Different applications and differentiated libraries for crystallographic fragment screening.

Macromolecular X-ray crystallography allows detection and characterisation of the binding of small, low-affinity chemical fragments. Here we review the utility of fragment screening for drug discovery, its potential for use in discovery science, as well as some of the distinct types of fragments that have been compiled into libraries.

Cryo-EM structure of the CDK2-cyclin A-CDC25A complex.

The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex.

Developing Parametric and Nonparametric Models for Model-Informed Precision Dosing: A Quality Improvement Effort in Vancomycin for Patients With Obesity.

Background: Both parametric and nonparametric methods have been proposed to support model-informed precision dosing (MIPD). However, which approach leads to better models remains uncertain. Using open-source software, these 2 statistical approaches for model development were compared using the pharmacokinetics of vancomycin in a challenging subpopulation of class 3 obesity.

Crystallographic fragment screening in academic cancer drug discovery.

Fragment-based drug discovery (FBDD) has brought several drugs to the clinic, notably to target proteins once considered to be challenging, or even undruggable. Screening in FBDD relies upon observing and/or measuring weak (millimolar-scale) binding events using biophysical techniques or crystallographic fragment screening. This latter structural approach provides no information about binding affinity but can reveal binding mode and atomic detail on protein-fragment interactions to accelerate hit-to-lead development.

Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1.

p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCF (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure.

Cyclin A and Cks1 promote kinase consensus switching to non-proline-directed CDK1 phosphorylation.

Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation.

Emerging approaches to CDK inhibitor development, a structural perspective.

Aberrant activity of the cyclin-dependent kinase family is frequently noted in a number of diseases identifying them as potential targets for drug development. However, current CDK inhibitors lack specificity owing to the high sequence and structural conservation of the ATP binding cleft across family members, highlighting the necessity of finding novel modes of CDK inhibition. The wealth of structural information regarding CDK assemblies and inhibitor complexes derived from X-ray crystallographic studies has been recently complemented through the use of cryo-electron microscopy.

Mapping Ligand Interactions of Bromodomains BRD4 and ATAD2 with FragLites and PepLites─Halogenated Probes of Druglike and Peptide-like Molecular Interactions.

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions.

Cell division drives DNA methylation loss in late-replicating domains in primary human cells.

DNA methylation undergoes dramatic age-related changes, first described more than four decades ago. Loss of DNA methylation within partially methylated domains (PMDs), late-replicating regions of the genome attached to the nuclear lamina, advances with age in normal tissues, and is further exacerbated in cancer. We present here experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication.

Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor.

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent and inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Near-IR Charge-Transfer Emission at 77 K and Density Functional Theory Modeling of Ruthenium(II)-Dipyrrinato Chromophores: High Phosphorescence Efficiency of the Emitting State Related to Spin-Orbit Coupling Mediation of Intensity from Numerous Low-Energy Singlet Excited States.

Efficient charge-transfer (CT) phosphorescence in the near-IR (NIR) spectral region is reported for four substituted Ru-(R-dipyrrinato) complexes, [Ru(bpy)(R-dipy)](PF), where bpy is 2,2'-bipyridine and the substituent R is phenyl (ph), 2,4,6-trimethylphenyl, 4-carboxyphenyl (HOOC-ph), or 4-pyridinyl. The experimentally determined phosphorescence efficiency, ι = /(ν) (where and ν are the phosphorescence rate constant and the phosphorescence frequency, respectively), of the [Ru(bpy)(R-dipy)] complexes was approximately double that of [Ru(bpy)(Am)] complexes (Am = ammine ligand) in the NIR region. Density functional theory (DFT) modeling indicated two strikingly different electronic configurations of the triplet emitting state (T) in the two types of complexes.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCF ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1.

Chatterboxes: the structural and functional diversity of cyclins.

Proteins of the cyclin family have divergent sequences and execute diverse roles within the cell while sharing a common fold: the cyclin box domain. Structural studies of cyclins have played a key role in our characterization and understanding of cellular processes that they control, though to date only ten of the 29 CDK-activating cyclins have been structurally characterized by X-ray crystallography or cryo-electron microscopy with or without their cognate kinases. In this review, we survey the available structures of human cyclins, highlighting their molecular features in the context of their cellular roles.

Low-Temperature Spectra and Density Functional Theory Modeling of Ru(II)-Bipyridine Complexes with Cyclometalated Ancillary Ligands: The Excited State Spin-Orbit Coupling Origin of Variations in Emission Efficiencies.

The 77 K emission spectra of cyclometalated ruthenium(II)-2,2'-bipyridine (CM-Ru-bpy) chromophores are very similar to those of related Ru-bpy complexes with am(m)ine or diimmine ancillary ligands, and density functional theory (DFT) modeling confirms that the lowest energy triplet metal to ligand charge transfer (MLCT) excited states of CM-Ru-bpy and related Ru-bpy complexes have very similar electronic configurations. However, the phosphorescence decay efficiencies of CM-Ru-bpy excited states are about twice those of the conventional Ru-bpy analogues. In contrast to the similar MLCT excited state electronic configurations of the two classes of complexes, the CM-Ru-bpy chromophores have much broader visible region MLCT absorptions resulting from several overlapping transitions, even at 87 K.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α.

A Retrospective Study of Early vs Delayed Home Dose Basal Insulin in the Acute Management of Diabetic Ketoacidosis.

Background: Insulin via continuous intravenous infusion (ICII) is a standard of care for treating patients with diabetic ketoacidosis (DKA). Once DKA is resolved, ICII is transitioned to subcutaneous therapy. However, recent guidelines recommend continuation of home dose subcutaneous basal insulin (HDBI) in patients with DKA.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites.

Articular cartilage and sternal fibrocartilage respond differently to extended microgravity.

The effects of spaceflight on cartilaginous structure are largely unknown. To address this deficiency, articular cartilage (AC) and sternal cartilage (SC) from mice exposed to 30 days of microgravity on the BION-M1 craft were investigated for pathological changes. The flight AC showed some evidence of degradation at the tissue level with loss of proteoglycan staining and a reduction in mRNA expression of mechano-responsive and structural cartilage matrix proteins compared to non-flight controls.

Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition.

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability.

Chemical Scavenging Yields for Short-Lived Products from the Visible Light Photoionization of the Tris(bipyridine)ruthenium(II) Triplet Metal-to-Ligand Charge-Transfer Excited State.

The rate of visible light photoionization of the tris(bipyridine)ruthenium(II) triplet metal-to-ligand charge-transfer excited state (MLCT) is very strongly dependent on the acid concentration in aqueous solution, and the pattern of this dependence is similar to that reported for the photoionization of iodide. With 405 nm visible irradiation of MLCT, less than 15% of the photoionized products appear as free solvated electrons in bulk solution, while more than 75% of the photoproducts appear to be solvent-separated, (oxidized substrate)-electron ion pairs that efficiently recombine with the photo-oxidized complex in the absence of an electron scavenger. The quantum yield of free solvated electrons generated by these 405 nm irradiations is approximately 0.