Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/53912
Title: Treatment of sarcopenia by antagonists of myostatin
Authors: Anissa Anindya Widjaja
Keywords: DRNTU::Science::Biological sciences::Biochemistry
Issue Date: 2013
Abstract: Sarcopenia is a profound loss of skeletal muscle mass, strength, and endurance that occurs with aging. Age-related muscle wasting has been reported even among healthy, physically active individual and the rate of muscle loss has been approximated to range 1-2% annually past the age of 50. Albeit, exercise and hormone replacement improve sarcopenia-associated decline of muscle performance, but due to personal awareness, cost and hormone side effects, a safer and more affordable pharmacological approach is necessary. Among the newer approaches towards sarcopenia treatment is inhibition of myostatin, a transforming regulator of muscle growth. The project seeks to develop a small molecule that can eventually be used for prevention and treatment of sarcopenia. First, we aim to unravel myostatin interaction with its possible receptors as to have insights into the mechanism of action of myostatin. Our SPR studies showed that myostatin interacts with the ECD of the members of TGFβ receptor type II i.e. ActRIIB, ActRIIA, BMPRII, and TGFβRII, with binding affinity (KD) of 257 pM, 6.4 nM, 69.6 nM, 61.1 nM, respectively. Furthermore, functional myoblast assay demonstrated that addition of ECD of TGFβ receptor type II members was able to neutralize myostatin inhibition on myoblast proliferation in a dose-dependent manner. Here, we also showed that, although follistatin, a known antagonist of several members of the TGFβ family, including myostatin, was able to directly bind to ActRIIB, the KD of the interaction (98 nM) was much weaker than the KD for myostatin-follistatin (584 pM). Second, we aim to identify the amino acids that contribute to high affinity binding of ActRIIB:myostatin complex. As ActRIIB:myostatin structure has not been elucidated, we adopted homology modelling to construct an ActRIIB:myostatin complex and performed an in silico alanine scanning to predict the amino acids responsible for the contact points of the interaction. Residues N83, Y95, D104 on myostatin & residues Y60, K74, W78, L79, and F101 on ActRIIB showed high binding free energy differences between the WT and alanine mutants. We also investigated the importance of R64 on ActRIIB, as the existence of its natural allelic variation was reported. Using SPR, we showed that there was a significant reduction in the binding affinity when either myostatin or ActRIIB mutants were involved in the interaction. Moreover, through functional myoblast assay we demonstrated that myostatin mutants had no ability to inhibit myoblast proliferation, whereas ActRIIB mutants overcame the myostatin inhibition on myoblast proliferation to a lesser extent as compared to WT ActRIIB. Hence, in this study we proposed that residues N83, Y95, and D104 of myostatin and residues Y60, R64, K74, W78, L79, and F101 of ActRIIB are essential for the stability and high affinity binding of the ActRIIB:myostatin complex, which renders the downstream signal transduction. Based on the findings on the functional epitope of ActRIIB:myostatin complex, we performed an in silico screening of small molecules library for their binding to myostatin interface for ActRIIB binding. We used SPR to screen the ability of the potential compounds to bind specifically to myostatin and we found that 31 molecules with good box-like sensorgram shape and no noticeable noise were specific to myostatin at 100 μM. From kinetic experiments, we found that 15 out of the 31 molecules bind with high specificity to myostatin and with expected affinity for small molecules. We then performed both myoblast proliferation assay and Smad3 luciferase assay and found that 5 compounds (15, 16, 23, 25, 38) were effective in neutralizing the myostatin-mediated proliferation inhibition effect via the Smad3-mediated signaling pathway.
URI: http://hdl.handle.net/10356/53912
Fulltext Permission: restricted
Fulltext Availability: With Fulltext
Appears in Collections:SBS Theses

Files in This Item:
File Description SizeFormat 
Tbsg0802751k.pdf
  Restricted Access
6.54 MBAdobe PDFView/Open

Page view(s) 1

533
checked on Oct 19, 2020

Download(s) 1

14
checked on Oct 19, 2020

Google ScholarTM

Check

Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.