A comprehensive study of Sansalvamide A derivatives: The structure–activity relationships of 78 derivatives in two pancreatic cancer cell lines

We report an extensive structure–activity relationship (SAR) of 78 compounds active against two pancreatic cancer cell lines. Our comprehensive evaluation of these compounds utilizes SAR that allow us to evaluate which features of potent compounds play a key role in their cytotoxicity. This is the first report of 19 new second-generation structures, where these new compounds were designed from the first generation of 59 compounds. These 78 structures were tested for their cytotoxicity and this is the first report of their activity against two pancreatic cancer cell lines. Our results show that out of 78 compounds, three compounds are worth pursuing as leads, as they show potency of ?55% in both cancer cell lines. These three compounds all have a common structural motif, two consecutive d-amino acids and an N-methyl moiety. Further, of these three compounds, two are second-generation structures, indicating that we can incorporate and utilize data from the first generation to design potency into the second generation. Finally, one analog is in the mid nanomolar range, and has the lowest IC₅₀ of any reported San A derivative. These analogs share no structural homology to current pancreatic cancer drugs, and are cytotoxic at levels on par with existing drugs treating other cancers. Thus, we have established Sansalvamide A as an excellent lead for killing multiple pancreatic cancer cell lines.     

Switch 1 Mutation S217A Converts Myosin V into a Low Duty Ratio Motor

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-P_i (inorganic phosphate) and actomyosin V-ADP-P_i and the rate of the hydrolysis step (myosin V-ATP → myosin V-ADP-P_i) were all ∼10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from ∼0.85 in WT myosin V to ∼0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.During the past 2 decades a considerable number of different myosins have been discovered (1). Myosin V is the best characterized among the so-called unconventional myosins (i.e. those not belonging to class II), and it serves as an important model molecule for studying actomyosin interactions and single molecule processive motility (2). Myosin V is a highly processive motor whose role is to transport cargo along actin filaments or bundles inside the cell (3–5). The kinetic mechanism of myosin V is significantly different from that of conventional myosins such as muscle myosin II, as it remains bound to actin (filamentous actin) through a number of ATPase cycles (6–8). Myosin V has a high duty ratio: a single-headed myosin V-S1 (myosin V, subfragment 1) is in the strongly bound AM-ADP state 80–90% of the time during ATP hydrolysis. An additional mechanism for promoting highly processive runs is the preferential release of ADP from the trail head because of mechanochemical gating, which causes a drastic reduction of the rate constant of ADP release from the lead head (9–11). Although there are significant differences between the ATPase mechanisms of the different myosins, the structure of the nucleotide binding pocket (composed of the switch 1 and 2 regions and the P-loop) is highly conserved. The position of the Ser²¹⁷ (Ser²³⁶ in Dictyostelium myosin II) residue of the switch 1 loop (the first serine in the NDNSSRFG sequence) is shown in Fig. 1. It had been shown previously by mutagenesis in Dictyostelium (12) and in smooth muscle myosin II (13) that the substitution of serine 236 to alanine retains at least partial enzymatic and motile function in these mutant myosins. Therefore, the OH group is not an essential part of the catalytic mechanism, but the rate of steady-state ATP hydrolysis is reduced several fold. However, neither of these studies includes a detailed kinetic analysis to determine which steps of the catalytic mechanism were altered by the mutation. Here we have exploited the higher affinity of myosin V-ADP-P_i for actin to determine the effect of the mutation on the rate constants of the product dissociation steps following the power stroke, which could not be determined using either Dictyostelium or smooth muscle myosin. We also conducted single molecule motility studies using total internal reflectance fluorescence (TIRF)⁵ microscopy to determine how the changes in the kinetic mechanism affect the motile properties of the molecule.Open in a separate windowFIGURE 1.Schematic representation of the critical residues in the ATP binding site of myosin based on the MgADP·VO₄ crystal of the Dictyostelium motor domain (Smith and Rayment (30)). The serine in position 217 was mutated to alanine for these kinetic studies. The small spheres are at the position of the oxygen of the water molecules.     

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110β isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway regulates glucose/nutrient homeostasis and cell survival and plays a central role in both normal metabolism and cancer. The PTEN tumor suppressor gene (29, 30, 54) negatively regulates the PI3K/AKT pathway by dephosphorylating the D3 hydroxyl subunit of phosphoinositide-3,4,5-trisphosphate, a key membrane phosphatidylinositol generated by PI3K (34). PTEN undergoes genetic or epigenetic inactivation in many malignancies, including glioblastoma, melanoma, and endometrial, prostate, and breast cancers, among others (6, 13, 22, 23, 47, 49-51, 55, 68). Similarly, germ line mutations of PTEN are associated with the development of hamartomatous neoplasias such as Cowden disease and Bannayan-Zonana syndrome (17, 21, 41).The tumor suppressor function of PTEN undergoes dynamic regulation involving both C-terminal phosphorylation and protein-protein interactions. Phosphorylation of serine and threonine residues at the PTEN C-terminal tail, mediated by kinases such as CK2 and glycogen synthase kinase 3β, alters its conformational structure and association with PDZ domain-containing proteins and attenuates PTEN enzymatic activity (1, 11, 20, 32, 45, 61-63, 66, 67, 71). Conversely, PTEN function is promoted in large part through its stabilization in unphosphorylated form by incorporation into a high-molecular-weight protein complex (the PTEN-associated complex [PAC]) (66). We first demonstrated the existence of the PAC through gel filtration studies of rat liver extracts, which identified PTEN within a high-molecular-mass peak (>600 kDa), as well as a low-molecular-mass peak (40 to 100 kDa) in which PTEN is monomeric and phosphorylated (66). Subsequently, several PDZ domain-containing proteins were shown to interact with PTEN, including MAGI-1b, MAGI-2, MAGI-3, ghDLG, hMAST205, MSP58/MCRS1, NHERF1, and NHERF2, which mediate indirect binding with platelet-derived growth factor (PDGF) receptor β (25, 36, 42, 57, 66). More recently, LKB1, a serine/threonine kinase tumor suppressor (7), was also found to interact with and phosphorylate PTEN in vitro (36). In aggregate, these data suggest that PTEN functional output is controlled by a complex interplay of protein interactions and regulation of C-terminal phosphorylation.Beyond these interactions, there is also evidence to support additional regulatory mechanisms by which the tumor suppressor function of PTEN is mediated. The herpesvirus-associated ubiquitin-specific protease was shown to interact directly with PTEN and promote its nuclear entry (53). Both ubiquitination and relocalization into the nucleus constitute important PTEN regulatory mechanisms (53, 64). In many tumors, PTEN nuclear exclusion has been associated with poor cancer prognosis and more aggressive cancer development (15, 44, 56). Moreover, successful treatment of acute promyelocytic leukemia was shown to be associated with an increase in monoubiquitinylation and relocation of PTEN into the nucleus (53).Like PTEN, the p85 regulatory subunit of PI3K serves as a prominent modulator of PI3K/AKT signaling. p85, which exists in three isoforms (α, β, and γ), targets the catalytic (110-kDa) PI3K subunit to the membrane, which brings it into proximity with membrane-associated phosphatidylinositol lipids. In the steady state, p85 forms a tight association with the catalytic PI3K subunit, usually p110α or p110β in nonhematopoietic cells, with p110δ predominating in leukocytes (19). Consistent with this notion, p85 and p110 exist in equimolar ratios in a wide variety of mammalian cell lines and tissues (19), although some studies have suggested a role for free p85 in cell signaling (33, 65).Several recent lines of evidence have begun to support a possible regulatory relationship between PTEN and p85 (reviewed in references 3 and 53). For example, liver-specific deletion of PIK3R1, which encodes the p85α regulatory subunit, reduces both the activation of PI3K and PTEN enzymatic activity in this context. As a result, p85α-deficient hepatic cells express elevated levels of phosphoinositide trisphosphate and exhibit prolonged AKT activation (60). In addition, both PTEN and p85 are regulated by small GTPase proteins such as RhoA, but PTEN coimmunoprecipitates with the RhoA effector Rock only in the presence of PI3K (18, 31, 37). Although only correlative in nature, these findings may suggest a possible role for PTEN in p85 regulation or vice versa, in addition to its known function as a direct antagonist of the PI3K/AKT pathway (3, 9, 52, 57, 60).In the present study, we demonstrate an endogenous association between p85 and PTEN. Using newly generated antibodies that selectively recognize the PTEN C-terminal tail in its unphosphorylated form, we demonstrate that this PTEN-p85 association preferentially involves the unphosphorylated form of PTEN. The specificity of this interaction was confirmed using multiple antibodies and through studies of both human cancer cells and murine embryonic fibroblasts (MEFs) deficient for specific p85 subunits. This association, which also engages p110β, is enhanced by trastuzumab treatment and correlates with diminished AKT phosphorylation. These results support a functional role for the PTEN-p85 association that may have important biological and therapeutic implications for PI3K/AKT pathway regulation.     

Extensibility of the Extended Tail Domain of Processive and Nonprocessive Myosin V Molecules

Myosin V is a single-molecule motor that moves organelles along actin. When myosin V pulls loads inside the cell in a highly viscous environment, the force on the motor is unlikely to be constant. We propose that the tether between the single-molecule motor and the cargo (i.e., the extended tail domain of the molecule) must be able to absorb the sudden mechanical motions of the motor and allow smooth relaxation of the motion of the cargo to a new position. To test this hypothesis, we compared the elastic properties of the extended tail domains of processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. The extended tail domain of these myosins consists of mechanically strong coiled-coil regions interspersed with flexible loops. In this work we explored the mechanical properties of coiled-coil regions using atomic force microscopy. We found that the processive and nonprocessive coiled-coil fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the atomic force microscopy stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V, suggesting that this elastic tether between the cargo and motor may play an important role in sustaining the processive motions of this single-molecule motor.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Risk of Ovarian Cancer and Inherited Variants in Relapse-Associated Genes

Background

We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk.

Methods and Findings

Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66–0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02–1.91).

Conclusions

Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.     

Essential elements of online information networks on invasive alien species

In order to be effective, information must be placed in the proper context and organized in a manner that is logical and (preferably) standardized. Recently, invasive alien species (IAS) scientists have begun to create online networks to share their information concerning IAS prevention and control. At a special networking session at the Beijing International Symposium on Biological Invasions, an online Eastern Asia–North American IAS Information Network (EA–NA Network) was proposed. To prepare for the development of this network, and to provide models for other regional collaborations, we compare four examples of global, regional, and national online IAS information networks: the Global Invasive Species Information Network, the Invasives Information Network of the Inter-American Biodiversity Information Network, the Chinese Species Information System, and the Invasive Species Information Node of the US National Biological Information Infrastructure. We conclude that IAS networks require a common goal, dedicated leaders, effective communication, and broad endorsement, in order to obtain sustainable, long-term funding and long-term stability. They need to start small, use the experience of other networks, partner with others, and showcase benefits. Global integration and synergy among invasive species networks will succeed with contributions from both the top-down and the bottom-up.