Discovery of Potent Azetidine-Benzoxazole MerTK Inhibitors with Target Engagement.

Inhibition of the receptor tyrosine kinase MerTK by small molecules has the potential to augment the immune response to tumors. Potent, selective inhibitors with high levels of target engagement are needed to fully evaluate the potential use of MerTK inhibitors as cancer therapeutics. We report the discovery and optimization of a series of pyrazinamide-based type 1.

Discovery of A-910, a Highly Potent and Orally Bioavailable Dual MerTK/Axl-Selective Tyrosine Kinase Inhibitor.

TAM receptor tyrosine kinases have emerged as promising therapeutic targets for cancer treatment due to their roles in both tumor intrinsic survival mechanisms and suppression of antitumor immunity within the tumor microenvironment. Inhibiting MerTK and Axl selectively is believed to hinder cancer cell survival, reverse the protumor myeloid phenotype, and suppress efferocytosis, thereby eliciting an antitumor immune response. In this study, we present the discovery of , a highly potent and selective dual MerTK/Axl inhibitor, achieved through a structure-based medicinal chemistry campaign.

Influence of the ligand-field on EPR parameters of cis- and trans-isomers in Mo systems relevant to molybdenum enzymes: Experimental and density functional theory study.

The electron paramagnetic resonance (EPR) investigation of mononuclear cis- and trans-(L1O)MoOCl complexes [L1OH = bis(3,5-dimethylpyrazolyl)-3-tert-butyl-2-hydroxy-5-methylphenyl)methane] reveals a significant difference in their spin Hamiltonian parameters which reflect different equatorial and axial ligand fields created by the heteroscorpionate donor atoms. Density functional theory (DFT) was used to calculate the values of principal components and relative orientations of the g and A tensors, and the molecular framework in four pairs of isomeric mononuclear oxo‑molybdenum(V) complexes (cis- and trans-(L1O)MoOCl, cis,cis- and cis,trans-(L-NS)MoOCl [L-NSH = N,N'-dimethyl-N,N'-bis(mercaptophenyl)ethylenediamine], cis,cis- and cis,trans-(L-NS)MoO(SCN), and cis- and trans-[(dt)MoO(OMe)] [dtH = 2,3-dimercapto-2-butene]). Scalar relativistic DFT calculations were conducted using three different exchange-correlation functionals.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery.

NMR-based fragment screening and lead discovery accelerated by principal component analysis.

Protein-based NMR spectroscopy has proven to be a very robust method for finding fragment leads to protein targets. However, one limitation of protein-based NMR is that the data acquisition and analysis can be time consuming. In order to streamline the scoring of protein-based NMR fragment screening data and the determination of ligand affinities using 2D NMR experiments we have developed a data analysis workflow based on principal component analysis (PCA) within the TREND NMR Pro software package.

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315.

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1.

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays.

The Role of N-α-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting.

Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting.

Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5.

The rapid spread of the recent Zika virus (ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 of Zika virus (ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively.

The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity.

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2.

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8.

SETD8 is a histone H4-K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent ( 50 nM, IC 0.

Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins.

An analog of BIX-01294 selectively inhibits a family of histone H3 lysine 9 Jumonji demethylases.

BIX-01294 and its analogs were originally identified and subsequently designed as potent inhibitors against histone H3 lysine 9 (H3K9) methyltransferases G9a and G9a-like protein. Here, we show that BIX-01294 and its analog E67 can also inhibit H3K9 Jumonji demethylase KIAA1718 with half-maximal inhibitory concentrations in low micromolar range. Crystallographic analysis of KIAA1718 Jumonji domain in complex with E67 indicated that the benzylated six-membered piperidine ring was disordered and exposed to solvent.

5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.

DNA methylation dynamics influence brain function and are altered in neurological disorders. 5-hydroxymethylcytosine (5-hmC), a DNA base that is derived from 5-methylcytosine, accounts for ∼40% of modified cytosine in the brain and has been implicated in DNA methylation-related plasticity. We mapped 5-hmC genome-wide in mouse hippocampus and cerebellum at three different ages, which allowed us to assess its stability and dynamic regulation during postnatal neurodevelopment through adulthood.

Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101.

Coordinated methyl-lysine erasure: structural and functional linkage of a Jumonji demethylase domain and a reader domain.

Both components of chromatin (DNA and histones) are subjected to dynamic postsynthetic covalent modifications. Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the epigenetic code. Known histone lysine demethylases include flavin-dependent monoamine oxidase lysine-specific demethylase 1 and α-ketoglutarate-Fe(II)-dependent dioxygenases containing Jumonji domains.

Structural basis for human PHF2 Jumonji domain interaction with metal ions.

PHF2 belongs to a class of α-ketoglutarate-Fe(2)(+)-dependent dioxygenases. PHF2 harbors a plant homeodomain (PHD) and a Jumonji domain. PHF2, via its PHD, binds Lys4-trimethylated histone 3 in submicromolar affinity and has been reported to have the demethylase activity of monomethylated lysine 9 of histone 3 in vivo.

A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability.

The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation.

Dynamics of histone lysine methylation: structures of methyl writers and erasers.

In Eukarya, the packaging of DNA into chromatin provides a barrier that allows for regulation of access to the genome. Chromatin is refractory to processes acting on DNA. ATP-dependent chromatin remodeling machines and histone-modifying complexes can overcome this barrier (or strengthen it in silencing processes).

hNaa10p contributes to tumorigenesis by facilitating DNMT1-mediated tumor suppressor gene silencing.

Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-alpha-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis.

Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases.

Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially 'read' by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2.

Molecular and mechanistic properties of the membrane-bound mitochondrial monoamine oxidases.

The past decade has brought major advances in our knowledge of the structures and mechanisms of MAO A and MAO B, which are pharmacological targets for specific inhibitors. In both enzymes, crystallographic and biochemical data show their respective C-terminal transmembrane helices anchor the enzymes to the outer mitochondrial membrane. Pulsed EPR data show both enzymes are dimeric in their membrane-bound forms with agreement between distances measured in their crystalline forms.

Development of spin-labeled pargyline analogues as specific inhibitors of human monoamine oxidases A and B.

Three TEMPO-conjugated pargyline analogues (ParSL-1, ParSL-2, and ParSL-3) have been synthesized and their inhibitory properties tested for the two human monoamine oxidase isoforms (hMAOA and hMAOB). The three analogues differ in flexibility and substituent positions (para or meta) of the linkers connecting the TEMPO group to the pargyline phenyl ring. ParSL-1 contains a flexible acetamido (-CH(2)-CO-NH-) linker connecting the two moieties at the para position.

Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294.

Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells.