Molecular targeting of protein arginine deiminases to suppress colitis and prevent colon cancer

Ulcerative colitis (UC) is a chronic disease, in which the lining of the colon becomes inflamed and develops ulcers leading to abdominal pain, diarrhea, and rectal bleeding. The extent of these symptoms depends on disease severity. The protein arginine deiminase (PAD) family of enzymes converts peptidyl-Arginine to peptidyl-Citrulline through citrullination. PADs are dysregulated, with abnormal citrullination in many diseases, including UC and colorectal cancer (CRC). We have developed the small molecule, pan-PAD inhibitor, Chlor-amidine (Cl-amidine), with multiple goals, including treating UC and preventing CRC. Building off our recent results showing that: 1) Cl-amidine suppresses colitis in vivo in a dextran sulfate sodium (DSS) mouse model; and 2) Cl-amidine induces microRNA (miR)-16 in vitro causing cell cycle arrest, we tested the hypothesis that Cl-amidine can prevent tumorigenesis and that miR-16 induction, by Cl-amidine, may be involved in vivo. Consistent with our hypothesis, we present evidence that Cl-amidine, delivered in the drinking water, prevents colon tumorigenesis in our mouse model of colitis-associated CRC where mice are given carcinogenic azoxymethane (AOM), followed by multiple cycles of 2% DSS to induce colitis. To begin identifying mechanisms, we examined the effects of Cl-amidine on miR-16. Results show miR-16 suppression during the colitis-to-cancer sequence in colon epithelial cells, which was rescued by drinking Cl-amidine. Likewise, Ki67 and cellular proliferation targets of miR-16 (Cyclins D1 and E1) were suppressed by Cl-amidine. The decrease in cell proliferation markers and increase in tumor suppressor miRNA expression potentially define a mechanism of how Cl-amidine is suppressing tumorigenesis in vivo

Potential role for PADI-mediated histone citrullination in preimplantation development.

BACKGROUND: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. RESULTS: In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. CONCLUSIONS: Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation

Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.

Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.

BACKGROUND: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects. METHODS: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes. RESULTS: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 x 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45alpha, and Ki67. CONCLUSION: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy

An improved synthesis of haloaceteamidine-based inactivators of protein arginine deiminase 4 (PAD4)

Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form citrulline and ammonia. This enzyme has been implicated in several disease states, e.g. rheumatoid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solution-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been developed. This synthesis proceeds in 80% yield over 4 steps at a significantly (12-fold) lower cost

Synthesis and evaluation of 2-ethynyl-adenosine-5′-triphosphate as a chemical reporter for protein AMPylation

Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate. One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death. Given the resurgent interest in this modification, there is a critical need for chemical tools that better facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the synthesis of 2-ethynyl-adenosine-5′-triphosphate (2eATP) and its utilization as a non-radioactive chemical reporter for protein AMPylation

1-adamantanethiol as a versatile nanografting tool

Strategies to regulate the self-assembly of adsorbates to create surface structures with molecularscale features and organization are of broad interest to nanoscience, biochemistry, and engineering. One approach utilizes molecules with tailored intermolecular interaction strengths and topologies to direct molecular self-assembly as exemplified by the adsorption of 1-adamantanethiol molecules on Au{111} substrates. 1-Adamantanethiolate self-assembled monolayers exhibit decreased packing densities and weaker intermolecular interaction strengths than n-alkanethiolate self-assembled monolayers, which result in their complete displacement upon exposure to n-alkanethiol molecules. Herein, we explore the capabilities of the atomic force microscopy-based lithographic technique, nanografting, to fabricate chemical patterns comprised of 1-adamantanethiolate monolayers. Positive 1-adamantanethiolate patterns are generated by nanografting 1-adamantanethiol molecules into preexisting n-alkanethiolate selfassembled monolayers, and negative 1-adamantanethiolate patterns are created by nanografting n-alkanethiol molecules into preexisting 1-adamantanethiolate self-assembled monolayers. The patterned 1-adamantanethiolate regions are displaced upon exposure to solutions of n-alkanethiol molecules. This two-step nanografting-displacement strategy minimizes pattern dissolution as 1-adamantanethiol molecules do not intercalate into the preexisting self-assembled monolayer during nanografting. 1-Adamantanethiol can be utilized create high-resolution sacrificial chemical patterns with feature sizes beyond those afforded other 1-adamantanethiol patterning strategies for applications such as resists for metallic and organic structures

Effects of surface water on organosilane nanostructure fabrication using particle lithography

Patterned organosilane self-assembled monolayers serve as molecular platforms for electronic, optical, and sensing applications. Among the numerous strategies to pattern organosilane monolayers, particle lithography offers a high throughput means to fabricate arrays of organosilane nanopatterns across large areas. Herein, we demonstrate that the utility of particle lithography for generating nanostructures can be further controlled by changes in sample preparation. Our systematic study of various drying conditions demonstrates a correlation between sample preparation and surface water and uses these findings to form nanopores, pillars, and rings within organosilane monolayers. Silica mesospheres deposited on Si substrates that are subjected to less rigorous drying conditions (3 h at room temperature) prior to organosilane deposition yield nanopores within decyltrichlorosilane monolayers that are significantly smaller than those produced on Si substrates that are prepared under more forcing conditions (12 h at room temperature and 2 h at 140 °C). This disparity in nanopore diameter can be rationalized by the presence or absence of water between the silica mesospheres and Si substrate. Sequential deposition of two organosilanes offers further evidence for the presence or absence of water beneath the silica mesospheres. For samples that are less rigorously dried, complete organosilane pillars are observed, and for samples that are more rigorously dried, organosilane rings are observed where the inner diameter is defined by the mesosphere-substrate contact geometry. The ability to produce varied organosilane nanostructures provides valuable insights about the water that is present on the surface and within the silica mesosphere template. These insights into the surface water and the effects of sample preparation on organosilane nanostructures enable greater hierarchical control over the fabrication process