A Tiny Pocket Packs a Punch: Leveraging Pyridones for the Discovery of CNS-Penetrant Aza-indazole IRAK4 Inhibitors.

We herein report the discovery, synthesis, and evolution of a series of indazoles and azaindazoles as CNS-penetrant IRAK4 inhibitors. Described is the use of structure-based and property-based drug design strategically leveraged to guide the property profile of a key series into a favorable property space while maintaining potency and selectivity. Our rationale that led toward functionalities with potency improvements, CNS-penetration, solubility, and favorable drug-like properties is portrayed.

Discovery of BIO-8169─A Highly Potent, Selective, and Brain-Penetrant IRAK4 Inhibitor for the Treatment of Neuroinflammation.

Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 () suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability.

The Discovery of 7-Isopropoxy-2-(1-methyl-2-oxabicyclo[2.1.1]hexan-4-yl)--(6-methylpyrazolo[1,5-]pyrimidin-3-yl)imidazo[1,2-]pyrimidine-6-carboxamide (BIO-7488), a Potent, Selective, and CNS-Penetrant IRAK4 Inhibitor for the Treatment of Ischemic Stroke.

Interleukin receptor-associated kinase 4 (IRAK4) is a key node of signaling within the innate immune system that regulates the production of inflammatory cytokines and chemokines. The presence of amage-ssociated olecular patterns (DAMPs) after tissue damage such as stroke or traumatic brain injury (TBI) initiates signaling through the IRAK4 pathway that can lead to a feed-forward inflammatory loop that can ultimately hinder patient recovery. Herein, we describe the first potent, selective, and CNS-penetrant IRAK4 inhibitors for the treatment of neuroinflammation.

Elucidation of the GSK3α Structure Informs the Design of Novel, Paralog-Selective Inhibitors.

Glycogen synthase kinase 3 (GSK3) remains a therapeutic target of interest for diverse clinical indications. However, one hurdle in the development of small molecule GSK3 inhibitors has been safety concerns related to pan-inhibition of both GSK3 paralogs, leading to activation of the Wnt/β-catenin pathway and potential for aberrant cell proliferation. Development of GSK3α or GSK3β paralog-selective inhibitors that could offer an improved safety profile has been reported but further advancement has been hampered by the lack of structural information for GSK3α.

Discovery of Phospholipase D Inhibitors with Improved Drug-like Properties and Central Nervous System Penetrance.

Phospholipase D (PLD) is a phospholipase enzyme responsible for hydrolyzing phosphatidylcholine into the lipid signaling molecule, phosphatidic acid, and choline. From a therapeutic perspective, PLD has been implicated in human cancer progression as well as a target for neurodegenerative diseases, including Alzheimer's. Moreover, knockdown of PLD rescues the ALS phenotype in multiple models of ALS (amyotrophic lateral sclerosis) and displays modest motor benefits in an SOD1 ALS mouse model.

Discovery of Potent Selective Nonzinc Binding Autotaxin Inhibitor BIO-32546.

Autotaxin (ATX) is a lysophospholipase D that is the main enzyme responsible for generating LPA in body fluids. Although ATX was isolated from a conditioned medium of melanoma cells, later it was discovered to play a critical role in vascular and neuronal development. ATX has also been implicated in primary brain tumor, fibrosis, and rheumatoid arthritis, as well as neurological diseases such as multiple sclerosis, Alzheimer's disease, and neuropathic pain.

Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1 (TTBK1) Inhibitors that Lower Tau Phosphorylation In Vivo.

Structural analysis of the known NIK inhibitor bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by (cell IC: 571 nM). Systematic optimization of this series of analogs led to the discovery of , a potent (cell IC: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat K: 0.32) for in vivo proof of pharmacology studies.

Mechanisms of Regulation and Diverse Activities of Tau-Tubulin Kinase (TTBK) Isoforms.

Tau-tubulin kinase 1 (TTBK1) is a CNS-specific, kinase that has been implicated in the pathological phosphorylation of tau in Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD). TTBK1 is a challenging therapeutic target because it shares a highly conserved catalytic domain with its homolog, TTBK2, a ubiquitously expressed kinase genetically linked to the disease spinocerebellar ataxia type 11. The present study attempts to elucidate the functional distinctions between the TTBK isoforms and increase our understanding of them as distinct targets for the treatment of neurodegenerative disease.

The crystal structure of the catalytic domain of tau tubulin kinase 2 in complex with a small-molecule inhibitor.

Tau proteins play an important role in the proper assembly and function of neurons. Hyperphosphorylation of tau by kinases such as tau tubulin kinase (TTBK) has been hypothesized to cause the aggregation of tau and the formation of neurofibrillary tangles (NFTs) that lead to the destabilization of microtubules, thereby contributing to neurodegenerative diseases such as Alzheimer's disease (AD). There are two TTBK isoforms with highly homologous catalytic sites but with distinct tissue distributions, tau phosphorylation patterns and loss-of-function effects.

Human PLD structures enable drug design and characterization of isoenzyme selectivity.

Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences.

Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution.

The evolution of cell-adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin.

Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand.

Background: The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent.

Molecular functions of the TLE tetramerization domain in Wnt target gene repression.

Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of β-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs.

Reactive oxygen species regulate nucleostemin oligomerization and protein degradation.

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers.

The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif.

Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells.

The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure.

Histone methylation regulates chromatin function dependent on the site and degree of the modification. In addition to creating binding sites for proteins, methylated lysine residues are likely to influence chromatin structure directly. Here we present crystal structures of nucleosomes reconstituted with methylated histones and investigate the folding behavior of resulting arrays.

A charged and contoured surface on the nucleosome regulates chromatin compaction.

Local nucleosome-nucleosome interactions in cis drive chromatin folding, whereas interactions in trans lead to fiber-fiber oligomerization. Here we show that peptides derived from the histone H4 tail and Kaposi's sarcoma herpesvirus LANA protein can replace the endogenous H4 tail, resulting in array folding and oligomerization. Neutralization of a LANA binding site on the histone surface enhanced rather than abolished nucleosome-nucleosome interactions.

Kaposi's sarcoma-associated herpesvirus LANA hitches a ride on the chromosome.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and has an etiologic role in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Survival in rapidly dividing cells depends on a carefully orchestrated chain of events. The viral genome, or episome, must replicate in concert with cellular genetic material, and then efficiently segregate to progeny nuclei.

The nucleosomal surface as a docking station for Kaposi's sarcoma herpesvirus LANA.

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates viral genome attachment to mitotic chromosomes. We find that N-terminal LANA docks onto chromosomes by binding nucleosomes through the folded region of histones H2A-H2B. The same LANA residues were required for both H2A-H2B binding and chromosome association.

Structure and dynamic properties of nucleosome core particles.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. Whereas earlier structural studies portrayed nucleosomes (the basic repeating unit of chromatin) as monolithic and static macromolecular assemblies, we now know that they are highly dynamic and capable of extensive crosstalk with the cellular machinery. Histone variants have evolved to locally alter chromatin structure, whereas histone chaperones and other cellular factors promote histone exchange and chromatin fluidity.

Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding.

Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position.