Identification of a small molecule inhibitor of Ebolavirus genome replication and transcription using in silico screening.

Ebola virus (EBOV) causes a severe haemorrhagic fever in humans and has a mortality rate over 50%. With no licensed drug treatments available, EBOV poses a significant threat. Investigations into possible therapeutics have been severely hampered by the classification of EBOV as a BSL4 pathogen. Here, we describe a drug discovery pathway combining in silico screening of compounds predicted to bind to a hydrophobic pocket on the nucleoprotein (NP); with a robust and rapid EBOV minigenome assay for inhibitor validation at BSL2. One compound (MCCB4) was efficacious (EC50 4.8 μM), exhibited low cytotoxicity (CC50 > 100 μM) and was specific, with no effect on either a T7 RNA polymerase driven firefly luciferase or a Bunyamwera virus minigenome. Further investigations revealed that this small molecule inhibitor was able to outcompete established replication complexes, an essential aspect for a potential EBOV treatment

Is the ADP ribose site of the Chikungunya virus NSP3 Macro domain a target for antiviral approaches?

Chikungunya virus (CHIKV) is a mosquito-transmitted virus of special concern as it causes Chikungunya fever, characterized by an acute febrile illness, rash, and arthralgia that can progress to chronic and debilitating arthritic symptoms. The effects of climate change on the geographic distribution of the mosquito vector has the potential to expose more of the globe to this virus. No antiviral agents or vaccines are currently available against CHIKV infection and the development of novel therapies that may lead to a future treatment is therefore necessary. In this context, the ADP-ribose binding site of the CHIKV nsP3 macro domain has been reported as a potential target for the development of antivirals. Mutations in the ADP-ribose binding site demonstrated decreased viral replication in cell culture and reduced virulence. In this study, 48,750 small molecules were screened in silico for their ability to bind to the ADP-ribose binding site of the CHIKV nsP3 macro domain. From this in silico analysis, 12 molecules were selected for in vitro analysis using a CHIKV subgenomic replicon in Huh-7 cells. Cell viability and CHIKV replication were evaluated and molecules C5 and C13 demonstrated 53 and 66% inhibition of CHIKV replication, respectively. By using a CHIKV-Dual luciferase replicon contain two reporter genes, we also demonstrated that the treatment with either compounds are probably interfering in the early replication rather than after RNA replication has occurred

The Growing Role of Electron Microscopy in Anti-parasitic Drug Discovery

Parasitic diseases are a huge burden on human health causing significant morbidity and mortality. However, parasitic structure based drug discovery programmes have been hindered by a lack of high resolution structural information from parasitic derived proteins and have largely relied upon homology models from mammalian systems. The recent renaissance in electron microscopy (EM) has caused a dramatic rise in the number of structures being determined at high resolution and subsequently enabled it to be thought of as a tool in drug discovery. In this review, we discuss the challenges associated with the structural determination of parasitic proteins. We then discuss the reasons behind the resurgence in EM, how it may overcome some of these challenges and provide examples of EM derived parasitic protein structures. Finally, we discuss the challenges which EM needs to overcome before it is used as a mainstream technique in parasitic drug discovery

Discrimination of Potent Inhibitors of Toxoplasma gondii Enoyl-Acyl Carrier Protein Reductase by a Thermal Shift Assay

Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD+) cofactor. We found that all of the inhibitors bind to a TgENR–NAD+ complex but that they differed in their dependence on NAD+ concentration. Ultimately, we were able to identify compounds that bind to the TgENR–NAD+ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites

Author Correction: New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections

Correction to: Scientific Reports https://doi.org/10.1038/srep29179, published online 14 July 201

Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with “Rosetta stone”-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease

From TgO/GABA-AT, GABA, and T-263 mutant to conception of Toxoplasma

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat’s oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with “Rosetta stone”-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite’s capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease

Discovery of a Benzimidazole-based Dual FLT3/TrKA Inhibitor Targeting Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) enzyme overexpression and mutations are the most common molecular abnormalities associated with acute myeloid leukemia (AML). In addition, recent studies investigated the role of tropomyosin receptor kinase A (TrKA) enzyme fusions in promoting AML growth and survival. Based on these premises, targeting both kinases using dual inhibitors would constitute a promising therapeutic approach to target resistant AML. Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP, that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC50 = 43.8, 97.2, and 92.5 nM respectively). Additionally, 4ACP demonstrated potent activity against colon cancer KM12 cell line (IC50 = 358 nM) and subsequent mechanistic deconvolution identified TrKA enzyme as a second plausible target (IC50 = 23.6 nM) for our compound. 4ACP manifested preferential antiproliferative activity against FLT3-ITD positive AML cell lines (MV4-11 IC50 = 38.8 ± 10.7 nM and MOLM-13 IC50 = 54.9 ± 4.1 nM), while lacking activity against FLT3-ITD negative AML cell lines. Western blot analysis confirmed 4ACP ability to downregulate ERK1/2 and mTOR signaling downstream of FLT3-ITD in AML cells. Furthermore, 4ACP prompted apoptotic and necrotic cell death and G0/G1 cell cycle arrest as indicated by cell cycle analysis. 4ACP did not show cytotoxic effects on normal BNL and H9c2 cells and demonstrated decreased activity against c-Kit enzyme, hence, indicating lower probability of synthetic lethal toxicity and a relatively safer profile. In light of these data, 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML