Management Approaches in WHO Grade III Meningioma: A National Oncology Trainees' Collaborative for Healthcare Research (NOTCH) UK Multi-Centre Retrospective Study.

AIMS: WHO Grade 3 (G3) meningiomas are rare tumours with limited data to guide management. This retrospective study documents UK management approaches across 14 centres over 11 years. MATERIALS AND METHODS: Patients with WHO G3 meningioma between 01/01/2008 and 31/12/2018 were identified. Data were collected on demographics, management strategy, adjuvant radiotherapy, approach in recurrence setting and survival. RESULTS: 84 patients were identified. 21.4% transformed from lower-grade disease. 96.4% underwent primary surgical resection, with 20.8% having evidence of residual disease on their post-op MRI. 59.3% of patients underwent adjuvant radiotherapy (RT) following surgical resection. Overall median PFS and OS were 12.6 months and 28.2 months, respectively. Median OS in the group who underwent complete surgical resection was 34.9 months, compared to 27.5 months for those who had incomplete resection (HR 0.58, 95% CI 0.27-1.23, p = 0.15). Median OS was 33.1 months for those who underwent adjuvant RT and 14.0 months for those who did not (HR 0.48, 95% CI 0.27-0.84, p = 0.004). Median adjuvant RT dose delivered was 60Gy (range 12Gy-60Gy), 45.8% of adjuvant RT was delivered using IMRT. At disease relapse, 31% underwent salvage surgery and 29.3% underwent salvage RT. Of those treated with salvage RT, 64.7% were re-treats and all were treated with hypofractionated RT. CONCLUSION: Surgery continues to be the preferred primary management strategy. Post-operative MRI within 48 hours is indicated to assess presence of residual disease and guide further surgical options. Adjuvant radiotherapy plays an important part of the management paradigm in these patients with the data supporting an attached survival advantage. Further surgery and re-irradiation is an option in the disease recurrence setting with radiosurgery frequently utilised in this context.

Emerging potential approaches in alkaline phosphatase (ALP) activatable cancer theranostics.

Alkaline phosphatase (ALP) is known as one of the most crucial members of the phosphatase family and encompasses the enormous ability to hydrolyze the phosphate group in various biomolecules; by this, it regulates several events in the pool of biological medium. Owing to its overexpression in various cancer cells, recently, its potential has evolved as a prominent biomarker in cancer research. In this article, we have underlined the recent advances (2019 onwards) of alkaline phosphatase in the arena of emerging cancer theranostics. Herein, we mainly focused on phosphate-locked molecular systems such as peptides, prodrugs, and aggregation-induced emission (AIE)-based molecules. When these theranostics encounter cancer cell-overexpressed ALP, it results in the hydrolysis of the phosphate group, which leads to the release of highly cytotoxic agents along with turn-on fluorophore/pre-existing fluorophore.

Ligand Dictated Photosensitization of Iridium(III) Dithiocarbamate Complexes for Photodynamic Therapy.

Organelle-targeted photosensitizers (PSs) for photodynamic therapy (PDT) are considered as an effective therapeutic strategy for the development of next generation PSs with the least side effects and high therapeutic efficacy. However, multiorganelle targeted PSs eliciting PDT via both type I and type II mechanisms are scarce. Herein, a series of cyclometalated iridium(III) complexes were formulated [Ir(C∧N)2(S∧S)] (C∧N = 2-phenylpyridine (ppy) and 2-(thiophen-2-yl)pyridine (thpy); S∧S = diethyldithiocarbamate (DEDTC), morpholine-N-dithiocarbamate (MORDTC) and methoxycarbonodithioate (MEDTC)) and the newly designed complexes Ir2@DEDTC and Ir1@MEDTC were characterized by single crystal X-ray crystallography. Complexes containing thpy as C∧N ligand exhibit excellent photophysical properties such as red-shifted emission, high singlet oxygen quantum yield (ϕΔ) and longer photoluminescence lifetime when compared with complexes containing ppy ligands. Ir2@DEDTC exhibits the highest Ï•Δ and photoluminescence lifetimes among the synthesized complexes. Therefore, Ir2@DEDTC was chosen to evaluate the photosensitizing ability to produce reactive oxygen species (ROS). Upon blue light irradiation (456 nm), it efficiently produces ROS, i.e., hydroxy radical (•OH) and singlet oxygen (1O2), which was confirmed by electron paramagnetic resonance (EPR) spectroscopy. In vitro photocytotoxicity toward HCT116, HeLa, and PC3 cell lines showed that out of all the synthesized complexes, Ir2@DEDTC has the highest photocytotoxic index (PI > 400) value. Ir2@DEDTC is efficiently taken up by the HCT116 cell line and accumulated mainly in the lysosome and mitochondria of the cells, and after PDT treatment, it elicits cell shrinkage, membrane blebbing, and DNA fragmentation. The phototherapeutic efficacy of Ir2@DEDTC has been investigated against 3D spheroids considering its ability to mimic some of the basic features of solid tumors. The morphology was drastically altered in the Ir2@DEDTC treated 3D spheroid after the light irradiation unleashed the potential of the Ir(III) dithiocarbamate complex as a superior PS for PDT. Hence, mitochondria and lysosome targeted photoactive cyclometalated Ir(III) dithiocarbamate complex exerting oxidative stress via both type I and type II PDT can be regarded as a dual-organelle targeted two-pronged approach for enhanced PDT.

Comparative Evaluation of the Relationship Between Airway Inadequacy, Head Posture, and Craniofacial Morphology in Mouth-Breathing and Nasal-Breathing Patients: A Cephalometric Observational Study.

BACKGROUND: The process of respiration is the primary factor of the posture of the jaws and tongue. Thus, a changed respiratory form like mouth breathing can change the posture of the head, jaw, and tongue. This, in turn, could change the equilibrium of pressure on the jaws and teeth thus affecting jaw growth and teeth positions. The influence of nasorespiratory function on craniofacial growth has stimulated interest and debate for more than a century. Mouth breathing is the reason for numerous orthodontic glitches such as a mouth breather's face evolving aberrantly because of of functional disruptions triggered by chronic airway obstruction. The relationship between nasorespiratory function and dentofacial development remains controversial despite the long-standing clinical concern of orthodontists, so there was a need to evaluate and compare the relationship between head posture, airway inadequacy, and craniofacial morphology in mouth breathers and nasal breathers. METHODOLOGY: Forty patients were selected and divided equally into two groups: mouth breathers and nasal breathers. Patients were diagnosed as mouth breathers based on physical examination and a history of chronic allergic rhinitis, adenoid, and tonsil enlargement. Lateral cephalograms were taken for all patients in the natural head position (NHP) with the Planmeca Proline XC Dimax3 x-ray machine (Planmeca, Helsinki-Uusimaa, Finland). All lateral cephalograms were traced and analysis was done to check airway, head posture, and craniofacial morphology. Descriptive statistics were performed to obtain the means and standard deviation of all the sample sizes. Unpaired t test was performed between nasal breathers and mouth breathers to check and evaluate the relationship. RESULT: Mouth-breathing patients varied from nasal-breathing patients in airway adequacy and craniofacial morphology. A little, but not statistically significant, difference was seen in head posture between the two groups. CONCLUSIONS: Early interception of mouth breathing in patients could be very helpful, as the postural changes in the mouth-breathing patients, if continued for a longer period of time, could be the reason for severe skeletal deformities as well as dental malocclusion.

Alkaline phosphatase (ALP) activatable small molecule-based prodrugs for cancer theranostics.

Highly water-soluble small molecule-based prodrugs (5-FUPD and SAHAPD) are formulated. They comprise a phosphate group to lock the active drug payload (5-fluorouracil and SAHA) along with a turn-on fluorophore consisting of a glutathione (GSH) depletory feature. Installation of the phosphate group along with purification of final product has been accomplished in an operationally facile manner. Activation of the prodrugs is facilitated by alkaline phosphatase (ALP)-mediated hydrolysis of the phosphate group followed by 1,8-elimination. The prodrugs were found to be highly effective against ALP flared human cervical cancer (HeLa) and liver cancer (HepG2) cell lines. Most notably, they were found to be innocuous to normal liver cells (WRL-68).

Development of a novel Bruton's tyrosine kinase inhibitor that exerts anti-cancer activities potentiates response of chemotherapeutic agents in multiple myeloma stem cell-like cells.

Despite recent improvements in multiple myeloma (MM) treatment, MM remains an incurable disease and most patients experience a relapse. The major reason for myeloma recurrence is the persistent stem cell-like population. It has been demonstrated that overexpression of Bruton's tyrosine kinase (BTK) in MM stem cell-like cells is correlated with drug resistance and poor prognosis. We have developed a novel small BTK inhibitor, KS151, which is unique compared to other BTK inhibitors. Unlike ibrutinib, and the other BTK inhibitors such as acalabrutinib, orelabrutinib, and zanubrutinib that covalently bind to the C481 residue in the BTK kinase domain, KS151 can inhibit BTK activities without binding to C481. This feature of KS151 is important because C481 becomes mutated in many patients and causes drug resistance. We demonstrated that KS151 inhibits in vitro BTK kinase activities and is more potent than ibrutinib. Furthermore, by performing a semi-quantitative, sandwich-based array for 71-tyrosine kinase phosphorylation, we found that KS151 specifically inhibits BTK. Our western blotting data showed that KS151 inhibits BTK signaling pathways and is effective against bortezomib-resistant cells as well as MM stem cell-like cells. Moreover, KS151 potentiates the apoptotic response of bortezomib, lenalidomide, and panobinostat in both MM and stem cell-like cells. Interestingly, KS151 inhibits stemness markers and is efficient in inhibiting Nanog and Gli1 stemness markers even when MM cells were co-cultured with bone marrow stromal cells (BMSCs). Overall, our results show that we have developed a novel BTK inhibitor effective against the stem cell-like population, and potentiates the response of chemotherapeutic agents.

Immunomodulatory Effects of a Concoction of Natural Bioactive Compounds-Mechanistic Insights.

Natural bioactive compounds derived from plant-based products are known for their biological immunomodulatory activities. They possess systemic pleiotropic effects, minimal side effects, and very low toxicities. Plant-based bioactive compounds have tremendous potential as natural therapeutic entities against various disease conditions and act as anti-inflammatory, antioxidant, anti-mutagenic, anti-microbial, anti-viral, anti-tumour, anti-allergic, neuroprotective, and cardioprotective agents. A herbal formulation extract including five biologically active compounds: Apigenin, Quercetin, Betulinic acid, Oleanolic acid, and ß-Sitosterol can impart several immunomodulatory effects. In this review, we systematically present the impact of these compounds on important molecular signaling pathways, including inflammation, immunity, redox metabolism, neuroinflammation, neutropenia, cell growth, apoptosis, and cell cycle. The review corroborates the beneficial effect of these compounds and shows considerable potential to be used as a safer, more cost-effective treatment for several diseases by affecting the major nodal points of various stimulatory pathways.

AIE material for photodynamic therapy.

Photodynamic therapy (PDT) is emerging as an excellent strategy to treat different types of cancers. The advantages of using PDT over other cancer treatment modalities are owing to its non-invasive nature, spatiotemporal precession, controllable photoactivity, and least side effects. The photosensitization ability of traditional photosensitizers (PSs) are severely curtailed by aggregation-induced quenching (ACQ). On the contrary, aggregation induced emission (AIE) molecules/fluorogens (AIEgens) show enhanced fluorescence emission and high reactive oxygen species (ROS)/singlet oxygen (1O2) production capability in the aggregated state. These unique characteristics of AIEgens make them potential AIE-PSs for fluorescence/luminescence image-guided combination PDT. In this chapter, we discussed the strategies that are developed to synthesize small molecule-based AIE-PSs, metal complex-based AIE-PSs, and AIE-PSs with two-photon absorbance (TPA) properties, polymer-based AIE-PSs, and nanoparticles based AIE-PSs for PDT. We have also discussed the rational design of targeting peptide conjugated AIE-PSs to selective target cancer cells over normal cells. Furthermore, recent findings on nanoparticle-based combination AIE-PSs are also discussed, where the combination AIE-PSs show synergistically improved anticancer activity and overcome the drug resistance. Finally, we shed light on the recent development, ongoing challenges, and future directions for designing better AIE-PS for PDT.

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

Role of Antizyme Inhibitor Proteins in Cancers and Beyond.

Polyamines are multivalent organic cations essential for many cellular functions, including cell growth, differentiation, and proliferation. However, elevated polyamine levels are associated with a slew of pathological conditions, including multiple cancers. Intracellular polyamine levels are primarily controlled by the autoregulatory circuit comprising two different protein types, Antizymes (OAZ) and Antizyme Inhibitors (AZIN), which regulate the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC). While OAZ functions to decrease the intracellular polyamine levels by inhibiting ODC activity and exerting a negative control of polyamine uptake, AZIN operates to increase intracellular polyamine levels by binding and sequestering OAZ to relieve ODC inhibition and to increase polyamine uptake. Interestingly, OAZ and AZIN exhibit autoregulatory functions on polyamine independent pathways as well. A growing body of evidence demonstrates the dysregulation of AZIN expression in multiple cancers. Additionally, RNA editing of the Azin1 transcript results in a "gain-of-function" phenotype, which is shown to drive aggressive tumor types. This review will discuss the recent advances in AZIN's role in cancers via aberrant polyamine upregulation and its polyamine-independent protein regulation. This report will also highlight AZIN interaction with proteins outside the polyamine biosynthetic pathway and its potential implication to cancer pathogenesis. Finally, this review will reveal the protein interaction network of AZIN isoforms by analyzing three different interactome databases.

Succination inactivates gasdermin D and blocks pyroptosis.

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD+, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 ± 2000 M-1 s-1) is at least as active as the TIR domain alone (kcat/KM = 1500 ± 300 M-1 s-1). Finally, we show that the SARM1 hydrolyzes NAD+ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.

Photoactive platinum(iv) complex conjugated to a cancer-cell-targeting cyclic peptide.

A conjugate of cancer-cell targeting cyclic disulphide nona-peptide c(CRWYDENAC) consisting of nine l-amino acids with the photoactive succinate platinum(iv) complex trans,trans-[Pt(N3)2(py)2(OH)(succinate)] (Pt-cP) has been synthesised and characterised. The conjugate was stable in dark, but released succinate-peptide and Pt(ii) species upon irradiation with visible light, and formed photoproducts with guanine. Conjugate Pt-cP exhibited higher photocytotoxicity than parent complex trans,trans,trans-[Pt(N3)2(OH)2(py)2] (FM-190) towards cancer cells, including ovarian A2780, lung A549 and prostate PC3 human cancer cells upon irradiation with blue light (465 nm, 17.28 J cm-2) with IC50 values of 2.8-22.4 µM and the highest potency for A549 cells. Even though the dark cellular accumulation of Pt-cP in A2780 cells was lower than that of parent FM-190, Pt from Pt-cP accumulated in cancer cells upon irradiation to a level >3× higher than that from FM-190. In addition, the cellular accumulation of Pt from Pt-cP was enhanced ca. 47× after irradiation.

Development of Activity-Based Proteomic Probes for Protein Citrullination.

Protein arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine to form peptidyl citrulline. This modification is increased in multiple inflammatory diseases and in certain cancers. PADs regulate a variety of signaling pathways including apoptosis, terminal differentiation, and transcriptional regulation. Activity-based protein profiling (ABPP) probes have been developed to understand the role of the PADs in vivo and to investigate the effect of protein citrullination in various pathological conditions. Furthermore, these ABPPs have been utilized as a platform for high-throughput inhibitor discovery. This review will showcase the development of ABPPs targeting the PADs. In addition, it provides a brief overview of PAD structure and function along with recent advances in PAD inhibitor development.

Photoactivatable Cell-Selective Dinuclear trans-Diazidoplatinum(IV) Anticancer Prodrugs.

A series of dinuclear octahedral PtIV complexes trans, trans, trans-[{Pt(N3)2(py)2(OH)(OC(O)CH2CH2C(O)NH)}2R] containing pyridine (py) and bridging dicarboxylate [R = -CH2CH2- (1), trans-1,2-C6H10- (2), p-C6H4- (3), -CH2CH2CH2CH2- (4)] ligands have been synthesized and characterized, including the X-ray crystal structures of complexes 1·2MeOH and 4, the first photoactivatable dinuclear PtIV complexes with azido ligands. The complexes are highly stable in the dark, but upon photoactivation with blue light (420 nm), they release the bridging ligand and mononuclear photoproducts. Upon irradiation with blue light (465 nm), they generate azidyl and hydroxyl radicals, detected using a 5,5-dimethyl-1-pyrroline N-oxide electron paramagnetic resonance spin trap, accompanied by the disappearance of the ligand-to-metal charge-transfer (N3 → Pt) band at ca. 300 nm. The dinuclear complexes are photocytotoxic to human cancer cells (465 nm, 4.8 mW/cm2, 1 h), including A2780 human ovarian and esophageal OE19 cells with IC50 values of 8.8-78.3 µM, whereas cisplatin is inactive under these conditions. Complexes 1, 3, and 4 are notably more photoactive toward cisplatin-resistant ovarian A2780cis compared to A2780 cells. Remarkably, all of the complexes were relatively nontoxic toward normal cells (MRC5 lung fibroblasts), with IC50 values >100 µM, even after irradiation. The introduction of an aromatic bridging ligand (3) significantly enhanced cellular uptake. The populations in the stages of the cell cycle remained unchanged upon treatment with complexes in the dark, while the population of the G2/M phase increased upon irradiation, suggesting that DNA is a target for these photoactivated dinuclear PtIV complexes. Liquid chromatography-mass spectrometry data show that the photodecomposition pathway of the dinuclear complexes results in the release of two molecules of mononuclear platinum(II) species. As a consequence, DNA binding of the dinuclear complexes after photoactivation in cell-free media is, in several respects, qualitatively similar to that of the photoactivated mononuclear complex FM-190. After photoactivation, they were 2-fold more effective in quenching the fluorescence of EtBr bound to DNA, forming DNA interstrand cross-links and unwinding DNA compared to the photoactivated FM-190.

Citrullination Inactivates Nicotinamide- N-methyltransferase.

Nicotinamide- N-methyltransferase (NNMT) catalyzes the irreversible methylation of nicotinamide (NAM) to form N-methyl nicotinamide using S-adenosyl methionine as a methyl donor. NNMT is implicated in several chronic disease conditions, including cancers, kidney disease, cardiovascular disease, and Parkinson's disease. Although phosphorylation of NNMT in gastric tumors is reported, the functional effects of this post-translational modification has not been investigated. We previously reported that citrullination of NNMT by Protein Arginine Deiminases abolished its methyltransferase activity. Herein, we investigate the mechanism of inactivation. Using tandem mass spectrometry, we identified three sites of citrullination in NNMT. With this information in hand, we used a combination of site-directed mutagenesis, kinetics, and circular dichoism experiments to demonstrate that citrullination of R132 leads to a structural perturbation that ultimately promotes NNMT inactivation.

The Rheumatoid Arthritis-Associated Citrullinome.

Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant.

Platinum(IV) Prodrugs.

This chapter is an overview of recent progress in the design of Pt(IV) prodrugs. These kinetically-inert octahedral prodrugs can be reduced in cancer cells to active squareplanar Pt(II) complexes, for example by intracellular reducing agents such as glutathione or by photoexcitation. The additional axial ligands in Pt(IV) complexes which are released on reduction, allow bioactive molecules to be delivered which can act synergistically with Pt(II) in killing cancer cells, or act as targeting vectors, allow attachment to polymer and nanoparticle delivery systems, or labelling with fluorescent probes. Pt(IV) prodrugs have yet to be approved for clinical use, although some offer the promise of increased efficacy and reduced side effects.

The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs).

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) and certain cancers. Given the clear link between increased PAD activity and human disease, the PADs are therapeutically relevant targets. Herein, we report the development of next generation cell permeable and "clickable" probes (BB-Cl-Yne and BB-F-Yne) for covalent labeling of the PADs both in vitro and in cell-based systems. Using advanced chemoproteomic technologies, we also report the off targets of both BB-Cl-Yne and BB-F-Yne. The probes are highly specific for the PADs, with relatively few off targets, especially BB-F-Yne, suggesting the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD inhibitors. Notably, these compounds can be used in a variety of modalities, including the identification of off targets of the parent compounds and as activity-based protein profiling probes in target engagement assays to demonstrate the efficacy of PAD inhibitors.