Showing papers by "Torrey Pines Institute for Molecular Studies published in 2012"

Recognizing Pitfalls in Virtual Screening: A Critical Review

Abstract: The aim of virtual screening (VS) is to identify bioactive compounds through computational means, by employing knowledge about the protein target (structure-based VS) or known bioactive ligands (ligand-based VS). In VS, a large number of molecules are ranked according to their likelihood to be bioactive compounds, with the aim to enrich the top fraction of the resulting list (which can be tested in bioassays afterward). At its core, VS attempts to improve the odds of identifying bioactive molecules by maximizing the true positive rate, that is, by ranking the truly active molecules as high as possible (and, correspondingly, the truly inactive ones as low as possible). In choosing the right approach, the researcher is faced with many questions: where does the optimal balance between efficiency and accuracy lie when evaluating a particular algorithm; do some methods perform better than others and in what particular situations; and what do retrospective results tell us about the prospective utility of a part...

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

Abstract: Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

The history of matrix metalloproteinases: milestones, myths, and misperceptions

Abstract: Since the discovery of tadpole collagenase in 1962, the matrix metalloproteinase (MMP) family has emerged as a significant proteinase group with recognized effects on the cardiovascular system. Over the last 40 years, many milestones have been achieved, from the identification of the first MMP, to the generation of the first MMP cDNA clone and null mouse, to the clinical approval of the first MMP inhibitor. Over the years, a few myths and misunderstandings have interwoven into the truths. In this review, we will discuss the major milestones of MMP research, as well as review the misinterpretations and misperceptions that have evolved. Clarifying the confusions and dispelling the myths will both provide a better understanding of MMP properties and functions and focus the cardiovascular field on the outstanding research questions that need to be addressed.

Brain Penetrant LRRK2 Inhibitor.

Abstract: Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are present in a subset of Parkinson's disease (PD) patients and may represent an attractive therapeutic target. Here, we report that a 2-anilino-4-methylamino-5-chloropyrimidine, HG-10-102-01 (4), is a potent and selective inhibitor of wild-type LRRK2 and the G2019S mutant. Compound 4 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1–0.3 μM in cells and is the first compound reported to be capable of inhibiting Ser910 and Ser935 phosphorylation in mouse brain following intraperitoneal delivery of doses as low as 50 mg/kg.

Type II natural killer T cells use features of both innate-like and conventional T cells to recognize sulfatide self antigens.

Abstract: Natural killer T cells (NKT cells) recognize lipid antigens presented by CD1d. Zajonc and Rossjohn and their colleagues describe molecular interactions between type II NKT cell antigen receptors and CD1d-ligand complexes, which demonstrate distinct modes of recognition used by the receptors.

The chemokine CCL18 causes maturation of cultured monocytes to macrophages in the M2 spectrum

Abstract: The observation that human monocytes cultured in the presence of the chemokine CCL18 showed increased survival, led us to profile cytokine expression in CCL18-stimulated versus control cultures. CCL18 caused significantly increased expression of chemokines (CXCL8, CCL2, CCL3 and CCL22), interleukin-10 (IL-10) and platelet-derived growth factor, but no up-regulation of M1 cytokines IL-1β or IL-12. CCL18-stimulated monocytes matured into cells with morphological resemblance to IL-4-stimulated macrophages, and expressed the monocyte marker CD14 as well the M2 macrophage markers CD206 and 15-lipoxygenase, but no mature dendritic cell markers (CD80, CD83 or CD86). Functionally, CCL18-stimulated macrophages showed a high capacity for unspecific phagocytosis and for pinocytosis, which was not associated with an oxidative burst. These findings suggest that CCL18-activated macrophages stand at the cross-roads between inflammation and its resolution. The chemokines that are produced in response to CCL18 are angiogenic and attract various leucocyte populations, which sustain inflammation. However, the capacity of these cells to remove cellular debris without causing oxidative damage and the production of the anti-inflammatory IL-10 will initiate termination of the inflammatory response. In summary, CCL18 induces an M2 spectrum macrophage phenotype in the absence of IL-4.

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis

Abstract: The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Nonneutralizing Epitopes

Abstract: Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong(,) K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ∼100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

Transcriptional responses of surface water marine microbial assemblages to deep-sea water amendment.

Abstract: Deep-water nutrient injection in the North Pacific Subtropical Gyre provides an aperiodic yet significant source of inorganic nutrients to the nutrient-limiting surface waters, and has been implicated in phytoplankton bloom formation. Here we examined short-term transcriptional responses of surface water picoplankton assemblages in a deep-sea water (DSW) mixing experiment. Both flow cytometric and transcriptomic analysis indicated stimulation of an Alteromonas-like population in the DSW-amended treatment after 12 h, relative to the control. Among the highly expressed alteromonad transcripts in DSW-treated samples, those encoding genes associated with chemotaxis, cell motility and carbon metabolism were most highly represented, relative to the control. Similarly, Prochlorococcus showed significantly higher levels of transcripts associated with carbon fixation and photosynthesis, as well as slightly increased cell density, relative to the control. Although other microbial taxa did not exhibit enhanced growth, DSW-stimulated changes in their genome-wide transcriptional profiles were still readily detectable. Cell-associated cyanophage DNA and cDNA profiles suggested DSW stimulation of phage-mediated cell lysis, in previously infected cells. Comparison of the DSW-responsive Alteromonas populations, to previously reported dissolved organic matter (DOM)-responding alteromonads (McCarren et al., 2010), revealed differential transcript abundances, predominantly among genes encoding mobile elements and phage-related genes. Transcript representation in other metabolic pathways differed significantly between Alteromonas populations in the two different treatments, suggesting perturbation-specific metabolic responses to DSW and DOM. In total, the results provide new insight into short-term responses of picoplankton to DSW mixing, which occur prior to the more well-studied, longer-term growth responses of larger phytoplankton species.

Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception

Abstract: Computational methods have led two groups to predict the endogenous presence of a highly conserved, amidated, 14-aa neuropeptide called either spexin or NPQ. NPQ/spexin is part of a larger prohormone that contains 3 sets of RR residues, suggesting that it could yield more than one bioactive peptide; however, no in vivo activity has been demonstrated for any peptide processed from this precursor. Here we demonstrate biological activity for two peptides present within proNPQ/spexin. NPQ/spexin (NWTPQAMLYLKGAQ-NH(2)) and NPQ 53-70 (FISDQSRRKDLSDRPLPE) have differing renal and cardiovascular effects when administered intracerebroventricularly or intravenously into rats. Intracerebroventricular injection of NPQ/spexin produced a 13 ± 2 mmHg increase in mean arterial pressure, a 38 ± 8 bpm decrease in heart rate, and a profound decrease in urine flow rate. Intracerebroventricular administration of NPQ 53-70 produced a 26 ± 9 bpm decrease in heart rate with no change in mean arterial pressure, and a marked increase in urine flow rate. Intraventricular NPQ/spexin and NPQ 53-70 also produced antinociceptive activity in the warm water tail withdrawal assay in mice (ED(50)<30 and 10 nmol for NPQ/spexin and NPQ 53-70, respectively). We conclude that newly identified peptides derived from the NPQ/spexin precursor contribute to CNS-mediated control of arterial blood pressure and salt and water balance and modulate nociceptive responses.

Unnatural Amino Acid Mutagenesis of Fluorescent Proteins

Abstract: Fluorescent proteins are useful probes of protein localization and function both in vitro and in vivo. As a result, considerable effort has focused on engineering new properties into fluorescent proteins. 4, 5] Both directed evolution and rational design have resulted in mutant proteins with altered photophysical properties, stabilities, and sensitivity to pH value, exogenous ligands, phosphorylation and the like. To further explore the effects of steric/electronic perturbations to the fluorophore of a fluorescent protein, we previously substituted tyrosine 66 of the fluorophore in green fluorescent protein (GFP) with a series of unnatural amino acids. Furthermore, others have reported the use of unnatural amino acid mutagenesis to study the spectral properties and folding behavior of GFP, and to generate biosensors. Herein we report the properties of four GFP (GFPUV, a GFP variant optimized for maximal fluorescence when excited by standard UV light; Clontech) mutants in which the hydroxy substituent of Tyr66 is replaced with boronate, azido, keto, and nitro substituents (Scheme 1).

Molecular Scaffold Analysis of Natural Products Databases in the Public Domain

Abstract: Natural products represent important sources of bioactive compounds in drug discovery efforts. In this work, we compiled five natural products databases available in the public domain and performed a comprehensive chemoinformatic analysis focused on the content and diversity of the scaffolds with an overview of the diversity based on molecular fingerprints. The natural products databases were compared with each other and with a set of molecules obtained from in-house combinatorial libraries, and with a general screening commercial library. It was found that publicly available natural products databases have different scaffold diversity. In contrast to the common concept that larger libraries have the largest scaffold diversity, the largest natural products collection analyzed in this work was not the most diverse. The general screening library showed, overall, the highest scaffold diversity. However, considering the most frequent scaffolds, the general reference library was the least diverse. In general, natural products databases in the public domain showed low molecule overlap. In addition to benzene and acyclic compounds, flavones, coumarins, and flavanones were identified as the most frequent molecular scaffolds across the different natural products collections. The results of this work have direct implications in the computational and experimental screening of natural product databases for drug discovery.

Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acid Linked Dolastatin Derivatives

Abstract: Disclosed herein are non-natural amino acids and dolastatin analogs that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The dolastatin analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Typically, the modified dolastatin analogs include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid dolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

Techniques for grounding agent-based simulations in the real domain: a case study in experimental autoimmune encephalomyelitis

Abstract: For computational agent-based simulation, to become a serious tool for investigating biological systems requires the implications of simulation-derived results to be appreciated in terms of the original system. However, epistemic uncertainty regarding the exact nature of biological systems can complicate the calibration of models and simulations that attempt to capture their structure and behaviour, and can obscure the interpretation of simulation-derived experimental results with respect to the real domain. We present an approach to the calibration of an agent-based model of experimental autoimmune encephalomyelitis (EAE), a mouse proxy for multiple sclerosis (MS), which harnesses interaction between a modeller and domain expert in mitigating uncertainty in the data derived from the real domain. A novel uncertainty analysis technique is presented that, in conjunction with a latin hypercube-based global sensitivity analysis, can indicate the implications of epistemic uncertainty in the real domain. These ...

Anti-HIV B Cell Lines as Candidate Vaccine Biosensors

Abstract: Challenge studies following passive immunization with neutralizing Abs suggest that an HIV vaccine could be efficacious were it able to elicit broadly neutralizing Abs (bNAbs). To better understand the requirements for activation of B cells producing bNAbs, we generated cell lines expressing bNAbs or their germline-reverted versions (gl-bNAbs) as BCRs. We then tested the abilities of the bNAb-expressing cells to recognize HIV pseudovirions and vaccine candidate proteins by binding and activation assays. The results suggest that HIV envelope (Env) Ag-expressing, infection-competent virions are poorly recognized by high-affinity bNAb-expressing cells, as measured by the inability of Ags to induce rapid increases in intracellular calcium levels. Other Ag forms appear to be highly stimulatory, in particular, soluble gp140 trimers and a multimerized, scaffolded epitope protein. Virions failed to efficiently activate bNAb-expressing B cells owing to delayed or inefficient BCR recognition, most likely caused by the low density of Env spikes. Importantly, B cells carrying gl-bNAb BCRs were not stimulated by any of the tested vaccine candidates. These data provide insight into why many HIV immunogens, as well as natural HIV infections, fail to rapidly stimulate bNAb responses and suggest that bNAb-expressing cell lines might be useful tools in evaluation of vaccine Ags for infectious diseases. Because soluble Env trimers or multimerized scaffolded epitopes are best at activating B cell-expressing bNAbs, these antigenic forms should be considered as preferred vaccine components, although they should be modified to better target naive gl-bNAb B cells.

Identification of distinct movement patterns in Pacific leatherback turtle populations influenced by ocean conditions

Abstract: Interactions with fisheries are believed to be a major cause of mortality for adult leatherback turtles (Dermochelys coriacea), which is of particular concern in the Pacific Ocean, where they have been rapidly declining. In order to identify where these interactions are occurring and how they may be reduced, it is essential first to understand the movements and behavior of leatherback turtles. There are two regional nesting populations in the East Pacific (EP) and West Pacific (WP), comprising multiple nesting sites. We synthesized tracking data from the two populations and compared their movement patterns. A switching state-space model was applied to 135 Argos satellite tracks to account for observation error, and to distinguish between migratory and area-restricted search behaviors. The tracking data, from the largest leatherback data set ever assembled, indicated that there was a high degree of spatial segregation between EP and WP leatherbacks. Area-restricted search behavior mainly occurred in the southeast Pacific for the EP leatherbacks, whereas the WP leatherbacks had several different search areas in the California Current, central North Pacific, South China Sea, off eastern Indonesia, and off southeastern Australia. We also extracted remotely sensed oceanographic data and applied a generalized linear mixed model to determine if leatherbacks exhibited different behavior in relation to environmental variables. For the WP population, the probability of area-restricted search behavior was positively correlated with chlorophyll-a concentration. This response was less strong in the EP population, but these turtles had a higher probability of search behavior where there was greater Ekman upwelling, which may increase the transport of nutrients and consequently prey availability. These divergent responses to oceanographic conditions have implications for leatherback vulnerability to fisheries interactions and to the effects of climate change. The occurrence of leatherback turtles within both coastal and pelagic areas means they have a high risk of exposure to many different fisheries, which may be very distant from their nesting sites. The EP leatherbacks have more limited foraging grounds than the WP leatherbacks, which could make them more susceptible to any temperature or prey changes that occur in response to climate change.

Scanning structure-activity relationships with structure-activity similarity and related maps: from consensus activity cliffs to selectivity switches.

Abstract: Systematic description of structure–activity relationships (SARs) of data sets and structure–property relationships (SPRs) is of paramount importance in medicinal chemistry and other research fields. To this end, structure–activity similarity (SAS) maps are one of the first tools proposed to describe SARs using the concept of activity landscape modeling. One of the major goals of the SAS maps is to identify activity cliffs defined as chemical compounds with high similar structure but unexpectedly very different biological activity. Since the first publication of the SAS maps more than ten years ago, these tools have evolved and adapted over the years to analyze various types of compound collections, including structural diverse and combinatorial sets with activity for one or multiple biological end points. The development of SAS maps has led to general concepts that are applicable to other activity landscape methods such as “consensus activity cliffs” (activity cliffs common to a series of representations...

Healing Touch With Guided Imagery for PTSD in Returning Active Duty Military: A Randomized Controlled Trial

Abstract: Post-traumatic stress disorder (PTSD) remains a significant problem in returning military and warrants swift and effective treatment. We conducted a randomized controlled trial to determine whether a complementary medicine intervention (Healing Touch with Guided Imagery [HT+GI]) reduced PTSD symptoms as compared to treatment as usual (TAU) returning combat-exposed active duty military with significant PTSD symptoms. Active duty military (n = 123) were randomized to 6 sessions (within 3 weeks) of HT+GI vs. TAU. The primary outcome was PTSD symptoms; secondary outcomes were depression, quality of life, and hostility. Repeated measures analysis of covariance with intent-to-treat analyses revealed statistically and clinically significant reduction in PTSD symptoms (p < 0.0005, Cohen's d = 0.85) as well as depression (p < 0.0005, Cohen's d = 0.70) for HT+GI vs. TAU. HT+GI also showed significant improvements in mental quality of life (p = 0.002, Cohen's d = 0.58) and cynicism (p = 0.001, Cohen's d = 0....

Synthesis and SAR of b-annulated 1,4-dihydropyridines define cardiomyogenic compounds as novel inhibitors of TGFβ signaling.

Abstract: A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface Because this is an unprecedented mechanism of action, we explored the series' structure-activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs We determined a pharmacophore and generated 1,4-DHPs with IC(50)s for TGFβ inhibition in the nanomolar range (eg, compound 28, 170 nM) Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (-) enantiomer This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells

Molecular modeling of inhibitors of human DNA methyltransferase with a crystal structure: discovery of a novel DNMT1 inhibitor.

Abstract: DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel anticancer drugs and other diseases. Molecular modeling and experimental approaches are being used to identify and develop inhibitors of human DNMTs. Most of the computational efforts conducted so far with DNMT1 employ homology models of the enzyme. Recently, a crystallographic structure of the methyltransferase domain of human DNMT1 bound to unmethylated DNA was published. Following on our previous computational and experimental studies with DNMTs, we herein present molecular dynamics of the crystal structure of human DNMT1. Docking studies of established DNMT1 inhibitors with the crystal structure gave rise to a structure-based pharmacophore model that suggests key interactions of the inhibitors with the catalytic binding site. Results had a good agreement with the docking and pharmacophore models previously developed using a homology model of the catalytic domain of DNMT1. The docking protocol was able to distinguish active DNMT1 inhibitors from, for example, experimentally known inactive DNMT1 inhibitors. As part of our efforts to identify novel inhibitors of DNMT1, we conducted the experimental characterization of aurintricarboxylic acid (ATA) that in preliminary docking studies showed promising activity. ATA had a submicromolar inhibition (IC(50)=0.68 μM) against DNMT1. ATA was also evaluated for Dnmt3a inhibition showing an IC(50)=1.4 μM. This chapter illustrates the synergy from integrating molecular modeling and experimental methods to further advance the discovery of novel candidates for epigenetic therapies.

Alzheimer's disease in a dish: promises and challenges of human stem cell models

Abstract: Human pluripotent stem cells can differentiate into disease-relevant cell types, which capture the unique genome of an individual patient and provide insight into pathological mechanisms of human disease. Recently, human stem cell models for Alzheimer's disease (AD), the most common neurodegenerative dementia, have been described. Stem cell-derived neurons from patients with familial and sporadic AD and Down's syndrome recapitulate human disease phenotypes such as amyloid β peptide production, hyperphosphorylation of tau protein and endosomal abnormalities. Treatment of human neurons with small molecules can modulate these phenotypes, demonstrating the utility of this system for drug development and screening. This review will highlight the current AD stem cell models and discuss the remaining challenges and potential future directions of this field.