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Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α / β -hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One family of intracellular systems are the guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activates Rho family G proteins, which, in turn, alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their highly conserved Dbl homology (DH) domains, Dbl GEFs are regulated in diverse ways, often involving the release of autoinhibition imposed by accessory domains. Among these domains, the pleckstrin homology (PH) domain is the most commonly observed and found immediately C-terminal to the DH domain. The domain has been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined that reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here, we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH domain might be exploited to achieve selective inhibition of Dbl family RhoGEFs by small-molecule therapeutics. SIGNIFICANCE STATEMENT: Dysregulation via overexpression or mutation of Dbl family Rho guanine nucleotide exchange factors (GEFs) contributes to cancer and neurodegeneration. Targeting the Dbl homology catalytic domain by small-molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for the development of selective therapeutics that modulate GEF activity., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.S. is the founder and scientific advisor of 7TM Antibodies GmbH, Jena, Germany. F.N. is an employee of 7TM Antibodies. All other authors declare no competing interests., (Copyright: © 2024 Walker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gβγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gβγ complexes in the presence of substrates/analogs, revealing two Gβγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gβγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gβγ binding sites and interdomain contacts altered by Gβγ binding suggest that Gβγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gβγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gα s , but their response to Gβγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gβγ and a dimeric form of AC5 that could be involved in its regulation. Gβγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C 1b ) that is known to be a hub for isoform-specific regulation. We confirmed the Gβγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gβγ and show reduced conditional activation by Gβγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gβγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase., Competing Interests: SR, SA, SL, CC, MC, JS, JC, JT No competing interests declared, (© 2023, Ravala et al.)

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC 50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and β -arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of G βγ , an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 "senses" CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and β -arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote β -arrestin recruitment, β -arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and β -arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating G βγ . This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions., (Copyright © 2023 by The Author(s).)

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo . Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo , but these responses were substantially reduced in Mrgprb2 -/- mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2 -/- mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC's immunomodulatory property through the activation of MRGPRX2/MRGPRB2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thapaliya, Amponnawarat, Tesmer and Ali.)

G protein-coupled receptor (GPCR) kinases (GRKs) and arrestins interact with agonist-bound GPCRs to promote receptor desensitization and downregulation. They also trigger signaling cascades distinct from those of heterotrimeric G proteins. Biased agonists for GPCRs that favor either heterotrimeric G protein or GRK/arrestin signaling are of profound pharmacological interest because they could usher in a new generation of drugs with greatly reduced side effects. One mechanism by which biased agonism might occur is by stabilizing receptor conformations that preferentially bind to GRKs and/or arrestins. In this review, we explore this idea by comparing structures of GPCRs bound to heterotrimeric G proteins with those of the same GPCRs in complex with arrestins and GRKs. The arrestin and GRK complexes all exhibit high conformational heterogeneity, which is likely a consequence of their unusual ability to adapt and bind to hundreds of different GPCRs. This dynamic behavior, along with the experimental tactics required to stabilize GPCR complexes for biophysical analysis, confounds these comparisons, but some possible molecular mechanisms of bias are beginning to emerge. We also examine if and how the recent structures advance our understanding of how arrestins parse the "phosphorylation barcodes" installed in the intracellular loops and tails of GPCRs by GRKs. In the future, structural analyses of arrestins in complex with intact receptors that have well-defined native phosphorylation barcodes, such as those installed by the two nonvisual subfamilies of GRKs, will be particularly illuminating., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

An increasing number of protein structures are being determined by cryogenic electron microscopy (cryo-EM). Although the resolution of determined cryo-EM density maps is improving in general, there are still many cases where amino acids of a protein are assigned with different levels of confidence. Here we developed a method that identifies potential misassignment of residues in the map, including residue shifts along an otherwise correct main-chain trace. The score, named DAQ, computes the likelihood that the local density corresponds to different amino acids, atoms, and secondary structures, estimated via deep learning, and assesses the consistency of the amino acid assignment in the protein structure model with that likelihood. When DAQ was applied to different versions of model structures in the Protein Data Bank that were derived from the same density maps, a clear improvement in the DAQ score was observed in the newer versions of the models. DAQ also found potential misassignment errors in a substantial number of deposited protein structure models built into cryo-EM maps., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio 1284 - 1959 , in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS -driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS / NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS / NRAS -driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by β-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR-MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR-AGO2-RAS signaling axis is essential for maintaining mutant HRAS and NRAS -driven malignancies., (© The Author(s) 2022. Published by Oxford University Press on behalf of the National Academy of Sciences.)

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein-coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs.

Oncogenic mutations in KRAS result in a constitutively active, GTP-bound form that in turn activates many proliferative pathways. However, because of its compact and simple architecture, directly targeting KRAS with small molecule drugs has been challenging. Another approach is to identify targetable proteins that interact with KRAS. Argonaute 2 (AGO2) was recently identified as a protein that facilitates RAS-driven oncogenesis. Whereas previous studies described the in vivo effect of AGO2 on cancer progression in cells harboring mutated KRAS, here we sought to examine their direct interaction using purified proteins. We show that full length AGO2 co-immunoprecipitates with KRAS using purified components, however, a complex between FL AGO2 and KRAS could not be isolated. We also generated a smaller N-terminal fragment of AGO2 (NtAGO2) which is believed to represent the primary binding site of KRAS. A complex with NtAGO2 could be detected via ion-mobility mass spectrometry and size exclusion chromatography. However, the data suggest that the interaction of KRAS with purified AGO2 (NtAGO2 or FL AGO2) is weak and likely requires additional cellular components or proteo-forms of AGO2 that are not readily available in our purified assay systems. Future studies are needed to determine what conformation or modifications of AGO2 are necessary to enrich KRAS association and regulate its activities., (© 2021 The Authors.)

G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization 1 . Although it is unclear how GRKs recognize these receptors 2-4 , a conserved region at the GRK N terminus is essential for this process 5-8 . Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR-GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate the desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating diseases such as heart failure and cancer. Previously, our work showed that Cys474, a GRK5 subfamily-specific residue located on a flexible loop adjacent to the active site, can be used as a covalent handle to achieve selective inhibition of GRK5 over GRK2 subfamily members. However, the potency of the most selective inhibitors remained modest. Herein, we describe a successful campaign to adapt an indolinone scaffold with covalent warheads, resulting in a series of 2-haloacetyl-containing compounds that react quickly and exhibit three orders of magnitude selectivity for GRK5 over GRK2 and low nanomolar potency. They however retain a similar selectivity profile across the kinome as the core scaffold, which was based on Sunitinib.

Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC 50 values less than 1 mM and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC 50 0.18 μM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared with in silico models based on pKa and ClogP, measures of protonation, and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Recombinant LPLA(2) and anti-LPLA(2) monoclonal antibodies are licensed to Echelon Biosciences by the University of Michigan., (Published by Elsevier Inc.)

The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G q -coupled receptors and constitutively active Gα q/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G q /phospholipase C (PLC) signaling. Here, we further characterized G q/11 regulation of SphK1. SphK1 translocation by the M 3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ 3 , but accelerated by wild-type PLCβ 3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M 3 receptor-stimulated inositol phosphate production, suggesting competition at Gα q . Embryonic fibroblasts from Gα q/11 double-deficient mice were used to show that amino acids W263 and T257 of Gα q , which interact directly with PLCβ 3 and p63RhoGEF, were important for bradykinin B 2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gα q binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M 3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G q/11 .

Phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 )-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP 3 , and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Single-particle cryoelectron microscopy (cryo-EM) continues to grow into a mainstream structural biology technique. Recent developments in data collection strategies alongside new sample preparation devices herald a future where users will collect multiple datasets per microscope session. To make cryo-EM data processing more automatic and user-friendly, we have developed an automatic pipeline for cryo-EM data preprocessing and assessment using a combination of deep-learning and image-analysis tools. We have verified the performance of this pipeline on a number of datasets and extended its scope to include sample screening by the user-free assessment of the qualities of a series of datasets under different conditions. We propose that our workflow provides a decision-free solution for cryo-EM, making data preprocessing more generalized and robust in the high-throughput era as well as more convenient for users from a range of backgrounds., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747 , which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the μ -opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A , which explains why GSK180736A -based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747 , which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the μ -opioid receptor most efficaciously because it has the ability to cross cell membranes., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phosphatidylinositol (3,4,5) trisphosphate (PIP 3 )-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein βγ subunits and the lipid PIP 3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP 3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP 3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Synthetic high-density lipoprotein (sHDL) nanoparticles composed of apolipoprotein A-I mimetic peptide and phospholipids have been shown to reduce atherosclerosis in animal models. Cholesterol is mobilized from atheroma macrophages by sHDL into the blood compartment and delivered to the liver for elimination. Historically, sHDL drug discovery efforts were focused on optimizing peptide sequences for interaction with cholesterol cellular transporters rather than understanding how both sHDL components, peptide and lipid, influence its pharmacokinetic and pharmacodynamic profiles. We designed two sets of sHDL having either identical phospholipid but variable peptide sequences with different plasma stability or identical peptide and phospholipids with variable fatty acid chain length and saturation. We found that sHDL prepared with proteolytically stable 22A-P peptide had 2-fold longer circulation half-time relative to the less stable 22A peptide. Yet, longer half-life did not translate into any improvement in cholesterol mobilization. In contrast, sHDL with variable phospholipid compositions showed significant differences in phospholipid PK, with distearoyl phosphatidylcholine-based sHDL demonstrating the longest half-life of 6.0 hours relative to 1.0 hour for palmitoyl-oleoyl phosphatidylcholine-based sHDL. This increase in half-life corresponded to an approx. 6.5-fold increase in the area under the curve for the mobilized cholesterol. Therefore, the phospholipid component in sHDL plays a major role in cholesterol mobilization in vivo and should not be overlooked in the design of future sHDL. SIGNIFICANCE STATEMENT: The phospholipid composition in sHDL plays a critical role in determining half-life and cholesterol mobilization in vivo., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made toward developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein, we describe the development of GRK5 subfamily selective inhibitors, 5 and 16d that covalently interact with a nonconserved cysteine (Cys474) unique to this subfamily. Compounds 5 and 16d feature a highly amenable pyrrolopyrimidine scaffold that affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and geometries. Our work thereby establishes a new pathway toward further development of subfamily selective GRK inhibitors and establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1-Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca 2+ ·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca 2+ ·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca 2+ ·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca 2+ ·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca 2+ ·CaM-dependent hypertrophy., Competing Interests: The authors declare no conflict of interest.

Lysosomal phospholipase A2 (PLA2G15) is a ubiquitous enzyme uniquely characterized by a subcellular localization to the lysosome and late endosome. PLA2G15 has an acidic pH optimum, is calcium independent, and acts as a transacylase in the presence of N-acetyl-sphingosine as an acceptor. Recent studies aided by the delineation of the crystal structure of PLA2G15 have clarified further the catalytic mechanism, sn-1 versus sn-2 specificity, and the basis whereby cationic amphiphilic drugs inhibit its activity. PLA2G15 has recently been shown to hydrolyze short chain oxidized phospholipids which access the catalytic site directly based on their aqueous solubility. Studies on the PLA2G15 null mouse suggest a role for the enzyme in the catabolism of pulmonary surfactant. PLA2G15 may also have a role in host defense and in the processing of lipid antigens for presentation by CD1 proteins. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau., (Published by Elsevier B.V.)

As part of a program toward making analogues of amlexanox (1), currently under clinical investigation for the treatment of type 2 diabetes and obesity, we have synthesized derivative 5 in which deuterium has been introduced into two sites of metabolism on the C-7 isopropyl function of amlexanox. The synthesis of 5 was completed in an efficient three-step process utilizing reduction of key olefin 7b to 8 by Wilkinson's catalyst to provide specific incorporation of di-deuterium across the double bond. Compound 5 displayed nearly equivalent potency to amlexanox (IC 50 , 1.1μM vs 0.6μM, respectively) against recombinant human TBK1. When incubated with human, rat, and mouse liver microsomes, amlexanox (1) and d 2 -amlexanox (5) were stable (t 1/2  > 60 minutes) with 1 showing marginally greater stability relative to 5 except for rat liver microsomes. These data show that incorporating deuterium into two sites of metabolism does not majorly suppress Cyp-mediated metabolism relative to amlexanox., (© 2019 John Wiley & Sons, Ltd.)

Lysosomal phospholipase A 2 (LPLA 2 /PLA2G15) is a key enzyme involved in lipid homeostasis and is characterized by both phospholipase A2 and transacylase activity and by an acidic pH optimum. Divalent cations such as Ca 2+ and Mg 2+ have previously been shown to have little effect on the activity of LPLA 2 , but the discovery of a novel crystal form of LPLA 2 with Zn 2+ bound in the active site suggested a role for this divalent cation in regulating enzyme activity. In this complex, the cation directly coordinates the serine and histidine of the α/β-hydrolase triad and stabilizes a closed conformation. This closed conformation is characterized by an inward shift of the lid loop, which extends over the active site and effectively blocks access to one of its lipid acyl chain binding tracks. Therefore, we hypothesized that Zn 2+ would inhibit LPLA 2 activity at a neutral but not acidic pH because histidine would be positively charged at lower pH. Indeed, Zn 2+ was found to inhibit the esterase activity of LPLA 2 in a noncompetitive manner exclusively at a neutral pH (between 6.5 and 8.0). Because lysosomes are reservoirs of Zn 2+ in cells, the pH optimum of LPLA 2 might allow it to catalyze acyl transfer unimpeded within the organelle. We conjecture that Zn 2+ inhibition of LPLA 2 at higher pH maintains a lower activity of the esterase in environments where its activity is not typically required.

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gα q/11 subfamily of heterotrimeric G proteins to the small guanosine triphosphatase (GTPase) RhoA, enabling Gα q/11 -coupled G protein-coupled receptors (GPCRs) to control downstream events, such as cell motility and gene transcription. This conserved signal transduction axis is crucial for tumor growth in uveal melanoma. Previous studies indicate that the GEF activity of the TrioC module is autoinhibited, with release of autoinhibition upon Gα q/11 binding. Here, we determined the crystal structure of TrioC in its basal state and found that the pleckstrin homology (PH) domain interacts with the Dbl homology (DH) domain in a manner that occludes the Rho GTPase binding site, thereby suggesting the molecular basis of TrioC autoinhibition. Biochemical and biophysical assays revealed that disruption of the autoinhibited conformation destabilized and activated the TrioC module in vitro. Last, mutations in the DH-PH interface found in patients with cancer activated TrioC and, in the context of full-length Trio, led to increased abundance of guanosine triphosphate-bound RhoA (RhoA·GTP) in human cells. These mutations increase mitogenic signaling through the RhoA axis and, therefore, may represent cancer drivers operating in a Gα q/11 -independent manner., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

P-Rex family Rho guanine-nucleotide exchange factors are important regulators of cell motility through their activation of a subset of small GTPases. Both P-Rex1 and P-Rex2 have also been implicated in the progression of certain cancers, including breast cancer and melanoma. Although these molecules display a high level of homology, differences exist in tissue distribution, physiological function, and regulation at the molecular level. Here, we sought to compare the P-Rex2 pleckstrin homology (PH) domain structure and ability to interact with PIP 3 with those of P-Rex1. The 1.9 Å crystal structure of the P-Rex2 PH domain reveals conformational differences in the loop regions, yet biochemical studies indicate that the interaction of the P-Rex2 PH domain with PIP 3 is very similar to that of P-Rex1. Binding of the PH domain to PIP 3 is critical for P-Rex2 activity but not membrane localization, as previously demonstrated for P-Rex1. These studies serve as a starting point in the identification of P-Rex structural features that are divergent between isoforms and could be exploited for the design of P-Rex selective compounds., Competing Interests: None., (© 2018 The Authors.)

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R 7 and R 8 A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC 50 values as low as 210 nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R 7 cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits I κ B kinase ε (IKK ε ) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes the inhibitory effects. IKK ε is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKK ε and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca 2+ concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca 2+ imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca 2+ , whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H 1 receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Lysosomal phospholipase A2 (LPLA 2 ) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N -acetyl-sphingosine. Prior structural analysis of LPLA 2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA 2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1- O -acyl- N -acetylsphingosine to measure the hydrolysis of sn -1 versus sn -2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA 2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain., (Copyright © 2018 Hinkovska-Galcheva et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Following ENU mutagenesis, a phenodeviant line was generated, termed the "Cartoon mouse," that exhibits profound defects in growth and development. Cartoon mice harbor a single S466P point mutation in the MT1-MMP hemopexin domain, a 200-amino acid segment that is thought to play a critical role in regulating MT1-MMP collagenolytic activity. Herein, we demonstrate that the MT1-MMP S466P mutation replicates the phenotypic status of Mt1-mmp -null animals as well as the functional characteristics of MT1-MMP -/- cells. However, rather than a loss-of-function mutation acquired as a consequence of defects in MT1-MMP proteolytic activity, the S466P substitution generates a misfolded, temperature-sensitive mutant that is abnormally retained in the endoplasmic reticulum (ER). By contrast, the WT hemopexin domain does not play a required role in regulating MT1-MMP trafficking, as a hemopexin domain-deletion mutant is successfully mobilized to the cell surface and displays nearly normal collagenolytic activity. Alternatively, when MT1-MMP S466P -expressing cells are cultured at a permissive temperature of 25 °C that depresses misfolding, the mutant successfully traffics from the ER to the trans -Golgi network (ER → trans -Golgi network), where it undergoes processing to its mature form, mobilizes to the cell surface, and expresses type I collagenolytic activity. Together, these analyses define the Cartoon mouse as an unexpected gain-of- abnormal function mutation, wherein the temperature-sensitive mutant phenocopies MT1-MMP -/- mice as a consequence of eliciting a specific ER → trans -Golgi network trafficking defect., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2-Gβγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)