Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

Accelerated Structural Prediction of Flexible Protein-Ligand Complexes: The SLICE Method.

Using existing and academically available software, we present a new method for the structural prediction of binding events containing flexible protein targets. SLICE (Selective Ligand-Induced Conformational Ensemble) combines opportunistic stochastic jumps of ligand position with standard molecular dynamics to model the induced-fit binding of ligands starting with unbound host coordinates. To induce the structural adaptations of the complex at the binding site, conformational jumps in ligand position are selected in SLICE from structures generated by a docking software. Multiple binding trajectories from the docking set are followed using molecular dynamics for a set time to relax the host structure and generate new host poses. A new configurational jump is made on the set of newly generated host poses. The process is then repeated. The method was implemented with AutoDock Vina as the docking method, Vina scores as the selection criterion, and Amber code for molecular dynamics and applied to several test systems. A system consisting of Chromobox protein homologue 8 (CBX8) and its small peptide ligand, H3K9Me3, for which the final (bound) configuration is known, is used for verifying SLICE in the present setup. The setup was also applied to several nonpeptide molecules on known difficult flexible targets exhibiting a large disparity between apo and holo host states. The SLICE simulations provide a promising approach to generate induced-fit configurations compared to existing long (microsecond) classical and accelerated dynamics approaches in all the test systems considered here. However, further optimization of SLICE parameters is required for replicating crystal structure coordinates for some systems. We discuss in the following pages the various SLICE parameters and how they can be optimized for the system at hand.

Structure of the Sac3 RNA-binding M-region in the Saccharomyces cerevisiae TREX-2 complex.

Transcription-export complex 2 (TREX-2, or THSC) facilitates localization of actively transcribing genes such as GAL1 to the nuclear periphery, contributes to the generation of export-competent mRNPs and influences gene expression through interactions with Mediator. TREX-2 is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31 and Sus1 bind and consists of three modules: the N-region (Sac3∼1-100), which binds mRNA export factor Mex67:Mtr2; the M-region, in which Thp1 and Sem1 bind to Sac3∼100-550; and the CID region in which Cdc31 and two Sus1 chains bind to Sac3∼720-805. Although the M-region of Sac3 was originally thought to encompass residues ∼250-550, we report here the 2.3Å resolution crystal structure of a complex containing Sac3 residues 60-550 that indicates that the TPR-like repeats of the M-region extend to residue 137 and that residues 90-125 form a novel loop that links Sac3 to Thp1. These new structural elements are important for growth and mRNA export in vivo. Although deleting Sac3 residues 1-90 produced a wild-type phenotype, deletion of the loop as well generated growth defects at 37°C, whereas the deletion of residues 1-250 impaired mRNA export and also generated longer lag times when glucose or raffinose was replaced by galactose as the carbon source.

Structural basis for the dimerization of Nab2 generated by RNA binding provides insight into its contribution to both poly(A) tail length determination and transcript compaction in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 5­7 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 5­7 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ß-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.

Identification of novel non-canonical RNA-binding sites in Gemin5 involved in internal initiation of translation.

Ribonucleic acid (RNA)-binding proteins are key players of gene expression control. We have shown that Gemin5 interacts with internal ribosome entry site (IRES) elements and modulates initiation of translation. However, little is known about the RNA-binding sites of this protein. Here we show that the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites, encompassing amino acids 1297-1412 (RBS1) and 1383-1508 (RBS2). While RBS1 exhibits greater affinity for RNA than RBS2, it does not affect IRES-dependent translation in G5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide G51383-1508, bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. A comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to that of the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of the protein being able to repress internal initiation of translation.

End-to-end continuous manufacturing of pharmaceuticals: integrated synthesis, purification, and final dosage formation.

A series of tubes: The continuous manufacture of a finished drug product starting from chemical intermediates is reported. The continuous pilot-scale plant used a novel route that incorporated many advantages of continuous-flow processes to produce active pharmaceutical ingredients and the drug product in one integrated system.

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.

Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology.

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

Molecular dynamics study of the interactions of ice inhibitors on the ice {001} surface.

The interactions of antifreeze protein (AFP) type I, antifreeze glycoproteins, polyvinyl pyrrolidone (PVP), and various amino acids with ice are investigated using Cerius2, a molecular modelling tool. Binding energies of these additives to a major ice crystal face {001} are computed. Binding energy comparison of threonine molecules (by themselves) and as threonine residues within AFP type I demonstrate their role in improving AFP's binding ability to the ice crystal face. The shifts in onset points of ice crystallization with AFP type I, PVP, and amino acids are measured using differential scanning calorimetry. These values when correlated with their respective binding energies reveal a direct proportionality and demonstrate AFP's effectiveness in inhibiting growth and nucleation of ice, over amino acids.

Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells.

Intracellular plasmid is rapidly incorporated into the nucleus of HeLa cells following cationic lipoplex transfection. CV1 cells are less effective in translocating plasmid to the nucleus and also express less transgene than HeLa cells. Cultured HeLa and CV1 cells and corresponding isolated nuclei were analyzed after transfection of a Cy3-labeled pGreenLantern plasmid (Cy3-pGL). Flow cytometry was used to measure both plasmid delivery and transgene expression from the plasmid encoding a CMV promoter-driven green fluorescent protein. During transfection, HeLa cells rapidly incorporated the plasmid, reaching a maximum of 80% Cy3-pGL positive cells 8 h posttransfection. The average Cy3-pGL-positive HeLa cell contained approximately 2470 plasmid copies. Forty-eight percent of the nuclei isolated from the transfected HeLa cells were positive for the plasmid marker after 8 h. In contrast to HeLa cells, fewer CV1 cells and CV1 nuclei incorporated plasmid DNA with peak transfection occurring after 12 h for 36% of the cells and after 8 h for 12% of the nuclei. However, the average Cy3-pGL-positive CV1 cell did not have a significantly different number of total cellular plasmid copies than the average positive HeLa cell. CV1 nuclei, however, had half as much nuclear associated plasmid as HeLa nuclei. HeLa cells are more efficient than CV1 cells at transporting plasmid from the cytoplasm to the nucleus. This study demonstrates the use of a novel quantitative method to study plasmid transport from the cytoplasm to the nucleus and the effect on transgene expression.