CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Benchmarking network algorithms for contextualizing genes of interest.

Computational approaches have shown promise in contextualizing genes of interest with known molecular interactions. In this work, we evaluate seventeen previously published algorithms based on characteristics of their output and their performance in three tasks: cross validation, prediction of drug targets, and behavior with random input. Our work highlights strengths and weaknesses of each algorithm and results in a recommendation of algorithms best suited for performing different tasks.

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions.

BACKGROUND: Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. METHODS: We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. RESULTS: We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P<1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P<1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P<6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. CONCLUSIONS: Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury.

Aldose reductase-mediated phosphorylation of p53 leads to mitochondrial dysfunction and damage in diabetic platelets.

BACKGROUND: Platelet abnormalities are well-recognized complications of diabetes mellitus. Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in diabetes mellitus. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in platelets in diabetes mellitus is unknown. METHODS AND RESULTS: Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase activation and subsequent reactive oxygen species production lead to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage, and rupture by sequestration of the antiapoptotic protein Bcl-xL. In a glucose dose-dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation, and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, aldose reductase activation, reactive oxygen species production, and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are 2 distinct states that depend on the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model. CONCLUSIONS: Aldose reductase contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for thrombotic complications in diabetes mellitus.

Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development.

RATIONALE: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. OBJECTIVE: This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. METHODS AND RESULTS: We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. CONCLUSIONS: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Biased suppression of TP homodimerization and signaling through disruption of a TM GxxxGxxxL helical interaction motif.

Thromboxane A2 (TXA2) contributes to cardiovascular disease (CVD) by activating platelets and vascular constriction and proliferation. Despite their preclinical efficacy, pharmacological antagonists of the TXA2 receptor (TP), a G protein-coupled receptor, have not been clinically successful, raising interest in novel approaches to modifying TP function. We determined that disruption of a GxxxGxxxL helical interaction motif in the human TP's (α isoform) fifth transmembrane (TM) domain suppressed TP agonist-induced Gq signaling and TPα homodimerization, but not its cell surface expression, ligand affinity, or Gq association. Heterodimerization of TPα with the functionally opposing prostacyclin receptor (IP) shifts TPα to signal via the IP-Gs cascade contributing to prostacyclin's restraint of TXA2 function. Interestingly, disruption of the TPα-TM5 GxxxGxxxL motif did not modify either IP-TPα heterodimerization or its Gs-cAMP signaling. Our study indicates that distinct regions of the TPα receptor direct its homo- and heterodimerization and that homodimerization is necessary for normal TPα-Gq activation. Targeting the TPα-TM5 GxxxGxxxL domain may allow development of biased TPα homodimer antagonists that avoid suppression of IP-TPα heterodimer function. Such novel therapeutics may prove superior in CVD compared with nonselective suppression of all TP functions with TXA2 biosynthesis inhibitors or TP antagonists.

An eicosanoid-centric view of atherothrombotic risk factors.

Cardiovascular disease is the foremost cause of morbidity and mortality in the Western world. Atherosclerosis followed by thrombosis (atherothrombosis) is the pathological process underlying most myocardial, cerebral, and peripheral vascular events. Atherothrombosis is a complex and heterogeneous inflammatory process that involves interactions between many cell types (including vascular smooth muscle cells, endothelial cells, macrophages, and platelets) and processes (including migration, proliferation, and activation). Despite a wealth of knowledge from many recent studies using knockout mouse and human genetic studies (GWAS and candidate approach) identifying genes and proteins directly involved in these processes, traditional cardiovascular risk factors (hyperlipidemia, hypertension, smoking, diabetes mellitus, sex, and age) remain the most useful predictor of disease. Eicosanoids (20 carbon polyunsaturated fatty acid derivatives of arachidonic acid and other essential fatty acids) are emerging as important regulators of cardiovascular disease processes. Drugs indirectly modulating these signals, including COX-1/COX-2 inhibitors, have proven to play major roles in the atherothrombotic process. However, the complexity of their roles and regulation by opposing eicosanoid signaling, have contributed to the lack of therapies directed at the eicosanoid receptors themselves. This is likely to change, as our understanding of the structure, signaling, and function of the eicosanoid receptors improves. Indeed, a major advance is emerging from the characterization of dysfunctional naturally occurring mutations of the eicosanoid receptors. In light of the proven and continuing importance of risk factors, we have elected to focus on the relationship between eicosanoids and cardiovascular risk factors.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Comprehensive biochemical analysis of rare prostacyclin receptor variants: study of association of signaling with coronary artery obstruction.

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.

Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition.

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen.

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.

Conserved rhodopsin intradiscal structural motifs mediate stabilization: effects of zinc.

Retinitis pigmentosa (RP), a neurodegenerative disorder, can arise from single point mutations in rhodopsin, leading to a cascade of protein instability, misfolding, aggregation, rod cell death, retinal degeneration, and ultimately blindness. Divalent cations, such as zinc and copper, have allosteric effects on misfolded aggregates of comparable neurodegenerative disorders including Alzheimer disease, prion diseases, and ALS. We report that two structurally conserved low-affinity zinc coordination motifs, located among a cluster of RP mutations in the intradiscal loop region, mediate dose-dependent rhodopsin destabilization. Disruption of native interactions involving histidines 100 and 195, through site-directed mutagenesis or exogenous zinc coordination, results in significant loss of receptor stability. Furthermore, chelation with EDTA stabilizes the structure of both wild-type rhodopsin and the most prevalent rhodopsin RP mutation, P(23)H. These interactions suggest that homeostatic regulation of trace metal concentrations in the rod outer segment of the retina may be important both physiologically and for an important cluster of RP mutations. Furthermore, with a growing awareness of allosteric zinc binding domains on a diverse range of GPCRs, such principles may apply to many other receptors and their associated diseases.

Prostacyclin receptor/thromboxane receptor interactions and cellular responses in human atherothrombotic disease.

Twenty-five years have passed since Vane and colleagues proposed a prostacyclin and thromboxane balance as critical to cardiovascular homeostasis. Prostacyclin prevents platelet aggregation and promotes vasodilatation, opposing the effects of thromboxane. Possible compensation by redundant functions, such as nitric oxide, long prevented appreciation of this balance. Effective use of low-dose aspirin in the secondary prevention of atherothrombosis suggested a clinical importance for the balance. However, it was not until the cyclooxygenase-2 inhibitor rofecoxib was withdrawn because of increased cardiovascular events that this critical balance was confirmed in humans. Moreover, clinical observations are supported by elegant animal receptor knockout experiments and subsequent human genetic variant studies. Combined, these findings provide valuable insight into the roles of these prostanoids in the development of atherothrombosis, emphasizing the need to reevaluate the use of selective prostacyclin- and thromboxane-based therapies in cardiovascular disease.

Arginine (CGC) codon targeting in the human prostacyclin receptor gene (PTGIR) and G-protein coupled receptors (GPCR).

The human prostacyclin receptor (hIP) has recently been recognized as an important seven transmembrane G-protein coupled receptor that plays critical roles in atheroprevention and cardioprotection. To date, four non-synonymous genetic variants have been identified, two of which occur at the same Arg amino acid position (R212H, R212C). This observation instigated further genetic screening for prostacyclin receptor variants on 1455 human genomic samples. A total of 31 distinct genetic variants were detected, with 6 (19%) involving Arg residues. Distinct differences in location and frequencies of genetic variants were noted between Caucasian, Asian, Hispanic and African Americans, with the most changes noted in the Asian cohort. From the sequencing results, three Arg-targeted changes at the same 212 position within the third cytoplasmic loop of the human prostacyclin (hIP) receptor were detected: 1) R212C (CGC-->TGC), 2) R212H (CGC-->CAC), and 3) R212R (CGC-->CGT). Three additional Arg codon variants (all exhibiting the same CGC to TGC change) were also detected, R77C, R215C, and R279C. Analysis (GPCR and SNP databases) of 200 other GPCRs, with recorded non-synonymous mutations, confirmed a high frequency of Arg-targeted missense mutations, particularly within the important cytoplasmic domain. Preferential nucleotide changes (at Arg codons), were observed involving cytosine (C) to thymine (T) (pyrimidine to pyrimidine), as well as guanine (G) to adenine (A) (purine to purine) (p<0.001, Pearson's goodness-of-fit test). Such targeting of Arg residues, leading to significant changes in coding amino acid size and/or charge, may have potentially-important structural and evolutionary implications on the hIP and GPCRs in general. In the case of the human prostacyclin receptor, such alterations may reduce the cardio-, vasculo-, and cytoprotective effects of prostacyclin.

Human prostacyclin receptor structure and function from naturally-occurring and synthetic mutations.

Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation (anti-thrombotic), and moderator of vascular smooth muscle cell proliferation-migration-differentiation (anti-atherosclerotic). These actions are mediated via a seven transmembrane-spanning G-protein coupled receptor (GPCR), known as the human prostacyclin receptor or hIP. Animal studies using prostacyclin receptor knock-out (IP-/-) mice have revealed increased propensities towards thrombosis, intimal hyperplasia, atherosclerosis, restenosis, as well as reperfusion injury. Of further importance has been the world-wide withdrawal of selective COX-2 inhibitors, due to their discriminating suppression of COX-2-derived PGI2 and its cardioprotective effects, leading to increased cardiovascular events, including myocardial infarction and thrombotic stroke. Over the last decade, mutagenesis studies of the IP receptor, in conjunction with in vitro functional assays and molecular modeling, have provided critical insights into the molecular mechanisms of both agonist binding and receptor activation. Most recently, the discovery of naturally-occurring and dysfunctional mutations within the hIP has provided additional insights into the proposed cardioprotective role of prostacyclin. The aim of this review is to summarize the most recent findings regarding hIP receptor structure-function that have developed through the study of both synthetic and naturally-occurring mutations.