Two disparate ligand-binding sites in the human P2Y1 receptor

Zhang, Dandan; Gao, Zhan‐Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

doi:10.1038/nature14287

Cited by 319 publications

(348 citation statements)

References 38 publications

(48 reference statements)

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“…To explore this possibility we selected a Class A G‐protein couple receptor (GPCRs) depositing onto the stage 0.4 nmoles of P2Y 1 , responsible for platelet aggregation and a key target for anti‐thrombotic therapy 27. After recording a native DESI spectrum in its apo form we added a cocktail of antagonists/agonists designed to target related GPCRs (Figure 3 d,e and Table S1).…”

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confidence: 99%

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

et al. 2017

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Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.

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confidence: 99%

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

et al. 2017

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“…Interestingly, previous mutation studies on P2Y 1 have indicated that some antagonists may bind deeper in the receptor at a site analogous to the P2Y 12 receptor (Ref. 40 and references cited therein). The most unexpected finding of the P2Y 1 structure is the binding site of the non-nucleotide antagonist BPTU, which was found to bind on the outside of the receptor on the lipid interface between TM1, TM2, and TM3 (Fig.…”

Section: Key Features Of Gpcr Structures Relevant To Drug Discoverymentioning

confidence: 99%

“…The structure of the P2Y 1 receptor, another platelet receptor involved in platelet aggregation, has also revealed multiple binding sites (40). The nucleotide antagonist MRS2500 binds to a site at the top of the transmembrane domain between TM6 and TM7 but also involving the N terminus and extracellular loop 2.…”

Section: Key Features Of Gpcr Structures Relevant To Drug Discoverymentioning

confidence: 99%

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From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

Jazayeri

Dias

Marshall

2015

Journal of Biological Chemistry

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A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.The process of drug discovery from bench to market is a high risk long term investment that has been estimated to cost about $1.8 billion per drug (1). Such high costs coupled with the pressure of patent expiry dates and increased regulatory constraints have propelled the pharmaceutical industry to increase efficiency of research and development to reduce the attrition rate. A key area of focus has been improvements in the quality of compounds that are discovered in the early stages of the drug discovery pipeline. The main driver for this effort has been the general observation that the hits identified from cell-based high throughput screening (HTS) 2 strategies are usually large lipophilic molecules that are difficult to optimize and carry a number of liabilities that significantly increase their failure rate (2). One of the main advances to tackle these issues has been the utilization of fragment-based drug discovery (FBDD) approaches that rely on screening small chemical fragments (100 -250 Da). Because of their small size, a significantly larger portion of chemical space can be explored with fewer compounds when compared with HTS. In addition, initial hits from a fragment screen bind more efficiently to their target and represent excellent starting points for medicinal chemists to grow and optimize these into lead and candidate molecules (3). The initial fragment hits exhibit low affinity, so they need to be screened at high concentrations that make them incompatible with biological assays. Instead, biophysical assays are used in FBDD cascades, and in addition, these approaches are combined with structural information derived primarily from x-ray crystallography. The application of structure-based drug design (SBDD) allows medicinal chemists to rationally convert fragment hits into larger compounds with higher affinity while maintaining the efficiency of binding and drug-like properties. Historically, SBDD methods gained traction with soluble proteins such as enzymes as routine generation of structural data is facilitated by their high stability in purified form (4 -6). This is in sharp contrast to membrane proteins such as G proteincoupled receptors (GPCRs) that have been refractory to SBDD due to the challenges associate...

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“…The fusion proteins themselves are highly crystalizable and feature N and C termini at the right distance for their insertion into GPCR loops without resulting in a significant distortion of the transmembrane bundle . These fusion proteins are typically T4L Jaakola et al, 2008;Wu et al, 2010) or a thermostabilized apocytochrome (b 562 RIL) Liu et al, 2012;Zhang et al, 2014), but more recently other proteins have also been used successfully as ICL3 fusions, such as the catalytic domain of Pyrococcus abyssi glycogen synthase in the orexin 2 receptor (OX 2 R) (Yin et al, 2015), or rubredoxin in C-C chemokine receptor type 5 (CCR5) (Tan et al, 2013) and the P2Y 1 receptor (Zhang et al, 2015a) (Supplemental Table 1). …”

Section: Molecular Biology Approaches To Facilitate Gpcr Crystallographymentioning

confidence: 99%

A Molecular Pharmacologist’s Guide to G Protein–Coupled Receptor Crystallography

et al. 2015

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G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fueled structure-based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist's guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage noncrystallographers to interrogate structures at the molecular level. Finally, we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function.

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Two disparate ligand-binding sites in the human P2Y1 receptor

Cited by 319 publications

References 38 publications

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

A Molecular Pharmacologist’s Guide to G Protein–Coupled Receptor Crystallography

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