Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis

Post-translational modifications (PTMs) of histone proteins are a hallmark of epigenetic regulation. They provide a mechanism to modulate chromatin structure and constitute the main features of the so-called “histone code”.1 The proposed function of this code is to integrate exogenous and endogenous signals into a diverse set of histone PTM patterns to enable the epigenetic control of gene expression. The key regulators of this process are the so-called “writers” and “erasers”, which act by dynamically modifying histones, and other chromatin-associated proteins, as well as the “readers”, which interpret these PTMs, thereby facilitating the downstream activation or repression of gene expression.2

The writers are histone-modifying enzymes that can be grouped according to their amino acid substrate preference, affecting mainly lysine, arginine, and serine residues.3 These enzymes can be further classified according to the type of covalent modification that they catalyze. Histone modifications include acetylation, methylation, phosphorylation, and the more recently described modifications of citrullination, ubiquitination, SUMOylation, proline isomerization, O-GlcNAcylation, and ADP-ribosylation.1b,3 On the basis of detailed mass spectrometric analyses, there are at least 15 different types of covalent histone modifications,4 and since histone proteins are modified at multiple sites, and different stoichiometries, the total number of histone marks is >160.5 Although our understanding of how histone modifications contribute to the epigenetic control of gene transcription has grown immensely over the past ∼15 years, the precise impact of this vast number of modifications, not to mention the crosstalk between them, has yet to be fully realized.

Histone proteins are small, highly basic proteins consisting of a globular domain and flexible N-terminal and C-terminal tails that protrude from the nucleosome. The core histone proteins (histones H2A, H2B, H3, and H4) form an octameric particle consisting of two H2A–H2B dimers and an H3–H4 tetramer, around which wrap two helical turns of DNA (∼150 bp).6 This structure, which is generally termed a nucleosome, comprises the basic building block of higher order chromatin structures that are further organized through the function of linker histones such as histone H1. On the basis of nucleosome positioning studies, around 80% of the yeast genome and even 99% of the mappable genome of human granulocytes is occupied by nucleosomes, thereby highlighting the importance of nucleosome-packaged DNA for eukaryotic cells.7 Importantly, while histone PTMs are found throughout the entire protein, they are most often clustered within the N-terminal tail. Although research on histone lysine modifications has drawn considerable attention and even resulted in the approval of novel anticancer drugs,8 the modification of histone arginine residues is a recently emerging nucleosomal mark of similar importance (Figure ​(Figure11).



Figure 1

N-terminal tails of histone proteins are the preferred targets of histone-modifying enzymes. The major modifications of histone arginine residues are citrullination and methylation. Abbreviations: Cit, citrulline; MMA, monomethylarginine; ADMA, asymmetric ...

Chemical biology of protein arginine modifications in epigenetic regulation.

Molecular similarity is a key concept in drug discovery. It is based on the assumption that structurally similar molecules frequently have similar properties. Assessment of similarity between small molecules has been highly effective in the discovery and development of various drugs. Especially, two-dimensional (2D) similarity approaches have been quite popular due to their simplicity, accuracy and efficiency. Recently, the focus has been shifted towards the development of methods involving the representation and comparison of three-dimensional (3D) conformation of small molecules. Among the 3D similarity methods, evaluation of shape similarity is now gaining attention for its application not only in virtual screening but also in molecular target prediction, drug repurposing and scaffold hopping. A wide range of methods have been developed to describe molecular shape and to determine the shape similarity between small molecules. The most widely used methods include atom distance-based methods, surface-based approaches such as spherical harmonics and 3D Zernike descriptors, atom-centered Gaussian overlay based representations. Several of these methods demonstrated excellent virtual screening performance not only retrospectively but also prospectively. In addition to methods assessing the similarity between small molecules, shape similarity approaches have been developed to compare shapes of protein structures and binding pockets. Additionally, shape comparisons between atomic models and 3D density maps allowed the fitting of atomic models into cryo-electron microscopy maps. This review aims to summarize the methodological advances in shape similarity assessment highlighting advantages, disadvantages and their application in drug discovery.

/pdf/advances-in-the-development-of-shape-similarity-methods-and-3rr7ev32me.pdf

Advances in the Development of Shape Similarity Methods and Their Application in Drug Discovery.

Dysregulated neutrophil activation contributes to the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Neutrophil-derived reactive oxygen species (ROS) and granule proteases are implicated in damage to and destruction of host tissues in both conditions (cartilage in RA, vascular tissue in SLE) and also in the pathogenic post-translational modification of DNA and proteins. Neutrophil-derived cytokines and chemokines regulate both the innate and adaptive immune responses in RA and SLE, and neutrophil extracellular traps (NETs) expose nuclear neoepitopes (citrullinated proteins in RA, double-stranded DNA and nuclear proteins in SLE) to the immune system, initiating the production of auto-antibodies (ACPA in RA, anti-dsDNA and anti-acetylated/methylated histones in SLE). Neutrophil apoptosis is dysregulated in both conditions: in RA, delayed apoptosis within synovial joints contributes to chronic inflammation, immune cell recruitment and prolonged release of proteolytic enzymes, whereas in SLE enhanced apoptosis leads to increased apoptotic burden associated with development of anti-nuclear auto-antibodies. An unbalanced energy metabolism in SLE and RA neutrophils contributes to the pathology of both diseases; increased hypoxia and glycolysis in RA drives neutrophil activation and NET production, whereas decreased redox capacity increases ROS-mediated damage in SLE. Neutrophil low-density granulocytes (LDGs), present in high numbers in the blood of both RA and SLE patients, have opposing phenotypes contributing to clinical manifestations of each disease. In this review we will describe the complex and contrasting phenotype of neutrophils and LDGs in RA and SLE and discuss their discrete roles in the pathogenesis of each condition. We will also review our current understanding of transcriptomic and metabolomic regulation of neutrophil phenotype in RA and SLE and discuss opportunities for therapeutic targeting of neutrophil activation in inflammatory auto-immune disease.

/pdf/neutrophils-in-the-pathogenesis-of-rheumatoid-arthritis-and-4mk7oe3ym6.pdf

Neutrophils in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus: Same Foe Different M.O

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC’s resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK) pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg) exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg). Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history of safe human use, our discovery of fluspirilene as a potential anti-HCC drug may present an immediately applicable clinical therapy.

/pdf/in-silico-identification-and-in-vitro-and-in-vivo-validation-160jd4r5qg.pdf

In Silico Identification and In Vitro and In Vivo Validation of Anti-Psychotic Drug Fluspirilene as a Potential CDK2 Inhibitor and a Candidate Anti-Cancer Drug

Three different macroporous resins (XAD7HP, DAX-8, and XAD4) were evaluated for their adsorption and desorption properties in preparing flavonoid-enriched oil palm (Elaeis guineensis Jacq.) leaf extract. The influences of initial concentration, solution pH, contact time, and desorption solvent (ethanol) concentration were determined by static sorption/desorption methods. The optimal condition for adsorption of flavonoids was achieved when the solution of the extract was adjusted to pH 7, reaching equilibrium after 1440 min at 298 K. The adsorption process was well described by a pseudo-second-order kinetics model, while the adsorption isotherm data fitted well with a Freundlich model. The adsorption by each resin was via an exothermic and physical adsorption process. Based on the static experiment results, XAD7HP was found to be the most appropriate adsorbent, while 80% ethanol was the best solvent for desorbent. Further evaluation of its dynamic adsorption and desorption characteristics on a packed glass column showed that XAD7HP could enrich the OPL total flavonoid content by a 3.57-fold increment. Moreover, UHPLC-UV/PDA and UHPLC-MS/MS analysis revealed that apigenin and luteolin derivatives were selectively adsorbed by XAD7HP. Additionally, both the crude OPL extract and the flavonoid-enriched fraction have good DPPH and NO free radical scavenging activities. Multiple interactions between the flavonoids and cross-linked polymeric XAD7HP resin through van der Waals forces and hydrogen bonding described the sorption processes. Therefore, by utilizing this method, the flavonoid-enriched fraction from crude OPL extract could be used as a potential bioactive ingredient in nutraceutical and pharmaceutical applications at minimum cost with optimum efficiency.

https://www.mdpi.com/1420-3049/25/4/778/pdf

Adsorption and Desorption Properties of Total Flavonoids from Oil Palm (Elaeis guineensis Jacq.) Mature Leaf on Macroporous Adsorption Resins.

Background
Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays.

/pdf/discovery-of-a-new-class-of-inhibitors-for-the-protein-40utm9xwpx.pdf

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening

Oil palm leaves (OPL) containing flavonoid C-glycosides are abundantly generated as oil palm byproducts. The performances of three macroporous resins with different physical and chemical properties for the enrichment of isoorientin, orientin, vitexin, and isovitexin from acid-hydrolyzed OPL (OPLAH) extract were screened. The XAD7HP resin exhibited the best sorption capacities for the targeted flavonoid C-glycosides and was thus selected for further evaluation. Static adsorption using the XAD7HP resin under optimal conditions (extract adjusted to pH 5, shaken at 298 K for 24 h) gave adsorption kinetics that fit well with a pseudo-second-order kinetic model. The adsorption of isoorientin and orientin was well described by Langmuir isotherms, while vitexin and isovitexin fit well with the Freundlich isotherms. Dynamic sorption trials using the column-packed XAD7HP resin produced 55–60-fold enrichment of isovitexin and between 11 and 25-fold enrichment of isoorientin, vitexin, and orientin using aqueous ethanol. The total flavonoid C-glycoside-enriched fractions (enriched OPLAH) with isoorientin (247.28–284.18 µg/mg), orientin (104.88–136.19 µg/mg), vitexin (1197.61–1726.11 µg/mg), and isovitexin (13.03–14.61 µg/mg) showed excellent antioxidant free radical scavenging activities compared with their crude extracts, with IC50 values of 6.90–70.63 µg/mL and 44.58–200.00 µg/mL, respectively. Hence, this rapid and efficient procedure for the preliminary enrichment of flavonoid C-glycosides by using macroporous resin may have practical value in OPL biomass waste utilization programs to produce high value-added products, particularly in the nutraceuticals, cosmeceuticals, pharmaceuticals, and fine chemicals industries.

Adsorption/Desorption Characteristics and Simultaneous Enrichment of Orientin, Isoorientin, Vitexin and Isovitexin from Hydrolyzed Oil Palm Leaf Extract Using Macroporous Resins

Protein Arginine Deiminase type 4 (PAD4) is a new therapeutic target for the treatment of rheumatoid arthritis. In this study, ligand-based virtual screening with the integration with drug repurposing strategy was applied to the discovery of PAD4 inhibitors. Ultrafast Shape Recognition (USR) was used to search for compounds with similar shape to a previously reported inhibitor with harmful side-effects, i.e., streptonigrin. Thirty five lead-like compounds and two existing drugs were obtained from virtual screening and their inhibitory activity was tested at fixed concentration of 100 μM. Five lead-like compounds showed significant inhibition on the enzymatic activity of PAD4. The potency of the best compound was investigated by carrying out IC50 study. Importantly, the structure of the best of these new active molecules was strikingly different from that of streptonigrin. Furthermore, this new PAD4 inhibitor is the most potent to date found by a computational approach and its structure can be optimized in the future for the design of an even better inhibitor of PAD4.

/pdf/ligand-based-virtual-screening-for-the-discovery-of-4tl931ncfp.pdf

Ligand-Based Virtual Screening for the Discovery of Inhibitors for Protein Arginine Deiminase Type 4 (PAD4)

Protein arginine deiminase type IV (PAD4) is responsible for the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullinated protein is the autoantigen in rheumatoid arthritis, and therefore, PAD4 is currently a promising therapeutic target for the disease. Recently, we reported the importance of the furan ring in the structure of PAD4 inhibitors. In this study, the furan ring was incorporated into peptides to act as the "warhead" of the inhibitors for PAD4. IC50 studies showed that the furan-containing peptide-based inhibitors were able to inhibit PAD4 to a better extent than the furan-containing small molecules that were previously reported. The best peptide-based inhibitor inhibited PAD4 reversibly and competitively with an IC50 value of 243.2 ± 2.4 μm. NMR spectroscopy and NMR-restrained molecular dynamic simulations revealed that the peptide-based inhibitor had a random structure. Molecular docking studies showed that the peptide-based inhibitor entered the binding site and interacted with the essential amino acids involved in the catalytic activity. The peptide-based inhibitor could be further developed into a therapeutic drug for rheumatoid arthritis.

/pdf/novel-furan-containing-peptide-based-inhibitors-of-protein-1fs8u7u3ji.pdf

Chian Ying Teo

Papers

Adsorption and Desorption Properties of Total Flavonoids from Oil Palm (Elaeis guineensis Jacq.) Mature Leaf on Macroporous Adsorption Resins.

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening

Adsorption/Desorption Characteristics and Simultaneous Enrichment of Orientin, Isoorientin, Vitexin and Isovitexin from Hydrolyzed Oil Palm Leaf Extract Using Macroporous Resins

Ligand-Based Virtual Screening for the Discovery of Inhibitors for Protein Arginine Deiminase Type 4 (PAD4)

Novel furan-containing peptide-based inhibitors of protein arginine deiminase type IV (PAD4).