The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.

The marine brown alga Halidrys siliquosa is known to produce compounds with antifouling activity against several marine bacteria. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of organic extracts obtained from the marine brown alga H. siliquosa against a focused panel of clinically relevant human pathogens commonly associated with biofilm-related infections. The partially fractionated methanolic extract obtained from H. siliquosa collected along the shores of Co. Donegal; Ireland; displayed antimicrobial activity against bacteria of the genus Staphylococcus; Streptococcus; Enterococcus; Pseudomonas; Stenotrophomonas; and Chromobacterium with MIC and MBC values ranging from 0.0391 to 5 mg/mL. Biofilms of S. aureus MRSA were found to be susceptible to the algal methanolic extract with MBEC values ranging from 1.25 mg/mL to 5 mg/mL respectively. Confocal laser scanning microscopy using LIVE/DEAD staining confirmed the antimicrobial nature of the antibiofilm activity observed using the MBEC assay. A bioassay-guided fractionation method was developed yielding 10 active fractions from which to perform purification and structural elucidation of clinically-relevant antibiofilm compounds.

In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the “Leishmania donovani box” were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the “Leishmania donovani box” using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds.