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.
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.
17Entamoeba histolytica is a protozoan parasite which infects approximately 50 million 18 people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E.19 histolytica infection has been successfully treated with metronidazole. However, drawbacks to 20 metronidazole therapy exist, including adverse effects, a long treatment course, and the need 21 for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome 22 with approximately 300 -400 members, some of which have been previously studied as 23 potential targets for the development of amoebicidal drug candidates. However, while these 24 efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in 25 approved drugs. In this study we took the alternative approach of testing a set of twelve 45 both amoebicidal and cysticidal activities may be used to treat amoebiasis, especially in cancer 46 patients or in life-threatening brain-and liver-infecting forms of the disease.47 65 which has been in use since the 1960s and has widespread use as a treatment against 66 anaerobic microbial infection (7, 8). However, while successful, metronidazole is not a perfect 67 solution to E. histolytica infection, with a few particularly notable existing issues. One of these is 68 problems with lack of patient compliance with the full course of treatment, leading to relapses 69 and increased disease spread (7). This is possibly due to factors such as drug adverse effects 70 or the need for continued dosing past the resolution of disease symptoms (9, 10). Another issue 71 is metronidazole's inability to kill the infective cyst stage of E. histolytica. Because of this, along 72 with its complete absorbance from the intestines, metronidazole must be followed by a 73 secondary luminal amoebicide such as paromomycin to prevent spread of the disease (11, 12). 74Also concerning is the potential for the emergence of resistance to metronidazole, which has 75 been previously observed in the laboratory (13). When considered together, these factors 76 comprise an unmet need for alternative amoebiasis therapies. 93 published a computational tool capable of such mapping for antineoplastic drugs, including a 94 large number of kinase inhibitors (20). We describe here the use of this tool to prioritize 95 molecules for screening against E. histolytica trophozoites based on initial hits from a small 96 primary screen. In total, 6 antineoplastic kinase inhibitors (AKIs) were found to have potent and 97 rapid anti-amoebic activity. The results of these experiments demonstrate the promise of using 98 target-based analysis to leverage compounds with multi-target pharmacology against a human 99 parasite. 100 101 Materials and Methods 5 102 103 E. histolytica cell culture 104 E. histolytica strain HM-1:IMSS trophozoites were maintained in 50ml culture flasks 105 (Greiner Bio-One) containing TYI-S-33 media, 10% heat-inactivated adult bovine serum 106 (Sigma), 1% MEM Vitamin Solution (Gibco), supplemented with penici...
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 80,000-compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound. 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 which may lead to an improved therapy against this parasite and in all of its life stages.
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, length of treatment, and the need for additional drugs to prevent transmission. All of these may decrease patient compliance and hence increase disease severity and spread of infection. In this study we identified the antimalarial drug mefloquine as possessing more potent, rapid, amoebicidal in vitro activity against E. histolytica trophozoites than metronidazole. We also showed that mefloquine could kill the cysts of a closely related reptilian parasite Entamoeba invadens unlike metronidazole. Additionally, mefloquine is known to possess a much longer half-life in human patients than metronidazole. This property, along with mefloquine's rapid and broad action against E. histolytica position it as a promising new drug candidate against this widespread and devastating disease.
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.
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