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Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches

SIMPLE SUMMARY: Rv1636 is a mycobacterial universal stress protein whose expression level increases in different type of stress conditions. This protein promotes the growth of Mycobacterium tuberculosis in the host derived stress conditions generated during infection. Therefore in this manuscipt, we...

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Autores principales: Beg, Md Amjad, Sadaf, Shamsi, Anas, Sahoo, Sibasis, Yousuf, Mohd, Najm, Mohammad Zeeshan, Almutawif, Yahya Ahmad, Islam, Asimul, Aloliqi, Abdulaziz A., Athar, Fareeda
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9405466/
https://www.ncbi.nlm.nih.gov/pubmed/36009841
http://dx.doi.org/10.3390/biology11081214
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author Beg, Md Amjad
Sadaf,
Shamsi, Anas
Sahoo, Sibasis
Yousuf, Mohd
Najm, Mohammad Zeeshan
Almutawif, Yahya Ahmad
Islam, Asimul
Aloliqi, Abdulaziz A.
Athar, Fareeda
author_facet Beg, Md Amjad
Sadaf,
Shamsi, Anas
Sahoo, Sibasis
Yousuf, Mohd
Najm, Mohammad Zeeshan
Almutawif, Yahya Ahmad
Islam, Asimul
Aloliqi, Abdulaziz A.
Athar, Fareeda
author_sort Beg, Md Amjad
collection PubMed
description SIMPLE SUMMARY: Rv1636 is a mycobacterial universal stress protein whose expression level increases in different type of stress conditions. This protein promotes the growth of Mycobacterium tuberculosis in the host derived stress conditions generated during infection. Therefore in this manuscipt, we are trying to target Rv1636 using natural inhibitor. Targeting essential Mycobacterial protein using natural prodect was hypothesized to generate a molecule with low toxic effects and high inhibitory activity. It was found that Rv1636 contains ATPase activity and its ATPase activity gets disturbed by addition of β-Amyrin in the reaction. β-Amyrin was forund to interfere with the ATP binding site of Rv1636 which was confirmed by molecular docking anad dynamic studies. In addition to the ATPase activity, Rv1636 was also contain the cAMP binding capacity and also involved in balancing the cAMP levels inside cells. So, targeting Rv1636 using β-Amyrin disrupts its ATPase activity and cAMP regulatory activity and these conditions might make Mycobacterium tuberculosis more susceptible to the host derived stress conditions. ABSTRACT: Mycobacterium tuberculosis has seen tremendous success as it has developed defenses to reside in host alveoli despite various host-related stress circumstances. Rv1636 is a universal stress protein contributing to mycobacterial survival in different host-derived stress conditions. Both ATP and cAMP can be bound with the Rv1636, and their binding actions are independent of one another. β-Amyrin, a triterpenoid compound, is abundant in medicinal plants and has many pharmacological properties and broad therapeutic potential. The current study uses biochemical, biophysical, and computational methods to define the binding of Rv1636 with β-Amyrin. A substantial interaction between β-Amyrin and Rv1636 was discovered by molecular docking studies, which helped decipher the critical residues involved in the binding process. VAL60 is a crucial residue found in the complexes of both Rv1636_β-Amyrin and Rv1636-ATP. Additionally, the Rv1636_β-Amyrin complex was shown to be stable by molecular dynamics simulation studies (MD), with minimal changes observed during the simulation. In silico observations were further complemented by in vitro assays. Successful cloning, expression, and purification of Rv1636 were accomplished using Ni-NTA affinity chromatography. The results of the ATPase activity assay indicated that Rv1636’s ATPase activity was inhibited in the presence of various β-Amyrin concentrations. Additionally, circular dichroism spectroscopy (CD) was used to examine modifications to Rv1636 secondary structure upon binding of β-Amyrin. Finally, isothermal titration calorimetry (ITC) advocated spontaneous binding of β-Amyrin with Rv1636 elucidating the thermodynamics of the Rv1636_β-Amyrin complex. Thus, the study establishes that β-Amyrin binds to Rv1636 with a significant affinity forming a stable complex and inhibiting its ATPase activity. The present study suggests that β-Amyrin might affect the functioning of Rv1636, which makes the bacterium vulnerable to different stress conditions.
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spelling pubmed-94054662022-08-26 Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches Beg, Md Amjad Sadaf, Shamsi, Anas Sahoo, Sibasis Yousuf, Mohd Najm, Mohammad Zeeshan Almutawif, Yahya Ahmad Islam, Asimul Aloliqi, Abdulaziz A. Athar, Fareeda Biology (Basel) Article SIMPLE SUMMARY: Rv1636 is a mycobacterial universal stress protein whose expression level increases in different type of stress conditions. This protein promotes the growth of Mycobacterium tuberculosis in the host derived stress conditions generated during infection. Therefore in this manuscipt, we are trying to target Rv1636 using natural inhibitor. Targeting essential Mycobacterial protein using natural prodect was hypothesized to generate a molecule with low toxic effects and high inhibitory activity. It was found that Rv1636 contains ATPase activity and its ATPase activity gets disturbed by addition of β-Amyrin in the reaction. β-Amyrin was forund to interfere with the ATP binding site of Rv1636 which was confirmed by molecular docking anad dynamic studies. In addition to the ATPase activity, Rv1636 was also contain the cAMP binding capacity and also involved in balancing the cAMP levels inside cells. So, targeting Rv1636 using β-Amyrin disrupts its ATPase activity and cAMP regulatory activity and these conditions might make Mycobacterium tuberculosis more susceptible to the host derived stress conditions. ABSTRACT: Mycobacterium tuberculosis has seen tremendous success as it has developed defenses to reside in host alveoli despite various host-related stress circumstances. Rv1636 is a universal stress protein contributing to mycobacterial survival in different host-derived stress conditions. Both ATP and cAMP can be bound with the Rv1636, and their binding actions are independent of one another. β-Amyrin, a triterpenoid compound, is abundant in medicinal plants and has many pharmacological properties and broad therapeutic potential. The current study uses biochemical, biophysical, and computational methods to define the binding of Rv1636 with β-Amyrin. A substantial interaction between β-Amyrin and Rv1636 was discovered by molecular docking studies, which helped decipher the critical residues involved in the binding process. VAL60 is a crucial residue found in the complexes of both Rv1636_β-Amyrin and Rv1636-ATP. Additionally, the Rv1636_β-Amyrin complex was shown to be stable by molecular dynamics simulation studies (MD), with minimal changes observed during the simulation. In silico observations were further complemented by in vitro assays. Successful cloning, expression, and purification of Rv1636 were accomplished using Ni-NTA affinity chromatography. The results of the ATPase activity assay indicated that Rv1636’s ATPase activity was inhibited in the presence of various β-Amyrin concentrations. Additionally, circular dichroism spectroscopy (CD) was used to examine modifications to Rv1636 secondary structure upon binding of β-Amyrin. Finally, isothermal titration calorimetry (ITC) advocated spontaneous binding of β-Amyrin with Rv1636 elucidating the thermodynamics of the Rv1636_β-Amyrin complex. Thus, the study establishes that β-Amyrin binds to Rv1636 with a significant affinity forming a stable complex and inhibiting its ATPase activity. The present study suggests that β-Amyrin might affect the functioning of Rv1636, which makes the bacterium vulnerable to different stress conditions. MDPI 2022-08-12 /pmc/articles/PMC9405466/ /pubmed/36009841 http://dx.doi.org/10.3390/biology11081214 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Beg, Md Amjad
Sadaf,
Shamsi, Anas
Sahoo, Sibasis
Yousuf, Mohd
Najm, Mohammad Zeeshan
Almutawif, Yahya Ahmad
Islam, Asimul
Aloliqi, Abdulaziz A.
Athar, Fareeda
Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title_full Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title_fullStr Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title_full_unstemmed Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title_short Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches
title_sort mechanistic insight into the enzymatic inhibition of β-amyrin against mycobacterial rv1636: in silico and in vitro approaches
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9405466/
https://www.ncbi.nlm.nih.gov/pubmed/36009841
http://dx.doi.org/10.3390/biology11081214
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