Design and Synthesis of New GS-6207 Subtypes for Targeting HIV-1 Capsid Protein.

HIV-1 capsid protein (CA) is the molecular target of the recently FDA-approved long acting injectable (LAI) drug lenacapavir (GS-6207). The quick emergence of CA mutations resistant to GS-6207 necessitates the design and synthesis of novel sub-chemotypes. We have conducted the structure-based design of two new sub-chemotypes combining the scaffold of GS-6207 and the N-terminal cap of PF74 analogs, the other important CA-targeting chemotype. The design was validated via induced-fit molecular docking. More importantly, we have worked out a general synthetic route to allow the modular synthesis of novel GS-6207 subtypes. Significantly, the desired stereochemistry of the skeleton C2 was confirmed via an X-ray crystal structure of the key synthetic intermediate 22a. Although the newly synthesized analogs did not show significant potency, our efforts herein will facilitate the future design and synthesis of novel subtypes with improved potency.

Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

mRNA-binding protein ZFP36 is expressed in atherosclerotic lesions and reduces inflammation in aortic endothelial cells.

OBJECTIVE: We studied the expression and function of an mRNA-binding protein, zinc finger protein-36 (ZFP36), in vascular endothelial cells in vivo and in vitro. We tested the hypotheses that ZFP36 regulates inflammation in vascular endothelial cells and that it functions through direct binding to target cytokine mRNAs. We also tested whether ZFP36 inhibits nuclear factor-κB-mediated transcriptional responses in vascular endothelial cells. APPROACH AND RESULTS: ZFP36 was minimally expressed in healthy aorta but was expressed in endothelial cells overlying atherosclerotic lesions in mice and humans. The protein was also expressed in macrophage foam cells of atherosclerosis. ZFP36 was expressed in human aortic endothelial cells in response to bacterial lipopolysaccharide, glucocorticoid, and forskolin, but not oxidized low-density lipoproteins or angiotensin II. Functional studies demonstrated that ZFP36 reduces the expression of inflammatory cytokines in target cells by 2 distinct mechanisms: ZFP36 inhibits nuclear factor-κB transcriptional activation and also binds to cytokine mRNAs, leading to reduced transcript stability. CONCLUSIONS: ZFP36 is expressed in vascular endothelial cells and macrophage foam cells where it inhibits the expression of proinflammatory mRNA transcripts. The anti-inflammatory effects of ZFP36 in endothelial cells occur via both transcriptional and posttranscriptional mechanisms. Our data suggest that enhancing vascular ZFP36 expression might reduce vascular inflammation.

Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Advance in sex differentiation in cucumber.

Cucumber belongs to the family Cucurbitaceae (melon genus) and is an annual herbaceous vegetable crop. Cucumber is an important cash crop that is grown all over the world. From morphology to cytology, from canonical genetics to molecular biology, researchers have performed much research on sex differentiation and its regulatory mechanism in cucumber, mainly in terms of cucumber sex determination genes, environmental conditions, and the effects of plant hormones, revealing its genetic basis to improve the number of female flowers in cucumber, thus greatly improving the yield of cucumber. This paper reviews the research progress of sex differentiation in cucumber in recent years, mainly focusing on sex-determining genes, environmental conditions, and the influence of phytohormones in cucumber, and provides a theoretical basis and technical support for the realization of high and stable yield cultivation and molecular breeding of cucumber crop traits.

Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies.

The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (∼55-fold), with major decrease in WA1 replicon fitness (∼20-fold), but not BA.1 (∼2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and ß-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through "wiggling and jiggling" accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a ß-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.

MAGI-3 competes with NHERF-2 to negatively regulate LPA2 receptor signaling in colon cancer cells.

BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. METHODS: We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. RESULTS: NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-ß3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. CONCLUSIONS: LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.

[Molecular characteristics and specific PCR detection of transgenic rice containing Cry1Ab].

Bt01 is a new type of rice that has been genetically modified to express Cry1Abprotein. This study confirmed that Cry1Abwas inserted into Bt01 as a single copy using Southern blotting analysis. TAIL-PCR method was further used to obtain its insertion site information. Specific PCR primers and TaqMan probes were designed based on the 5'-integration junction sequence of transgenic rice Bt01. The results showed that the limit of detection (LOD) was ten copies in qualitative PCR. The quantitative PCR assay showed that the LOD was five copies, and the limit of quantification (LOQ) was ten cop-ies. In addition, the accuracy of the established quantitative PCR was verified by detecting two samples containing 3% and 0.5%Bt01, respectively. The quantitative PCR analysis showed the results were 2.7% and 0.47%, respectively.The above results indicated that the event-specific PCR methods developed have high specificity and good sensitivity, which could be effective methods for identifying and testing the genetically modified Bt01 rice.

Lysophosphatidic acid stimulates the intestinal brush border Na(+)/H(+) exchanger 3 and fluid absorption via LPA(5) and NHERF2.

BACKGROUND & AIMS: Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na(+)/H(+) exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na(+)/H(+) exchanger activity and Na(+)-dependent fluid absorption in the intestine. METHODS: We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na(+)/H(+) exchanger regulatory factor 2 (NHERF2; Nherf2(-/-)) or LPA(2) (Lpa(2)(-/-)). Roles of LPA(5) and NHERF2 were determined by analysis of heterologous expression. RESULTS: Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA(5) was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA(5) signaling required coexpression of NHERF2, which interacted with LPA(5). LPA-mediated intestinal fluid absorption was impaired in Nherf2(-/-) mice, demonstrating the requirement for NHERF2 in LPA(5) activity. However, fluid absorption was unaltered in Lpa(2)(-/-) mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. CONCLUSIONS: LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA(5). Regulation by LPA(5) depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Discovery of New Small Molecule Hits as Hepatitis B Virus Capsid Assembly Modulators: Structure and Pharmacophore-Based Approaches.

Hepatitis B virus (HBV) capsid assembly modulators (CpAMs) have shown promise as potent anti-HBV agents in both preclinical and clinical studies. Herein, we report our efforts in identifying novel CpAM hits via a structure-based virtual screening against a small molecule protein-protein interaction (PPI) library, and pharmacophore-guided compound design and synthesis. Curated compounds were first assessed in a thermal shift assay (TSA), and the TSA hits were further evaluated in an antiviral assay. These efforts led to the discovery of two structurally distinct scaffolds, ZW-1841 and ZW-1847, as novel HBV CpAM hits, both inhibiting HBV in single-digit µM concentrations without cytotoxicity at 100 µM. In ADME assays, both hits displayed extraordinary plasma and microsomal stability. Molecular modeling suggests that these hits bind to the Cp dimer interfaces in a mode well aligned with known CpAMs.

Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability.

Of all known small molecules targeting human immunodeficiency virus (HIV) capsid protein (CA), PF74 represents by far the best characterized chemotype, due to its ability to confer antiviral phenotypes in both early and late phases of viral replication. However, the prohibitively low metabolic stability renders PF74 a poor antiviral lead. We report herein our medicinal chemistry efforts toward identifying novel and metabolically stable small molecules targeting the PF74 binding site. Specifically, we replaced the inter-domain-interacting, electron-rich indole ring of PF74 with less electron-rich isosteres, including imidazolidine-2,4-dione, pyrimidine-2,4-dione, and benzamide, and identified four potent antiviral compounds (10, 19, 20 and 26) with markedly improved metabolic stability. Compared to PF74, analog 20 exhibited similar submicromolar potency, and much longer (51-fold) half-life in human liver microsomes (HLMs). Molecular docking corroborated that 20 binds to the PF74 binding site, and revealed distinct binding interactions conferred by the benzamide moiety. Collectively, our data support compound 20 as a promising antiviral lead.

Potency and metabolic stability: a molecular hybrid case in the design of novel PF74-like small molecules targeting HIV-1 capsid protein.

PF74 (1) is a potent and well-characterized prototypical small molecule targeting human immunodeficiency virus type 1 (HIV-1) capsid protein (CA), but not a viable antiviral lead due to the lack of metabolic stability. We report herein our molecular hybridization-based medicinal chemistry efforts toward potent and metabolically stable PF74-like small molecules. The design of the new sub-chemotype 4 rationally combines binding features of two recently reported PF74-like compounds 2 and 3. The subsequent confirmation and structure-activity relationship (SAR) of hit 4a entailed the chemical synthesis of 37 novel analogs, most of which showed modest but meaningful thermal shift, and low µM antiviral activity. The most potent analogs (4a, 4d, 4o, and 4r) all exhibited noticeably improved metabolic stability over PF74. Molecular modeling suggests that these new analogs bind to the PF74 binding site. Overall, our work demonstrated that the molecular hybridization approach is suitable for designing compounds with balanced potency and metabolic stability.

Design, Synthesis and Characterization of HIV-1 CA-Targeting Small Molecules: Conformational Restriction of PF74.

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 µM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.

Novel HIV-1 capsid-targeting small molecules of the PF74 binding site.

The PF74 binding site in HIV-1 capsid protein (CA) is a compelling antiviral drug target. Although PF74 confers mechanistically distinct antiviral phenotypes by competing against host factors for CA binding, it suffers from prohibitively low metabolic stability. Therefore, there has been increasing interest in designing novel sub-chemotypes of PF74 with similar binding mode and improved metabolic stability. We report herein our efforts to explore the inter-domain interacting indole moiety for designing novel CA-targeting small molecules. Our design includes simple substitution on the indole ring, and more importantly, novel sub-chemotypes with the indole moiety replaced with a few less electron-rich rings. All 56 novel analogs were synthesized and evaluated for antiviral activity, cytotoxicity, and impact on CA hexamer stability. Selected analogs were tested for metabolic stability in liver microsomes. Molecular modeling was performed to verify compound binding to the PF74 site. In the end, 5-hydroxyindole analogs (8,9 and 12) showed improved potency (up to 20-fold) over PF74. Of the novel sub-chemotypes, α- and ß-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.

Toward Structurally Novel and Metabolically Stable HIV-1 Capsid-Targeting Small Molecules.

HIV-1 capsid protein (CA) plays an important role in many steps of viral replication and represents an appealing antiviral target. Several CA-targeting small molecules of various chemotypes have been studied, but the peptidomimetic PF74 has drawn particular interest due to its potent antiviral activity, well-characterized binding mode, and unique mechanism of action. Importantly, PF74 competes against important host factors for binding, conferring highly desirable antiviral phenotypes. However, further development of PF74 is hindered by its prohibitively poor metabolic stability, which necessitates the search for structurally novel and metabolically stable chemotypes. We have conducted a pharmacophore-based shape similarity search for compounds mimicking PF74. We report herein the analog synthesis and structure-activity relationship (SAR) of two hits from the search, and a third hit designed via molecular hybridization. All analogs were characterized for their effect on CA hexamer stability, antiviral activity, and cytotoxicity. These assays identified three active compounds that moderately stabilize CA hexamer and inhibit HIV-1. The most potent analog (10) inhibited HIV-1 comparably to PF74 but demonstrated drastically improved metabolic stability in liver microsomes (31 min vs. 0.7 min t1/2). Collectively, the current studies identified a structurally novel and metabolically stable PF74-like chemotype for targeting HIV-1 CA.

Chemical profiling of HIV-1 capsid-targeting antiviral PF74.

The capsid protein (CA) of HIV-1 plays essential roles in multiple steps of the viral replication cycle by assembling into functional capsid core, controlling the kinetics of uncoating and nuclear entry, and interacting with various host factors. Targeting CA represents an attractive yet underexplored antiviral approach. Of all known CA-targeting small molecule chemotypes, the peptidomimetic PF74 is particularly interesting because it binds to the same pocket used by a few important host factors, resulting in highly desirable antiviral phenotypes. However, further development of PF74 entails understanding its pharmacophore and mitigating its poor metabolic stability. We report herein the design, synthesis, and evaluation of a large number of PF74 analogs aiming to provide a comprehensive chemical profiling of PF74 and advance the understanding on its detailed binding mechanism and pharmacophore. The analogs, containing structural variations mainly in the aniline domain and/or the indole domain, were assayed for their effect on stability of CA hexamers, antiviral activity, and cytotoxicity. Selected analogs were also tested for metabolic stability in liver microsomes, alone or in the presence of a CYP3A inhibitor. Collectively, our studies identified important pharmacophore elements and revealed additional binding features of PF74, which could aid in future design of improved ligands to better probe the molecular basis of CA-host factor interactions, design strategies to disrupt them, and ultimately identify viable CA-targeting antiviral leads.

MAGI-3 regulates LPA-induced activation of Erk and RhoA.

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA(2) contains a carboxyl-terminal motif that allows interaction with PDZ domain-containing proteins, such as NHERF2 and PDZ-RhoGEF. To identify additional interacting partners of LPA(2), the LPA(2) carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA(2)-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA(2) and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA(2), but not to LPA(1) and LPA(3). This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA(2), and mutational alteration of the carboxyl-terminal sequences of LPA(2) severely attenuated its ability to bind MAGI-3. LPA(2) also associated with MAGI-3 in cells as determined by co-affinity purification. Overexpression of MAGI-3 in SW480 cells showed no apparent effect on LPA-induced activation of Erk and Akt. In contrast, silencing of MAGI-3 expression by siRNA drastically inhibited LPA-induced Erk activation, suggesting that the lack of an effect by overexpression was due to the high endogenous MAGI-3 level in these cells. Previous studies have shown that the cellular signaling elicited by LPA results in activation of the small GTPase RhoA by Galpha(12/13) - as well as Galpha(q)-dependent pathways. Overexpression of MAGI-3 stimulated LPA-induced RhoA activation, whereas silencing of MAGI-3 by siRNA resulted in a small but statistically significant decrease in RhoA activation. These results demonstrate that MAGI-3 interacts directly with LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA.

Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway.

Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxia-responsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1alpha. 103D5R markedly decreased HIF-1alpha protein levels induced by hypoxia or cobaltous ions in a dose- and time-dependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1beta, IkappaBalpha, and beta-actin. The inhibitory activity of 103D5R against HIF-1alpha was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1alpha protein synthesis, whereas HIF-1alpha mRNA levels and HIF-1alpha degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stress-activated protein kinase/c-jun-NH(2)-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1alpha for therapeutic purposes.

The mRNA-binding protein Zfp36 is upregulated by ß-adrenergic stimulation and represses IL-6 production in 3T3-L1 adipocytes.

Obesity produces a chronic inflammatory state that contributes to the development of diabetes and atherosclerosis. In obese humans, fat depot adipocytes and macrophages produce inflammatory cytokines and other factors which exert unfavorable local and systemic immune responses. The expression of many cytokines is modulated at the post-transcriptional level by mRNA-binding proteins which recognize AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of these transcripts. One such protein, zinc finger protein 36 (Zfp36), is known to destabilize target mRNAs leading to decreased cytokine expression. Few regulators of Zfp36 expression in adipocytes have been described and mRNA targets of Zfp36 in adipocytes are largely unknown. We found that macrophage-derived inflammatory stimuli enhanced endogenous Zfp36 expression in 3T3-L1 adipocytes. Furthermore, the ß-adrenergic receptor agonist isoproterenol (Iso) and the glucocorticoid dexamethasone (Dex) each enhanced Zfp36 expression in adipocytes, the former most likely via a cyclic adenosine monophosphate (cAMP)-dependent pathway. By contrast, Zfp36 expression in murine macrophages (RAW 264.7) was not enhanced by exposure to Dex but was stimulated by retinoic acid (RA). Zfp36 inhibited basal and lipopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) expression in adipocytes. These data reveal important and cell type-specific modulators of Zfp36 expression in adipocytes and macrophages and identify Zfp36 as a potent repressor of adipocyte-derived IL-6. Furthermore, this work identifies new factors that stimulate adipocyte Zfp36 expression that are neither classically inflammatory nor mitogenic. Upregulating an mRNA-binding protein for therapeutic purposes may provide a novel mechanistic approach with which to treat diverse inflammatory disorders including common conditions associated with obesity.

IRBIT, inositol 1,4,5-triphosphate (IP3) receptor-binding protein released with IP3, binds Na+/H+ exchanger NHE3 and activates NHE3 activity in response to calcium.

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.