Wesley H. Brooks

Epigenetics and autoimmunity.

Advances in genetics, such as sequencing of the human genome, have contributed to identification of susceptible genetic patterns in autoimmune diseases (AID). However, genetics is only one aspect of the diseases that does not reflect the influence of environment, sex or aging. Epigenetics, the control of gene packaging and expression independent of alterations in the DNA sequence, is providing new directions linking genetics and environmental factors. Recent findings have contributed to our understanding of how epigenetic modifications could influence AID development, showing differences between AID patients and healthy controls but also showing how one disease differs from another. With regards to epigenetic abnormalities, DNA methylation and histone modifications could be affected leading to large spatial and temporal changes in gene regulation. Other epigenetic processes, such as the influence of the ionic milieu around chromatin and DNA supercoiling stresses may be suspected also. The newly described role of microRNAs in control of gene expression is important by promoting or suppressing autoreactivity in AID. As a consequence control of cellular processes is affected becoming conducive, for example, to the development of autoreactive lymphocytes in systemic lupus erythematosus, synoviocyte proliferation in rheumatoid arthritis, or neural demyelination in multiple sclerosis. Application of epigenetics to AID is in its infancy and requires new hypotheses, techniques, tools, and collaborations between basic epigenetic researchers and autoimmune researchers in order to improve our comprehension of AID. From this will arise new therapeutics, means for early intervention, and perhaps prevention.

DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus.

Abstract Systemic lupus erythematosus (SLE) disease is an autoimmune disease of unknown aetiology that affects predominantly women of child bearing age. Since previous studies, including ours, have demonstrated that CD4+ T cells and B cells from SLE patients are defective in their ability to methylate their DNA upon antigen stimulation, the aim of this study was to investigate whether DNA demethylation affects the transcription of HRES-1 in B cells. HRES-1 is the prototype of Human Endogenous Retrovirus (HERV) overexpressed in SLE. We have observed that SLE B cells were characterized by their incapacity to methylate the HRES-1 promoter, both in unstimulated and in anti-IgM stimulated B cells. In turn, HRES-1/p28 expression was increased in SLE B cells after B cell receptor engagement, but not in controls. In SLE B cells the Erk/DNMT1 pathway was defective. In addition, blocking the autocrine-loop of IL-6 in SLE B cells with an anti-IL-6 receptor monoclonal antibody restores DNA methylation and control of HRES-1/p28 expression became effective. As a consequence, a better understanding of HERV dysregulation in SLE reinforces our comprehension of the disease and opens new therapeutic perspectives.

X chromosome inactivation and autoimmunity.

Autoimmune diseases appear to have multiple contributing factors including genetics, epigenetics, environmental factors, and aging. The predominance of females among patients with autoimmune diseases suggests possible involvement of the X chromosome and X chromosome inactivation. X chromosome inactivation is an epigenetic event resulting in multiple levels of control for modulation of the expression of X-linked genes in normal female cells such that there remains only one active X chromosome in the cell. The extent of this control is unique among the chromosomes and has the potential for problems when regulation is disrupted. Here we discuss the X chromosome inactivation process and how the X chromosome and X chromosome inactivation may be involved in development of autoimmune disorders.

Computational Validation of the Importance of Absolute Stereochemistry in Virtual Screening

Consideration of stereochemistry early in the identification and optimization of lead compounds can improve the efficiency and efficacy of the drug discovery process and reduce the time spent on subsequent drug development. These improvements can result by focusing on specific enantiomers that have the desired potential therapeutic effect (eutomers), while removing from consideration enantiomers that may have no, or even undesirable, effects (distomers). A virtual screening campaign that correctly takes stereochemical information into account can, in theory, be utilized to provide information about the relative binding affinities of enantiomers. Thus, the proper enumeration of the relevant stereoisomers in general, and enantiomeric pairs in particular, of chiral compounds is crucial if one is to use virtual screening as an effective drug discovery tool. As is obvious, in cases where no stereochemical information is provided for chiral compounds in a 2D chemical database, then each possible stereoisomer should be generated for construction of the subsequent 3D database to be used for virtual screening. However, acute problems can arise in 3D database construction when relative stereochemistry is encoded in a 2D database for a chiral compound containing multiple stereogenic atoms but absolute stereochemistry is not implied. In this case, we report that generation of enantiomeric pairs is imperative in database development if one is to obtain accurate docking results. A study is described on the impact of the neglect of enantiomeric pairs on virtual screening using the human homolog of murine double minute 2 (MDM2) protein, the product of a proto-oncogene, as the target. Docking in MDM2 with GLIDE 4.0 was performed using the NCI Diversity Set 3D database and, for comparison, a set of enantiomers we created corresponding to mirror image structures of the single enantiomers of chiral compounds present in the NCI Diversity Set. Our results demonstrate that potential lead candidates may be overlooked when databases contain 3D structures representing only a single enantiomer of racemic chiral compounds.

The contribution of epigenetics in Sjögren’s Syndrome

Sjögren’s syndrome (SS) is a chronic autoimmune epithelitis that combines exocrine gland dysfunctions and lymphocytic infiltrations. While the pathogenesis of SS remains unclear, its etiology is multifunctional and includes a combination of genetic predispositions, environmental factors, and epigenetic factors. Recently, interest has grown in the involvement of epigenetics in autoimmune diseases. Epigenetics is defined as changes in gene expression, that are inheritable and that do not entail changes in the DNA sequence. In SS, several epigenetic mechanisms are defective including DNA demethylation that predominates in epithelial cells, an abnormal expression of microRNAs, and abnormal chromatin positioning-associated with autoantibody production. Last but not least, epigenetic modifications are reversible as observed in minor salivary glands from SS patients after B cell depletion using rituximab. Thus epigenetic findings in SS open new perspectives for therapeutic approaches as well as the possible identification of new biomarkers.

Discovery and Synthesis of Hydronaphthoquinones as Novel Proteasome Inhibitors

Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed, and compound 1 was synthesized in-house to confirm the structure and activity (IC(50) = 3.0 ± 1.6 μM [n = 25]). Novel hydronaphthoquinone derivatives of 1 were designed, synthesized, and evaluated as proteasome inhibitors. The structure-activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties, were the most active compounds with up to 20-fold greater CT-L inhibition than compound 1 (compounds 15e, 15f, 15h, 15j, IC(50) values around 200 nM, and compound 29, IC(50) = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC(50) = 0.44 to 1.01 μM) than 1 (IC(50) = 3.54 to 7.22 μM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analogue 39 inhibited proteasome CT-L activity irreversibly.

Epigenetics and Sjogren’s Syndrome

There is growing evidence that epigenetics, the study of heritable changes in gene expression that do not involve mutations in the DNA itself, may play an essential role in autoimmune diseases (AID). In Sjögrens syndrome (SS), a chronic AID characterized by an epithelis of the exocrine glands, epigenetic studies have focused on three mechanisms: DNA methylation and its consequences including human endogenous retrovirus (HERV) expression; microRNA expression; and protein post-translational modifications associated with autoantibody production. Although in its infancy, comprehension of the epigenetic (dys)regulation in SS may help us to understand: why SS affects predominantly middle-aged women; why genetically predisposed individuals develop SS but not others; why flare-ups occur; why treatment responses differ between patients; and why some patients develop lymphoma. From these studies will arise a better comprehension of the pathophysiology of SS as well as development of new diagnostic and prognostic biomarkers, and novel therapeutics for prevention and perhaps early intervention.

Autoimmune Diseases and Polyamines

Genetics and environmental factors have important roles in autoimmune diseases but neither has given us sufficient understanding of these mysterious diseases. Therefore, we are now looking closer at epigenetics, an interface between genetics and environmental factors. Epigenetics can be defined as reversible heritable changes to chromatin that can alter gene expression without altering the gene’s DNA sequence. Methylation of DNA and histones are primary means of epigenetic control. By adding methyl groups to DNA and histones, it can limit accessibility of the underlying gene thereby altering the amount of gene expression. The methyl group is derived from an essential molecule in the cell, S-adenosylmethionine (SAM). However, a group of small molecules called polyamines also require SAM for their synthesis. Polyamines are essential for many cellular functions and polyamine activity is increased in many autoimmune diseases. Presented here is the “polyamine hypothesis” in which increased polyamine synthesis competes with cellular methylation (epigenetic control) for SAM. It is proposed that increased polyamine activity can cause disruption of cellular methylation, which can lead to abnormal expression of previously sequestered genes and disruption of other methylation-dependent cellular processes.

New Insights into the Design of Inhibitors of Human S-Adenosylmethionine Decarboxylase: Studies of Adenine C8 Substitution in Structural Analogues of S-Adenosylmethionine

S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C8-substituted adenine analogues bound in the active site.

Increased Polyamines Alter Chromatin and Stabilize Autoantigens in Autoimmune Diseases

Polyamines are small cations with unique combinations of charge and length that give them many putative interactions in cells. Polyamines are essential since they are involved in replication, transcription, translation, and stabilization of macro-molecular complexes. However, polyamine synthesis competes with cellular methylation for S-adenosylmethionine, the methyl donor. Also, polyamine degradation can generate reactive molecules like acrolein. Therefore, polyamine levels are tightly controlled. This control may be compromised in autoimmune diseases since elevated polyamine levels are seen in autoimmune diseases. Here a hypothesis is presented explaining how polyamines can stabilize autoantigens. In addition, the hypothesis explains how polyamines can inappropriately activate enzymes involved in NETosis, a process in which chromatin is modified and extruded from cells as extracellular traps that bind pathogens during an immune response. This polyamine-induced enzymatic activity can lead to an increase in NETosis resulting in release of autoantigenic material and tissue damage.