High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds.

Entamoeba histolytica is a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection with E. histolytica is referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 81,664 compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound. The best compound in this series, JNJ001, showed excellent inhibition activity against E. histolytica trophozoites with EC50 values at 0.29 µM, which is better than the current approved treatment, metronidazole. Further experimentation confirmed the activity of this compound, as well as that of several structurally related compounds, originating from both the Janssen Jump-stARter library, and from chemical vendors, thus highlighting a new structure-activity relationship (SAR). In addition, we confirmed that the compound inhibited E. histolytica survival as rapidly as the current standard of care and inhibited transmissible cysts of the related model organism Entamoeba invadens. Together these results constitute the discovery of a novel class of chemicals with favorable in vitro pharmacological properties. The discovery may lead to an improved therapy against this parasite and in all of its life stages.

ARN25068, a versatile starting point towards triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau-related neurological disorders.

The human kinome plays a crucial role in several pathways. Its dysregulation has been linked to diverse central nervous system (CNS)-related disorders with a drastic impact on the aging population. Among them, tauopathies, such as Alzheimer's Disease (AD) and Frontotemporal Lobar degeneration (FTLD-tau), are neurodegenerative disorders pathologically defined by the presence of hyperphosphorylated tau-positive intracellular inclusions known as neurofibrillary tangles (NFTs). Compelling evidence has reported the great potential of the simultaneous modulation of multiple protein kinases (PKs) involved in abnormal tau phosphorylation through a concerted pharmacological approach to achieve a superior therapeutic effect relative to classic "one target, one drug" approaches. Here, we report on the identification and characterization of ARN25068 (4), a low nanomolar and well-balanced dual GSK-3ß and FYN inhibitor, which also shows inhibitory activity against DYRK1A, an emerging target in AD and tauopathies. Computational and X-Ray studies highlight compound 4's typical H-bonding pattern of ATP-competitive inhibitors at the binding sites of all three PKs. In a tau phosphorylation assay on Tau0N4R-TM-tGFP U2OS cell line, 4 reduces the extent of tau phosphorylation, promoting tau-stabilized microtubule bundles. In conclusion, this compound emerges as a promising prototype for further SAR explorations to develop potent and well-balanced triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau hyperphosphorylation.

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds.

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.

Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells.

Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.