From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

RCSB Protein Data Bank (RCSB.org): delivery of experimentally-determined PDB structures alongside one million computed structure models of proteins from artificial intelligence/machine learning.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), founding member of the Worldwide Protein Data Bank (wwPDB), is the US data center for the open-access PDB archive. As wwPDB-designated Archive Keeper, RCSB PDB is also responsible for PDB data security. Annually, RCSB PDB serves >10 000 depositors of three-dimensional (3D) biostructures working on all permanently inhabited continents. RCSB PDB delivers data from its research-focused RCSB.org web portal to many millions of PDB data consumers based in virtually every United Nations-recognized country, territory, etc. This Database Issue contribution describes upgrades to the research-focused RCSB.org web portal that created a one-stop-shop for open access to ∼200 000 experimentally-determined PDB structures of biological macromolecules alongside >1 000 000 incorporated Computed Structure Models (CSMs) predicted using artificial intelligence/machine learning methods. RCSB.org is a 'living data resource.' Every PDB structure and CSM is integrated weekly with related functional annotations from external biodata resources, providing up-to-date information for the entire corpus of 3D biostructure data freely available from RCSB.org with no usage limitations. Within RCSB.org, PDB structures and the CSMs are clearly identified as to their provenance and reliability. Both are fully searchable, and can be analyzed and visualized using the full complement of RCSB.org web portal capabilities.

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease.

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.

The RNA polymerase "switch region" is a target for inhibitors.

The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), the US data center for the global PDB archive and a founding member of the Worldwide Protein Data Bank partnership, serves tens of thousands of data depositors in the Americas and Oceania and makes 3D macromolecular structure data available at no charge and without restrictions to millions of RCSB.org users around the world, including >660 000 educators, students and members of the curious public using PDB101.RCSB.org. PDB data depositors include structural biologists using macromolecular crystallography, nuclear magnetic resonance spectroscopy, 3D electron microscopy and micro-electron diffraction. PDB data consumers accessing our web portals include researchers, educators and students studying fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. During the past 2 years, the research-focused RCSB PDB web portal (RCSB.org) has undergone a complete redesign, enabling improved searching with full Boolean operator logic and more facile access to PDB data integrated with >40 external biodata resources. New features and resources are described in detail using examples that showcase recently released structures of SARS-CoV-2 proteins and host cell proteins relevant to understanding and addressing the COVID-19 global pandemic.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first 6 months of the COVID-19 pandemic.

Understanding the molecular evolution of the SARS-CoV-2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three-dimensional structures of SARS-CoV-2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein-protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein-protein and protein-nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Chemogenetics defines receptor-mediated functions of short chain free fatty acids.

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.

RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, rcsb.org), the US data center for the global PDB archive, serves thousands of Data Depositors in the Americas and Oceania and makes 3D macromolecular structure data available at no charge and without usage restrictions to more than 1 million rcsb.org Users worldwide and 600 000 pdb101.rcsb.org education-focused Users around the globe. PDB Data Depositors include structural biologists using macromolecular crystallography, nuclear magnetic resonance spectroscopy and 3D electron microscopy. PDB Data Consumers include researchers, educators and students studying Fundamental Biology, Biomedicine, Biotechnology and Energy. Recent reorganization of RCSB PDB activities into four integrated, interdependent services is described in detail, together with tools and resources added over the past 2 years to RCSB PDB web portals in support of a 'Structural View of Biology.'

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Complex Pharmacology of Free Fatty Acid Receptors.

G protein-coupled receptors (GPCRs) are historically the most successful family of drug targets. In recent times it has become clear that the pharmacology of these receptors is far more complex than previously imagined. Understanding of the pharmacological regulation of GPCRs now extends beyond simple competitive agonism or antagonism by ligands interacting with the orthosteric binding site of the receptor to incorporate concepts of allosteric agonism, allosteric modulation, signaling bias, constitutive activity, and inverse agonism. Herein, we consider how evolving concepts of GPCR pharmacology have shaped understanding of the complex pharmacology of receptors that recognize and are activated by nonesterified or "free" fatty acids (FFAs). The FFA family of receptors is a recently deorphanized set of GPCRs, the members of which are now receiving substantial interest as novel targets for the treatment of metabolic and inflammatory diseases. Further understanding of the complex pharmacology of these receptors will be critical to unlocking their ultimate therapeutic potential.

The RCSB protein data bank: integrative view of protein, gene and 3D structural information.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, http://rcsb.org), the US data center for the global PDB archive, makes PDB data freely available to all users, from structural biologists to computational biologists and beyond. New tools and resources have been added to the RCSB PDB web portal in support of a 'Structural View of Biology.' Recent developments have improved the User experience, including the high-speed NGL Viewer that provides 3D molecular visualization in any web browser, improved support for data file download and enhanced organization of website pages for query, reporting and individual structure exploration. Structure validation information is now visible for all archival entries. PDB data have been integrated with external biological resources, including chromosomal position within the human genome; protein modifications; and metabolic pathways. PDB-101 educational materials have been reorganized into a searchable website and expanded to include new features such as the Geis Digital Archive.

Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.-Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

EMDataBank unified data resource for 3DEM.

Three-dimensional Electron Microscopy (3DEM) has become a key experimental method in structural biology for a broad spectrum of biological specimens from molecules to cells. The EMDataBank project provides a unified portal for deposition, retrieval and analysis of 3DEM density maps, atomic models and associated metadata (emdatabank.org). We provide here an overview of the rapidly growing 3DEM structural data archives, which include maps in EM Data Bank and map-derived models in the Protein Data Bank. In addition, we describe progress and approaches toward development of validation protocols and methods, working with the scientific community, in order to create a validation pipeline for 3DEM data.

Probe-Dependent Negative Allosteric Modulators of the Long-Chain Free Fatty Acid Receptor FFA4.

High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date, a single molecule, 4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide (AH-7614), has been described as an FFA4 antagonist; however, its mechanism of antagonism remains unknown. We synthesized AH-7614 and a chemical derivative and demonstrated these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signaling induced by a range of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anticancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, 4-methyl-N-(9H-xanthen-9-yl)benzamide (TUG-1387), will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells toward a more mature, adipocyte-like phenotype.

Non-equivalence of Key Positively Charged Residues of the Free Fatty Acid 2 Receptor in the Recognition and Function of Agonist Versus Antagonist Ligands.

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled free fatty acid 2 (FFA2) receptor, and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabeled FFA2 antagonist to probe ligand binding to FFA2, and in combination with mutagenesis and molecular modeling studies, we define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, although agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.

Using Biosensors to Study Free Fatty Acid Receptor Pharmacology and Function.

The free fatty acid (FFA) family of G protein coupled receptors (GPCRs) has generated significant interest for exploiting its members as potential drug targets. However, unravelling the complex pharmacology of this family of receptors has proven challenging. In recent years the use of biosensor technologies capable of assessing biological functions in living cells, and in real time, has greatly enhanced our ability to study GPCR pharmacology and function. These include genetically encoded sensors that change the intensity or wavelength of light emitted from a bioluminescent or fluorescent protein in response to a stimulus, as well as non-genetically encoded sensors able to measure more global cellular changes, such as mass redistribution within a cell. This chapter will examine how these sensors can be used to study GPCRs, and in particular how they are helping uncover the pharmacology of the FFA family of receptors.

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120.

It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.

The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120).

The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases.