Interrogating the Roles of Post-Translational ... - ACS Publications

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Interrogating the Roles of Post-Translational Modifications of NonHistone Proteins Miniperspective Zakey Yusuf Buuh, Zhigang Lyu, and Rongsheng E. Wang* Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States ABSTRACT: Post-translational modifications (PTMs) allot versatility to the biological functions of highly conserved proteins. Recently, modifications to non-histone proteins such as methylation, acetylation, phosphorylation, glycosylation, ubiquitination, and many more have been linked to the regulation of pivotal pathways related to cellular response and stability. Due to the roles these dynamic modifications assume, their dysregulation has been associated with cancer and many other important diseases such as inflammatory disorders and neurodegenerative diseases. For this reason, we present a review and perspective on important posttranslational modifications on non-histone proteins, with emphasis on their roles in diseases and small molecule inhibitors developed to target PTM writers. Certain PTMs’ contribution to epigenetics has been extensively expounded; yet more efforts will be needed to systematically dissect their roles on non-histone proteins, especially for their relationships with nononcological diseases. Finally, current research approaches for PTM study will be discussed and compared, including limitations and possible improvements.

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INTRODUCTION Despite the homogeneity associated with many proteins, diversity in the gene expressions and the biological pathways in which these proteins partake in vary from cell to cell. These varied functionalities are largely attributed to the posttranslational modifications (PTMs) of these proteins. Studies have shown that through site-specific modifications, histone configuration can be tuned to activate or repress transcription, DNA repair, and replication.1,2 With regard to non-histone proteins, a wide variety of modifications have been accredited to the initiation and regulation of pathways related to apoptosis, metabolism, and many other cell signaling cascades. To date, more than 200 types of PTMs have been identified,2 with the most common ones shown in Figure 1. PTMs are added to substrates by specific enzymes (“writer”) and then recognized by another family of proteins (“reader”) that often initiate a cascade of downstream signaling. Most PTMs can also be removed by enzymes called “eraser” at the end of PTM-induced signaling. All these regulating proteins are considered PTM modulators. Yet certain residue motifs can undergo a multitude of PTMs, and sometimes an enzyme can take part in multiple roles. Phosphorylation of the “substrate inhibitor of cyclindependent protein kinase-1” (Sic1), for example, allows for the binding of E3 ubiquitin ligase. These phosphorylated residues are consequently ubiquitinylated, thereby allowing for recognition by the ubiquitin-binding domain (UBD) that initiates degradation of Sic1.3 Nevertheless, dysregulated PTM enzymes have been disclosed to be pivotal to the pathogenesis of many © 2017 American Chemical Society

diseases, such as tumorigenesis, inflammation, autoimmune disorders, neurodegeneration, and virus infection, etc.4−9 Thus, there is an intensive research interest in PTM writers, readers, and erasers, which are regarded as “druggable” therapeutic targets. Inhibitors that effectively target epigenetic modulators such as histone deacetylases have already been approved by FDA for cancer therapy.10 However, most research efforts have focused on modifications of histone proteins and the study of their relationships with cancer, which have been well summarized by several comprehensive reviews.7,10−12 In recent years, PTMs on non-histone proteins have been increasingly observed,13 and there is culminated interest to study their impact on diseases other than oncology.14−21 Accordingly, a number of inhibitors were developed to probe these PTMs’ effects on cellular proteins, particularly by targeting PTM writers. Herein, we will discuss the roles of these PTMs on nonhistone proteins, with an eye toward the representative PTM inhibitors reported since 2015 and their application as potential therapeutics.

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METHYLATION Methylation is commonly observed on lysine, arginine, and histidine residues and has varied intensities including mono-, di-, and trimethylation. Happening to a large extent on nonhistone substrates, methylation is mediated by a variety of Received: December 11, 2016 Published: May 15, 2017 3239

DOI: 10.1021/acs.jmedchem.6b01817 J. Med. Chem. 2018, 61, 3239−3252

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of non-histone PTMs. Methylation has been observed on lysine and arginine and is demonstrated here on lysine. Phosphorylation has been observed on serine, threonine, and tyrosine and is demonstrated here on serine. O-GlcNAcylation has been observed on serine, threonine and is demonstrated here on serine. Although its chemical structure is not simple enough to be portrayed here, the macromoleculebased ubiquitination is also one of the most common non-histone PTMs.

about 50% of cancer cases.23 Mazur et al. demonstrated that the methylation of lysine 260 on MAP3K2 by methyltransferase SMYD3 upregulated the Ras pathway, leading to increased cell proliferation and pancreatic tumorigenisis.23,31 This methylation did not directly alter the activity of MAP3K2 but instead obstructed its recognition by a protein phosphatase PP2A that can dephosphorylate, deactivate MAP3K2 and thereby antagonize the Ras pathway. Thus, the methylation by upregulated SMYD led to the hyperactivation of MAP3K2 and as a result intensified Ras signaling.23,25,31 A follow-up SMYD3 knockdown in mice successfully inhibited pancreatic tumorigenesis.31 Methylation Inhibitors. Most methylation inhibitors reported so far target methyltransferases. For instance, SMYD2 is overexpressed in cancer cell lines and patients and is found to monomethylate the aforementioned p53 to inhibit its transcriptional activities. The most recent SMYD2 inhibitor, aminopyrazoline-based 1 (BAY-598) (Figure 2), was identified through initial screening with scintillation proximity assay and appears to be selective, potent, and cell-active (IC50 ≈ 58 nM). Notably, this compound significantly reduced the methylation of protein AHNAK in vivo at doses starting from 30 mg kg−1 day−1.21 Combined administration of 1 with doxorubicin also effectively inhibited tumor growth in the KYSE-150 xenograft model.21

methyltransferases such as SET 7/9 or SET and MYND domain containing protein (SMYD).11,22,23 Many of these “writers” work in tandem with demethylases to attain a reversible mechanism by which biologically relevant pathways are regulated.24,25 Nonetheless, methylation has been particularly hijacked by cancer cells to silence tumor suppressors and to promote pathways leading to aberrant cell proliferation. One famous non-histone substrate is p53, a tumor suppressing transcription factor that is linked to the regulation of transcriptional targets involved in apoptosis and cell cycle arrest.26,27 Under homeostatic conditions, p53 exists in its unmodified form. Because of its instability and low half-life, p53 remains at low concentrations and is considered inactive.26,27 Stresses including DNA damage and oncogene activation lead to a cascade of signal mediations that promote the stability of p53, thereby increasing p53’s concentration and activity. Among these signal mediators, PTM such as methylation has been linked to both the promotion and repression of p53’s activity. Huang et al. reported that the monomethylation of lysine 370 inhibited p53’s activity while the dimethylation at the same residue had contrary effects. 28 LSD1, a lysine demethylase, was observed acting as the regulatory eraser between mono- and dimethylation of lysine 370 to promote the inhibition of p53. Thus, LSD1 acts as a site-specific antagonist toward p53’s DNA damage response.28,29 Another important substrate is mitogen-activated protein-3 kinase-2 (MAP3K2), which activates ERK5, JNK, MEK5, and many other proteins in response to certain growth factors.30 MAP3K2 mediates Ras-ERK, a complex pathway that promotes cell proliferation, and abnormality in this pathway is present in

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ACETYLATION Similar to methylation, acetylation controls gene expression and affects a diverse set of disease signaling.32 While the acetylation of histones is critical to keeping them “unwrapped”,5 3240



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Figure 2. Chemical structures of recently developed inhibitors of non-histone PTMs (1−18).

both microtubules and microtentacles. While other types of mutations such as detyrosination were proposed to contribute to microtubule stabilization, only the enhanced acetylation was noted to be present in metastatic cell lines.13 Further, mutation of lysine 40 destabilized microtubules and resulted in an inhibition of metathesis.13 Friedmann et al. showed that αTAT1 was the major acetyltransferase involved, and aberrant αTAT1 levels have been associated with multiple cancer cell lines.13,38,40 Surprisingly, another substrate of acetylation is tau protein, with its aggregation likely driving the onset of Alzheimer’s diseases. Normally, tau complexes with microtubules in a stabilizing manner and exists at the cytoskeleton of the neuronal system.20 Although the mechanism by which protein aggregation transpires is unknown, the disassociation of tau from its microtubule complex is required to initiate the

acetyltransferase’s effects at the lysine and arginine residues of cellular proteins are also fundamental to protein stability and downstream cell signaling. Kaypee et al. uncovered that acetylation essentially “capped” these residues and obstructed their sequential ubiquitination, which otherwise leads to proteasomal degradation of substrate proteins.4 There are many reported cases in which the altered protein stability, along with dysregulations of acetyltransferase and/or deacetylase, directly correlated to cancer, neurodegeneration, or autoimmune diseases.13,33−37 One important substrate is α-tubulin, which forms a dimer with β-tubulin, and their polymerized form consists of microtubules. Boggs et al. discovered that the migration and attachment of invasive breast cancers heavily depended on the stability of microtubule and microtentacles.13,38,39 Enhanced αtubulin acetylation, specifically at lysine 40, was identified in 3241



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aggregation.20 Due to the highly conserved nature of the tau protein, PTMs, specifically phosphorylation, have been involved in controlling its function. However, recent discovery by Cohen et al. connected acetylation with tau’s aggregation.35 Acetylation of lysine 18 and threonine 40 at the microtubulebinding region of tau made it dissociate from microtubules and led to the formation of tau fibrils. Additional proteomic studies revealed hyperacetylation of tau at multiple sites, with consequent mutation studies confirming that acetylation at lysine 280 disrupts Coulombic interaction between tau and microtubules.35 Thus, lysine acetylation (especially at Lys280) could be another biomarker for Alzheimer’s disease and a possible point of inhibition as a therapeutic target. Acetylation also decreases the activity of FoxO, a forkhead based family of transcription factors (TF), which modulates apoptosis and oxidative stress response.38,41 Daitoku et al. disclosed that CBP/p300-catalyzed acetylation of FoxO was followed by the phosphorylation and detainment of FoxO in the cytoplasm, thereby rendering it inactive. The presence of radical oxygen species (ROS), which initiates many carcinogenic pathways, enabled the disulfide covalent linkage between FoxO and CPB/p300 acetyltransferase.34 Subsequently, Bim, a proapoptotic protein with its expression stimulated by FoxO, is downregulated upon the acetylation and cytoplasmic trapping of FoxO.34 While this Bim/FoxO inactivation is important to cancer, its connection to autoimmune diseases such as type I diabetes is also prevalent. Chuang et al. showed that diabetic patients have lowered expression levels of Sirt1, a deacetylase specific for FoxO.37 Treatment with acetyltransferase inhibitors, on the other hand, significantly increased Bim levels and induced apoptosis of diabetic cells.37 Acetylation Inhibitors. To prohibit acetylation, one popular target is lysine acetyltransferase p300, which has more than 70 substrate proteins. Acetylation by p300 is required to activate a significant amount of non-histone substrates such as p53 and survivin proteins that are related to cancer,42 NF-κB that facilitates inflammation, and the aforementioned FoxO that is metabolism-related.43 Given these observations, there has been considerable interest in developing p300-targeted inhibitors. Several natural product inhibitors including curcumin have been identified in the past. More recently, compound 2 (garcinol) (Figure 2) was reported to inhibit p300 in vitro (IC50 ≈ 7 μM) and induce the apoptosis of HeLa cells through downregulating their global gene expression.44 High-throughput screening also led to the identification of compound 3 (L002, NSC764414) which docked into the active site of p300 with an IC50 value of 1.98 μM.45 3 was specific toward leukemia, lymphoma, and breast cancer cell lines and suppressed acetylation and the associated tumor growth in MDA-MB-468 breast cancer xenografts.45 Another potent lead, 4 (C646), was screened out as a competitor with acetyl-coA cofactor (Ki ≈ 400 nM) and displayed a high selectivity against p300 compared to other acetyltransferases.46 Notably, this compound inhibited the growth of melanoma and lung cancer cell lines through G1/S cell cycle arrest, although its in vivo efficacy remains to be determined.

threonine, and tyrosine are the common residues for phosphorylation. Besides the allosteric regulation of substrate protein’s stability and activity, phosphorylation serves as a point of recognition by ubiquitin-ligase for proteolysis or by signalcascade inducing proteins for cell cycle and migration.3,47,49 Depending on the site and substrate sequence, phosphorylation can be enhanced or antagonized by tandem modifications in the dynamic PTM world. Nonetheless, dysregulated phosphorylation has been associated with many diseases due to the diverse types of substrates, kinases and phosphatases. Oligo2, a transcription factor in the central nervous system, has been linked to the invasive migration of glioblastoma.17 Akin to the α-tubulin-dependent invasion of breast cancer, hyperphosphorylation of serines at the N-terminus of Oligo2 is responsible for the progression of glioma to invasive glioblastoma. Phosphorylation-activated Oligo2 increased the Oligo2-induced expression of ZEB1 and promoted the TGF-β2 signaling pathway that accounts for cancer invasiveness.17,50 As opposed to Oligo2, phosphorylation on retinoblastoma protein (pRb) suppressed its activity.51 pRb acts as a negative regulator of cell growth by blocking S-phase progression and shifting cell cycle from G1 to S phase.52 Thus, pRb functions as a tumor suppressor protein in cancer cells, and its inactivity has been linked to many tumor malignancies. Cyclin-dependent kinase (CDK) phosphorylated pRb at more than 13 sites,53 which altered its structural conformation, thereby inhibiting pRb’s activity. Phosphorylation also appears key to Parkinson’s disease (PD), in which α-synuclein (AS) was phosphorylated by leucine rich repeat protein kinase (LRRK2) at serine 128 and formed conglomerates dubbed as “Lewy bodies”.54 Qing et al. discovered that LRRK2 was upregulated in Lewy body deposits and the majority was mutated at G2019 → S. Unexpectedly, this mutation enhanced LRRK2’s kinase activity, making the mutated kinase a possible biomarker for the onset of PD.54 Phosphorylation Inhibitors. Inhibition of kinases has been widely adapted to disease treatments, since most kinase substrates are non-histone proteins that participate in an array of cell signaling events related to cancer, hepatotoxicity, Alzheimer’s, Parkinson’s, and numerous other diseases.55−57 Thus, kinases have emerged as one of the most successful families of therapeutic targets since the late 80s.58 Over the past, several review articles have comprehensively summarized the development of kinase inhibitors.58,59 Herein, we will focus on discussing only recently developed inhibitors, particularly on their treatment of nononcologic diseases. For example, Alzheimer’s disease is characteristic of deficiencies in protein phosphatase 2A (PP2A), which results in an accumulation of hyperphosphorylated tau protein in the brain.57 Treatment with compound 5 (morroniside), a natural product isolated from Cornus of f icinalis (50−200 μM), decreased the phosphorylation of the upstream Src kinase, which increased PP2A’s activity and thereby inhibited tau’s hyperphosphorylation.57 The receptorinteracting protein kinase 2 (RIPK2) plays pivotal roles in inflammatory disorders through its mediation of proinflammatory signals initiated by bacterial sensors NOD.56 Activation of RIPK2, however, requires its autophosphorylation and subsequent ubiquitination.56 The most potent inhibitor of RIPK2, 6 (ponatinib), bound to its expanded hydrophobic pocket and thereby blocked phosphorylation and ubiquitination as well as the resulting NF-κB signaling (IC50 ≈ 6.7 nM).56 More recently, the phosphorylation of many mitochondrial proteins was revealed to be the underlying mechanism of C-Jun

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PHOSPHORYLATION The reversible phosphorylation, mediated by protein kinases and phosphatases, is undoubtedly the most biologically significant PTM as a result of its role in the regulation of metabolism, cell cycle, apoptosis, and autophagy.33,47,48 Serine, 3242



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Figure 3. Ubiquitin−proteasome pathway (“Ub”, ubiquitin; “E1”, ubiquitin-activating enzyme E1; “E2”, ubiquitin-activating enzyme E2; “E3”, ubiquitin-activating enzyme E3).

decreased in most neurodegenerative diseases, which is due to the suppressive binding of ATP synthetase by amyloid-β (Aβ).67 Martin et al. summarized that sometimes the competitive modification between glycotransferases and protein kinases may displace the hyperphosphorylation of tau. As such, site-specific O-GlcNAcylation on serine 356 deterred tau’s selfaggregation.20 Thus, the duplicity of glycosylation on tau aggregation is likely site-dependent. Similarly, there is the linkage of N-acetylglucosamine to the amide nitrogen of asparagine, thereby noted as N-GlcNAcylation.68 For this reason, it is seen that O-GlcNAcylation is more analogous to phosphorylation than N-GlcNAcylation. In addition, intramembranous proteins are known to undergo both phosphorylation and O-GlcNAcylation but not N-GlcNAcylation..63,68 Glycosylation Inhibitors. One famous inhibitor of Nlinked glycosylation is compound 9 (2-DG) (Figure 2), which abrogated cell surface expression of c-KIT and FTL3-ITD, affected STAT3- and MAPK-ERK signaling, and induced apoptosis of acute myeloid leukemia both in vitro and in vivo. Compound 9 was also noted to down-regulate the expression of Sp1 transcription factor through the inhibition of O-glycosylation.69 On the other hand, 10 (Thiamet G) is a potent and selective inhibitor for GlcNAcase (Ki ≈ 21 nM).69 Synthesized as a transition state analog of O-GlcNAcase, compound 10 exhibited 37 000-fold selectivity for O-GlcNAcase over human lysosomal β-hexosaminidase. At a fixed concentration of 500 μM, compound 10 showed no inhibition of five other glycoside hydrolases. It also effectively induced OGlcNAcylation intracellularly (EC50 ≈ 30 nM)69 and in rats (intravenous injection at dosages higher than 2 mg/kg). Further study suggested that 10 is orally bioavailable and can effectively cross the blood−brain barrier.69 In addition to the treatment of Alzheimer’s disease,67 10 can induce global OGlcNAcylation on proteins including p53 in ovarian cancer cells.65 As an adjuvant, coadministration of 10 with chemotherapeutic cisplatin enhanced tumor cell cycle arrest.65 Moreover, 10 was recently shown to significantly inhibit the growth of Thermobaculum terrenum,64 suggesting its potential application as an antibacteria agent. Similar to the observation in ovarian cancer cell lines,65 a time-dependent increase of O-GlcNAc levels was reported in PC-12 cells upon treatment with 10. While no signs of phenotypical toxicity were observed yet, the onset of global O-

N-terminal protein kinase (JNK) induced mitochondrial dysfunction and acute liver injury.55 One of the potent and selective JNK inhibitors, compound 7 (SU3327, IC50 ≈ 0.7 μM) was observed to significantly decrease the levels of mitochondrial phosphoproteins and liver damage in the mouse model of acute liver injury.55 Phosphorylation is also the switch for activation of RAS-RAF-MEK-ERK kinase cascade.60 This MAPK signal transduction is aberrantly activated in cancer cells, contributing to increased cell division, enhanced motility, and paused apoptosis.60 One member of the RAF kinase family, BRAF, activated ERK through phosphorylation and was detected in 50% of human malignant melanomas and 15% of colorectal cancers.60 The small molecule inhibitor, 8 (BGB283), inhibited BRAF activity with an IC50 value of 23 nM and preferentially inhibited the growth of cancer cells bearing BRAF and EGFR mutation or amplification. In both cell-line-derived and human colorectal cancer xenografts, this small molecule potently inhibited tumor growth at a dosage as low as 3 mg/kg. Currently, compound 8 is under phase I clinical trials and holds the promise of being a BRAF-selective drug candidate for colorectal cancer.60

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GLYCOSYLATION

As a ubiquitous PTM, glycosylation has been considered equally important as phosphorylation in terms of the many biological signaling it is enrolled in. O-GlcNAcylation, which adds O-linked N-acetylglucosamine to threonine or serine residues, has been recognized as one of the most common and important PTMs.61−63 This transfer is mediated by O-GlcNActransferase (OGT), while O-GlcNAcase (OGA) can reverse the process.64 More than a thousand non-histone proteins are substrates of O-GlcNAcylation, including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2, and lamin-B1, most of which are cytoplasmic proteins.65 OGlcNAcylation affects protein localization, stability, and activity,64 with its level upregulated in primary breast and colorectal cancers.65 Wang et al. demonstrated that glycosylation also contributes to tau protein aggregation and deglycosylation of tau straightened filament clusters in Alzheimer’s disease.66 Notably, deglycosylation coupled with dephosphorylation triggered the reattachment of tau to microtubules, thereby relieving Alzheimer-like symptoms.66 Interestingly, O-GlcNAcylation on ATP synthetase was 3243



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based screening utilizing the NF-κB luciferase reporter, compound 14 (NSC697923) was selected, which inhibited the activity of ubiquitin enzyme E2 complex (Ubc13-Uev1A) via blockage of its thioester conjugation with ubiquitin.73 14 demonstrated significant cytotoxicity to multiple diffuse large Bcell lymphoma cell lines73 and multiple neuroblastoma cell lines,78 with EC50 values of ∼0.5−3 μM. Notably, loss of p53 function is also believed to be mediated by ubiquitin-enzyme E2, and treatment of neuroblastoma cells with 14 upregulated p53 activities.78 Nevertheless, E2 enzyme’s ubiquitination on p27 can be blocked by a small molecule 15 (CC0651, Figure 4)

GlcNAcylation as a result of general O-GlcNAcase inhibition may raise some concerns with regard to unwanted side effects.69 Thus, selective inhibition of certain OGA isoforms has been investigated despite minimal discrepancies being noted with regard to each isoform’s catalytic hydrolase activity. There are a couple of GlcNAcase isoforms such as vOGA, fOGA, and sOGA. Kim et al. have recently developed compound 11 (α-GlcNAc thiolsulfonate), which demonstrated 4-fold potency toward sOGA with respect to fOGA.70

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UBIQUITINATION Ubiquitination leads to the degradation of most proteins in cells and thereby regulates almost every aspect of eukaryotic cellular function. Ubiquitin is a conserved 76-residue protein, with the glycine on its carboxyl terminal being recognized and attached to the internal cysteine of the ubiquitin-activating enzyme E1 through thiol esterification (Figure 3). This attachment utilizes 1 equiv of ATP. Ubiquitin-conjugation enzyme E2 then replaces E1 through cysteine exchange and starts to recognize protein substrates. Under the catalysis by ubiquitin-ligase E3, the ubiquitin tag is transferred from E2 to the lysine amino acid on the substrate protein to complete the ubiquitination that eventually labels the substrate via an amide bond. The ubiquitination cycle is then repeated to add more ubiquitin molecules in a sequential mode. Eventually, the polyubiquitinated substrate is recognized by proteasome to initiate unfolding and degradation. This ubiquitin−proteasome pathway not only removes damaged proteins but also maintains the levels of many signaling proteins that are key to cellular functions. Excessive proteolysis of tumor-suppressor proteins such as p27 happens inside various tumor cells.71,72 Further, Lys63-mediated ubiquitination of κB kinase β (IKKβ) and upstream proteins including NEMO and TAK1 initiated NF-κB signaling, which is upregulated in multiple melanoma and inflammatory diseases.73,74 Ubiquitin-dependent degradation appeared also necessary for the development and maturation of synapses before reaching adulthood.75 Yet, dephosphorylation and hyperubiquitination of LRRK2 are believed to be the phenotypic cause of inherited Parkinson’s disease.76 Another interesting aspect is that a majority of ubiquitination is phosphorylation-dependent. Taking the protein p27 as an example, phosphorylation at threonine 187 or serine 10 is a prerequisite for the recognition by ubiquitin ligase E3 in the nucleus and cytoplasm, respectively.48 Inspired by the ubiquitin−proteasome pathway, a notable genetic knockdown technique has been recently developed that can remove aberrant proteins. The proteolysis targeting chimeric molecules (PROTACs) are small molecules consisting of a pharmacophore selective for the proteins of interest, and they are chemically linked to a protein moiety capable of docking to E3 ligase. Upon small molecule’s binding to target proteins, E3 ligase is recruited close by which would immediately degrade targets via poly-ubiquitination. PROTACs offer an efficiency parallel to other knockdown assays but appear to be more convenient and time-saving.77 Ubiquitination Inhibitors. A fair amount of research effort is focused on developing therapeutics targeting the ubiquitin system. For instance, a cell-based high-throughput screening of small molecules that prevent p27 degradation identified compounds 12 (NSC624306) and 13 (PYR-41), both of which selectively blocked the ubiquitin−thioester formation required for the activation of ubiquitin E1.71 In another cell-

Figure 4. Structure of 15 bound to ubiquitin and E2 enzyme Cdc34A (PDB code 4MDK). (A) Chemical structure of compound 15. (B) Cartoon representation of Cdc34A in cyan and ubiquitin in orange. Compound 15 was shown in sticks mode (green).

(IC50 ≈ 1.7 μM).79 Incubation of human cancer cell lines with 15 caused the accumulation of p27 and suppressed cell proliferation. Crystal structure studies (Figure 4B) suggested that this molecule inserted into a cryptic pocket coformed by E2 enzyme and ubiquitin.79,80 The binding by 15 trapped the weak interaction between E2 and ubiquitin and thereby inhibited the spontaneous hydrolysis of their thioester linkage, which eventually interfered with the discharge of ubiquitin from E2 to the substrate protein.79,80 Along with E1 and E2, E3 ligases such as Skp2 and Pirh2 have been popular therapeutic targets as well, with their overexpression being observed in oral, head, and neck cancers.81 One E3 ligase, linear ubiquitin chain assembly complex (LUBAC), is responsible for catalyzing the formation of linear ubiquitin chains and is involved in IκB kinase-mediated NF-κB activation, the abnormal activation of which closely relates to tumor malignancy and inflammatory diseases.82 Through an in silico screening, compound 16 was found to bind to F-box domain of Skp2 and it selectively inhibited Skp2mediated ubiquitination of p27 and Akt proteins.83 Moreover, 16 exhibited potent in vivo effects in lung or prostate tumor xenografts at dosages as low as 40 mg kg−1 day−1.83 In another independent study, 17 (Gliotoxin), a natural metabolite of fungus, has been known for its inhibition of NF-κB signaling but was only until recently discovered to function through a 3244



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Figure 5. Chemical structures of recently developed inhibitors of non-histone PTMs (19−32).

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selective inhibition of LUBAC.82 The compound selectively bound to the catalytic center of LUBAC and inhibited enzymatic activity with an IC50 value of ∼0.51 μM.82 Additionally, certain compounds function through simultaneous inhibition of more than one ubiquitin enzyme. 18 (BAY 11-7082) is a famous immunosuppressive and induced the apoptosis of B-cell lymphoma and leukemic T cells. The underlying mechanism, of which was just uncovered, showed that 18 exerted these effects through inactivation of both the E2 conjugation enzyme and E3 ligase LUBAC.84 In particular, the compound serves as a Michael acceptor to block the active cysteines on E2 enzymes.84

NEDDYLATION Some ubiquitin-like proteins, such as neural precursor cell expressed developmentally downregulated protein 8 (NEDD8), are also involved in functionally important post-translational modifications.85 Neddylation is a process analogous to ubiquitination, in which NEDD8 is conjugated to proteins under the sequential catalysis by NEDD8-activating enzyme E1, E2, and NEDD8-E3 ligase.85,86 Cullins, especially the cullin subunits of E3 ligases, are the neddylated protein substrates, which require neddylation for proper protein assembly and for promoting the degradation of other proteins that are crucial for cell viability, growth, and development.86 The upregulated 3245



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inhibitor is 20 (Cl-amidine, Figure 5) that belongs to the haloacetamidine class and is believed to exert most of its therapeutic effects via inhibition of PAD4.94 20 has an intracellular EC50 of ∼160 μM and showed modest efficacy (at 10 mg kg−1 day−1) in various animal models of inflammatory disorders including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis.93 To improve its efficacy, a stepwise structural optimization has been recently pursued. The first attempt resulted in the C-terminal tetrazole analog 21, which possesses improved intracellular potency and selectivity (EC50 ≈ 10 μM).94 Then, Cl-amidine’s C-terminal was replaced with benzimidazole and its N-terminal was replaced with biphenyl moiety to finally make compound 22 (BB-Clamidine).93 This compound, with an EC50 of ∼8.8 μM, was over 20-fold more potent than the parent Cl-amidine in cell assay. Although the plasma concentration at 1 mg kg−1 day−1 was below the stated cellular potency over a span of 8 h,93 the compound was quite effective in vivo by successfully reversing joint inflammation in the mouse model of collagen-induced arthritis.95 PAD inhibition by BB-Cl-amidine in various lupus models also resulted in significant reduction of NET and protection of organs from lupus-related damage.93

neddylation pathway contributes to tumorigenesis and the pathogenesis of Alzheimer’s disease, making targeting neddylation a therapeutical approach for cancer and neurodegenerative disorders.86 Neddylation Inhibitors. The first-in-class small molecule inhibitor is compound 19 (MLN4924) (Figure 5) that has been under multiple clinical trials. By forming an adduct with NEDD8, 19 selectively blocked NEDD8-activating enzymes (NAEs) (IC50 ≈ 4.7 nM), promoted DNA damage, and induced apoptosis of tumor cells.86,87 Although structurally related to AMP, 19 was believed to be distinct from AMP in terms of its unique deazapurine base, carbocycyle, and sulfamate functional groups.87 With a panel of selectivity assays against other ubiquitin- or SUMO-activating enzymes (UAE, SAE, UBA6, and ATG7), 19 appeared to bear excellent selectivity (IC50 values of >1 μM). However, it remains to be seen whether 19 can be inactive toward kinases that use AMP or methyltransferases that use SAM cofactor. At tolerable dosages starting from 30 mg kg−1 day−1, this inhibitor effectively suppressed the tumor growth in multiple xenografts.86,87 Besides single-agent treatment, compound 19 was recently shown to overcome tumor cells’ resistance to common chemotherapy, suggesting its potential role as a cancer adjuvant as well.50 Notably, NEDD8 was commonly observed in neurons, and treatment of rat hippocampal neurons with 19 caused reduced synaptic strength, downregulated glutamate receptors, and decreased dendritic spine width and density.88 These observations suggested the necessary role of neddylation in maintaining synapse and the possibility of targeting high levels of neddylation in neurodegenerative diseases.88

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PRENYLATION Prenylation (also known as “isoprenylation”) is the addition of lipophilic components consisting of prenyl groups to Cterminal cysteine(s) of a protein.96,97 It can be further categorized into farnesylation, which is the addition of a farnesyl moiety by farnesyltransferase (FTase), or geranylgeranylation which is the attachment of geranylgeranyl isoprene units by geranylgeranyl transferase (GGTase).96,97 Prenylation promotes protein−protein interactions and protein−membrane association. CAAX proteins such as Ras, Rho, and Rac are the substrates of prenylation, which play pivotal roles in the growth regulation and carcinogenesis of mammalian cells.96 These proteins feature a specific amino acid sequence (CAAX) at Cterminal and exist primarily at the cytoplasmic surface of cellular membranes.98 Most CAAX proteins do not have a transmembrane domain and will thereby need prenylation to anchor them to the plasma membrane followed by transportation to other intracellular membranes.98 For example, prenylation of oncogenic Ras is required for its transforming activity including the localization of Ras into the plasma membrane.96,97 Farnesylation of p21ras was found to be important for inflammatory gene expression.99 Prenylation is also necessary for virus particle assembly. The host-mediated post-translational change is crucial for the hepatitis D virus (HDV) life cycle.100 Prenylation Inhibitors. Prenylation inhibitors hold promise in treating not only cancer but also virus infection and inflammatory diseases. Although dual inhibitors of FTase and GGTase were identified from natural mycotoxin in the early time,101 there is increasing demand for targeted narrowspectrum therapy. Early FTase inhibitors are peptidomimietics of substrates (Figure 5), such as 23 (FTI-2148) (IC50 ≈ 1.4 nM)102 and 24 (FTI-276 and FTI-277) (IC50 ≈ 0.5 nM).103 Small molecule inhibitors were later identified through highthroughput screening followed by medicinal chemistry optimization. Representative FTase inhibitors are 25 (BZA5B) (IC50 ≈ 41 nM), 26 (Lonafranib, Figure 6) (IC50 ≈ 1.9 nM), 27 (Tipifarnib) (IC50 ≈ 7.9 nM), and 28 (BMS-214662) (IC50 ≈ 1.35 nM), most of which are highly selective for FTase, with IC50 values much higher than 1000 nM for GGTase.97

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CITRULLINATION Citrullination (also known as “deimination”) is generated through the hydrolysis of arginine and is catalyzed by protein arginine deiminase (PAD), a “writer” that has five different isoforms.89 The conversion of arginine to citrulline results in an increase in molecular weight by 1 Da and the loss of a positive charge, which bears considerable effects on protein functions and protein−protein interactions.89,90 Most citrullinated proteins are related to the central/peripheral nervous system, and their citrullination is believed to be the cause of autoimmune diseases.90,91 For example, citrullinated proteins in the brain and spinal cord were commonly observed in multiple sclerosis (MS) and the mouse model (experimental autoimmune encephalomyelitis).89,90 Citrullination on the protein components of myelin (e.g., myelin basic protein) interfered with the proteins’ interaction with lipids, thereby leading to the instability and degradation of myelin in MS patients.90 Rheumatoid arthritis (RA) is another chronic inflammatory disease that happens at synovial joints and are induced by the serum autoantibodies against citrullinated fibrins.92 High levels of citrullinated collagen and fibrinogen were observed in the plasma and synovial specimens from RA patients.92 In addition to these high levels of citrullination of substrate proteins in autoimmune diseases, the upregulation of PAD enzymes was detected in the inflamed joints of RA patients.90,91 PAD’s dysregulation and translocation promoted the formation of neutrophil extracellular traps (NETs), leading to the development of other inflammatory diseases including lupus.93 Citrullination Inhibitors. Inhibition of citrullination, especially by deactivating PAD, is expected to be a potential treatment of inflammatory diseases. The most widely used PAD 3246



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reduced Cys and Leu with 2-naphthyl-4-aminobenzoic acid resulted in derivative 31a (GGTI-297) and its methyl ester 31b (GGTI-298), which had IC50 values of around 50 nM for GGTase I and were less active against FTase (IC50 values of ∼200 nM).96 Notably, compound 31b successfully blocked in vitro growth and invasion of human tumor cell lines through blocking geranylgeranylation of the involved proteins (e.g., RhoA).107,108

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OTHER PTMS Besides prenylation, palmitoylation and myristoylation consist of the lipidation of non-histone proteins to enhance their hydrophobicity and to increase protein−protein interactions. Palmitoylation is usually added to cysteine and sometimes serine or threonine residues, while myrisotylation is attached exclusively to the α-amino group of the N-terminal glycine.109 Their protein substrates span from Lck kinase to Ras proteins, heterotrimeric G proteins, and the protein components of bacterial/microbial cell wall, etc.110 Inhibitors of palmitoylation have been summarized in a recent review, highlighting the correlation between palmitoylation and oncogenic growth signaling.111 For myristoylation, the enzyme N-myristoyltransferase (NMT) is considered a potential therapeutic target, being vital to protozoa and yeasts.109 Pyrazole sulfonamide inhibitors (e.g., 32 (DDD86481), IC50 ≈ 12 nM) were screened out to be fungicidal toward pathogen Aspergillus f umigatus.109 Over the past 2 years, other rare PTMs have been identified in a diverse set of diseases, warranting further investigation in the future. For instance, lysine succinylation is widespread in metabolic pathways and is revealed to be key to heart injury.112 In addition, s-nitrosylation of liver kinase B1 is found to be important for LPS-induced septic shock.113 The emergence of new PTMs relevant to disease represents a new class of possible therapeutic targets and will need further biological studies to elucidate their related mechanisms.

Figure 6. Structure of 26 bound to FTPase (PDB code 1O5M). (A) Chemical structure of compound 26. (B) Cartoon representation of FTPase with chain A in light blue and chain B in light orange. Compound 26 was in sticks mode (green).

Among them, 26 and 27 have advanced into clinical trials for patients with metastatic solid tumors or hematologic malignancies. There were some convincing efficacies achieved for single agent treatment on patients with blood cancers such as chronic myeloid leukemia, advanced myelodysplastic syndrome, and acute myeloid leukemia.97 Yet, most FTase inhibitors, specifically those targeting the Ras protein driven cancers pathways, failed in clinical trials in the early 2000s.104 One reason is due to the presence of multiple subgroups of Ras proteins. Many preclinical FTase inhibitors were modeled for the inhibition of H-Ras despite the presence of more mutagenic N-Ras and K-Ras isoforms. Second, inhibition of farnesylation did not prevent geranylgeranylation of Ras proteins, which still allowed the subsequent Ras signaling and membrane localization.105 Third, Ras proteins that are not the sole substrates for FTase and FTase inhibitors’ effects on tumors may be contributing to their inhibition of other farnesylated proteins. The tumor-suppressing function of certain farnesylated Ras family GTPases could also be compromised by FTase inhibition. Therefore, FTase inhibitors are no longer pursued for cancer but are increasingly investigated for treating nononcologic diseases. For instance, compound 29 (LB42708) (IC50 ≈ 0.8 nM) suppressed p21ras-dependent NF-κB activation and cytokine release in collagen-induced arthritic mouse model.99 Inhibition of FTase also dosedependently abolished virus particle production,100 with 26 significantly reducing the virus levels in HDV patients in a recent phase IIa clinical study.100 Despite intensive research efforts in the development of FTase inhibitors, progress toward discovering selective GGTase inhibitors has lagged. Most GGTase-specific inhibitors are peptidomimetics, and there have been few in vivo studies. One representative inhibitor, 30a (GGTI-287), potently inhibited GGTase I (IC50 ≈ 5 nM) in vitro, and its methyl ester derivative, 30b (GGTI-286), was selective in the whole cell assay for inhibiting the processing of geranylgeranylated and oncogenic k-Ras4B (IC50 ≈ 2 μM) rather than the farnesylated H-Ras (IC50 > 30 μM).106 Further structural design to link the

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DISCUSSION AND CONCLUSION With the discovery of a fast growing number of non-histone substrates, there is an emerging concept that the posttranslational modification of non-histone proteins can substantially alter protein functions.21 Thus, PTM’s regulatory roles can be extended to multiple cellular pathways besides histone-mediated transcription,21 including development, metabolism, memory, inflammation, cancer, and viral infection.43 Accordingly, small molecule inhibitors have been developed in recent years to target PTM on non-histone proteins, some of which, as being reviewed here, have demonstrated convincing effects in animal models and even clinical trials. Most inhibitors developed to date are against PTM writers but not PTM readers or substrates. Many PTM writers such as methyltransferase or acetyltransferase are upstream in cell signaling. Moreover, they tend to work on diverse types of substrate proteins. Taken together, general inhibition of PTM writers may also block other desired biological processes, thereby bringing in unwanted side effects. Hockly et al. expected the use of deacetylase inhibitor such as SAHA to increase acetylation in the brain, thereby alleviating the transcriptional dysregulation observed in Huntington’s disease.114 However, global acetylation in the related mouse model resulted in issues with severe toxicity that even offset the desired improvement of cell differentiation and apoptosis. Given the recent progress of antibody−drug conjugates,115 a 3247



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groups modifies both the steric size and polarity of the adduct site, which can perturb PTM precursor/cofactor recognition sites, thereby impacting subsequent modification by the PTM enzymes and PTM-induced downstream signaling.134,135 Nonetheless, more PTM enzyme targets will emerge from the advancement of innovative bioorthogonal reactions and the rapid development of instrumental bioanalysis. PTM-related drug discovery, especially on diseases associated with nonhistone proteins, is arising and will turn out to be a fruitful approach in multiple areas of human therapy.

possible solution could be the targeted delivery of PTM inhibitors to specific organs to improve the therapeutics’ specificity. For instance, one can potentially conjugate PTM inhibitors onto bispecific antibodies capable of penetrating the blood−brain barrier and selectively target β-amyloid for Alzheimer’s disease or α-synuclein for Parkinson’s disease.116,117 To treat cancer, PTM inhibitors can be conjugated to antibodies that solely recognizes biomarkers specific to the surface of tumor cells.118,119 Another approach could be to develop inhibitors targeting PTM substrates or readers that are relatively downstream to writers. Given that PTM’s modulating effects on the same substrate are site-dependent,20,35,48,54,66 it may be highly desirable to generate sequence-specific inhibitors that recognize not only PTM but also the site of modification. Accordingly, one could conceivably synthesize a peptide possessing both the desired sequence and PTM and use it as the target for highthroughput screening. Since a protein’s repertoire could be important for specific targeting, the whole substrate protein can be alternatively expressed as the target and can potentially bear the site-specific PTM through the amber-suppression mediated genetic incorporation of unnatural amino acid.120 As for “readers”, there has recently been a growing interest into the development of reader inhibitors due to readers having inherent selectivity for the PTMs and PTM substrates.121 For example, bromodomain inhibitors can block the recognition of acetylated lysines on nuclear factor κB to reduce graft-versushost disease in an animal model121 or on p53 protein to protect cardiomyocytes from ischemia-induced apoptosis.122,123 Given the continuous discovery of promising PTM inhibitors, focus over the next decades could be on how to develop these inhibitors into clinical drugs. The number of marketable small molecule drugs used to be relatively small compared to the explosion of new leads, presumably due to the failure of translating preclinical effects to humans.124 Key issues such as off-target toxicity, metabolic inactivation, and failure to reach target tissues remain as challenges for developing drugs against PTMs.125,126 In this regard, the aforementioned ontarget specificity would be pivotal to ensure that the observed in vivo efficacies can be attributed to the inhibition of PTMinitiated pathways. Thus, careful preclinical studies (e.g., selectivity profiling using microarrays, proteomics, or cell lines;59 in vivo pharmacokinetic and pharmacodynamics studies) are needed to move forward toward clinical trials. One obstacle for drug development in this field is the availability of targets (PTM enzymes). More PTM modulators remain to be uncovered. Yet this process is difficult to advance definitively, especially for discoveries regarding non-histone substrates.127,128 The PTM-mediated interactions can be weak and transient, making it challenging to characterize them with traditional approaches such as immunoprecipitation or affinity pull down.127 The recent trend of using bait peptides or proteins that contain photoaffinity groups could significantly break this dilemma by converting noncovalent interactions to irreversible photo-cross-linking.127,128 As for discovering PTM substrates, the traditional immunoprecipitation is largely limited by the affinity and selectivity of antibody which usually led to inefficient enrichment and a high background of unmodified proteins.129,130 Chemical proteomics may help by making use of alkyne/azide-based “click” chemistry to tag substrate proteins and thereby enabling instant labeling, detection, and pull down of target proteins within complex proteomes.131−133 However, the addition of the alkyne/azide

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AUTHOR INFORMATION

Corresponding Author

*Phone: (215) 204-1855. E-mail: [email protected]. ORCID

Rongsheng E. Wang: 0000-0002-5749-7447 Notes

The authors declare no competing financial interest. Biographies Zakey Yusuf Buuh studied organic chemistry at the College of Holy Cross and obtained his B.Sc. in 2016. He started his Ph.D. study in 2016 and joined the research group of Professor Rongsheng (Ross) Wang. His main interests are medicinal chemistry and the development of novel chemical probes for post-translational modifications. Zhigang Lyu received his B.Sc. from Zhengzhou University (China) in 2013 and his Master’s degree at the same university in 2016. In 2016, he started his Ph.D. at the Department of Chemistry, Temple University. His current research focuses on imaging agents and drug design that target post-translational modification-associated enzymes. Rongsheng E. Wang obtained his Ph.D. in Bioorganic Chemistry in 2010 from the Department of Chemistry, Washington University in St. Louis (USA), where he worked with Professor John-Stephen Taylor to develop inhibitors of heat shock protein 70s for cancer therapy. From 2010 to 2012, he was a research scientist at Mediomics, LLC. From 2012 to 2016, he did a postdoc with Professor Peter G. Schultz at The Scripps Research Institute (CA, USA), where he developed novel therapeutic proteins, UAA-containing antibody drug conjugates for treatment of inflammatory diseases. Since 2016, he became a faculty member at Department of Chemistry, Temple University (USA). His current research interests lie in the identification of post-translational modification-related protein targets that are key to human diseases, and structure-based therapeutic design.

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ACKNOWLEDGMENTS We thank the generous financial support from the Temple University startup fund. ABBREVIATIONS USED PTM, post-translational modification; Sic1, substrate inhibitor of cyclin-dependent protein kinase 1; UBD, ubiquitin-binding domain; FDA, U.S. Food and Drug Administration; SET, Drosophila proteins Su(var)3-9, Enhancer-of-zeste, and Trithorax; MYND, Myeloid-Nervy-DEAF1; SMYD, SET and MYND domain containing protein; LSD1, lysine-specific demethylase 1; MAP3K2, mitogen-activated protein-3 kinase2; ERK, extracellular signal-regulated kinase; JNK, c-Jun Nterminal kinase; MEK, MAPK/ERK kinase; PP2A, protein phosphatase 2; AHNAK, neuroblast differentiation-associated protein; αTAT1, α-tubulin acetyltransferase 1; FoxO, a forkhead box protein O; TF, transcription factor; CBP, CREB (cAMP response element binding protein)-binding 3248



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protein; ROS, radical oxygen species; NF-κB, nuclear factor κB; ZEB1, zinc finger E-box binding homeobox 1; TGF-β, transforming growth factor β; pRb, retinoblastoma protein; CDK, cyclin-dependent kinase; PD, Parkinson’s disease; AS, αsynuclein; LRRK2, leucine rich repeat protein kinase; RIPK, receptor-interacting protein kinase; NOD, nucleotide-binding oligomerization domain; BRAF, B-Raf proto-oncogene; EGFR, epidermal growth factor receptor; O-GlcNAc, O-linked Nacetylglucosamine; OGA, O-GlcNAcase; Aβ, amyloid-β; c-KIT, mast/stem cell growth factor receptor; FTL3-ITD, fms like tyrosine kinase-3-internal tandem duplication; STAT3, signal transducer and activator of transcription 3; IKKβ, κB kinase β; NEMO, NF-κB essential modulator; TAK1, transforming growth factor β-activated kinase 1; LUBAC, linear ubiquitin chain assembly complex; NEDD8, neural precursor cell expressed developmentally downregulated protein 8; NAE, NEDD8-activating enzyme; PAD, protein arginine deiminase; MS, multiple sclerosis; RA, rheumatoid arthritis; NET, neutrophil extracellular trap; FTase, farnesyltransferase; GGTase, geranylgeranyl transferase; HDV, hepatitis D virus; NMT, N-myristoyltransferase; LPS, lipopolysaccharide; SAHA, suberanilohydroxamic acid

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NOTE ADDED AFTER ASAP PUBLICATION This Perspective was published ASAP on May 24, 2017. The Table of Contents graphic, Abstract graphic, and Figure 1 have been updated. The corrected version was reposted on September 29, 2017. 3252


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