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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Twenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC₅₀values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment.</p> <p>Conclusion</p> <p>Three compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.</p

Chemical Biology of Protein Arginine Modifications in Epigenetic Regulation

Review article on histone citrullination (arginine deimination), histone arginine methylation, and noncanonical histone arginine modifications

Protein Arginine Methylation and Citrullination in Epigenetic Regulation

The post-translational modification of arginine residues represents a key mechanism for the epigenetic control of gene expression. Aberrant levels of histone arginine modifications have been linked to the development of several diseases including cancer. In recent years, great progress has been made in understanding the physiological role of individual arginine modifications and their effects on chromatin function. The present review aims to summarize the structural and functional aspects of histone arginine modifying enzymes and their impact on gene transcription. We will discuss the potential for targeting these proteins with small molecules in a variety of disease states

Impact of GSK199 and GSK106 binding on protein arginine deiminase IV stability and flexibility: a computational approach

Protein arginine deiminase IV (PAD4) is a potential target for diseases including rheumatoid arthritis and cancers. Currently, GSK199 is a potent, selective yet reversible PAD4 inhibitor. Its derivative, GSK106, on the other hand, was reported as an inactive compound when tested against PAD4 assay. Although they had similar skeleton, their impact towards PAD4 structural and flexibility is unknown. In order to fill the research gap, the impact of GSK199 and GSK106 binding towards PAD4 stability and flexibility is investigated via a combination of computational methods. Molecular docking indicates that GSK199 and GSK106 are capable to bind at PAD4 pocket by using its back door with − 10.6 kcal/mol and − 9.6 kcal/mol, respectively. The simulations of both complexes were stable throughout 100 ns. The structure of PAD4 exhibited a tighter packing in the presence of GSK106 compared to GSK199. The RMSF analysis demonstrates significant changes between the PAD4-GSK199 and PAD4-GSK106 simulations in the regions containing residues 136, 160, 220, 438, and 606. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis shows a marked difference in binding free energies, with − 11.339 kcal/mol for the PAD4-GSK199 complex and 1.063 kcal/mol for the PAD4-GSK106 complex. The hydrogen bond analysis revealed that the GSK199 and GSK106 binding to PAD4 are assisted by six hydrogen bonds and three hydrogen bonds, respectively. The visualisation of the MD simulations revealed that GSK199 remained in the PAD4 pocket, whereas GSK106 shifted away from the catalytic site. Meanwhile, molecular dockings of benzoyl arginine amide (BAEE) substrate have shown that BAEE is able to bind to PAD4 catalytic site when GSK106 was present but not when GSK199 occupied the site. Overall, combination of computational approaches successfully described the behaviour of binding pocket of PAD4 structure in the presence of the active and inactive compounds

Discovery of new inhibitor for the Protein Arginine Deiminase Type 4 (PAD4) by Rational Design of α-Enolase-derived Peptides

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.5–1.0% of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35% with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors

Specificity Determining Features at the Interface of Biomolecular Complexes as Regulators of Biological Functions

Amino acid residues at the biomolecular interface play essential roles in many biological and cellular processes; relevant to this thesis, protein-protein interactions regulate signaling pathways and enzymatic activity, whereas protein-DNA interactions control gene expression, and protein-peptide interactions are central to the immune system. Biomolecular recognition and binding stability are largely determined by residues at the molecular interface. In this thesis, we focused on three biological datasets that are related to humans and human health: 1) dysregulated citrullination in the inflamed joints of rheumatoid arthritis patients, 2) a novel family of PRD-like transcription factors critical to the first few cell divisions in human life, and 3) epitopes that likely activate a cytotoxic T cell-mediated immune response against SARS-CoV-2 infection. For each dataset, in order to study the structural and functional consequences of molecular interactions, we applied a wide range of bioinformatics techniques to analyze sequences, structures and biological data retrieved from various databases, as well as taking into account experimental results from collaborators and from the literature. In rheumatoid arthritis, normally cytoplasmic peptidylarginine deiminase (PAD) enzymes citrullinate arginine residues in extracellular matrix (ECM) proteins. To examine specificity determining features that regulate the citrullination activity, we analyzed the sequence and structure data of the ECM proteins that were found citrullinated in chronically inflamed human joints. For citrullination, we found that an arginine side chain needs to be exposed to solvent but can arise from β-strands, α-helices, loops and β-turns. Moreover, there is no sequence motif linked to enzymatic activity. In addition, we studied the effect of citrullination on proteins important for a normal ECM, focusing on integrin binding to fibronectin and transforming growth factor-β (TGF-β). Citrullination of these proteins was found to inhibit cell attachment and spreading since PAD-treatment of the isoDGR motif in fibronectin and the RGD motif in TGF-β significantly reduced their binding with integrin αVβ3 and αVβ6, respectively. The expression of the human paired (PRD)-like transcription factors (TFs) are limited to the period of embryonic genome activation up to the 8-cell stage. We identified that one of these PRD-like TFs, LEUTX, binds to a TAATCC sequence motif. Sequence comparisons revealed that LEUTX protein is comprised of two domains: the DNA-binding homeodomain and a Leutx domain containing a transactivation domain. We identified specificity determining residues in the LEUTX homeodomain that are important for recognition of the TAATCC-containing 36 bp DNA motif enriched in genes involved in embryonic genome activation. We demonstrated using molecular models why a heterozygotic missense mutation A54V at the DNA-specificity determining position of LEUTX has significantly reduced overall transcriptional activity, as well as why the double mutant – I47T and A54V – form of LEUTX restores binding to the DNA motif similarly to that seen in the I47T mutation alone. At the onset of the COVID-19 pandemic we sought to understand the molecular factors that trigger the cytotoxic T cell-mediated immune response against the SARS-CoV-2 virus, taking advantage of binding data and 3D structures for related viruses and other pathogenic organisms. We first predicted the MHC class I (MHC-I)-specific immunogenic epitopes of length 8- to 11 amino acids from the SARS-CoV-2 proteins. Next, we predicted that the 9-mer epitopes would have the highest potential to elicit a strong immune response. For experimental validation, the predicted 9-mer epitopes were matched with the SARS-CoV-derived epitopes that are known to elicit an effective T cell response in vitro. Furthermore, our observations provide a structural explanation for the binding of SARS-CoV-2 epitopes to MHC-I molecules, identifying conserved immunogenic epitopes essential for understanding the pathogenesis of COVID-19. The three investigated datasets were made in concert with collaborative experimental studies and/or considering publicly available experimental data. The experimental studies generally provided the starting point for the in silico studies, which in turn had the objective of providing a detailed explanation of the experimental results. Furthermore, the in silico results could be used to devise novel and focused experiments, suggesting that bioinformatics predictions and wet-laboratory experimental investigations optimally take place with multiple advantages. Overall, this thesis demonstrates the synergy that is possible by applying this interdisciplinary approach to understanding the consequences of molecular interactions.Aminosyror i kontaktytan mellan olika biomolekyler spelar en viktig roll i många biologiska och cellulära processer; relevanta interaktioner för den här avhandlingen är protein-protein interaktioner som reglerar signaleringsrutter och enzymatisk aktivitet, protein-DNA interaktioner som kontrollerar genexpression, samt protein-peptid interaktioner som har en central roll i immunförsvaret. Biomolekylär igenkänning och bindningsstabilitet beror till stor del på de aminosyror som finns i den molekylära kontaktytan. I den här avhandlingen fokuserade vi på tre biologiska dataset som är relaterade till människor och människors hälsa: 1) felreglerad citrullinering i inflammerade leder hos patienter med reumatoid artrit, 2) en nyupptäckt familj av PRD (human paired)-lika transkriptionsfaktorer som är nödvändiga för de första celldelningarna i människolivet, och 3) epitoper som troligen aktiverar en cytotoxisk T-cell-förmedlad immunrespons mot SARS-CoV-2 infektioner. För att studera de strukturella och funktionella konsekvenserna av de molekylära interaktionerna i varje dataset, användes en mängd olika bioinformatiska tekniker för att analysera sekvenser, strukturer och biologiska data från olika databaser och dessutom beaktades experimentella resultat från samarbetspartners och från litteraturen. I reumatoid artrit citrullinerar vanligen PAD (cytoplasmatiska peptidyl arginin deiminas)-enzymer arginin-aminosyror i proteiner i det extracellulära matrixet (ECM). För att undersöka egenskaper som avgör specificiteten hos citrullineringsaktiviteten analyserade vi sekvens- och strukturdata för ECM-proteiner som blir citrullinerade i kroniskt inflammerade leder hos människor. Vi upptäckte att en argininsidokedja måste vara i kontakt med det omgivande lösningsmedlet för att kunna citrullineras, att de kan finnas i beta-strängar, alfa-helixar och beta-svängar, samt att det inte finns några sekvensmotiv som är kopplade till enzymatisk aktivitet. Utöver detta studerade vi effekten av citrullinering på proteiner som är viktiga för normal extracellulär matrix, med fokus på integrinbinding till fibronektin och TGF-β (transforming growth factor-β). Citrullinering av dessa proteiner upptäcktes inhibera cellvidhäftning och spridning eftersom PAD-behandling av isoDGR-motivet i fibronektin och RGD-motivet i TGF-β ordentligt reducerar deras bindning till integrin αVβ3 och αVβ6, respektive. Expressionsnivåerna av PRD-lika transkriptionsfaktorer (TF) är begränsade till perioden av zygotens genomaktivering upp till 8-cells stadiet. Vi identifierade att en av dessa PRD-lika transkriptionsfaktorer, LEUTX, binder till ett TAATCC sekvensmotiv. Sekvensjämförelser avslöjade att LEUTX proteinet består av två domäner, det DNA-bindande homeodomänet och en leutx-domän som innehåller en transaktiveringsdomän. Vi identifierade specificitetsbestämmande aminosyror i LEUTX homeodomänen som är viktiga för igenkänning av TAATCC-innehållande 36 baspars DNA-motivet som är berikad med gener involverade i zygotens genomaktivering. Vi använde molekylära modeller för att visa varför en heterozygotisk missense-mutation, A54V, i DNA-specificitetsbestämmande positionen i LEUTX har ordentligt minskad generell transkriptionsaktivitet, och varför dubbelmutanten I47T och A54V återställer bindning till DNA-motivet på samma sätt som observerats i enbart I47T mutationen. När COVID-19 pandemin inleddes försökte vi förstå de molekylära faktorer som startar den cytotoxiska T-cell-förmedlade immunresponsen mot SARS-CoV-2 viruset, genom att utnyttja bindningsdata och 3D strukturer för relaterade virus och andra patogena organismer. Vi förutspådde först MHC klass I (MHC-I)-specifika immunogena epitoper av längden 8 till 11 aminosyror från SARS-CoV-2 proteiner. Därefter förutspådde vi att epitoper bestående av 9 aminosyror hade den högsta potentialen att orsaka en stark immunrespons. För experimentell validering matchades de 9 aminosyror långa epitoperna med epitoper från SARS-CoV som man vet att orsakar en effektiv T-cell respons in vitro. Våra observationer bidrar också med en strukturell förklaring för bindningen av SARS-CoV-2 epitoper till MHC-I molekyler, vilket identifierar konserverade immunogena epitoper som är nödvändiga för att förstår patogenesen hos COVID-19. De tre undersökta dataseten gjordes i samarbete med experimentella studier och/eller genom att ta allmänt tillgängliga experimentella data i beaktande. De experimentella studierna gav en startpunkt för in silico-studierna, vilka i sin tur hade som mål att ge en detaljerad förklaring till de experimentella resultaten. In silico-resultaten kan också användas för att utveckla nya och fokuserade experiment, vilket indikerar att bioinformatiska förutspåelser och experimentella studier optimalt sker med många fördelar. Över lag visar denna avhandling synergin som är möjlig genom att använda detta interdisciplinära arbetssätt för att förstå konsekvenserna av molekylära interaktioner

Characterising the Role of the Calcium-dependent Citrullinating Enzyme Peptidyl Arginine Deiminase 2 in Ovarian Cancer

Epithelial ovarian cancer (EOC) represents the fifth most common cause of cancer mortality among women worldwide and accounts for the highest fatalities amongst gynaecological malignancies. The dysregulation of calcium-dependent peptidyl arginine deiminase 2 (PADI2) plays a key role in the tumorigenesis of several cancers; however, the role of PADI2 in the pathogenesis of EOC is yet to be investigated. Using RNA-seq and microarray data from primary serous ovarian cancers (The Cancer Genome Atlas data set) combined with survival data, the expression of PADI2 was assessed using each platform (n=262 and n=564). Kaplan-Meier analysis and Log-rank tests showed an association of PADI2 mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of PADI2 was associated with improved survival. Expression studies were used to examine the overexpression and knockout (CRISPR/Cas9 editing) of PADI2 expression, respectively, in the human-derived high-grade serous OVCAR-4 and mouse-derived EOC ID8-Luc2 cell lines. In OVCAR-4 cells, PADI2 overexpression reduced proliferation (22%) and cellular aggregation (94%), while increasing apoptosis (34%) and autophagy (38%), in a Ca2+- and citrullination-dependent manner, at 72 hours. PADI2 overexpression also induced cisplatin cytotoxicity by 10% at 72 hours. In ID8-Luc2 cells, PADI2 overexpression significantly decreased cisplatin cytotoxicity by 24%, independently of exogenous Ca2+ supplementation or induced citrullination, at 72 hours. In addition, qRT-PCR validation of TCGA gene co-expression data indicated that PADI2 overexpression correlated with expression of a number of genes. This was confirmed in functional studies where there was a 1.69-fold increase in ARHGEF10L and 0.75-fold decrease in FZD5 expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of PADI2 showed that higher PADI2 confers decreased survival of EOC patients. Conversely, PADI2 overexpression in vitro, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating FZD5 and ARHGEF10L. Collectively, this work suggests that PADI2 may serve as a potential therapeutic target in human EOC. Codon based selection analyses were used to test for evidence of positive selection in PADI2. The phylogenetic maximum likelihood analysis of PADI2 orthologues retrieved from 31 species indicated that there was no evidence of positive selection in PADI2 codons, but numerous residues were under evolutionary constraint

Characterising the Role of the Calcium-dependent Citrullinating Enzyme Peptidyl Arginine Deiminase 2 in Ovarian Cancer

Epithelial ovarian cancer (EOC) represents the fifth most common cause of cancer mortality among women worldwide and accounts for the highest fatalities amongst gynaecological malignancies. The dysregulation of calcium-dependent peptidyl arginine deiminase 2 (PADI2) plays a key role in the tumorigenesis of several cancers; however, the role of PADI2 in the pathogenesis of EOC is yet to be investigated. Using RNA-seq and microarray data from primary serous ovarian cancers (The Cancer Genome Atlas data set) combined with survival data, the expression of PADI2 was assessed using each platform (n=262 and n=564). Kaplan-Meier analysis and Log-rank tests showed an association of PADI2 mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of PADI2 was associated with improved survival. Expression studies were used to examine the overexpression and knockout (CRISPR/Cas9 editing) of PADI2 expression, respectively, in the human-derived high-grade serous OVCAR-4 and mouse-derived EOC ID8-Luc2 cell lines. In OVCAR-4 cells, PADI2 overexpression reduced proliferation (22%) and cellular aggregation (94%), while increasing apoptosis (34%) and autophagy (38%), in a Ca2+- and citrullination-dependent manner, at 72 hours. PADI2 overexpression also induced cisplatin cytotoxicity by 10% at 72 hours. In ID8-Luc2 cells, PADI2 overexpression significantly decreased cisplatin cytotoxicity by 24%, independently of exogenous Ca2+ supplementation or induced citrullination, at 72 hours. In addition, qRT-PCR validation of TCGA gene co-expression data indicated that PADI2 overexpression correlated with expression of a number of genes. This was confirmed in functional studies where there was a 1.69-fold increase in ARHGEF10L and 0.75-fold decrease in FZD5 expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of PADI2 showed that higher PADI2 confers decreased survival of EOC patients. Conversely, PADI2 overexpression in vitro, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating FZD5 and ARHGEF10L. Collectively, this work suggests that PADI2 may serve as a potential therapeutic target in human EOC. Codon based selection analyses were used to test for evidence of positive selection in PADI2. The phylogenetic maximum likelihood analysis of PADI2 orthologues retrieved from 31 species indicated that there was no evidence of positive selection in PADI2 codons, but numerous residues were under evolutionary constraint