Nutritional Supplements, Ginseng and Leucine, Increase Erythropoiesis in Diamond Blackfan Anemia Models through Inhibition of Nemo-like Kinase

Abstract Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is typically associated with mutations in one of at least 20 ribosomal genes and is associated with anemia, congenital abnormalities, and cancer predisposition. The current treatment for DBA is associated with toxicities including iron overload from repeated transfusions, immune suppression from chronic corticosteroid therapy or consequences from stem cell transplantation. Developing new therapies for DBA remains a challenge, since it is a rare disease and the connection between faulty ribosomes and defects in erythropoiesis is still being explored. As such, repurposing existing, approved drugs is one approach to find new ways to treat this disease. We recently identified that Nemo-like Kinase (NLK) is hyper-activated and contributes to disease pathogenesis in the erythroid progenitors of patients with DBA, irrespective of the genetic mutation. NLK is an atypical member of the MAPK family of kinases. Due to a high degree of conservation, kinase inhibitors that specifically target NLK are not currently available, although several small molecules inhibit NLK as an off-target. We are actively pursuing a number of these compounds as potential therapies for DBA. In addition to known kinase inhibitors, we have examined the active components of widely available nutritional supplements. Although rigorous clinical evaluation is required, these supplements are well tolerated and offer an alternative or a complement to conventional drug therapy for DBA. Ginsenoside Rb1, an active component of ginseng, increases erythropoiesis in in vitro models of DBA (2.6-fold) at 50mM (p=0.048) with an EC 50 of 2.3mM. Importantly, ginsenoside Rb1 does not impact healthy erythropoiesis or other hematopoietic lineages and is nontoxic to normal cells. Our results demonstrate that ginsenoside Rb1 does not inhibit NLK kinase activity directly, but rather induces a microRNA (miR-208) that binds to NLK mRNA leading to degradation of transcript by 35.9% (p=0.007) before the protein can be translated. Another nutritional supplement that has been indicated to improve erythropoiesis (in DBA and non-diseased models) and is currently in clinical trials is the amino acid leucine. In DBA progenitors, 5mM leucine increases erythropoiesis from 8.8 to 16.3% of healthy controls. Leucine acts by stimulating the activity of mTORC1 but similar to erythropoiesis, mTORC1 activity is only stimulated from 26.4 to 57.2% of controls in DBA progenitors. Our results demonstrate that leucine does not impact NLK expression or activity directly. Aberrantly activated NLK phosphorylates and inhibits the activation of mTORC1 (target of leucine). The suppression of NLK by 50mM ginsenoside Rb1 restored mTORC1 activity to basal (94.7% of non-diseased control) but also restored leucine sensitivity (from 57.2 to 88.1% of non-diseased controls). Importantly, combining 5mM leucine and 50mM ginsenoside Rb1 increased erythropoiesis from 1.9-fold and 2.6-fold when used alone, to 8.9-fold (or 78.3% of healthy controls) together in our in vitro models of DBA (CI=0.31). Ginseng and leucine offer promising alternatives to steroids and other immunosuppressive drugs for DBA patients. The goal of these studies is to raise the hemoglobin in DBA patients to avoid the need for red cell transfusions. As nutritional supplements are widely available and well tolerated, this class of compounds provides alternatives to currently approved drugs to treat DBA. Disclosures Glader: Agios: Consultancy.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Glucocorticoids Reduce Expression Of p53 Target Genes and Reverse Anemia In a Mouse Model Of Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v122.21.3703.3703 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3703-3703

Author(s):

Sara Sjögren ◽

Pekka Jaako ◽

Stefan Karlsson ◽

Johan Flygare

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Fetal Liver ◽

Bone Marrow Failure ◽

Erythroid Progenitors ◽

Diamond Blackfan Anemia ◽

Therapeutic Mechanism ◽

Fetal Liver Cells ◽

Responsive Gene

Abstract Diamond-Blackfan anemia (DBA) is a rare congenital disease of macrocytic anemia, increased cancer predisposition and progressive bone marrow failure. Mutations in ribosomal protein genes are responsible for most cases and the most commonly mutated gene, RPS19 is affected in around 25% of DBA patients. Although glucocorticoid (GC) administration has been used to stimulate erythropoiesis in DBA patients for decades, the therapeutic mechanism of GC treatment is poorly understood. Since it has been shown that bone marrow failure in DBA is caused by p53 activation we hypothesized the therapeutic mechanism of GC involves reduced p53 activity. To assess possible connections between p53 activity and GC administration in DBA we used a doxycycline-inducible mouse model for RPS19-deficient DBA (Jaako et al, Blood 2011). In these mice, doxycycline administration leads to reduced Rps19 levels, defective ribosomal biogenesis, proliferative arrest, increased apoptosis of erythroid progenitors and p53-dependent bone marrow failure. In this study we show that Rps19-deficient mice respond to treatment with the GC prednisolone, with a significant increase in red blood cell count as well as in hemoglobin concentration and hematocrit compared to Rps19-deficient controls (p-values<0.05). Also, prednisolone treated Rps19-deficient mice show a significant increase in the Ter119+ erythroid cell fraction in the bone marrow compared to Rps19-deficient controls. Gene expression analysis of c-Kit+ fetal liver cells from E14.5-15.5 of Rps19-deficient embryos cultured in vitro in the presence of doxycycline for 3 days, showed an increase in p53 responsive gene expression of multiple p53 targets such as p21, Bax, Ccng1, Phlda3 and Prl-3. However, upon administration of the synthetic GC dexamethasone all these genes significantly failed to up-regulate, even in the presence of doxycycline (p-values< 0.02). Also, dexamethasone treated RPS19-deficient c-Kit+ fetal liver cells show an increase in proliferation compared to RPS19-deficient controls (p-value< 0.05), that starts already after 2 days of in vitro culture, being more pronounced over time. These results indicate that administration of GC dampens p53 responsive gene expression of genes known to promote cell cycle arrest as well as apoptosis. Although GCs are known to regulate many functions within an organism, our results indicate that the therapeutic effect of GC in DBA patients at least in part is explained by inhibition of p53 responsive gene activation, leading to increased proliferation and survival of erythroid progenitors. Disclosures: No relevant conflicts of interest to declare.

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High Throughput Virtual Screening to Discover Inhibitors of the Main Protease of the Coronavirus SARS-CoV-2

Molecules ◽

10.3390/molecules25143193 ◽

2020 ◽

Vol 25 (14) ◽

pp. 3193 ◽

Cited By ~ 8

Author(s):

Olujide O. Olubiyi ◽

Maryam Olagunju ◽

Monika Keutmann ◽

Jennifer Loschwitz ◽

Birgit Strodel

Keyword(s):

Natural Products ◽

Kinase Inhibitors ◽

Amino Acid Residues ◽

Enzyme Dynamics ◽

Hydrogen Bond Formation ◽

Protease Enzyme ◽

Main Protease ◽

Starting Point ◽

Approved Drugs

We use state-of-the-art computer-aided drug design (CADD) techniques to identify prospective inhibitors of the main protease enzyme, 3CLpro of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19. From our screening of over one million compounds including approved drugs, investigational drugs, natural products, and organic compounds, and a rescreening protocol incorporating enzyme dynamics via ensemble docking, we have been able to identify a range of prospective 3CLpro inhibitors. Importantly, some of the identified compounds had previously been reported to exhibit inhibitory activities against the 3CLpro enzyme of the closely related SARS-CoV virus. The top-ranking compounds are characterized by the presence of multiple bi- and monocyclic rings, many of them being heterocycles and aromatic, which are flexibly linked allowing the ligands to adapt to the geometry of the 3CLpro substrate site and involve a high amount of functional groups enabling hydrogen bond formation with surrounding amino acid residues, including the catalytic dyad residues H41 and C145. Among the top binding compounds we identified several tyrosine kinase inhibitors, which include a bioflavonoid, the group of natural products that binds best to 3CLpro. Another class of compounds that decently binds to the SARS-CoV-2 main protease are steroid hormones, which thus may be endogenous inhibitors and might provide an explanation for the age-dependent severity of COVID-19. Many of the compounds identified by our work show a considerably stronger binding than found for reference compounds with in vitro demonstrated 3CLpro inhibition and anticoronavirus activity. The compounds determined in this work thus represent a good starting point for the design of inhibitors of SARS-CoV-2 replication.

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Diamond-blackfan anemia: in vitro response of erythroid progenitors to the ligand for c-kit

Blood ◽

10.1182/blood.v78.9.2198.2198 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2198-2202 ◽

Cited By ~ 51

Author(s):

JL Abkowitz ◽

KM Sabo ◽

B Nakamoto ◽

CA Blau ◽

FH Martin ◽

...

Keyword(s):

Mononuclear Cells ◽

Therapeutic Trial ◽

Erythroid Progenitors ◽

Primary Defect ◽

Diamond Blackfan Anemia ◽

Kit Receptor ◽

In Vitro Response ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Abstract To provide insights into the pathogenesis of Diamond-Blackfan anemia, we examined the in vitro response of erythroid progenitors to the recently isolated ligand for c-kit (stem cell factor, SCF). For these studies, marrow or blood mononuclear cells from 10 Diamond-Blackfan patients were cultured with erythropoietin (Ep), Ep and interleukin-3, Ep and granulocyte-macrophage colony-stimulating factor, or Ep and lymphocyte conditioned media (LCM). These combinations were tested in the presence or absence of SCF. The mean number of cells per erythroid burst increased 5 to 50-fold in cultures containing SCF. Furthermore, many additional erythroid bursts were seen (mean increment 3.2 x baseline values). Although burst-forming unit-erythroid (BFU-E) from all patients responded, there were differences among individuals in the sensitivity of their BFU-E to SCF. In six patients and all control studies, plateau frequencies of erythroid bursts were achieved with less than or equal to 10 ng/mL SCF, whereas in studies from the other four patients, over 50 ng/mL SCF was required. These data invite speculation that the c-kit receptor/ligand axis is involved in the pathogenesis of Diamond-Blackfan anemia. More importantly and regardless of whether the observed patterns of response reflect the primary defect or an epiphenomenon, our data strongly support a therapeutic trial of SCF in patients with Diamond-Blackfan anemia.

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Combating Acquired Resistance to Tyrosine Kinase Inhibitors in Lung Cancer

American Society of Clinical Oncology Educational Book ◽

10.14694/edbook_am.2015.35.e165 ◽

2015 ◽

pp. e165-e173 ◽

Cited By ~ 8

Author(s):

Christine M. Lovly

Keyword(s):

Lung Cancer ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Acquired Resistance ◽

Initial Response ◽

In Vitro Models ◽

Tumor Biopsy

The prospective identification and therapeutic targeting of oncogenic tyrosine kinases with tyrosine kinase inhibitors (TKIs) has revolutionized the treatment for patients with non–small cell lung cancer (NSCLC). TKI therapy frequently induces dramatic clinical responses in molecularly defined cohorts of patients with lung cancer, paving the way for the implementation of precision medicine. Unfortunately, acquired resistance, defined as tumor progression after initial response, seems to be an inevitable consequence of this treatment approach. This brief review will provide an overview of the complex and heterogeneous problem of acquired resistance to TKI therapy in NSCLC, with a focus on EGFR-mutant and ALK-rearranged NSCLC. In vitro models of TKI resistance and analysis of tumor biopsy samples at the time of disease progression have generated breakthroughs in our understanding of the spectrum of mechanisms by which a tumor can thwart TKI therapy and have provided an important rationale for the development of novel approaches to delay or overcome resistance. Numerous ongoing clinical trials implement strategies, including novel, more potent TKIs and rational combinations of targeted therapies, some of which have already proven effective in surmounting therapeutic resistance.

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Lenalidomide Up-Regulates SPARC and Inhibits In Vitro Growth of the Malignant Clone in Myelodysplastic Syndrome Patients with 5q Deletion.

Blood ◽

10.1182/blood.v108.11.855.855 ◽

2006 ◽

Vol 108 (11) ◽

pp. 855-855

Author(s):

Andrea Pellagatti ◽

Martin Jädersten ◽

Ann-Mari Forsblom ◽

Helen Cattan ◽

Birger Christensson ◽

...

Keyword(s):

Gene Expression ◽

Myelodysplastic Syndrome ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Expression Profiles ◽

Activin A ◽

Healthy Controls ◽

Erythroid Progenitors ◽

Normal Cells

Abstract The immunomodulatory drug lenalidomide induces cytogenetic remissions in 75% of patients with myelodysplastic syndrome (MDS) and del(5)(q31) through unknown mechanisms. We investigated the in vitro effects of lenalidomide on growth and maturation in differentiating erythroblasts from MDS patients with del(5)(q31) (n=13) and from healthy controls (n=10). Lenalidomide selectively inhibited growth of del(5q) erythroblasts, while not affecting normal cells, including cytogenetically normal cells from MDS del(5q) patients. The inhibitory effect was more pronounced in erythroid than in myeloid cells. In order to gain insight into the mode of action of lenalidomide and to identify the molecular targets of this drug, we have investigated the gene expression profiles of the lenalidomide-treated and untreated intermediate erythroblasts from MDS del(5q) patients (n=9) and from healthy controls (n=8). GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), covering over 47,000 transcripts representing 39,000 human genes, were used. Treatment with lenalidomide significantly influenced the pattern of gene expression in del(5q) intermediate erythroblasts, with up-regulation of VSIG4, PPIC, TPBG, and SPARC in all samples, and down-regulation of many genes involved in erythropoiesis, including HBA2, GYPA, and KLF1, in most samples. Up-regulation of SPARC (median 4.4-fold, range 2.4–9.5) is of particular interest since SPARC, a gene with known tumor suppressor functions, is both anti-proliferative and anti-angiogenic, and is located at 5q31–q32, within the commonly deleted region in MDS 5q- syndrome. Activin A was one of the most significant differentially expressed genes between lenalidomide-treated cells of MDS del(5q) patients and healthy controls. Activin A is a member of the transforming growth factor-beta superfamily, with pleiotropic functions including apoptosis of hemopoietic cells. We conclude that lenalidomide specifically inhibits growth of del(5q) erythroid progenitors, while not affecting cytogenetically normal cells. These novel findings suggest that up-regulation of SPARC and Activin A may underlie the potent effects of lenalidomide, in particular growth inhibition and anti-angiogenesis, in MDS with del(5)(q31). The localization of the SPARC gene to the CDR of the 5q- syndrome is intriguing and, in relation to the findings of the present study, we suggest that SPARC may well play a role in the molecular pathogenesis of the 5q- syndrome.

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Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: Evidence from RPL11-deficient in vivo and in vitro models

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2017.12.019 ◽

2018 ◽

Vol 495 (2) ◽

pp. 1839-1845 ◽

Cited By ~ 4

Author(s):

Anirban Chakraborty ◽

Tamayo Uechi ◽

Yukari Nakajima ◽

Hanna T. Gazda ◽

Marie-Françoise O'Donohue ◽

...

Keyword(s):

Cross Talk ◽

In Vitro Models ◽

Diamond Blackfan Anemia

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Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis

Blood ◽

10.1182/blood-2010-11-318543 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2296-2304 ◽

Cited By ~ 52

Author(s):

Anupama Narla ◽

Shilpee Dutt ◽

J. Randall McAuley ◽

Fatima Al-Shahrour ◽

Slater Hurst ◽

...

Keyword(s):

Bone Marrow ◽

Expression Profiles ◽

Bone Marrow Failure ◽

Gene Expression Profiles ◽

Absolute Number ◽

Red Blood Cell Transfusion ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Cd34 Cells

Abstract Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.

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Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes

Blood ◽

10.1182/blood.v80.12.3000.bloodjournal80123000 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3000-3008

Author(s):

BP Alter ◽

ME Knobloch ◽

L He ◽

AP Gillio ◽

RJ O'Reilly ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Factor ◽

Mononuclear Cells ◽

Bone Marrow Failure ◽

Dyskeratosis Congenita ◽

Normal Numbers ◽

Erythroid Progenitors ◽

Bone Marrow Failure Syndromes

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

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

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008425 ◽

2021 ◽

Vol 15 (2) ◽

pp. e0008425

Author(s):

Conall Sauvey ◽

Gretchen Ehrenkaufer ◽

Da Shi ◽

Anjan Debnath ◽

Ruben Abagyan

Keyword(s):

Kinase Inhibitors ◽

Model Organism ◽

Protozoan Parasite ◽

Drug Candidates ◽

New Class ◽

Alternative Approach ◽

The 1960S ◽

Approved Drugs ◽

Micromolar Range

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.

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