Multiple Phase Regulation Enables Efficient and Bright Quasi-2D Perovskite Light-Emitting Diodes.

Quasi-2D perovskites, multiquantum well materials with the energy cascade structure, exhibit impressive optoelectronic properties and a wide range of applications in various optoelectronic devices. However, the insufficient exciton energy transfer caused by the excess of small-n phases that induce nonradiative recombination and the spatially random phase distribution that impedes charge transport severely inhibit the device performance of light-emitting diodes (LEDs). Here, a faster energy transfer process and efficient carrier recombination are achieved by introducing the multifunctional additive 2-(methylsulfonyl)-4-(trifluoromethyl)benzoic acid (MTA) to manipulate the crystallization process of perovskites. The introduction of MTA not only constrains the PEA and restrains the formation of small-n phases to improve the energy transfer process but also optimizes the crystal orientation to promote charge transport. As a result, highly efficient pure green quasi-2D perovskite LEDs with a peak EQE of 25.9%, a peak current efficiency of 108.1 cd A-1, and a maximum luminance of 288798 cd m-2 are achieved.

Discovery of non-boronic acid Arginase 1 inhibitors through virtual screening and biophysical methods.

Inhibiting Arginase 1 (ARG1), a metalloenzyme that hydrolyzes l-arginine in the urea cycle, has been demonstrated as a promising therapeutic avenue in immuno-oncology through the restoration of suppressed immune response in several types of cancers. Most of the currently reported small molecule inhibitors are boronic acid based. Herein, we report the discovery of non-boronic acid ARG1 inhibitors through virtual screening. Biophysical and biochemical methods were used to experimentally profile the hits while X-ray crystallography confirmed a class of trisubstituted pyrrolidine derivatives as optimizable alternatives for the development of novel classes of immuno-oncology agents targeting this enzyme.

Discovery of a novel class of non-ATP site DFG-out state p38 inhibitors utilizing computationally assisted virtual fragment-based drug design (vFBDD).

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide.

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli.

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.

Structure Determination of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas in A New Monoclinic Crystal Form.

Basic phospholipase A(2) from the venom of Agkistrodon halys Pallas ( Agkistrodin blomhoffii brevicaudus ) exhibits hemolytic and anti-coagulant activities. A new monoclinic crystal form with four molecules per asymmetric unit was grown in the absence of n-octyl beta-o-glucopyranoside (beta-OG). The enzyme structure was determined by the molecular replacement method. The combined analysis of self- and cross- rotation function was used and non-crystallographic symmetry restraints were imposed to the structure refinement. The final model gave an acceptable crystallographic R factor and reasonable stereochemistry. Two molecules formed an interfacial-recognition-site linked dimer and two such dimers constituted a tetramer having pseudo 222 symmetry. Structural comparison with previously reported monoclinic forms, in which beta-OG was bound, showed that the variation of crystallization conditions had effects on the crystal packing, leading to significant changes of the cell parameters. Nevertheless, the structures of both the dimer and tetramer in the two crystal forms closely resembled to each other, indicating that the oligomers found in the monoclinic crystal forms were stable.

High-resolution structure of the p53 core domain: implications for binding small-molecule stabilizing compounds.

The p53 transcriptional regulator is the most frequently mutated protein in human cancers and the majority of tumor-derived p53 mutations map to the central DNA-binding core domain, with a subset of these mutations resulting in reduced p53 stability. Here, the 1.55 A crystal structure of the mouse p53 core domain with a molecule of tris(hydroxymethyl)aminomethane (Tris) bound through multiple hydrogen bonds to a region of p53 shown to be important for repair of a subset of tumor-derived p53-stability mutations is reported. Consistent with the hypothesis that Tris binding stabilizes the p53 core domain, equilibrium denaturation experiments are presented that demonstrate that Tris binding increases the thermodynamic stability of the mouse p53 core domain by 3.1 kJ mol(-1) and molecular-dynamic simulations are presented revealing an overall reduction in root-mean-square deviations of the core domain of 0.7 A when Tris is bound. It is also shown that these crystals of the p53 core domain are suitable for the multiple-solvent crystal structure approach to identify other potential binding sites for possible core-domain stabilization compounds. Analysis of the residue-specific temperature factors of the high-resolution core-domain structure, coupled with a comparison with other core-domain structures, also reveals that the L1, H1-S5 and S7-S8 core-domain loops, also shown to mediate various p53 activities, harbor inherent flexibility, suggesting that these regions might be targets for other p53-stabilizing compounds. Together, these studies provide a molecular scaffold for the structure-based design of p53-stabilization compounds for development as possible therapeutic agents.

Two classes of p38alpha MAP kinase inhibitors having a common diphenylether core but exhibiting divergent binding modes.

Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.

Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding.

The targeted acetylation status of histones and several other transcriptional regulatory proteins plays an important role in gene expression, although the mechanism for this is not well understood. As a model to understand how targeted acetylation may effect transcription, we determined the x-ray crystal structure of the chromatin protein Alba from Archaeoglobus fulgidus, a substrate for the Sir2 protein that deacetylates it at lysine 11 to promote DNA binding by Alba. The structure reveals a dimer of dimers in which the dimer-dimer interface is stabilized by several conserved hydrophobic residues as well as the lysine 11 target of Sir2. We show that, in solution, the mutation of these hydrophobic residues or lysine 11 disrupts dimer-dimer formation and decreases DNA-binding affinity. We propose that the in vivo deacetylation of lysine 11 of archaeal Alba by Sir2 promotes protein oligomerization for optimal DNA binding. Implications for the mechanism by which histone acetylation modulates gene expression are discussed.

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)-dependent Sir2 histone/protein deacetylases.

Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD(+) and acetyllysine within protein targets and generate lysine, 2'-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD(+) analogue, carba-NAD(+), as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD(+) reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2'-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved beta1-alpha2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD(+) reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD(+) cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules.

Structure and autoregulation of the yeast Hst2 homolog of Sir2.

Yeast Hst2 (yHst2) is a member of the silencing information regulator 2 (Sir2) family of NAD(+)-dependent protein deacetylases that are implicated in transcriptional silencing, DNA repair, genome stability and longevity. The X-ray crystal structure of the full-length yHst2 protein reveals a central catalytic core domain fold that is characteristic of the other Sir2 homologs, and C- and N-terminal extensions that interact with the NAD(+) and acetyl-lysine substrate-binding sites, respectively, suggesting an autoregulatory function for these domains. Moreover, the N-terminal extension mediates formation of a homotrimer within the crystal lattice. Enzymatic and sedimentation equilibrium studies using deletion constructs of yHst2 support the involvement of the N- and C-terminal yHst2 regions and trimer formation in catalysis by yHst2. Together, these studies indicate that the sequence-divergent N- and C-terminal regions of the eukaryotic Sir2 proteins may have a particularly important role in their distinct substrate-binding properties, biological activities or both.

Improved expression, purification, and crystallization of p38alpha MAP kinase.

p38alpha mitogen-activated protein (MAP) kinase is widely expressed in many mammalian tissues and is activated as a part of signal transduction cascades that respond to inflammatory stimuli. The activation of p38 is known to trigger various biological effects, including cell death, differentiation, and proliferation. The central role played by p38alpha in cellular signaling events, including those that control a wide range of inflammatory and autoimmune diseases, makes it an attractive drug target. To develop optimized small molecule therapeutics targeting p38alpha, different techniques must be employed for the detailed biochemical, biophysical, and structural characterization of the interactions of p38alpha with lead compounds. These methods typically require large quantities of highly purified p38alpha protein. We describe here an improved expression and purification method for recombinant p38alpha production that reproducibly yields over 70 mg of highly purified protein per liter of shake flask bacterial culture. This yield is significantly higher than that previously reported for p38alpha production in Escherichia coli. We achieved a significant increase in soluble p38alpha protein expression by using the genetically modified E. coli strain BL21 DE3 Rosetta, which is optimized for expression of eukaryotic proteins with codons rarely used in E. coli. The p38alpha protein was purified to near homogeneity using a simple two-step procedure including nickel-chelating Sepharose chromatography followed by anion-exchange chromatography using MonoQ resin. Purified p38alpha was characterized using the standard commercially available small molecule inhibitor SB-203580. The binding association and dissociation rate constants determined by Biacore are in excellent agreement with previously reported values. The purified p38alpha protein was efficiently activated by MKK6 kinase to yield phosphorylated p38alpha. Purified p38alpha protein was also successfully crystallized, producing crystals diffracting to 1.9 angstroms, exceeding the highest resolution for p38alpha reported in the Protein DataBank. The simplicity and efficiency of this approach should prove useful for many laboratories that are interested in production of p38alpha for biochemical and biophysical studies and structure-based drug design.

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity.

p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers.