scholarly journals RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site

2020 ◽  
Author(s):  
Katarzyna Z Haza ◽  
Heather L Martin ◽  
Ajinkya Rao ◽  
Amy L Turner ◽  
Sophie E Saunders ◽  
...  
Keyword(s):  
Signalling Pathways ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Ras Mutations ◽  
Human Cancers ◽  
Pharmacophore Identification ◽  
Covalent Inhibitor

ABSTRACTRAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. We have identified two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signalling pathways with distinct isoform and mutant profiles. Affimer K6 is the first biologic to bind in the SI/SII pocket, whilst Affimer K3 is the first non-covalent inhibitor of the SII region, revealing a novel RAS conformer with a large, druggable SII/α3 pocket. This work demonstrates the potential of using biologics with small interface surfaces to select novel druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

scholarly journals RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Katarzyna Z. Haza ◽  
Heather L. Martin ◽  
Ajinkya Rao ◽  
Amy L. Turner ◽  
Sophie E. Saunders ◽  
...  
Keyword(s):  
Small Molecules ◽  
Signaling Pathways ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Allosteric Site ◽  
Ras Mutations ◽  
Downstream Signaling ◽  
Human Cancers ◽  
Pharmacophore Identification ◽  
Covalent Inhibitor

AbstractRAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

DLX6-AS1: a putative lncRNA candidate in multiple human cancers

2021 ◽  
Vol 23 ◽  
Author(s):  
Mohsen Sheykhhasan ◽  
Yaghoub Ahmadyousefi ◽  
Reihaneh Seyedebrahimi ◽  
Hamid Tanzadehpanah ◽  
Hamed Manoochehri ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Critical Analysis ◽  
Molecular Mechanisms ◽  
Signalling Pathways ◽  
Biological Functions ◽  
Initial Development ◽  
Human Cancers ◽  
Expression Of Genes ◽  
Non Coding Rnas ◽  
Research Studies

Abstract Long non-coding RNAs (lncRNAs) have important roles in regulating the expression of genes and act as biomarkers in the initial development of different cancers. Increasing research studies have verified that dysregulation of lncRNAs occurs in various pathological processes including tumorigenesis and cancer progression. Among the different lncRNAs, DLX6-AS1 has been reported to act as an oncogene in the development and prognoses of different cancers, by affecting many different signalling pathways. This review summarises and analyses the recent research studies describing the biological functions of DLX6-AS1, its overall effect on signalling pathways and the molecular mechanisms underlying its action on the expression of genes in multiple human cancers. Our critical analysis suggests that different signalling pathways associated to this lncRNA may be used as a biomarker for diagnosis, or targets of treatment in cancers.

scholarly journals Development of Noonan syndrome by deregulation of allosteric SOS autoactivation

2020 ◽  
Vol 295 (39) ◽  
pp. 13651-13663 ◽  
Author(s):  
Hope Gloria Umutesi ◽  
Hanh My Hoang ◽  
Hope Elizabeth Johnson ◽  
Kwangho Nam ◽  
Jongyun Heo
Keyword(s):  
Noonan Syndrome ◽  
Genetic Disorder ◽  
Active Form ◽  
The Body ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Allosteric Site ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Ras Family

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants—V14I, T58I, and G60E—populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant–mediated development of Noonan syndrome.

scholarly journals Dysregulation of Rho GTPases in Human Cancers

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1179 ◽  
Author(s):  
Haiyoung Jung ◽  
Suk Ran Yoon ◽  
Jeewon Lim ◽  
Hee Jun Cho ◽  
Hee Gu Lee
Keyword(s):  
Cancer Progression ◽  
Rho Gtpases ◽  
Cell Cycle Progression ◽  
Rho Gtpase ◽  
Current Knowledge ◽  
New Drugs ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Human Cancers ◽  
Cytoskeletal Dynamics

Rho GTPases play central roles in numerous cellular processes, including cell motility, cell polarity, and cell cycle progression, by regulating actin cytoskeletal dynamics and cell adhesion. Dysregulation of Rho GTPase signaling is observed in a broad range of human cancers, and is associated with cancer development and malignant phenotypes, including metastasis and chemoresistance. Rho GTPase activity is precisely controlled by guanine nucleotide exchange factors, GTPase-activating proteins, and guanine nucleotide dissociation inhibitors. Recent evidence demonstrates that it is also regulated by post-translational modifications, such as phosphorylation, ubiquitination, and sumoylation. Here, we review the current knowledge on the role of Rho GTPases, and the precise mechanisms controlling their activity in the regulation of cancer progression. In addition, we discuss targeting strategies for the development of new drugs to improve cancer therapy.

scholarly journals Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence

2015 ◽  
Vol 291 (4) ◽  
pp. 1703-1718 ◽  
Author(s):  
Uybach Vo ◽  
Navratna Vajpai ◽  
Liz Flavell ◽  
Romel Bobby ◽  
Alexander L. Breeze ◽  
...  
Keyword(s):  
Intrinsic Fluorescence ◽  
The Novel ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Site Specific ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Catalytic Sites ◽  
Son Of Sevenless

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

scholarly journals Rho GTPase–independent regulation of mitotic progression by the RhoGEF Net1

2013 ◽  
Vol 24 (17) ◽  
pp. 2655-2667 ◽  
Author(s):  
Sarita Menon ◽  
Wonkyung Oh ◽  
Heather S. Carr ◽  
Jeffrey A. Frost
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Rho Gtpase ◽  
Spindle Assembly ◽  
Nucleotide Exchange ◽  
Altered Expression ◽  
Human Cancers ◽  
Nucleotide Exchange Factor ◽  
Chromosome Congression ◽  
P21 Activated Kinase

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily–specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

RAS mutations in human cancers: Roles in precision medicine

2019 ◽  
Vol 59 ◽  
pp. 23-35 ◽  
Author(s):  
Avaniyapuram Kannan Murugan ◽  
Michele Grieco ◽  
Nobuo Tsuchida
Keyword(s):  
Precision Medicine ◽  
Ras Mutations ◽  
Human Cancers

Role of Epac in brain and heart

2012 ◽  
Vol 40 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Anne-Coline Laurent ◽  
Magali Breckler ◽  
Magali Berthouze ◽  
Frank Lezoualc'h
Keyword(s):  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Signalling Pathways ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
New Sensors ◽  
Kinase A

Epacs (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors for the Ras-like small GTPases Rap1 and Rap2. Epacs were discovered in 1998 as new sensors for the second messenger cAMP acting in parallel to PKA (protein kinase A). As cAMP regulates many important physiological functions in brain and heart, the existence of Epacs raises many questions regarding their role in these tissues. The present review focuses on the biological roles and signalling pathways of Epacs in neurons and cardiac myocytes. We discuss the potential involvement of Epacs in the manifestation of cardiac and central diseases such as cardiac hypertrophy and memory disorders.

scholarly journals Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, elF2B

2017 ◽  
Author(s):  
Alisa F. Zyryanova ◽  
Félix Weis ◽  
Alexandre Faille ◽  
Akeel Abo Alard ◽  
Ana Crespillo-Casado ◽  
...  
Keyword(s):  
Stress Response ◽  
Cellular Response ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Exchange Factor ◽  
Interaction Sites ◽  
Nucleotide Exchange Factor

AbstractThe Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the β and δ regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

scholarly journals Biophysical characterization of the human K0513 protein

2020 ◽  
Author(s):  
Ndivhuwo Nemukondeni ◽  
Afolake Arowolo ◽  
Addmore Shonhai ◽  
Tawanda Zininga ◽  
Adélle Burger
Keyword(s):  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Limited Proteolysis ◽  
Malignant Tumour ◽  
Water Soluble ◽  
Cancer Prognosis ◽  
Signalling Pathways ◽  
Nucleotide Exchange ◽  
Three Dimensional Model ◽  
Structural Characterisation

ABSTRACTGlioblastoma multiforme (GBM) is an aggressive grade IV primary malignant tumour which accounts for 78 % of all brain tumours. K0513 is a GBM biomarker that is upregulated in the invasive phenotype. K0513 is expressed ubiquitously and is reportedly enriched in the cerebral cortex of the brain. K0513 is further implicated in signalling pathways involving neuroplasticity, cytoskeletal regulation and apoptosis. The protein encoded by K0513 is a prospective biomarker for pancreatic cancer prognosis. However, the gene product of K0153 is not well characterised. This study focused on structure-function characterisation of human K0513 protein. To this end, we employed bioinformatics analysis and biophysical approaches to characterize the protein. In silico structural characterisation of the human K0513 protein suggests the presence of a SET binding factor 2 (SBF2) domain and a transmembrane region. The SBF2 domain is found in the Myotubularin-related protein 13 (MTMR13), which may function as a nucleotide exchange factor for the RAS-associated GTPase, Rab28. K0513 was predicted to interact with RAS-associated GTPase, Rab3a. Secondary structure prediction revealed K0513 to be predominantly α-helical in nature. The predicted three-dimensional model of K0513 showed a globular fold, suggesting that the protein is water-soluble. K0513 was heterologously expressed in E. coli XL1-Blue cells and subsequent purification yielded 80 % soluble protein. Biophysical characterisation using tryptophan-based fluorescence spectroscopy and limited proteolysis showed the conformation of K0513 is mostly unperturbed in the presence of nucleotides. Interestingly, K0513 was detected in lung carcinoma, fibrosarcoma and cervical adenocarcinoma cells, supporting its possible role in cancer signalling pathways.

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