic stability, excellent oral bioavailability (100%), and strong in vivo efficacy in a visceral leishmaniasis mouse model (97% parasite load reduction at 25 mg/kg).

Previous investigation of the potent antileishmanial properties of antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines with biaryl side chains led to our development of a new clinical candidate for visceral leishmaniasis (DNDI-0690). Within a collaborative backup program, a racemic monoaryl lead (3) possessing comparable activity in mice but a greater hERG liability formed the starting point for our pursuit of efficacious second generation analogues having good solubility and safety. Asymmetric synthesis and appraisal of its enantiomers first established that chiral preferences for in vivo efficacy were species dependent and that neither form afforded a reduced hERG risk. However, in line with our findings in a structurally related series, less lipophilic heteroaryl ethers provided significant solubility enhancements (up to 16-fold) and concomitantly attenuated hERG inhibition. One promising pyridine derivative (49) displayed 100% oral bioavailability in mice and delivered a 96% parasite burden reduction when dosed at 50 mg/kg in a Leishmania donovani mouse model of visceral leishmaniasis.

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development., Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo. Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

The nitroimidazole DNDI-0690 is a clinical drug candidate for visceral leishmaniasis (VL) that also shows potent in vitro and in vivo activity against cutaneous leishmaniasis (CL). To support further development of this compound into a patient-friendly oral or topical formulation for the treatment of CL, we investigated the free drug exposure at the dermal site of infection and subsequent elimination of the causative Leishmania pathogen., The nitroimidazole DNDI-0690 is a clinical drug candidate for visceral leishmaniasis (VL) that also shows potent in vitro and in vivo activity against cutaneous leishmaniasis (CL). To support further development of this compound into a patient-friendly oral or topical formulation for the treatment of CL, we investigated the free drug exposure at the dermal site of infection and subsequent elimination of the causative Leishmania pathogen. This study evaluates the pharmacokinetics (PK) and pharmacodynamics (PD) of DNDI-0690 in mouse models of CL. Skin microdialysis and Franz diffusion cell permeation studies revealed that DNDI-0690 permeated poorly (

Objectives Three new chemical series (bicyclic nitroimidazoles, aminopyrazoles and oxaboroles) were selected by Drugs for Neglected Diseases initiative as potential new drug leads for leishmaniasis. Pharmacodynamics studies included both in vitro and in vivo efficacy, cross-resistance profiling against the current antileishmanial reference drugs and evaluation of their cidal activity potential. Methods Efficacy against the reference laboratory strains of Leishmania infantum (MHOM/MA(BE)/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) was evaluated in vitro on intracellular amastigotes and in vivo in the early curative hamster model. Cidal activity was assessed over a period of 15 days in an in vitro ‘time-to-kill’ assay. Cross-resistance was assessed in vitro on a panel of L. infantum strains with different degrees of resistance to either antimony, miltefosine or paromomycin. Results All lead compounds showed potent and selective in vitro activity against the Leishmania strains tested and no cross-resistance could be demonstrated against any of the current antileishmanial drugs. Cidal activity was obtained in vitro for all series within 15 days of exposure with some differences noted between L. donovani and L. infantum. When evaluated in vivo, all lead compounds showed high efficacy and no adverse effects were observed. Conclusions The new lead series were shown to have cidal pharmacodynamic activity. The absence of cross-resistance with any of the current antileishmanial drugs opens possibilities for combination treatment to reduce the likelihood of treatment failures and drug resistance., Graphical abstract Image 1, Highlights • Good efficacy was evaluated for all series in vitro and in vivo. • No cross-resistance towards current anti-leishmanial drugs was observed. • Cidal activity was obtained in vitro for all series within 15 days of exposure. • Some differences were observed between L. infantum and L. donovani.

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series

With the aim of improving the available drugs for the treatment of Chagas disease, individual enantiomers of nifurtimox were characterized. The results indicate that the enantiomers are equivalent in their in vitro activity against a panel of Trypanosoma cruzi strains; in vivo efficacy in a murine model of Chagas disease; in vitro toxicity and absorption, distribution, metabolism, and excretion characteristics; and in vivo pharmacokinetic properties. There is unlikely to be any therapeutic benefit of an individual nifurtimox enantiomer over the racemic mixture.

Sulfonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytoremediation process to remove sulfonated aromatic compounds from industrial effluents, the most promising results have been obtained with Rheum rabarbarum (rhubarb), a plant species producing natural anthraquinones. Rhubarb is not only able to accumulate, but also to transform, these xenobiotic chemicals. Even if the biochemical mechanisms involved in the detoxification of sulfonated anthraquinones are not yet fully understood, they probably have cross talks with secondary metabolism, redox processes, and plant energy metabolism. Therefore, key problems of xenobiotic detoxification are addressed, as well as the potential role of the apoplast, often neglected, and the possible links with plant sulfur metabolism.

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using non-animal host models.