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A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia

Siva, Kavitha LU ; Ek, Fredrik LU ; Chen, Jun LU ; Ghani Alattar, Abdul LU ; Sigmundsson, Kristmundur; Olsson, Roger LU ; Wlodarski, Marcin; Lundbäck, Thomas and Flygare, Johan LU (2019) In SLAS discovery : advancing life sciences R & D 24(3). p.304-313
Abstract

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of... (More)

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
DBA, DYRK inhibitor, Harmine, screening assay, small-molecule libraries
in
SLAS discovery : advancing life sciences R & D
volume
24
issue
3
pages
10 pages
publisher
SAGE Publications Inc.
external identifiers
  • scopus:85061871298
ISSN
2472-5552
DOI
10.1177/2472555218823531
language
English
LU publication?
yes
id
e56addfc-f225-42a3-9aff-456b1827e379
date added to LUP
2019-03-01 11:58:04
date last changed
2019-04-10 04:20:22
@article{e56addfc-f225-42a3-9aff-456b1827e379,
  abstract     = {<p>Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to &lt;10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.</p>},
  author       = {Siva, Kavitha and Ek, Fredrik and Chen, Jun and Ghani Alattar, Abdul and Sigmundsson, Kristmundur and Olsson, Roger and Wlodarski, Marcin and Lundbäck, Thomas and Flygare, Johan},
  issn         = {2472-5552},
  keyword      = {DBA,DYRK inhibitor,Harmine,screening assay,small-molecule libraries},
  language     = {eng},
  number       = {3},
  pages        = {304--313},
  publisher    = {SAGE Publications Inc.},
  series       = {SLAS discovery : advancing life sciences R & D},
  title        = {A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia},
  url          = {http://dx.doi.org/10.1177/2472555218823531},
  volume       = {24},
  year         = {2019},
}