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Article ; Online: Conformational insight into multi-protein signaling assemblies by hydrogen-deuterium exchange mass spectrometry.

Harrison, Rane A / Engen, John R

2016 Volume 41, Page(s) 187–193

Abstract: Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide information about proteins that can be challenging to obtain by other means. Structure/function relationships, binding interactions, and the effects of modification have all been ... ...

Abstract	Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide information about proteins that can be challenging to obtain by other means. Structure/function relationships, binding interactions, and the effects of modification have all been measured with HDX MS for a diverse and growing array of signaling proteins and multiprotein signaling complexes. As a result of hardware and software improvements, receptors and complexes involved in cellular signaling-including those associated with membranes-can now be studied. The growing body of HDX MS studies of signaling complexes at membranes is particularly exciting. Recent examples are presented to illustrate what can be learned about signaling proteins with this technique.
Language	English
Publishing date	2016-12
Publishing country	England
Document type	Review ; Journal Article
ZDB-ID	1068353-7
ISSN	1879-033X ; 0959-440X
ISSN (online)	1879-033X
ISSN	0959-440X
DOI	10.1016/j.sbi.2016.08.003
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Article ; Online: KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry.

Lu, Jia / Harrison, Rane A / Li, Lianbo / Zeng, Mei / Gondi, Sudershan / Scott, David / Gray, Nathanael S / Engen, John R / Westover, Kenneth D

Structure (London, England : 1993)

2017 Volume 25, Issue 9, Page(s) 1442–1448.e3

Abstract: KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an ... ...

Abstract	KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.
MeSH term(s)	Cell Line ; Crystallography, X-Ray ; Deuterium Exchange Measurement ; Drug Design ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Humans ; Mass Spectrometry ; Models, Molecular ; Molecular Conformation ; Mutation ; Proto-Oncogene Proteins p21(ras)/chemistry ; Proto-Oncogene Proteins p21(ras)/genetics ; Quinazolines/chemistry ; Quinazolines/pharmacology ; Structure-Activity Relationship
Chemical Substances	Enzyme Inhibitors ; KRAS protein, human ; Quinazolines ; Proto-Oncogene Proteins p21(ras) (EC 3.6.5.2)
Language	English
Publishing date	2017-08-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ZDB-ID	1213087-4
ISSN	1878-4186 ; 0969-2126
ISSN (online)	1878-4186
ISSN	0969-2126
DOI	10.1016/j.str.2017.07.003
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Zs.A 4141: Show issues

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Article ; Online: ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance.

Communications biology

2021 Volume 4, Issue 1, Page(s) 497

Abstract: Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct ... ...

Abstract	Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8
MeSH term(s)	Animals ; Extracellular Vesicles/physiology ; Female ; Immunologic Surveillance ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tumor Microenvironment/physiology
Language	English
Publishing date	2021-04-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-021-02004-5
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Article ; Online: Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

Harrison, Rane A / Lu, Jia / Carrasco, Martin / Hunter, John / Manandhar, Anuj / Gondi, Sudershan / Westover, Kenneth D / Engen, John R

Journal of molecular biology

2016 Volume 428, Issue 23, Page(s) 4723–4735

Abstract: Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and ... ...

Abstract	Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.
MeSH term(s)	Animals ; Humans ; Mass Spectrometry ; Molecular Dynamics Simulation ; Nucleotides/metabolism ; ras Proteins/chemistry ; ras Proteins/metabolism ; rho GTP-Binding Proteins/chemistry ; rho GTP-Binding Proteins/metabolism
Chemical Substances	Nucleotides ; ras Proteins (EC 3.6.5.2) ; rho GTP-Binding Proteins (EC 3.6.5.2)
Language	English
Publishing date	2016-11-20
Publishing country	England
Document type	Journal Article
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2016.10.017
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Article ; Online: ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance

Communications Biology, Vol 4, Iss 1, Pp 1-

2021 Volume 17

Abstract: Su Chul Jang et al. develop exoSTING, consisting of an engineered extracellular vesicle loaded with a potent cyclic dinucleotide (CDN) agonist of the STING pathway. They find that exoSTING shows more than 100-fold increased potency in in vivo tumor ... ...

Abstract	Su Chul Jang et al. develop exoSTING, consisting of an engineered extracellular vesicle loaded with a potent cyclic dinucleotide (CDN) agonist of the STING pathway. They find that exoSTING shows more than 100-fold increased potency in in vivo tumor models and has increased tumor retention and lower levels of systemic inflammatory cytokine production as compared to free CDN.
Keywords	Biology (General) ; QH301-705.5
Language	English
Publishing date	2021-04-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Therapeutic extracellular vesicle production is substantially increased by inhibition of cellular cholesterol biosynthesis.

Martin, Shelly / McConnell, Russell / Harrison, Rane / Jang, Su Chul / Sia, Chang Ling / Kamerkar, Sushrut / Duboff, Anna / Jacob, Lisa / Finn, Jonathan / Estes, Scott

Biotechnology and bioengineering

2023 Volume 120, Issue 9, Page(s) 2685–2699

Abstract: Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of ...

Abstract	Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of the manufacturing process are rich with opportunities to enhance the ability to deliver these medicines to patients. With the goal of improving cell culture EV productivity, we have utilized high throughput siRNA screens to identify the underlying genetic pathways that regulate EV productivity to inform rational host cell line engineering and media development approaches. The screens identified multiple metabolic pathways of potential interest; one of which was validated and shown to be a ready implementable, cost-effective strategy to increase EV titers. We show that both EV volumetric and specific productivity from HEK293 and CHO-S were increased in a dose and cell line-dependent manner up to ninefold when cholesterol synthesis was inhibited by the inclusion of statins in the cell culture media. In addition, we show in response to statin treatment, elevation of EV markers in mesenchymal stem cell (MSC) cell culture media suggesting this approach can also be applicable to MSC EVs. Furthermore, we show that the EVs produced from statin-treated HEK293 cultures are effectively loaded by both endogenous and exogenous loading methods and have equivalent in vitro or in vivo potency relative to EVs from untreated cultures.
MeSH term(s)	Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism ; HEK293 Cells ; Extracellular Vesicles/metabolism ; Cell Culture Techniques ; Cholesterol/metabolism
Chemical Substances	Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2023-04-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	280318-5
ISSN	1097-0290 ; 0006-3592
ISSN (online)	1097-0290
ISSN	0006-3592
DOI	10.1002/bit.28401
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Article: Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching

Harrison, Rane A / Jia Lu / Martin Carrasco / John Hunter / Anuj Manandhar / Sudershan Gondi / Kenneth D. Westover / John R. Engen

Journal of Molecular Biology. 2016 Nov. 20, v. 428

2016

Abstract	Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogenâdeuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.
Keywords	deuterium ; mass spectrometry ; molecular dynamics ; mutants ; proteins ; signal transduction
Language	English
Dates of publication	2016-1120
Size	p. 4723-4735.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2016.10.017
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Dooley, Kevin / McConnell, Russell E / Xu, Ke / Lewis, Nuruddeen D / Haupt, Sonya / Youniss, Madeleine R / Martin, Shelly / Sia, Chang Ling / McCoy, Christine / Moniz, Raymond J / Burenkova, Olga / Sanchez-Salazar, Jorge / Jang, Su Chul / Choi, Bryan / Harrison, Rane A / Houde, Damian / Burzyn, Dalia / Leng, Charan / Kirwin, Katherine /

Ross, Nikki L / Finn, Jonathan D / Gaidukov, Leonid / Economides, Kyriakos D / Estes, Scott / Thornton, James E / Kulman, John D / Sathyanarayanan, Sriram / Williams, Douglas E

Molecular therapy : the journal of the American Society of Gene Therapy

2021 Volume 29, Issue 5, Page(s) 1729–1743

Abstract: Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for ... ...

Abstract	Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.
MeSH term(s)	Animals ; Cell Communication ; Drug Delivery Systems ; Extracellular Vesicles/genetics ; Extracellular Vesicles/metabolism ; Extracellular Vesicles/transplantation ; Female ; HEK293 Cells ; Humans ; Membrane Proteins/genetics ; Membrane Proteins/metabolism ; Mice ; Neoplasm Proteins/genetics ; Neoplasm Proteins/metabolism ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Proteins/administration & dosage ; Repressor Proteins/genetics ; Repressor Proteins/metabolism
Chemical Substances	BASP1 protein, human ; Membrane Proteins ; Neoplasm Proteins ; Nerve Tissue Proteins ; PTGFRN protein, human ; Proteins ; Repressor Proteins
Language	English
Publishing date	2021-01-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2010592-7
ISSN	1525-0024 ; 1525-0016
ISSN (online)	1525-0024
ISSN	1525-0016
DOI	10.1016/j.ymthe.2021.01.020
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Zs.A 5640: Show issues

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Article ; Online: Modulating the strength of hydrogen bond acceptors to achieve low Caco2 efflux for oral bioavailability of PARP inhibitors blocking centrosome clustering.

Gu, Chungang / Lamb, Michelle L / Johannes, Jeffrey W / Sylvester, Mark A / Eisman, Mark S / Harrison, Rane A / Hu, Haiqing / Kazmirski, Steven / Mikule, Keith / Peng, Bo / Su, Nancy / Wang, Wenxian / Ye, Qing / Zheng, Xiaolan / Lyne, Paul D / Scott, David A

Bioorganic & medicinal chemistry letters

2016 Volume 26, Issue 19, Page(s) 4775–4780

Abstract: During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral ... ...

Abstract	During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral bioavailability. Conversely, compounds with lower efflux had reduced potency. The project team was able to improve the bioavailability by reducing efflux through systematic modifications to the strength of the HBA by changing the electronic properties of neighboring groups, whilst maintaining sufficient acceptor strength for potency. Additionally, it was observed that enantiomers with different potency showed similar efflux, which is consistent with the promiscuity of efflux transporters. Eventually, a balance between potency and low efflux was achieved for a set of lead compounds with good bioavailability which allowed the project to progress towards establishing in vivo pharmacokinetic/pharmacodynamic relationships.
MeSH term(s)	Administration, Oral ; Animals ; Biological Availability ; Caco-2 Cells ; Centrosome/metabolism ; Dogs ; Humans ; Hydrogen Bonding ; Madin Darby Canine Kidney Cells ; Mice ; Poly(ADP-ribose) Polymerase Inhibitors/administration & dosage ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacokinetics ; Rats
Chemical Substances	Poly(ADP-ribose) Polymerase Inhibitors
Language	English
Publishing date	2016--01
Publishing country	England
Document type	Journal Article
ZDB-ID	1063195-1
ISSN	1464-3405 ; 0960-894X
ISSN (online)	1464-3405
ISSN	0960-894X
DOI	10.1016/j.bmcl.2016.08.030
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Zs.A 3203: Show issues

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Article ; Online: Patient Characteristics and Real-World Treatment Patterns Among Early Users of PCSK9 Inhibitors.

Rane, Pallavi B / Patel, Jeetvan / Harrison, David J / Shepherd, Jason / Leith, Andrea / Bailey, Hollie / Piercy, James

American journal of cardiovascular drugs : drugs, devices, and other interventions

2017 Volume 18, Issue 2, Page(s) 103–108

Abstract: Background: Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) reduce low-density lipoprotein cholesterol (LDL-C) levels and are approved for patients with familial hypercholesterolemia or atherosclerotic cardiovascular disease who ... ...

Abstract	Background: Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) reduce low-density lipoprotein cholesterol (LDL-C) levels and are approved for patients with familial hypercholesterolemia or atherosclerotic cardiovascular disease who require additional LDL-C lowering. Objective: Our objective was to characterize patients receiving PCSK9i medications in real-world practice and describe physician-reported treatment patterns among dyslipidemia patients using PCSK9i or other lipid-lowering therapy. Methods: We analyzed data from a point-in-time Adelphi dyslipidemia disease-specific programme (DSP) survey conducted in the USA in 2016. Physicians provided treatment history, laboratory values, patient characteristics, and comorbidities for treated patients. To ensure sufficient numbers of PCSK9i-treated patients, we conducted systematic oversampling of patients being prescribed PCSK9i. Outcomes included patient characteristics and physician-reported treatment patterns. Results: The DSP included 159 physicians, who provided information on 1522 patients (304 PCSK9i; 1218 non-PCSK9i). Mean ± standard deviation (SD) baseline LDL-C levels were 180.0 ± 39.7 mg/dl for PCSK9i patients and 159.2 ± 40.5 mg/dl for non-PCSK9i patients. Prior statin use was reported in 69.1% of PCSK9i patients and 19.5% of non-PCSK9i patients, and physician-reported statin intolerance was observed in 31.6% of PCSK9i and 5.3% of non-PCSK9i patients. Use of statins only was reported in 40.5% of PCSK9i and 88.8% of non-PCSK9i patients. The most common physician-reported reasons for change to PCSK9i were lack of efficacy (70.2%) and muscle-related symptoms (myalgia 28.6%; myopathy 11.1%). Conclusions: Physicians surveyed appeared to prescribe PCSK9i medications appropriately. PCSK9i-treated patients had higher rates of cardiovascular comorbidities and physician-determined statin intolerance, had higher LDL-C levels, and received more lines of therapy than non-PCSK9i patients.
MeSH term(s)	Anticholesteremic Agents/therapeutic use ; Atherosclerosis/drug therapy ; Atherosclerosis/metabolism ; Cholesterol, LDL ; Dyslipidemias/drug therapy ; Dyslipidemias/metabolism ; Female ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use ; Hyperlipoproteinemia Type II/drug therapy ; Hyperlipoproteinemia Type II/metabolism ; Hypolipidemic Agents/therapeutic use ; Male ; Middle Aged ; Proprotein Convertase 9/antagonists & inhibitors
Chemical Substances	Anticholesteremic Agents ; Cholesterol, LDL ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Hypolipidemic Agents ; PCSK9 protein, human (EC 3.4.21.-) ; Proprotein Convertase 9 (EC 3.4.21.-)
Language	English
Publishing date	2017-08-28
Publishing country	New Zealand
Document type	Journal Article
ZDB-ID	2052547-3
ISSN	1179-187X ; 1175-3277
ISSN (online)	1179-187X
ISSN	1175-3277
DOI	10.1007/s40256-017-0246-z
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