Towards a Better Understanding of On and Off Target Effects of the Lymphocyte-Specific Kinase LCK for the Development of Novel and Safer Pharmaceuticals

In this work we have developed a multi-tiered computational platform to study protein-drug interactions. At the beginning of the workflow more efficient and less accurate methods are used to enable large libraries of proteins in many conformations and massive chemical libraries to be screened. At each subsequent step in the workflow a subset of input data is investigated with increased accuracy and more computationally expensive methods. We demonstrate the developed workflow with the investigation of the lymphocyte-specific kinase LCK, which is implicated as a drug target in many cancers and also known to have toxic effects when unintentionally targeted. Several LCK states and conformations are investigated using molecular docking and generalized Born and surface area continuum solvation (MM/GBSA). Different variations in the drug screening process provide unique results that may elucidate the biological mechanisms underlying the drug interactions

Non-invasive monitoring of arthritis treatment response via targeting of tyrosine-phosphorylated annexin A2 in chondrocytes

BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation

Epigenetic understanding of gene-environment interactions in psychiatric disorders: a new concept of clinical genetics

Epigenetics is a mechanism that regulates gene expression independently of the underlying DNA sequence, relying instead on the chemical modification of DNA and histone proteins. Although environmental and genetic factors were thought to be independently associated with disorders, several recent lines of evidence suggest that epigenetics bridges these two factors. Epigenetic gene regulation is essential for normal development, thus defects in epigenetics cause various rare congenital diseases. Because epigenetics is a reversible system that can be affected by various environmental factors, such as drugs, nutrition, and mental stress, the epigenetic disorders also include common diseases induced by environmental factors. In this review, we discuss the nature of epigenetic disorders, particularly psychiatric disorders, on the basis of recent findings: 1) susceptibility of the conditions to environmental factors, 2) treatment by taking advantage of their reversible nature, and 3) transgenerational inheritance of epigenetic changes, that is, acquired adaptive epigenetic changes that are passed on to offspring. These recently discovered aspects of epigenetics provide a new concept of clinical genetics

DNA methylation, the early-life social environment and behavioral disorders

One of the outstanding questions in behavioral disorders is untangling the complex relationship between nurture and nature. Although epidemiological data provide evidence that there is an interaction between genetics (nature) and the social and physical environments (nurture) in a spectrum of behavioral disorders, the main open question remains the mechanism. Emerging data support the hypothesis that DNA methylation, a covalent modification of the DNA molecule that is a component of its chemical structure, serves as an interface between the dynamic environment and the fixed genome. We propose that modulation of DNA methylation in response to environmental cues early in life serves as a mechanism of life-long genome adaptation. Under certain contexts, this adaptation can turn maladaptive resulting in behavioral disorders. This hypothesis has important implications on understanding, predicting, preventing, and treating behavioral disorders including autism that will be discussed

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function

Molecular Dynamics Studies of Calcium Binding to Beta Parvalbumin

Parvalbumin (PV) is a globular calcium-binding protein expressed primarily in skeletal muscle and secondarily in neuronal tissue. While defects in PV function have been correlated with a variety of severe pathological conditions, including epileptic seizures, engineered sequences have been shown to mitigate cardiac dysfunction in animal models, which could potentially benefit heart patients in Kentucky and the United States. Our computational studies of the beta PV isoform seek to understand calcium binding at the protein’s pseudo and canonical EF hand secondary structures. Specifically, we have employed molecular dynamics (MD) simulations to understand why calcium binds tightly in wild-type PV and even more tightly upon mutating an amino acid (Leucine-85-Phenylalanine) far from the calcium binding sites. Our MD simulations were analyzed to reveal changes in PV’s three dimensional structure, including alpha helical angles and interhelical distances, as well as their influence on the density of protein oxygens that directly bind calcium. These data may provide a thermodynamic basis for how mutations vary calcium affinity and more importantly, could guide re-engineering of PV to mitigate defective calcium signaling in heart cells. Broadly speaking, since the EF hand is common to a large class of proteins, we anticipate that our findings could shed light on related calcium-dependent proteins that modulate a wide range of physiological functions

Understanding Ion Binding Affinity and Selectivity in Beta Parvalbumin Using Molecular Dynamics and Mean Sphere Approximation Theory

Parvalbumin (PV) is a globular calcium-binding protein expressed primarily in skeletal muscle and secondarily in neuronal tissue. Defects in PV function have been correlated with a variety of severe pathological conditions, including epileptic seizures, while engineered sequences have been shown to mitigate cardiac dysfunction in animal models. Our computational studies of the beta PV isoform seek to quantify thermodynamic drivers of high affinity and selective calcium (Ca2+) binding at the pseudo and canonical EF structural motifs. Specifically, we employed molecular dynamics (MD) simulations and Mean Sphere Approximation (MSA) theory to quantify the structural and thermodynamic factors favoring Ca2+-binding relative to other common intracellular electrolytes in both EF-hands. Our MD simulations provided the coordination geometry and the density of metal-chelating oxygens within the EF-hand scaolds for both calcium and magnesium. These structural data inform the MSA model, from which the free energy and chemical potential within the metal binding site are predicted. This approach provided a thermodynamic basis for ion affinity and selectivity in beta-PV over a broad range of electrolyte compositions and concentrations that would be difficult to ascertain by MD alone. The minimal computational cost of MSA theory relative to MD further offers the potential to predict key thermodynamics quantities across a wide range of PV sequence homologs and Ca2+- binding proteins

Understanding Ion Binding Affinity and Selectivity in β‑Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory

Parvalbumin (PV) is a globular calcium (Ca²⁺)-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca²⁺ versus magnesium (Mg²⁺) binding at the protein’s two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the “local” thermodynamics of Ca²⁺ binding selectivity over Mg²⁺. Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca²⁺ and Mg²⁺, as well the conformational strain induced by Mg²⁺ relative to Ca²⁺ binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg²⁺ and Ca²⁺, yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca²⁺ versus Mg²⁺, strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue–residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca²⁺- and Mg²⁺-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca²⁺ binding proteins

Sex-dimorphism in human serum endocannabinoid and n-acyl ethanolamine concentrations across the lifespan

Abstract The endocannabinoid (ECB) system has recently been considered a potential treatment target for various clinical disorders. However, research around age- and sex-related changes within the ECB system is relatively limited. To improve our understanding of these changes, the current study measured arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG), oleoylethanolamine (OEA), palmitoylethanolamine (PEA), arachidonic acid (AA), cortisol, and progesterone in pooled serum samples stratified by sex (male and female) and age groups (5–15; 15–30; 30–45; 45–60; 60–75; 85+), using liquid-chromatography tandem mass spectrometry. Serum progesterone levels significantly increased in females of the 15–30 and 30–45 age groups, before declining. Significantly higher cortisol, AEA, 2-AG, OEA, and PEA were found in males and in older age, while significantly higher AA was found in females. Our results indicate that ECBs and related hormones exhibit sexual dimorphism in the age ranges that correspond with female pregnancy, menopause, and post menopause. Male testosterone levels most likely influences male ECB changes throughout the lifespan. Future research could capitalise on these findings by performing repeated measurements in individuals in a longitudinal style, to further refine the temporal profile of age-specific changes to the ECB system identified here

Neural and behavioral epigenetics; what it is, and what is hype

The ability to examine epigenetic mechanisms in the brain has become readily available over the last 20 years. This has led to an explosion of research and interest in neural and behavioral epigenetics. Of particular interest to researchers, and indeed the lay public, is the possibility that epigenetic processes, such as changes in DNA-methylation and histone modification, may provide a biochemical record of environmental effects. This has led to some fascinating insights into how molecular changes in the brain can control behavior. However, some of this research has also attracted controversy and, as is dealt with here, some overblown claims. This latter problem is partly linked to the shifting sands of what is defined as 'epigenetics'. In this review, I provide an overview of what exactly epigenetics is, and what is hype, with the aim of opening up a debate as to how this exciting field moves forward