Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development.

Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq.

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.

The CDK9 tail determines the reaction pathway of positive transcription elongation factor b.

CDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.

Nongenomic STAT5-dependent effects on Golgi apparatus and endoplasmic reticulum structure and function.

We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.

Halogen bonds form the basis for selective P-TEFb inhibition by DRB.

Cdk9, the kinase of the positive transcription elongation factor b, is required for processive transcription elongation by RNA polymerase II. Cdk9 inhibition contributes to the anticancer activity of many Cdk inhibitors under clinical investigation and hence there is interest in selective Cdk9 inhibitors. DRB (5,6-dichlorobenzimidazone-1-ß-D-ribofuranoside) is a commonly used reagent for Cdk9 inhibition in cell biology studies. The crystal structures of Cdk9 and Cdk2 in complex with DRB reported here describe the molecular basis for the DRB selectivity toward Cdk9. The DRB chlorine atoms form halogen bonds that are specific for the Cdk9 kinase hinge region. Kinetic and thermodynamic experiments validate the structural findings and implicate the C-terminal residues of Cdk9 in contributing to the affinity for DRB. These results open the possibility to exploit halogen atoms in inhibitor design to specifically target Cdk9.

Insights from soft X-rays: the chlorine and sulfur sub-structures of a CK2alpha/DRB complex.

The diffraction pattern of a protein crystal is normally a product of the interference of electromagnetic waves scattered by electrons of the crystalline sample. The diffraction pattern undergoes systematic changes in case additionally X-ray absorption occurs, meaning if the wavelength of the primary X-ray beam is relatively close to the absorption edge of selected elements of the sample. The resulting effects are summarized as "anomalous dispersion" and can be always observed with "soft" X-rays (wavelength around 2 A) since they match the absorption edges of sulfur and chlorine. A particularly useful application of this phenomenon is the experimental detection of the sub-structures of the anomalous scatterers in protein crystals. We demonstrate this here with a crystal of a C-terminally truncated variant of human CK2alpha to which two molecules of the inhibitor 5,6-dichloro-1-beta-D-ribo-furanosyl-benzimidazole (DRB) are bound. The structure of this co-crystal has been solved recently. For this study we measured an additional diffraction data set at a wavelength of 2 A which showed strong anomalous dispersion effects. On the basis of these effects we detected all sulfur atoms of the protein, the two liganded DRB molecules and a total of 16 additional chloride ions some of them emerging at positions filled with water molecules in previous structure determinations. A number of chloride ions are bound to structural and functional important locations fitting to the constitutive activity and the acidophilic substrate specificity of the enzyme.

Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening.

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

The identification of phosphorylation sites of pp32 and biochemical purification of a cellular pp32-kinase.

The versatile phosphoprotein pp32 is involved in important physiological processes, including cell proliferation, apoptosis, mRNA transport, and transcription. We have previously reported that pp32, through histone masking, inhibits histone acetylation and transcriptional activation by histone acetyltransferases. However, how pp32 itself is regulated remained largely unknown. Although pp32 is a phosphoprotein, neither the phosphorylation sites nor the cellular kinase has been identified. In this report, utilizing an in vitro kinase assay and a biochemical purification scheme, we identify casein kinase II as a cellular pp32-kinase. Our deletion and site-specific mutagenesis studies identify serines 158 and 204 as the sites of phosphorylation. Generation and utilization of antibodies with higher affinity for phospho-pp32 demonstrate that pp32 is indeed phosphorylated in vivo at these two sites. Mutagenesis studies on pp32 suggest a role for serines 158 and 204 in its function. The identification of the pp32 kinase and the sites of pp32 phosphorylation as well as the generation of antibodies with higher affinity for phospho-pp32 should now provide key information and tools for future studies on pp32 regulation.

Casein kinase II-mediated phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation.

Alpha-synuclein is a phosphoprotein that accumulates as a major component of Lewy bodies in the brains of patients with Parkinson disease. Synphilin-1, which is also present in Lewy bodies, binds with alpha-synuclein and forms cytoplasmic inclusions in transfected cells. Yet the molecular determinants of this protein-protein interaction are unknown. Here we report that casein kinase II (CKII) phosphorylates synphilin-1 and that the beta subunit of this enzyme complex binds to synphilin-1. Additionally, both CKII alpha and beta subunits are present within cytoplasmic inclusions in cells that overexpress synphilin-1. Notably, the interaction between synphilin-1 and alpha-synuclein is markedly dependent on phosphorylation. Inhibition of CKII activity by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocks the binding between these two proteins and significantly reduces the percentage of cells that contain eosinophilic cytoplasmic inclusions. Mutation of the major CKII phosphorylation site in alpha-synuclein (S129A) has no significant impact on the binding between alpha-synuclein and synphilin-1 or on the formation of synphilin-1/alpha-synuclein-positive inclusions. These data suggest that the CKII-mediated phosphorylation of synphilin-1 rather than that of alpha-synuclein is critical in modulating their tendency to aggregate into inclusions. These observations collectively indicate that a ubiquitous post-translational modification such as phosphorylation can regulate inclusion body formation in the context of alpha-synuclein and synphilin-1 interaction.

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.