Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

Discovery of Novel Allosteric Inhibitors of Deoxyhypusine Synthase.

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.

New Series of Potent Allosteric Inhibitors of Deoxyhypusine Synthase.

Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.

Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode.

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.

Discovery of 3-Benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea Derivatives as Novel and Selective Cyclin-Dependent Kinase 12 (CDK12) Inhibitors.

Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.

Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.

The modulation of pre-mRNA splicing is proposed as an attractive anti-neoplastic strategy, especially for the cancers that exhibit aberrant pre-mRNA splicing. Here, we discovered that T-025 functions as an orally available and potent inhibitor of Cdc2-like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T-025 reduced CLK-dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive-associated biomarker of T-025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T-025 treatment. MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti-tumor efficacy of T-025 in an allograft model of spontaneous, MYC-driven breast cancer, at well-tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC-driven cancer patients.

Potent Inhibition of hERG Channels by the Over-the-Counter Antidiarrheal Agent Loperamide.

OBJECTIVES: The aim of this study was to determine the in vitro electrophysiological properties of loperamide. The authors' hypothesis was that loperamide is a potent blocker of the current carried by the human ether-à-go-go-related gene (hERG) potassium channel. BACKGROUND: Loperamide is a peripherally-acting µ-opioid agonist available worldwide as an over-the-counter treatment for diarrhea. Like most opioids, it is not currently known to be proarrhythmic. Recent cases of torsade de pointes in association with high-dose loperamide raise concern given its structural similarity to methadone, another synthetic opioid with an established arrhythmia risk. METHODS: Effects of loperamide on blockade of the hERG potassium channel ion current were assessed in Chinese Hamster Ovary (CHO) cells stably expressing hERG to elucidate current amplitude and kinetics. The concentration required to produce 50% inhibition of hERG current was assessed from the amplitude of tail currents and the impact on action potential duration was assessed in isolated swine ventricular cardiomyocytes. RESULTS: The 50% inhibitory concentration for loperamide inhibition of hERG ionic tail currents was approximately 40 nmol/l. In current-voltage measurements, loperamide reduced steady and tail currents and shifted the current activation to more negative potentials. Loperamide (10 nmol/l) also increased the action potential duration, assessed at 90% of repolarization, in ventricular myocytes by 16.4 ± 1.7% (n = 6; p < 0.004). The maximum rate of rise of phase 0 of the action potential, however, was not significantly altered at any tested concentration of loperamide. CONCLUSIONS: Loperamide is a potent hERG channel blocker. It significantly prolongs the action potential duration and suggests a causal association between loperamide and recent clinical cases of torsade de pointes.

Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate.

Stearoyl-coenzyme A desaturase-1 (SCD1) has an important role in lipid metabolism, and SCD1 inhibitors are potential therapeutic agents for the treatment of metabolic diseases and cancers. Here we report the 3.25-Å crystal structure of human SCD1 in complex with its substrate, stearoyl-coenzyme A, which defines the new SCD1 dimetal catalytic center and reveals the determinants of substrate binding to provide insights into the catalytic mechanism of desaturation of the stearoyl moiety. The structure also provides a mechanism for localization of SCD1 in the endoplasmic reticulum: human SCD1 folds around a tight hydrophobic core formed from four long α-helices that presumably function as an anchor spanning the endoplasmic reticulum membrane. Furthermore, our results provide a framework for the rational design of pharmacological inhibitors targeting the SCD1 enzyme.

Olfactory and visual responses of the longlegged chafer Hoplia spectabilis Medvedev (Coleoptera: Scarabaeidae) in Qinghai Province, China.

BACKGROUND: Monitoring traps and control methods are needed for the long-legged chafer, Hoplia spectabilis Medvedev, which has recently reached outbreak numbers in pastureland of Qinghai Province, China. RESULTS: Field trapping experiments, using cross-pane funnel (barrier) traps, showed that H. spectabilis adults were not significantly attracted to branches of the host plant Dasiphora fructicosa (L.) Rydb. However, beetles were slightly attracted to similar host plant branches infested by conspecific beetles, possibly owing to weakly attractive volatiles, primarily (Z)-3-hexenyl acetate, released from beetle-damaged host leaves. This compound was weakly attractive when released from traps. However, H. spectabilis beetles showed strong visual responses to yellow- or white-painted trap panes, with weaker responses to blue, red or green panes, and least response to black panes. Black traps at 0.2-1.5 m above ground intercepted significantly more beetles than traps at 2.5 m. The mean flight height based on trap catches was 0.88 m (SD = 0.76), yielding an effective flight layer of 1.9 m. Flight response of beetles to colored barrier traps occurred between 10:00 and 18:00, and peaked between 12:00-14:00, when daily temperatures reached their maximum. CONCLUSION: Unbaited yellow or white cross-pane funnel traps are recommended for both monitoring and mass-trapping programs against this economically and ecologically important scarab beetle.

Structural determinants underlying photoprotection in the photoactive orange carotenoid protein of cyanobacteria.

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum.

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.

Activation of (Na+ + K+)-ATPase modulates cardiac L-type Ca2+ channel function.

Cellular Ca(2+) signaling underlies diverse vital biological processes, including muscle contractility, memory encoding, fertilization, cell survival, and cell death. Despite extensive studies, the fundamental control mechanisms that regulate intracellular Ca(2+) movement remain enigmatic. We have found recently that activation of the (Na(+)+K(+))-ATPase markedly potentiates intracellular Ca(2+) transients and contractility of rat heart cells. Little is known about the pathway responsible for the activation of the (Na(+)+K(+))-ATPase-initiated Ca(2+) signaling. Here, we demonstrate a novel mechanism in which activation of the (Na(+)+K(+))-ATPase is coupled to increased L-type Ca(2+) channel function through a signaling cascade involving Src and ERK1/2 but not well established regulators of the channel, such as adrenergic receptor system or activation of PKA or CaMKII. We have also identified Ser(1928), a phosphorylation site for the alpha1 subunit of the L-type Ca(2+) channel that may participate in the activation of the (Na(+)+K(+))-ATPase-mediated Ca(2+) signaling. The findings reported here uncover a novel molecular cross-talk between activation of the (Na(+)+K(+))-ATPase and L-type Ca(2+) channel and provide new insights into Ca(2+) signaling mechanisms for deeper understanding of the nature of cellular Ca(2+) handling in heart.

Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase.

The minichromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. Whereas the eukaryotic complex consists of 6 homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-A crystal structure of the near-full-length ssoMCM. The structure shows an elongated fold, with 5 subdomains that are organized into 2 large N- and C-terminal domains. A near-full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual beta-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Our mutational analysis of residues at the intersubunit interfaces and around the side channels demonstrates their critical roles for hexamerization and helicase function. These structural and biochemical results provide a basis for future study of the helicase mechanisms of the archaeal and eukaryotic MCM complexes in DNA replication.

The structure of a DnaB-family replicative helicase and its interactions with primase.

Helicases are essential enzymes for DNA replication, a fundamental process in all living organisms. The DnaB family are hexameric replicative helicases that unwind duplex DNA and coordinate with RNA primase and other proteins at the replication fork in prokaryotes. Here, we report the full-length crystal structure of G40P, a DnaB family helicase. The hexamer complex reveals an unusual architectural feature and a new type of assembly mechanism. The hexamer has two tiers: a three-fold symmetric N-terminal tier and a six-fold symmetric C-terminal tier. Monomers with two different conformations, termed cis and trans, come together to provide a topological solution for the dual symmetry within a hexamer. Structure-guided mutational studies indicate an important role for the N-terminal tier in binding primase and regulating primase-mediated stimulation of helicase activity. This study provides insights into the structural and functional interplay between G40P helicase and DnaG primase.

Mechanisms of resistance and adaptation to thapsigargin in androgen-independent prostate cancer PC3 and DU145 cells.

Cells with increasing resistance to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), ranging from 60-fold (PC3/TG(10) cells) to 1350-fold (PC3/TG(2000) cells), were derived from PC3 cells. SERCA2 is overexpressed in all PC3/TG cells but retains sensitivity to TG. siRNA-mediated downregulation of SERCA completely or partially reverses TG resistance in PC3/TG(10) or PC3/TG(2000) cells, respectively; thus SERCA overexpression mediates resistance in PC3/TG(10) cells but is not the only resistance mechanism in PC3/TG(2000) cells. By contrast, SERCA is not overexpressed in TG-resistant DU145/TG cells derived from DU145 cells. DU145/TG cells retain resistance while in PC3/TG cells resistance decreases upon removal of TG selection. The transport proteins PGP/BCRP/MRP1 and anti-apoptotic proteins Bcl2/Bcl(XL) are not involved in mediating resistance in either cell line. PARP and caspase 3 cleavage in response to other drugs demonstrate that the apoptotic pathways tested remain intact in these cells. Further, no cross-resistance occurs to other drugs. Thus, novel TG-specific resistance mechanisms are recruited by these cancer cells.

The APOBEC-2 crystal structure and functional implications for the deaminase AID.

APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long alpha-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation.

Structure of the Epstein-Barr virus major envelope glycoprotein.

Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses.

Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.

The transformation potential of Simian Virus 40 depends on the activities of large T-antigen (LTag), which interacts with several cellular tumor suppressors including the important "guardian" of the genome, p53. Inhibition of p53 function by LTag is necessary for both efficient viral replication and cellular transformation. We determined the crystal structure of LTag in complex with p53. The structure reveals an unexpected hexameric complex of LTag binding six p53 monomers. Structure-guided mutagenesis of LTag and p53 residues supported the p53-LTag interface defined by the complex structure. The structure also shows that LTag binding induces dramatic conformational changes at the DNA-binding area of p53, which is achieved partially through an unusual "methionine switch" within p53. In the complex structure, LTag occupies the whole p53 DNA-binding surface and likely interferes with formation of a functional p53 tetramer. In addition, we showed that p53 inhibited LTag helicase function through direct complex formation.

Characterization of ryanodine receptor-mediated calcium release in human B cells: relevance to diagnostic testing for malignant hyperthermia.

BACKGROUND: Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH. METHODS: Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+). RESULTS: EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals). CONCLUSIONS: RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.

Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes.

We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+ -ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.