Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

CRISPR Screen Reveals BRD2/4 Molecular Glue-like Degrader via Recruitment of DCAF16.

Molecular glues (MGs) are monovalent small molecules that induce an interaction between proteins (native or non-native partners) by altering the protein-protein interaction (PPI) interface toward a higher-affinity state. Enhancing the PPI between a protein and E3 ubiquitin ligase can lead to degradation of the partnering protein. Over the past decade, retrospective studies of clinical drugs identified that immunomodulatory drugs (e.g., thalidomide and analogues) and indisulam exhibit a molecular glue effect by driving the interaction between non-native substrates to CRBN and DCAF15 ligases, respectively. Ensuing reports of phenotypic screens focused on MG discovery have suggested that these molecules may be more common than initially anticipated. However, prospective discovery of MGs remains challenging. Thus, expanding the repertoire of MGs will enhance our understanding of principles for prospective design. Herein, we report the results of a CRISPR/Cas9 knockout screen of over 1000 ligases and ubiquitin proteasome system components in a BRD4 degradation assay with a JQ1-based monovalent degrader, compound 1a. We identified DCAF16, a substrate recognition component of the Cul4 ligase complex, as essential for compound activity, and we demonstrate that compound 1a drives the interaction between DCAF16 and BRD2/4 to promote target degradation. Taken together, our data suggest that compound 1a functions as an MG degrader between BRD2/4 and DCAF16 and provides a foundation for further mechanistic dissection to advance prospective MG discovery.

Exsolution-Driven Surface Transformation in the Host Oxide.

Exsolution synthesizes self-assembled metal nanoparticle catalysts via phase precipitation. An overlooked aspect in this method thus far is how exsolution affects the host oxide surface chemistry and structure. Such information is critical as the oxide itself can also contribute to the overall catalytic activity. Combining X-ray and electron probes, we investigated the surface transformation of thin-film SrTi0.65Fe0.35O3 during Fe0 exsolution. We found that exsolution generates a highly Fe-deficient near-surface layer of about 2 nm thick. Moreover, the originally single-crystalline oxide near-surface region became partially polycrystalline after exsolution. Such drastic transformations at the surface of the oxide are important because the exsolution-induced nonstoichiometry and grain boundaries can alter the oxide ion transport and oxygen exchange kinetics and, hence, the catalytic activity toward water splitting or hydrogen oxidation reactions. These findings highlight the need to consider the exsolved oxide surface, in addition to the metal nanoparticles, in designing the exsolved nanocatalysts.

Targeting IRAK3 for Degradation to Enhance IL-12 Pro-inflammatory Cytokine Production.

Interleukin-1 receptor-associated kinase 3 (IRAK3) is a pseudokinase mediator in the human inflammatory pathway, and ablation of its function is associated with enhanced antitumor immunity. Traditionally, pseudokinases have eluded "druggability" and have not been considered tractable targets in the pharmaceutical industry. Herein we disclose a CRISPR/Cas9-mediated knockout of IRAK3 in monocyte-derived dendritic cells that results in an increase in IL-12 production upon lipopolysaccharide (LPS) stimulation. Furthermore, we disclose and characterize Degradomer D-1, which displays selective proteasomal degradation of IRAK3 and reproduces the 1L-12p40 increases observed in the CRISPR/Cas9 knockout.

Proton irradiation-decelerated intergranular corrosion of Ni-Cr alloys in molten salt.

The effects of ionizing radiation on materials often reduce to "bad news". Radiation damage usually leads to detrimental effects such as embrittlement, accelerated creep, phase instability, and radiation-altered corrosion. Here we report that proton irradiation decelerates intergranular corrosion of Ni-Cr alloys in molten fluoride salt at 650 °C. We demonstrate this by showing that the depth of intergranular voids resulting from Cr leaching into the salt is reduced by proton irradiation alone. Interstitial defects generated from irradiation enhance diffusion, more rapidly replenishing corrosion-injected vacancies with alloy constituents, thus playing the crucial role in decelerating corrosion. Our results show that irradiation can have a positive impact on materials performance, challenging our view that radiation damage usually results in negative effects.

Compensating film stress in thin silicon substrates using ion implantation.

Future space telescopes, especially X-ray telescopes, will require thin mirrors to achieve high optical throughput. Thin mirrors are more difficult to fabricate than thick mirrors, but recent advances have made accurate fabrication of thin mirrors possible. However, mirrors must have a reflective coating, which typically has non-repeatable and non-uniform intrinsic stress that deforms a thin mirror. Reducing coating stress by controlling deposition parameters typically reduces reflectivity. Non-uniform integrated stress compensation (NISC) methods, in which spatially controlled stress is applied to the mirror substrate backside to balance the frontside coating stress, decouple the film stress from the reflectivity. Ion implantation is one NISC method, where high-energy ions are implanted into a glass or silicon substrate to generate stress near the substrate surface. In this paper, we demonstrate the use of ion implantation for stress compensation of 30 nm thick chromium films applied to the front of five silicon wafers. The reflective films have mean integrated stress between -8 and -35 N/m, which cause deformations between 400 and 1600 nm RMS. We demonstrate that these wafers can be restored to the pre-coating shape to within 60 nm RMS, in most cases within 1/20th of the coating deformation.

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. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,ß-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

SAR and characterization of non-substrate isoindoline urea inhibitors of nicotinamide phosphoribosyltransferase (NAMPT).

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.

Structure activity optimization of 6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazines as Jak1 kinase inhibitors.

Previous work investigating tricyclic pyrrolopyrazines as kinase cores led to the discovery that 1-cyclohexyl-6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (12) had Jak inhibitory activity. Herein we describe our initial efforts to develop orally bioavailable analogs of 12 with improved selectivity of Jak1 over Jak2.

Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor.

BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.

(S)-N-(5-Chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol a Notch-1-sparing gamma-secretase inhibitor.

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

Discovery of begacestat, a Notch-1-sparing gamma-secretase inhibitor for the treatment of Alzheimer's disease.

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors.

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Synthesis and activity of N-cyanoguanidine-piperazine P2X7 antagonists.

A novel series of cyanoguanidine-piperazine P2X(7) antagonists were identified and structure-activity relationship (SAR) studies described. Compounds were assayed for activity at human and rat P2X(7) receptors in addition to their ability to inhibit IL-1 beta release from stimulated human whole blood cultures. Compound 27 possesses potent activity (0.12 microM) in this latter assay and demonstrates moderate clearance in-vivo.

Discovery of a novel series of Notch-sparing gamma-secretase inhibitors.

Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.

Synthesis and in vitro activity of N'-cyano-4-(2-phenylacetyl)-N-o-tolylpiperazine-1-carboximidamide P2X7 antagonists.

A novel series of cyanoguanidine-piperazine P2X(7) antagonists was designed based upon the structure of A-740003. Structure-activity relationship (SAR) studies focused on the piperazine moiety and the right hand side substitution. Compounds were assayed for activity at human and rat P2X(7) receptors and compound 29 was found to possess potent activity (IC(50)=30-60 nM) at both species.

Psychological reactance: examination across age, ethnicity, and gender.

Psychological reactance is a motivational state aroused when real or perceived personal freedoms are threatened, reduced, or eliminated. Although psychological reactance theory has existed for almost 40 years, there is still dissent over some of its most basic characteristics. Research on age and ethnicity is scant, and research on gender has not produced a clear pattern of results. We attempted to clarify the relationships of these variables to psychological reactance. A total of 3,499 undergraduates completed the Therapeutic Reactance Scale and a brief demographic questionnaire. We found a curvilinear relationship between age and reactance, with older and younger participants exhibiting higher reactance than the middle age group. African Americans, Asians, and Hispanics exhibited higher total, behavioral, and verbal reactance than Caucasians and Native Americans. Men produced higher total, behavioral, and verbal reactance scores than women. We discuss the implications of these findings and make suggestions for further research.

Total Synthesis of the Alkoxydioxines (+)- and (-)-Chondrillin and (+)- and (-)-Plakorin via Singlet Oxygenation/Radical Rearrangement.

The sequential application of singlet oxygenation and peroxyl radical rearrangement provides an asymmetric entry to 4-peroxy-2-enols and 4-peroxy-2-enones. Enantiomerically enriched 2-hydroperoxy-3-alkenols, obtained via hydroxyl-directed addition of (1)O(2) to Z-allylic alcohols, undergo stereospecific radical rearrangement to form 4-hydroperoxy-2-alkenols. The yields of the rearrangement are improved in the presence of excess tert-butyl hydroperoxide, which limits dimerization of the substrate peroxyl radicals. However, the rearrangement equilibrium is unaffected by the presence of polar co-solvents or by the incorporation of a group able to selectively hydrogen bond to the product hydroperoxide. Photoisomerization of the (E)-4-hydroperoxy-2-enone rearrangement products results in irreversible ring closure to furnish diastereomeric mixtures of enantiomerically enriched dioxinols. The strategy is applied to the total synthesis of the alkoxydioxine natural products chondrillin and plakorin. Comparison of the optical rotation of the synthetic material against literature reports indicates that the natural products are either enantiomerically pure or highly enriched in one enantiomer. In addition, our results conclusively demonstrate that the reported configuration of chondrillin is in error.