7-Amino-3-phenyl-2-methyl-pyrazolopyrimidine derivatives inhibit human rhinovirus replication.

Small molecules that exhibit broad-spectrum enteroviral inhibitory activity by targeting viral replication proteins are highly desired in antiviral drug discovery studies. To discover new human rhinovirus (hRV) inhibitors, we performed a high-throughput screening of 100,000 compounds from the Korea Chemical Bank library. This search led to identification of two phosphatidylinositol-4-kinase IIIß (PI4KIIIß) inhibitors having the pyrazolo-pyrimidine core structure, which display moderate anti-rhinoviral activity along with mild cytotoxicity. The results of a study aimed at optimizing the activity of the hit compounds showed that the pyrazolo-pyrimidine derivative 6f exhibits the highest activity (EC50 = 0.044, 0.066, and 0.083 µM for hRV-B14, hRV-A16, and hRV-A21, respectively) and moderate toxicity (CC50 = 31.38 µM). Furthermore, 6f has broad-spectrum activities against various hRVs, coxsackieviruses and other enteroviruses, such as EV-A71, EV-D68. An assessment of kinase inhibition potencies demonstrated that 6f possesses a high and selective kinase inhibition activity against PI4KIIIß (IC50 value of 0.057 µM) and not against PI4KIIIα (>10 µM). Moreover, 6f exhibits modest hepatic stability (46.9 and 55.3 % remaining after 30 min in mouse and human liver microsomes, respectively). Finally, an in vivo study demonstrated that 6f possesses a desirable pharmacokinetic profile reflected in low systemic clearance (0.48 L∙h-1 kg-1) and modest oral bioavailability (52.4 %). Hence, 6f (KR-26549) appears to be an ideal lead for the development of new antiviral drugs.

PI4KIIIß-Mediated Phosphoinositides Metabolism Regulates Function of the VTA Dopaminergic Neurons and Depression-Like Behavior.

Phosphoinositides, including phosphatidylinositol-4,5-bisphosphate (PIP2), play a crucial role in controlling key cellular functions such as membrane and vesicle trafficking, ion channel, and transporter activity. Phosphatidylinositol 4-kinases (PI4K) are essential enzymes in regulating the turnover of phosphoinositides. However, the functional role of PI4Ks and mediated phosphoinositide metabolism in the central nervous system has not been fully revealed. In this study, we demonstrated that PI4KIIIß, one of the four members of PI4Ks, is an important regulator of VTA dopaminergic neuronal activity and related depression-like behavior of mice by controlling phosphoinositide turnover. Our findings provide new insights into possible mechanisms and potential drug targets for neuropsychiatric diseases, including depression. Both sexes were studied in basic behavior tests, but only male mice could be used in the social defeat depression model.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles.

The formation of extracellular vesicles (EVs) is induced by the sphingolipid ceramide. How this pathway is regulated is not entirely understood. Here, we report that the ceramide transport protein (CERT) mediates a non-vesicular transport of ceramide between the endoplasmic reticulum (ER) and the multivesicular endosome at contact sites. The process depends on the interaction of CERT's PH domain with PI4P generated by PI4KIIα at endosomes. Furthermore, a complex is formed between the START domain of CERT, which carries ceramide, and the Tsg101 protein, which is part of the endosomal sorting complex required for transport (ESCRT-I). Inhibition of ceramide biosynthesis reduces CERT-Tsg101 complex formation. Overexpression of CERT increases EV secretion while its inhibition reduces EV formation and the concentration of ceramides and sphingomyelins in EVs. In conclusion, we discovered a function of CERT in regulating the sphingolipid composition and biogenesis of EVs, which links ceramide to the ESCRT-dependent pathway.

Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication.

Picornaviruses are a diverse and major cause of human disease, and their genomes replicate with intracellular membranes. The functionality of these replication organelles depends on the activities of both viral nonstructural proteins and co-opted host proteins. The mechanism by which viral-host interactions generate viral replication organelles and regulate viral RNA synthesis is unclear. To elucidate this mechanism, enterovirus A71 (EV-A71) was used here as a virus model to investigate how these replication organelles are formed and to identify the cellular components that are critical in this process. An immunoprecipitation assay was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify 172 cellular proteins and four viral proteins associating with viral 3A protein. Secretory carrier membrane protein 3 (SCAMP3) was one of the host proteins we selected for further investigation. Here, we demonstrate by immunoprecipitation assay that SCAMP3 associates with 3A protein and colocalizes with 3A protein during virus infection. SCAMP3 knockdown or knockout in infected cells decreases synthesis of EV-A71 viral RNA, viral proteins, and viral growth. Furthermore, the viral 3A protein associates with SCAMP3 and phosphatidylinositol-4-kinase type III ß (PI4KIIIß) as shown by immunoprecipitation assay and colocalizes to the replication complex. Upon infection of cells with a SCAMP3 knockout construct, PI4KIIIß and phosphatidylinositol-4-phosphate (PI4P) colocalization with EV-A71 3A protein decreases; viral RNA synthesis also decreases. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication. The 3A and SCAMP3 interaction is also important for the replication of coxsackievirus B3 (CVB3). SCAMP3 also associates with 3A protein of CVB3 and enhances viral replication but does not regulate dengue virus 2 (DENV2) replication. Taken together, the results suggest that enterovirus 3A protein, SCAMP3, PI4KIIIß, and PI4P form a replication complex and positively regulate enterovirus replication. IMPORTANCE Virus-host interaction plays an important role in viral replication. 3A protein of enterovirus A71 (EV-A71) recruits other viral and host factors to form a replication complex, which is important for viral replication. In this investigation, we utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that secretory carrier membrane protein 3 (SCAMP3) is a novel host factor that associates with enterovirus 3A protein, phosphatidylinositol-4-kinase type III ß (PI4KIIIß), and phosphatidylinositol-4-phosphate (PI4P) to form a replication complex and positively regulates viral replication. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication.

Novel phosphatidylinositol 4-kinases III beta (PI4KIIIß) 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 IIIß (PI4KIIIß) 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 PI4KIIIß, demonstrating the importance of both acceptor and donor hydrogen bonding groups for forming interactions with binding site residues Val598 and Lys549.

ATG9A supplies PtdIns4P to the autophagosome initiation site.

The identity of the platform supporting the initiation and formation of the nascent autophagosome, the phagophore, is not fully understood. Nucleation and expansion of the phagophore membrane requires a coordinated flux or activation of specific proteins and membrane lipids at the initiation site. The transmembrane protein ATG9A is essential for macroautophagy/autophagy and proposed to be an initiator of the phagophore by directing or facilitating the delivery of proteins and lipids to the initiation site. Upon amino acid starvation, ATG9A-containing vesicles are formed from the Golgi complex and endosomal compartments and translocate to the initiation site. Unravelling the complement of proteins and lipids brought by ATG9A vesicles to the forming autophagosome is essential to further understand the initiation of autophagy.

Modeling sphingomyelin synthase 1 driven reaction at the Golgi apparatus can explain data by inclusion of a positive feedback mechanism.

Here we present a minimal mathematical model for the sphingomyelin synthase 1 (SMS1) driven conversion of ceramide to sphingomyelin based on chemical reaction kinetics. We demonstrate via mathematical analysis that this model is not able to qualitatively reproduce experimental measurements on lipid compositions after altering SMS1 activity. We prove that a positive feedback mechanism from the products to the reactants of the reaction is one possible model extension to explain these specific experimental data. The proposed mechanism in fact exists in vivo via protein kinase D and the ceramide transfer protein CERT. The model is further evaluated by additional observations from the literature.

Identification of a series of compounds with potent antiviral activity for the treatment of enterovirus infections.

Rhinovirus (genus enterovirus) infections are responsible for many of the severe exacerbations of asthma and chronic obstructive pulmonary disease. Other members of the genus can cause life-threatening acute neurological infections. There is currently no antiviral drug approved for the treatment of such infections. We have identified a series of potent, broad-spectrum antiviral compounds that inhibit the replication of the human rhinovirus, Coxsackie virus, poliovirus, and enterovirus-71. The mechanism of action of the compounds has been established as inhibition of a lipid kinase, PI4KIIIß. Inhibition of hepatitis C replication in a replicon assay correlated with enterovirus inhibition.