Pliability in the m6A-Binding Region Extends Druggability of YTH Domains.

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6-Methyladenosine-Modified RNAs.

YTHDF proteins bind the N 6-methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.

Identification of a BAZ2A-Bromodomain Hit Compound by Fragment Growing.

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 µM and a good selectivity over the paralog BAZ2B bromodomain.

Identification of a BAZ2A Bromodomain Hit Compound by Fragment Joining.

The bromodomains of BAZ2A and BAZ2B (bromodomain adjacent to zinc finger domain proteins 2) are among the most hard to drug of the 61 human bromodomains. While little is known about the role of BAZ2B, there is strong evidence for the opportunity of targeting BAZ2A in various cancers. Here, a benzimidazole-triazole fragment that binds to the BAZ2A acetyl lysine pocket was identified by a molecular docking campaign and validated by competitive binding assays and X-ray crystallography. Another ligand was observed in close proximity by soaking experiments using the BAZ2A bromodomain preincubated with the benzimidazole-triazole fragment. The crystal structure of BAZ2A with the two ligands was employed to design a few benzimidazole-triazole derivatives with increased affinity. We also present the engineering of a BAZ2A bromodomain mutant for consistent, high-resolution crystallographic studies.

A novel class of selective CK2 inhibitors targeting its open hinge conformation.

Protein kinase CK2 sustains cancer growth, especially in hematological malignancies. Its inhibitor SRPIN803, based on a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold, showed notable specificity. Our synthesis of the initially proposed SRPIN803 resulted in its constitutional isomer SRPIN803-revised, where the 2-cyano-2-propenamide group does not cyclise and fuse to the thiadiazole ring. Its crystallographic structure in complex with CK2α identifies the structural determinants of the reported specificity. SRPIN803-revised explores the CK2 open hinge conformation, extremely rare among kinases, also interacting with side chains from this region. Its optimization lead to the more potent compound 4, which inhibits endocellular CK2, significantly affects viability of tumour cells and shows remarkable selectivity on a panel of 320 kinases.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

Biochemical and cellular mechanism of protein kinase CK2 inhibition by deceptive curcumin.

Protein kinase CK2 is an antiapoptotic cancer-sustaining protein. Curcumin, reported previously as a CK2 inhibitor, is too bulky to be accommodated in the CK2 active site and rapidly degrades in solution generating various ATP-mimetic inhibitors; with a detailed comparative analysis, by means of both protein crystallography and enzymatic inhibition, ferulic acid was identified as the principal curcumin degradation product responsible for CK2 inhibition. The other curcumin derivatives vanillin, feruloylmethane and coniferyl aldehyde are weaker CK2 inhibitors. The high instability of curcumin in standard buffered solutions flags this compound, which is included in many commercial libraries, as a possible source of misleading interpretations, as was the case for CK2. Ferulic acid does not show any cytotoxicity and any inhibition of cellular CK2, due to its poor cellular permeability. However, curcumin acts as a prodrug in the cellular context, by generating its degradation products inside the treated cells, thus rescuing CK2 inhibition and consequently inducing cell death. Through the intracellular release of its degradation products, curcumin is expected to affect various target families; here, we identify the first bromodomain of BRD4 as a new target for those compounds. DATABASE: Structural data are available in the PDB database under the accession numbers 6HOP (CK2α/curcumin), 6HOQ (CK2α/ferulic acid), 6HOR (CK2α/feruloylmethane), 6HOT (CK2α/ferulic aldehyde), 6HOU (CK2α/vanillin) and 6HOV (BRD4/ferulic acid).

Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell-Cell Adhesion.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell-cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell-cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell-cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

Structural Analysis of Small-Molecule Binding to the BAZ2A and BAZ2B Bromodomains.

The bromodomain-containing protein BAZ2A is a validated target in prostate cancer research, whereas the function of its paralogue BAZ2B is still undefined. The bromodomains of BAZ2A and BAZ2B have a similar binding site for their natural ligand, the acetylated lysine side chain. Here, we present an analysis of the binding modes of eight compounds belonging to three distinct chemical classes. For all compounds, the moiety mimicking the natural ligand engages in essentially identical interactions in the BAZ2A and BAZ2B bromodomains. In contrast, the rest of the molecule is partially solvent-exposed and adopts different orientations with different interactions in the two bromodomains. Some of these differences could be exploited for designing inhibitors with selectivity within the BAZ2 bromodomain subfamily.

Discovery of Inhibitors of Four Bromodomains by Fragment-Anchored Ligand Docking.

The high-throughput docking protocol called ALTA-VS (anchor-based library tailoring approach for virtual screening) was developed in 2005 for the efficient in silico screening of large libraries of compounds by preselection of only those molecules that have optimal fragments (anchors) for the protein target. Here we present an updated version of ALTA-VS with a broader range of potential applications. The evaluation of binding energy makes use of a classical force field with implicit solvent in the continuum dielectric approximation. In about 2 days per protein target on a 96-core compute cluster (equipped with Xeon E3-1280 quad core processors at 2.5 GHz), the screening of a library of nearly 77 000 diverse molecules with the updated ALTA-VS protocol has resulted in the identification of 19, 3, 3, and 2 µM inhibitors of the human bromodomains ATAD2, BAZ2B, BRD4(1), and CREBBP, respectively. The success ratio (i.e., number of actives in a competition binding assay in vitro divided by the number of compounds tested) ranges from 8% to 13% in dose-response measurements. The poses predicted by fragment-based docking for the three ligands of the BAZ2B bromodomain were confirmed by protein X-ray crystallography.

Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members.

Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels.

Crystal Structure of Human E-Cadherin-EC1EC2 in Complex with a Peptidomimetic Competitive Inhibitor of Cadherin Homophilic Interaction.

Cadherins are transmembrane cell adhesion proteins whose aberrant expression often correlates with cancer development and proliferation. We report the crystal structure of an E-cadherin extracellular fragment in complex with a peptidomimetic compound that was previously shown to partially inhibit cadherin homophilic adhesion. The structure reveals an unexpected binding mode and allows the identification of a druggable cadherin interface, thus paving the way to a future structure-guided design of cell adhesion inhibitors against cadherin-expressing solid tumors.

Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate-bound complex.

Amadoriases, also known as fructosyl amine oxidases (FAOX), are enzymes that catalyze the de-glycosylation of fructosyl amino acids. As such, they are excellent candidates for the development of enzyme-based diagnostic and therapeutic tools against age- and diabetes-induced protein glycation. However, mostly because of the lack of a complete structural characterization of the different members of the family, the molecular bases of their substrate specificity have yet to be fully understood. The high resolution crystal structures of the free and the substrate-bound form of Amadoriase I shown herein allow for the identification of key structural features that account for the diverse substrate specificity shown by this class of enzymes. This is of particular importance in the context of the rather limited and partially incomplete structural information that has so far been available in the literature on the members of the FAOX family. Moreover, using molecular dynamics simulations, we describe the tunnel conformation and the free energy profile experienced by the ligand in going from bulk water to the catalytic cavity, showing the presence of four gating helices/loops, followed by an "L-shaped" narrow cavity. In summary, the tridimensional architecture of Amadoriase I presented herein provides a reference structural framework for the design of novel enzymes for diabetes monitoring and protein deglycation. Proteins 2016; 84:744-758. © 2016 Wiley Periodicals, Inc.

The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway.

Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a further snapshot in the yet-to-be-achieved complete description of the highly dynamic cadherin dimerization pathway. Moreover, this is the first multidomain cadherin fragment to be crystallized and structurally characterized in its closed conformation that does not carry any extra N-terminal residues before the naturally occurring aspartic acid at position 1. Finally, two clear alternate conformations are observed for the critical Trp2 residue, suggestive of a transient, metastable state. The P-cadherin structure and packing arrangement shown here provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway.