Topoisomerase inhibitor
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Transcript of Topoisomerase inhibitor
Hsien-Sung Huang , John A. Allen, Angela M. Mabb, Ian F. King, Jayalakshmi Miriyala, Bonnie Taylor-Blake, Noah Sciaky, J. Walter Dutton Jr, Hyeong-Min Lee., Xin Chen, Jian Jin, Arlene S. Bridges, Mark J. Zylka, Bryan L. Roth and Benjamin D. Philpot
National Institute of Mental Health, Psychoactive Drug Screening Program, Department of Pharmacology, University of North CarolinaSchool of Medicine, Chapel Hill, Nor th Carolina 27599, USA.
Prafulla S. KatkarPhD XVIIII year.
What the paper is dealing all about ?
Possible future therapeutic strategy for the angelmon syndrome.
Angelmon syndrome :
Neurodevelopment disorder caused by deletion or mutation of maternal alleleof ubiquitin protein ligase E3A (UBE3A).
1st report – Henry Angelmon 1965
Year 1965-1997 – Study of mechanism and implications in Human
21st century - Not yet any possible therapy available.
This article work is first of all, worked out treatment strategy.
Albrecht et al (1997)
Normal individual has monoallelic expression of Ube3a from mother.
Symptoms :1. Intellectual disability 2. ataxia3. absent speech 4. jerky arm movement5. inappropriate laughter
Ube3a is a HECT domain protein that enables ubiqitin and target protein to come in proximity and allow for the attachment of ubiquitin molecules.
BD Pathway 855 imaging Microscope
Auto fluorescent
Ube3a (M+/P
YFP) neurons DIV -7
10µM drugs 72 hrs.
Ube3a (M+/P
YFP) neurons DIV -7
10µM drugs 72 hrs.
Array scan Software
BD Pathway High content Image Microscopy
Irinotecan identified as unsilencer of paternally inherited Ube3a allele
Identified active (FDI Approved )
Irinotecan upregulate the Ube3a protein level in wild (M+/P+) and angelmon trait (M-/P+)
Ube3a (M+/P
YFP) neurons DIV -7
10µM drugs /0.02% DMSO 72 hrs.
Identified active (FDI approved)
Ube3a (M+/P
YFP) neurons DIV -7
300 nM drugs /0.02% DMSO 72 hrs.Dose dependant
Time dependant
Topotecan and Irinotecan upregulate the paternal Ube3a-YFP in a time and dose dependant manner.
Topotecan is 20 time more potent than irinotecan
Topotecan 300nm-72hr Restoration to wild type
level
Catalytically active
versus Ube3a
Topotecan treatment restore the paternal Ube3a protein at its catalytic active form
on genomic level
Antisense sequence overlaps and inhibit the expression of paternal Ube3a
Quantitative RT PCR
RNA from cortical neurons (M-/P
+) treated
With 300nM topotecan
Data are normalize with housekeeping genes rpL22 and Act6
Checking methylation :
CAST/EIJ C57BL/6 and neuron from the resulting hybrid were Treated with 300nm topotecan for 72 hr
Genomic DNA isolated after 72hrs and bisulphite conversion done and amplified by PCR
500µm
in vivo
Topotecan (3.74 µg/h) in lateral ventricle of Ube3a(M
-/P
YFP) or Ube3a (M
-/P
+)
I.C.V. injection for 2 weeks, then killed mice immediately or 5 hr after drug delivery.
No activity in cerebellum
I.C.V.
Zeiss LSM 510 confocal microscope
In cerebellum
Pharmacokinetics :
Topotecan (21.6 µg/h)For 5 days
Topotecan conc. declines after 5hr drug delivery
•Topotecan does not persist and metabolize in brain.
• Unsilencing of Ube3a is directly correlated with drug conc. In brain.
•Topotecan unsilence the paternal Ube3a allele throughout the nervous system.
(Purkinje cells)
Cerebral cortex
: transient or long lasting
i.t. delivery 50nm/day in 5µl for 10 out of 14 days
Topotecan and irinotecan unsilenceLarge population of lumbar spinalNeurons.So, quantification of all Ube3a-YFPCells is possible.
M- P
YFP
MY
FPP
+
500µm
Topotecan unsilence the paternal Ube3a in spinal neurons and permanently modify the expression of Ube3a.