Related Experiment Video

Updated: Jul 25, 2025

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates
11:28

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

Published on: March 31, 2012

16.6K

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors

Muhammad S Hussain1, Degang Liu1, Warren J Alilain2

  • 1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States.

ACS Chemical Neuroscience
|June 29, 2023
PubMed

Insights

Researchers identified small-molecule inhibitors targeting RhoA (Ras homolog gene family member A), a key protein hindering nerve repair after injury. These inhibitors show promise for developing new treatments for central nervous system injuries.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Neuroscience

Background:

  • RhoA (Ras homolog gene family member A) is a GTPase regulating the actin cytoskeleton.
  • RhoA inhibits axon growth, impeding recovery from spinal cord and traumatic brain injuries.
  • No small-molecule RhoA inhibitors currently exist despite extensive research.

Purpose of the Study:

  • To screen cysteine electrophiles for potential RhoA inhibitors.
  • To investigate covalent bond formation at Cys-107 for RhoA inhibition.
  • To identify starting points for developing novel RhoA covalent inhibitors.

Main Methods:

  • Screening a library of cysteine electrophiles.
  • Assessing inhibition of RhoA nucleotide exchange by the guanine exchange factor Trio.
  • Utilizing wild-type RhoA and a Cys107Ser RhoA mutant.
  • Conducting time- and concentration-dependent inhibition studies.
  • Evaluating selectivity against Rac1 and KRAS GTPases.

Main Results:

  • Two fragments, propiolamide (ACR-895) and acrylamide (ACR-917), inhibited RhoA nucleotide exchange by Trio.
  • Inhibitors formed covalent bonds with wild-type RhoA at Cys-107, but not the mutant.
  • Inhibition occurred in a time- and concentration-dependent manner with single-digit hour half-lives.
  • One fragment demonstrated selectivity for RhoA over Rac1 and did not affect KRAS.
  • Fragments did not inhibit RhoA binding to the ROCK effector protein.

Conclusions:

  • Cys-107 is a viable target site for RhoA inhibition.
  • Fragment starting points for developing RhoA covalent inhibitors were identified.
  • These inhibitors hold potential for treating central nervous system injuries.
Keywords:
Rac1Rho GTPasesRhoAcovalent inhibitorguanine exchange factorsmall molecule

More Related Videos

Comparing the Affinity of GTPase-binding Proteins using Competition Assays10:37

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Published on: October 8, 2015

9.3K
RhoC GTPase Activation Assay09:58

RhoC GTPase Activation Assay

Published on: August 22, 2010

12.5K

Related Concept Videos

Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
4.0K
Rab Proteins01:14

Rab Proteins

Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
4.0K
Rab Cascades01:25

Rab Cascades

Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
2.7K
GTPases and their Regulation02:14

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins,...
8.4K
Activation and Inactivation of G Proteins01:22

Activation and Inactivation of G Proteins

Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
7.3K
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a...
6.3K