Phosphatidylinositol 4-kinase IIIβ Research Articles

N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIβ (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIβ (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

Activation of neuronal AT 1 R exacerbates hypertension‐induced cognitive impairment through decreasing neuronal function

Hypertension has now been closely associated to cognitive impairment and considered as one of the risk factors of Alzheimer's disease (AD), due to its contribution to cerebrovascular dysfunction. To investigate whether there is neuronal mechanism, hypertension was induced in C57BL/6j male mice (6 months) by DOCA-salt treatment (1.5 mg/g, s.c., 28 days). Firstly, behavioral study, which contains novel object recognition (NOR) and nesting score tests, was performed to assess cognitive function. DOCA plus 1% NaCl in the drinking water significantly increased the blood pressure (BP) of treated control mice (normal type, NT), before vs. after DOCA (tail-cuff, systolic BP): 114.4 ±2.0 vs. 130.8 ±1.9 mmHg. At the end of DOCA-salt treatment, both NOR% [(timenovel-timeold)/(timenovel+timeold)×100%] and nesting scores were markedly reduced in NT mice, compared to their baselines, indicating impaired cognitive function (NOR%: 10.83 ±7.413% vs. 39.46 ±8.926%, n=13; nesting scores: 3.1 ±0.23 vs. 5, n=10). Later on, brain tissues were collected, and markers for neuronal function were measured via qRT-PCR. In both of the prefrontal cortex and hippocampus, it was found that the mRNA levels of BDNF (brain-derived neurotrophic factor) and PI4KIIIβ (phosphatidylinositol 4-kinase IIIβ) were significantly down-regulated, in those NT mice with DOCA-salt hypertension (cortical Bdnf: P<0.001 vs. sham, cortical Pi4k: P<0.001 vs. sham, hippocampal Bdnf: P<0.001 vs. sham, hippocampal Pi4k: P<0.05 vs. sham, n=7). Noteworthy, PI4K has been linked to the progression of neurodegenerative disorders, especially AD, therefore suggesting that reduction of neuronal function could be a part of the etiology of hypertension-related cognitive decline. We previously demonstrated that neuronal AT1R plays pivotal role in the maintenance of neurogenic hypertension, and here we hypothesized that activation of AT1R could also exacerbate hypertension-induced reduction in neuronal function. In mice with DOCA-salt hypertension, the function of cortical neurons was shown to be improved by selective deletion of neuronal AT1aR, as evidenced by significantly higher mRNA levels of BDNF and PI4KIIIβ, compared to the controls (P<0.05 vs. sham, n=6). To further study the possible involvement of neuronal AT1R in AD, 5×FAD mice were bred with mice with neuronal AT1R deletion (AT1NKO). AD-associated reduction of ACE2 protein, mainly in neurons, was found to be slightly ameliorated in the prefrontal cortex of 5×FAD-AT1NKO, compared to the age/sex-matched 5×FAD, showing by immunocytochemistry (24610 ±4182 vs. 13420 ±3720 AFU, n=6 slices). Although the detailed mechanism is still unknown, our data suggests that, neuron-expressing AT1R could participate in the development of hypertension-associated cognitive impairment, independently of vascular AT1R.

Drosophila phosphatidylinositol-4 kinase fwd promotes mitochondrial fission and can suppress Pink1/parkin phenotypes.

Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson’s disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. We sought additional factors that impinge on mitochondrial dynamics and which may also suppress Pink1/parkin phenotypes. We found that the Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, we identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, we found that fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration.

Structural Cues for Understanding eEF1A2 Moonlighting.

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the "canonical" one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.

Novel phosphatidylinositol 4-kinases III beta (PI4KIIIβ) inhibitors discovered by virtual screening using free energy models.

Herein, the LASSBio Chemical Library is presented as a valuable source of compounds for screening to identify hits suitable for subsequent hit-to-lead optimization stages. A feature of the LASSBio Chemical Library worth highlighting is the fact that it is a smart library designed by medicinal chemists with pharmacological activity as the main priority. The great majority of the compounds part of this library have shown in vivo activity in animal models, which is an indication that they possess overall favorable bioavailability properties and, hence, adequate pharmacokinetic profiles. This, in turn, is supported by the fact that approximately 85% of the compounds are compliant with Lipinski’s rule of five and ca. 95% are compliant with Veber’s rules, two important guidelines for oral bioavailability. In this work it is presented a virtual screening methodology combining a pharmacophore-based model and an empirical Gibbs free energy-based model for the ligand–protein interaction to explore the LASSBio Chemical Library as a source of new hits for the inhibition of the phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) enzyme, which is related to the development of viral infections (including enteroviruses, SARS coronavirus, and hepatitis C virus), cancers and neurological diseases. The approach resulted in the identification of two hits, LASSBio-1799 (7) and LASSBio-1814 (10), which inhibited the target enzyme with IC50 values of 3.66 μM and IC50 and 6.09 μM, respectively. This study also enabled the determination of the structural requirements for interactions with the active site of PI4KIIIβ, demonstrating the importance of both acceptor and donor hydrogen bonding groups for forming interactions with binding site residues Val598 and Lys549.Electronic supplementary materialThe online version of this article (10.1007/s10822-020-00327-9) contains supplementary material, which is available to authorized users.

ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ.

ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid-starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.

Emerging Roles of Neuronal Ca2+ Sensor-1 in Cardiac and Neuronal Tissues: A Mini Review.

The EF-hand calcium (Ca2+)-binding protein, neuronal Ca2+ sensor-1 (NCS-1/frequenin), is predominantly expressed in neuronal tissues and plays a crucial role in neuronal functions, including synaptic transmission and plasticity. NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIβ (PI-4K-β) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.

Phosphatidylinositol 4-kinase III\u03b2 (PI4KB) forms highly flexible heterocomplexes that include ACBD3, 14-3-3, and Rab11 proteins

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.

Protein kinase D1 and oxysterol-binding protein form a regulatory complex independent of phosphorylation.

Protein kinase D (PKD) controls secretion from the trans-Golgi network (TGN) by phosphorylating phosphatidylinositol 4-kinase IIIβ and proteins that bind and/or transfer phosphatidylinositol 4-phosphate (PtdIns-4P), such as oxysterol-binding protein (OSBP) and ceramide transfer protein. Here, we investigated the consequences of PKD phosphorylation of OSBP at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCS). Results with OSBP phospho-mutants revealed that PKD phosphorylation did not affect sterol and PtdIns-4P binding, activation of sphingomyelin (SM) synthesis at Golgi-ER MCS or other OSBP phospho-sites. Instead, an interaction was identified between the N-terminal region of OSBP and PKD1 that was independent of kinase activity and OSBP phosphorylation status. S916 autophosphorylation of PKD1 was inhibited by OSBP expression suggesting the interaction negatively regulates PKD1 activity. Stimulation of PKD1 activity by phorbol ester promoted the Golgi-localization of wild-type and phospho-mutants of OSBP but did not affect OSBP-dependent SM synthesis. Only when wild-type or kinase-dead PKD1 was overexpressed was 25-hydroxycholesterol-activated SM synthesis inhibited. We conclude that OSBP and PKD1 form a complex that inhibits both the oxysterol-dependent activity of OSBP at the ER-Golgi and activation of PKD1. Formation of the complex was independent of PKD1 activity and phosphorylation of OSBP.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

The protein kinase D1-mediated classical protein secretory pathway regulates the Ras oncogene-induced senescence response.

Senescent cells develop a senescence-associated secretory phenotype (SASP). The factors secreted by cells with a SASP have multiple biological functions that are mediated in an autocrine or paracrine manner. However, the status of the protein kinase D1 (PKD1; also known as PRKD1)-mediated classical protein secretory pathway, from the trans-Golgi network (TGN) to the cell surface, during cellular senescence and its role in the cellular senescence response remain unknown. Here, we show that the activities or quantities of critical components of this pathway, including PKD1, ADP-ribosylation factor 1 (ARF1) and phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), at the TGN are increased in senescent cells. Blocking of this pathway decreases IL-6 and IL-8 (hereafter IL-6/IL-8) secretion and results in IL-6/IL-8 accumulation in SASP-competent senescent cells. Inhibition of this pathway reduces IL-6/IL-8 secretion during Ras oncogene-induced senescence (OIS), retards Ras OIS and alleviates its associated ER stress and autophagy. Finally, targeting of this pathway triggers cell death in SASP factor-producing senescent cells due to the intracellular accumulation of massive amounts of IL-6/IL-8. Taken together, our results unveil the hyperactive state of the protein secretory pathway in SASP-competent senescent cells and its critical functions in mediating SASP factor secretion and the Ras OIS process, as well as in determining the fate of senescent cells.

Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase IIIβ-ACBD3 Interaction.

Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIβ (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIβ, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication.

Structural analysis of phosphatidylinositol 4-kinase IIIβ (PI4KB) - 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro.

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is responsible for the synthesis of the Golgi and trans-Golgi network (TGN) pool of phosphatidylinositol 4-phospahte (PI4P). PI4P is the defining lipid hallmark of Golgi and TGN and also serves as a signaling lipid and as a precursor for higher phosphoinositides. In addition, PI4KB is hijacked by many single stranded plus RNA (+RNA) viruses to generate PI4P-rich membranes that serve as viral replication organelles. Given the importance of this enzyme in cells, it has to be regulated. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB recognition by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. We discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. Surprisingly, the enzymatic activity of PI4KB is not directly modulated by 14-3-3 proteins. However, 14-3-3 proteins protect PI4KB from proteolytic degradation in vitro. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. It also predicted no direct modulation of PI4KB enzymatic activity by 14-3-3 proteins and that 14-3-3 binding will not interfere with PI4KB recruitment to the membrane by the ACBD3 protein. In addition, the structural analysis explains the observed protection from degradation; it revealed that several disordered regions of PI4KB become protected from proteolytical degradation upon 14-3-3 binding. All the structural predictions were subsequently biochemically validated.

Rational Design of Novel Highly Potent and Selective Phosphatidylinositol 4-Kinase IIIβ (PI4KB) Inhibitors as Broad-Spectrum Antiviral Agents and Tools for Chemical Biology.

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

Purine analogs as phosphatidylinositol 4-kinase IIIβ inhibitors

We report on an extensive structure–activity relationship study of novel PI4K IIIβ inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIβ inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.

Highly Selective Phosphatidylinositol 4-Kinase IIIβ Inhibitors and Structural Insight into Their Mode of Action.

Phosphatidylinositol 4-kinase IIIβ is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIβ-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIβ, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.

Modulation of phosphatidylinositol 4-phosphate levels by CaBP7 controls cytokinesis in mammalian cells.

Calcium and phosphoinositide signaling regulate cell division in model systems, but their significance in mammalian cells is unclear. Calcium-binding protein-7 (CaBP7) is a phosphatidylinositol 4-kinaseIIIβ (PI4KIIIβ) inhibitor required during cytokinesis in mammalian cells, hinting at a link between these pathways. Here we characterize a novel association of CaBP7 with lysosomes that cluster at the intercellular bridge during cytokinesis in HeLa cells. We show that CaBP7 regulates lysosome clustering and that PI4KIIIβ is essential for normal cytokinesis. CaBP7 depletion induces lysosome mislocalization, extension of intercellular bridge lifetime, and cytokinesis failure. These data connect phosphoinositide and calcium pathways to lysosome localization and normal cytokinesis in mammalian cells.

New small-molecule inhibitors effectively blocking picornavirus replication.

Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIIIβ remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C- and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Diverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

Phosphatidylinositol 4-Phosphate in the Golgi Apparatus Regulates Cell–Cell Adhesion and Invasive Cell Migration in Human Breast Cancer

Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIβ, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus.