Publications by authors named "Manoj K Rathinaswamy"

7 Publications

  • Page 1 of 1

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Elife 2021 Mar 4;10. Epub 2021 Mar 4.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.64691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955810PMC
March 2021

Defining How Oncogenic and Developmental Mutations of PIK3R1 Alter the Regulation of Class IA Phosphoinositide 3-Kinases.

Structure 2020 02 9;28(2):145-156.e5. Epub 2019 Dec 9.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada. Electronic address:

The class I phosphoinositide 3-kinases (PI3Ks) are key signaling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572 truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals unique insight into isoform-specific regulation of p110s by p85α.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2019.11.013DOI Listing
February 2020

Class I phosphoinositide 3-kinase (PI3K) regulatory subunits and their roles in signaling and disease.

Adv Biol Regul 2020 01 28;75:100657. Epub 2019 Sep 28.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 2Y2, Canada. Electronic address:

The Class I phosphoinositide 3-kinases (PI3Ks) are a group of heterodimeric lipid kinases that regulate crucial cellular processes including proliferation, survival, growth, and metabolism. The diversity in functions controlled by the various catalytic isoforms (p110α, p110β, p110δ, and p110γ) depends on their abilities to be activated by distinct stimuli such as receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and the Ras family of small G-proteins. A major factor determining the ability of each p110 enzyme to be activated is the presence of regulatory binding partners. Given the overwhelming evidence for the involvement of PI3Ks in diseases such as cancer, inflammation, immunodeficiency and diabetes, an understanding of how these regulatory proteins influence PI3K function is essential. This article highlights research deciphering the role of regulatory subunits in PI3K signaling and their involvement in human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbior.2019.100657DOI Listing
January 2020

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

J Med Chem 2018 12 28;61(23):10463-10472. Epub 2018 Nov 28.

Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography , University of California, San Diego , La Jolla , California 92093 , United States.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688905PMC
December 2018

Conformational sampling of membranes by Akt controls its activation and inactivation.

Proc Natl Acad Sci U S A 2018 04 9;115(17):E3940-E3949. Epub 2018 Apr 9.

Department of Structural and Computational Biology, Max F. Perutz Laboratories, 1030 Vienna, Austria;

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1716109115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924885PMC
April 2018

Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States.

Cell Chem Biol 2017 12 12;24(12):1455-1466.e14. Epub 2017 Oct 12.

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA. Electronic address:

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2017.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915340PMC
December 2017

Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas.

J Biol Chem 2017 07 17;292(29):12256-12266. Epub 2017 May 17.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada. Electronic address:

Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras-PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras-PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110α and p110δ isoforms but does not activate p110β. The p110α and p110δ activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen-deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110α and p110δ. Together, these results afford detailed molecular insight into the Ras-PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras-PI3K interactions in a native membrane context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.789263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519374PMC
July 2017