Discovery of Potent Isoindolinone Inhibitors that Target an Active Conformation of PARP1 Using DNA-Encoded Libraries.

Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair enzyme, has proven to be a successful strategy for the treatment of various cancers. With the appropriate selection conditions and protein design, DNA-encoded library (DEL) technology provides a powerful avenue to identify small molecules with the desired mechanism of action towards a target of interest. However, DNA-binding proteins, such as PARP1, can be challenging targets for DEL screening due to non-specific protein-DNA interactions. To overcome this, we designed and screened a PARP1 catalytic domain construct without the autoinhibitory helical domain. This allowed us to interrogate an active, functionally-relevant form of the protein resulting in the discovery of novel isoindolinone PARP1 inhibitors with single-digit nanomolar potency. These inhibitors also demonstrated little to no PARP1-DNA trapping, a property that could be advantageous in the clinic.

Phomoxanthone A Targets ATP Synthase.

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.

A phase transition enhances the catalytic activity of SARM1, an NAD+ glycohydrolase involved in neurodegeneration.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to stress. NAD+ depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.

Identification of the first noncompetitive SARM1 inhibitors.

Sterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) is a key therapeutic target for diseases that exhibit Wallerian-like degeneration; Wallerian degeneration is characterized by degeneration of the axon distal to the site of injury. These diseases include traumatic brain injury, peripheral neuropathy, and neurodegenerative diseases. SARM1 promotes neurodegeneration by catalyzing the hydrolysis of NAD+ to form a mixture of ADPR and cADPR. Notably, SARM1 knockdown prevents degeneration, indicating that SARM1 inhibitors will likely be efficacious in treating these diseases. Consistent with this hypothesis is the observation that NAD+ supplementation is axoprotective. To identify compounds that block the NAD+ hydrolase activity of SARM1, we developed and performed a high-throughput screen (HTS). This HTS assay exploits an NAD+ analog, etheno-NAD+ (ENAD) that fluoresces upon cleavage of the nicotinamide moiety. From this screen, we identified berberine chloride and zinc chloride as the first noncompetitive inhibitors of SARM1. Though modest in potency, the noncompetitive mode of inhibition, suggests the presence of an allosteric binding pocket on SARM1 that can be targeted for future therapeutic development. Additionally, zinc inhibition and site-directed mutagenesis reveals that cysteines 629 and 635 are critical for SARM1 catalysis, highlighting these sites for the design of inhibitors targeting SARM1.

Calcium Regulates the Nuclear Localization of Protein Arginine Deiminase 2.

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.

Synthesis and Anticancer Activity of Structure Simplified Naturally-Inspired Dimeric Chromenone Derivatives.

Select dimeric chromenones exhibit low micromolar cyctotoxicity toward lymphoma and leukemia cell lines, L5178Y and HL60, respectively. The bioactive dimeric chromenones were identified from a focused library of structurally-simplified derivatives of naturally-occurring dimeric chromenones and tetrahydroxanthones that was prepared as part of this study. The simple dimeric chromenone scaffolds contain no stereogenic centers, are easily synthesized, and may be utilized as lead compounds in cancer research and drug discovery.

Photochemical Control of Protein Arginine Deiminase (PAD) Activity.

Protein arginine deiminases (PADs) play an important role in the pathogenesis of various diseases, including rheumatoid arthritis, multiple sclerosis, lupus, ulcerative colitis, and breast cancer. Therefore, the development of PAD inhibitors has drawn significant research interest in recent years. Herein, we describe the development of the first photoswitchable PAD inhibitors. These compounds possess an azobenzene photoswitch to optically control PAD activity. Screening of a series of inhibitors structurally similar to BB-Cl-amidine afforded compounds 1 and 2 as the most promising candidates for the light-controlled inhibition of PAD2; the cis isomer of 1 is 10-fold more potent than its trans isomer, whereas the trans isomer of 2 is 45-fold more potent than the corresponding cis isomer. The altered inhibitory potency upon photoisomerization has been confirmed in a competitive activity-based protein profiling (ABPP) assay. Further investigations indicate that the trans isomer of 2 is an irreversible inhibitor, whereas the cis isomer acts as a competitive inhibitor. In cells, the trans isomer of compound 1 is completely inactive, whereas the cis isomer inhibits histone H3-citrullination in a dose-dependent manner. Taken together, 1 serves as the foundation for developing photopharmaceuticals that can be activated at the desired tissue, using light, to treat diseases where PAD activity is dysregulated.

Development of a Selective Inhibitor of Protein Arginine Deiminase 2.

Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy (30a). Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.

Dispersing Zwitterions into Comb Polymers for Nonviral Transfection: Experiments and Molecular Simulation.

Polymer-based gene delivery vehicles benefit from the presence of hydrophilic groups that mitigate the inherent toxicity of polycations and that provide tunable polymer-DNA binding strength and stable complexes (polyplexes). However, hydrophilic groups screen charge, and as such can reduce cell uptake and transfection efficiency. We report the effect of embedding zwitterionic sulfobetaine (SB) groups in cationic comb polymers, using a combination of experiments and molecular simulations. Ring-opening metathesis polymerization (ROMP) produced comb polymers with tetralysine (K4) and SB pendent groups. Dynamic light scattering, zeta potential measurements, and fluorescence-based experiments, together with coarse-grained molecular dynamics simulations, described the effect of SB groups on the size, shape, surface charge, composition, and DNA binding strength of polyplexes formed using these comb polymers. Experiments and simulations showed that increasing SB composition in the comb polymers decreased polymer-DNA binding strength, while simulations indicated that the SB groups distributed throughout the polyplex. This allows polyplexes to maintain a positive surface charge and provide high levels of gene expression in live cells. Notably, comb polymers with nearly 50 mol % SB form polyplexes that exhibit positive surface charge similarly as polyplexes formed from purely cationic comb polymers, indicating the ability to introduce an appreciable amount of SB functionality without screening surface charge. This integrated simulation-experimental study demonstrates the effectiveness of incorporating zwitterions in polyplexes, while guiding the design of new and effective gene delivery vectors.

Design, synthesis, and biological evaluation of tetrazole analogs of Cl-amidine as protein arginine deiminase inhibitors.

Protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs.

Polymer-peptide delivery platforms: effect of oligopeptide orientation on polymer-based DNA delivery.

The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.

Promoting cell adhesion on slippery phosphorylcholine hydrogel surfaces.

The growing interest in regenerative medicine has created a need for superior polymer matrices that suit multiple physical, mechanical, and biological requirements. While the phospholipid bilayer of a cell membrane is considered optimal for interacting with biologics, polymeric materials composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) offer a cell membrane-like synthetic alternative. In this work, thiol-containing phosphorylcholine polymers were synthesized by radical copolymerization of a lipoic acid-functionalized methacrylate with MPC. The canonical cell adhesion oligopeptide (GRGDS) was incorporated into the polymers by copolymerization of a GRGDS-containing methacrylamide prepared by solid phase peptide synthesis. The relative amounts of phosphorylcholine, lipoic acid and oligopeptide were controlled by the monomer feed ratios, and the polymers were characterized by NMR spectroscopy and aqueous gel permeation chromatography (GPC). These multifunctional polymers formed hydrogels rapidly (<10 minutes) by Michael addition when poly(ethylene glycol)diacrylate (PEGDA) was added at pH 9 - an initiator-free gelation performed in a completely aqueous environment. Two cell lines, live mouse skeletal muscle myoblasts (C2C12) and human ovarian cancer (SKOV3) cells, were observed to specifically attach, spread and proliferate only on hydrogels containing the GRGDS peptide sequence, with a notable dependence on peptide concentration. The remarkable hydrophilicity and biocompatibility attributed to polyMPC combined with the facile gelation conditions of these polymers affords a platform of new bio-cooperative materials suitable for cell studies.

Interleukin-27 gene delivery for modifying malignant interactions between prostate tumor and bone.

We have examined the role of a novel cytokine, interleukin-27 (IL-27), in mediating interactions between prostate cancer and bone. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models, including prostate cancer. Prostate cancer is frequently associated with metastases to the bone, where the tumor induces a vicious cycle of communication with osteoblasts and osteoclasts to induce bone lesions, which are a significant cause of pain and skeletal-related events for patients, including a high fracture risk. We describe our findings in the effects of IL-27 gene delivery on prostate cancer cells, osteoblasts, and osteoclasts at different stages of differentiation. We applied the IL-27 gene delivery protocol in vivo utilizing sonoporation (sonodelivery) with the goal of treating and reducing the growth of prostate cancer at a bone metastatic site in vivo. We used a new model of immune-competent prostate adenocarcinoma and characterized the tumor growth reduction, gene expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth, can help normalize bone structure, and can promote enhanced accumulation of effector cells in prostate tumors. These results are promising, because they are relevant to developing a novel IL-27-based strategy that can treat both the tumor and the bone, by using this simple and effective sonodelivery method for treating prostate tumor bone metastases.

Disulfide cross-linked phosphorylcholine micelles for triggered release of camptothecin.

A series of block copolymers based on 2-methacryloyloxyethyl phosphorylcholine (MPC) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Incorporation of dihydrolipoic acid (DHLA) into the hydrophobic block led to formation of block copolymer micelles in water. The micelles were between 15 and 30 nm in diameter, as characterized by dynamic light scattering (DLS), with some size control achieved by adjusting the hydrophobic/hydrophilic balance. Cross-linked micelles were prepared by disulfide formation, and observed to be stable in solution for weeks. The micelles proved amenable to disassembly when treated with a reducing agent, such as dithiothreitol (DTT), and represent a potential delivery platform for chemotherapeutic agents. As a proof-of-concept, camptothecin (CPT) was conjugated to the polymer scaffold through a disulfide linkage, and release of the drug from the micelle was monitored by fluorescence spectroscopy. These CPT-loaded prodrug micelles showed a reduction in release rate compared to physically encapsulated CPT. The use of disulfide conjugation facilitated drug release under reducing conditions, with a half-life (t1/2) of 5.5 h in the presence of 3 mM DTT, compared to 28 h in PBS. The toxicity of the micellar prodrugs was evaluated in cell culture against human breast (MCF7) and colorectal (COLO205) cancer cell lines.

PolyMPC-doxorubicin prodrugs.

We demonstrate the conjugation of the cancer drug doxorubicin (DOX) to poly(methacryloyloxyethyl phosphorylcholine) (polyMPC), linked by hydrazone groups, using (1) a one-pot ATRP/click sequence, and (2) a post-polymerization conjugation strategy. While the one-pot method gave polyMPC-DOX conjugates in a facile single step, post-polymerization conjugation gave higher-molecular-weight polymers with very high DOX loadings. DOX release from the polyMPC backbone was pH-dependent (faster at pH 5.0 than at pH 7.4) owing to the hydrazone linkage. Half-life values of DOX release ranged from 2 to 40 h at pH 5.0. Cell culture experiments showed that highly loaded polyMPC-DOX conjugates exhibited higher intracellular drug accumulation and lower half-maximal inhibitory concentration (IC(50)) values, while a polymer with 30 wt % drug loading showed a maximum tolerated dose in the range of 30-50 mg/kg DOX equivalent weight in healthy mice.

The parkin-like human homolog of Drosophila ariadne-1 (HHARI) can induce aggresome formation in mammalian cells and is immunologically detectable in Lewy bodies.

Loss of functional Parkin is responsible for the death of midbrain dopaminergic neurons in human autosomal recessive juvenile parkinsonism. Since no cells express functional Parkin, it is unclear why other neuronal and non-neuronal populations are not also endangered. One possible explanation is that other neurons express a redundant ubiquitin-protein ligase (E3) that is absent from dopaminergic neurons. In this study, we demonstrate that human homolog of Drosophila ariadne-1 (HHARI) is a candidate for such a redundant function. In in vitro assays, HHARI binds to many of the same proteins as parkin, including CDCrel-1, synphilin-1, and CASK. In cell culture studies, HHARI forms aggresomes that are indistinguishable from those formed by parkin in terms of morphology, subcellular localization, incorporation of ubiquitin-proteasome components, and dependence on microtubules. In addition, endogenous HHARI is found in human Lewy bodies in both Parkinson's disease and diffuse Lewy body disorder. Taken together, these data suggest that HHARI, and perhaps other Parkin-like E3 ligases, may serve redundant roles for parkin in different cell types.

Reconfiguring polylysine architectures for controlling polyplex binding and non-viral transfection.

Poly(L-lysine) (PLL) is a cationic polyelectrolyte of interest for many applications, including in therapeutic biology for DNA complexation and transfection. Several non-lysine based polycations have been shown to afford more efficient transfection in live cells than has been achieved with PLL. We find that reconfiguring polylysine into short oligolysine grafts, strung from a hydrophobic polymer backbone, gives transfection reagents greatly superior to PLL, despite having the identical cationic functional groups (i.e., exclusively primary amines). Altering the oligolysine graft length modulates DNA-polymer interactions and transfection efficiency, while incorporating the PKKKRKV heptapeptide (the Simian virus SV40 large T-antigen nuclear localization sequence) pendent groups onto the polymer backbone led to even greater transfection efficiency over the oligolysine-grafted structures. Protein expression levels obtained with these novel polymer transfection reagents were higher than, or comparable to, expression seen in the cases of JetPEI™, FuGENE® 6 and Lipofectamine™ 2000, the later being notorious for cytotoxicity that accompanies high transfection efficiency. The relative strength of the polymer-DNA complex is key to the transfection performance, as judged by serum stability and PicoGreen analysis. Moreover, polyplexes formed from our graft copolymer structures exhibit low cytotoxicity, contributing to the therapeutic promise of these novel reagents.

Polymeric phosphorylcholine-camptothecin conjugates prepared by controlled free radical polymerization and click chemistry.

Novel polymer-drug conjugates, consisting of zwitterionic poly(methacryloyloxyethyl phosphorylcholine) (polyMPC) as the polymer component, and camptothecin (CPT) as the drug, were prepared by two methods. In one case, CPT was transformed by acylation into a functional initiator for copper catalyzed atom transfer radical polymerization (ATRP), and polyMPC was grown from this therapeutic initiator. In the other case, a one-pot ATRP-"click" conjugation strategy was employed to synthesize novel polyMPC structures containing multiple copies of the drug pendant to the zwitterionic polymer chain. The latter method allows polyMPC-graft-CPT conjugates to be prepared with a high weight percent drug loading (up to 14% CPT) with excellent solubility in pure water (>250 mg/mL). The linkage chemistry chosen between the polyMPC backbone and the pendant drugs proved critically important for assuring drug release within a time frame reasonable to consider these structures as a platform for injectable cancer therapeutics. Liberation of the drug from the polymer backbone was monitored by high-performance liquid chromatography, using size-exclusion and reverse-phase columns, and the toxicity of the polymer-drug conjugates was examined in cell culture against breast (MCF7), ovarian (OVCAR-3), and colorectal (COLO 205) cancer cell lines.

Cell death in myoblasts and muscles.

One of the hallmarks of development is that many more cells are produced than are ultimately needed for organogenesis. In the case of striated skeletal muscle, large numbers of myoblasts are generated in the somites and then migrate to take up residence in the limbs and the trunk. A subset of these cells fuses to form multinucleated skeletal muscle fibers, while a second group, known as satellite cells, exits the cell cycle and persists as a pool of lineage-restricted stem cells that can repair damaged muscle. The remaining cells initiate apoptosis and are rapidly lost. Primary myoblasts and established satellite cell lines are powerful tools for dissecting the regulatory events that mediate differentiative decisions and have proven to be important models. As well, muscle diseases represent debilitating and often fatal disorders. This chapter provides a general background for muscle development and then details a variety of assays for monitoring the differentiation and the death of muscle. While some of these methods are specialized to address the phenotypic properties of skeletal muscle, others can be employed with a wide variety of cell types.

Polyester-graft-phosphorylcholine prepared by ring-opening polymerization and click chemistry.

Phosphorylcholine (PC)-substitution on aliphatic polyesters is accomplished by click chemistry with PC-substituted azides.