CG -SLENP: A chemical genetics strategy to selectively label existing proteins and newly synthesized proteins.

Protein synthesis and subsequent delivery to the target locations in cells are essential for their proper functions. Methods to label and distinguish newly synthesized proteins from existing ones are critical to assess their differential properties, but such methods are lacking. We describe the first chemical genetics-based approach for selective labeling of existing and newly synthesized proteins that we termed as CG -SLENP. Using HaloTag in-frame fusion with lamin A (LA), we demonstrate that the two pools of proteins can be selectively labeled using CG -SLENP in living cells. We further employ our recently developed selective small molecule ligand LBL1 for LA to probe the potential differences between newly synthesized and existing LA. Our results show that LBL1 can differentially modulate these two pools of LA. These results indicate that the assembly states of newly synthesized LA are distinct from existing LA in living cells. The CG -SLENP method is potentially generalizable to study any cellular proteins.

Protein arginine methyltransferase 5 is essential for oncogene product EWSR1-ATF1-mediated gene transcription in clear cell sarcoma.

Transcription dysregulation is common in sarcomas driven by oncogenic transcription factors. Clear cell sarcoma of soft tissue (CCSST) is a rare sarcoma with poor prognosis presently with no therapy. It is characterized by a balanced t(12;22) (q13;q12) chromosomal translocation, resulting in a fusion of the Ewing's sarcoma gene EWSR1 with activating transcription factor 1 (ATF1) to give an oncogene EWSR1-ATF1. Unlike normal ATF1, whose transcription activity is dependent on phosphorylation, EWSR1-ATF1 is constitutively active to drive ATF1-dependent gene transcription to cause tumorigenesis. No EWSR1-ATF1-targeted therapies have been identified due to the challenges in targeting intracellular transcription factors. Through proteomics screening to identify potential druggable targets for CCSST, we discovered protein arginine methyltransferase 5 (PRMT5) as a novel protein to interact with EWSR1-ATF1. PRMT5 is a type II protein arginine methyltransferase to symmetrically dimethylate arginine residues in substrate proteins to regulate a diverse range of activities including gene transcription, RNA splicing, and DNA repair. We found that PRMT5 enhances EWSR1-ATF1-mediated gene transcription to sustain CCSST cell proliferation. Genetic silencing of PRMT5 in CCSST cells resulted in severely impaired cell proliferation and EWSR1-ATF1-driven transcription. Furthermore, we demonstrate that the clinical-stage PRMT5 inhibitor JNJ-64619178 potently and efficaciously inhibited CCSST cell growth in vitro and in vivo. These results provide new insights into PRMT5 as a transcription regulator and warrant JNJ-64619178 for further clinical development to treat CCSST patients.

A clickable photoaffinity probe of betulinic acid identifies tropomyosin as a target.

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge, unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid.

Design, Synthesis and Biological Evaluation of Prodrugs of 666-15 as Inhibitors of CREB-Mediated Gene Transcription.

cAMP-response element binding protein (CREB) is a transcription factor involved in multiple cancers. Chemical inhibitors of CREB represent potential anticancer agents. We previously identified 666-15 as a potent CREB inhibitor. While 666-15 showed efficacious anticancer activity in vivo through intraperitoneal (IP) injection, its oral bioavailability is limited. To increase its oral bioavailability, we describe synthesis and evaluation of prodrugs based on 666-15. The amino acid esters were attempted, but they were not stable for detailed characterization. The corresponding sulfate and phosphates were prepared. The sulfate of 666-15 was too stable to release 666-15 while the phosphates were converted into 666-15 with half-lives of ∼2 h. Phosphate 3 was also a potent CREB inhibitor with anti-breast cancer activity. Furthermore, compound 3 showed much improved oral bioavailability at 38%. These studies support that 3 can be used as an oral CREB inhibitor while IP administration of 666-15 is preferred for in vivo applications.

Mechanistic insights into the activation of ester prodrugs of 666-15.

cAMP-response element binding protein (CREB) is an oncogenic transcription factor implicated in many different types of cancer. We previously reported the discovery of 666-15 as a potent inhibitor of CREB-mediated gene transcription. In an effort to improve the aqueous solubility of 666-15, amino ester prodrugs 1 and 4 were designed and synthesized. Detailed chemical and biological studies of 1 and 4 revealed that a small portion of the prodrugs were converted into 666-15 through intermediate 3 instead of a long-range O,N-acyl transfer reaction that was initially proposed. These results provide unique insights into the activation of these ester prodrugs.

A suite of bioassays to evaluate CREB inhibitors.

The cyclic-AMP response element binding protein (CREB) is an important nuclear transcription factor and has been shown to be overexpressed and/or over-activated in many different cancer types, suggesting that targeting CREB is a novel approach for developing cancer therapies. Our lab discovered the first cell-permeable small molecule inhibitor of CREB, from which we further developed a potent CREB inhibitor with in vivo anti-cancer activity. In this article, we detailed our biochemical and cell-based bioassays to assess different small molecule CREB inhibitors.

Identification of lamins as the molecular targets of LBL1 using a clickable photoaffinity probe.

Phenotypic screening is a powerful approach to discover small molecules targeting pathways or disease biology with complex genetic causes. Following the initial discovery of these small molecules is their target identification, which is at the cornerstone in addressing their biological and clinical utility. Yet, finding the needle in the haystack remains a challenge. Nuclear lamins are type V intermediate filament proteins that form a filamentous structure underneath the inner nuclear envelope to support the mechanical stability of the mammalian cell nucleus. They also participate a myriad of other cellular signaling processes with incompletely understood molecular mechanisms. Small molecules that can directly bind to nuclear lamins will be incredible tools to address lamins' roles in different aspects of biology. However, these small molecules did not exist until recently. We previously discovered an acylpyrroloquinazoline called LBL1 that selectively killed breast cancer cells without harming normal human cells. To help understand the mechanism of action of LBL1, we recently took an unbiased chemical proteomics approach to identify its direct binding targets from the entire human cellular proteome. In this chapter, we describe our detailed methods to identify and validate lamins as the direct targets of LBL1. In this approach, we developed a clickable photoaffinity probe called LBL1-P that contains acylpyrroloquinazoline, trifluoromethyldiazirine and alkyne groups. Furthermore, we described a fluorescence microscopic method to validate that LBL1 directly targets lamin A in living cells. When properly designed, this approach should be broadly applicable to other bioactive small molecules.

Discovery of a Synergistic Inhibitor of cAMP-Response Element Binding Protein (CREB)-Mediated Gene Transcription with 666-15.

CREB is a transcription factor implicated in the pathogenesis of multiple cancers. Targeting CREB is a promising strategy to develop potential cancer therapeutics. Previously, we identified 666-15 as a potent CREB inhibitor. Herein, we designed an ester prodrug of 666-15 through a long-range O,N-acyl transfer reaction for improved aqueous solubility. Unexpectedly, we discovered a small molecule 11 (653-47) that can potentiate the CREB inhibitory activity of 666-15 although 653-47 alone does not inhibit CREB.

A Lamin-Binding Ligand Inhibits Homologous Recombination Repair of DNA Double-Strand Breaks.

Nuclear lamins are type V intermediate filament proteins. Lamins, including LA, LB1, LB2, and LC, are the major protein components forming the nuclear lamina to support the mechanical stability of the mammalian cell nucleus. Increasing evidence has shown that LA participates in homologous recombination (HR) repair of DNA double-strand breaks (DSBs) . However, the mechanisms underlying this process are incompletely understood. We recently identified the first lamin-binding ligand 1 (LBL1) that directly binds LA and inhibited cancer cell growth. We provided here further mechanistic investigations of LBL1 and revealed that LA interacts with the HR recombinase Rad51 to protect Rad51 from degradation. LBL1 inhibits LA-Rad51 interaction leading to accelerated proteasome-mediated degradation of Rad51, culminating in inhibition of HR repair of DSBs. These results uncover a novel post-translational regulation of Rad51 by LA and suggest that targeting the LA-Rad51 axis may represent a promising strategy to develop cancer therapeutics.

Toward Developing Chemical Modulators of Hsp60 as Potential Therapeutics.

The 60 kDa heat shock protein (Hsp60) is classically known as a mitochondrial chaperonin protein working together with co-chaperonin 10 kDa heat shock protein (Hsp10). This chaperonin complex is essential for folding proteins newly imported into mitochondria. However, Hsp60, and/or Hsp10 have also been shown to reside in other subcellular compartments including extracellular space, cytosol, and nucleus. The proteins in these extra-mitochondrial compartments may possess a wide range of functions dependent or independent of its chaperoning activity. But the mechanistic details remain unknown. Mutations in Hsp60 gene have been shown to be associated with neurodegenerative disorders. Abnormality in expression level and/or subcellular localization have also been detected from different diseased tissues including inflammatory diseases and various cancers. Therefore, there is a strong interest in developing small molecule modulators of Hsp60. Most of the reported inhibitors were discovered through various chemoproteomics strategies. In this review, we will describe the recent progress in this area with reported inhibitors from both natural products and synthetic compounds. The former includes mizoribine, epolactaene, myrtucommulone, stephacidin B, and avrainvillamide while the latter includes o-carboranylphenoxyacetanilides and gold (III) porphyrins. The potencies of the known inhibitors range from low micromolar to millimolar concentrations. The potential applications of these inhibitors include anti-cancer, anti-inflammatory diseases, and anti-autoimmune diseases.

Anticancer Pyrroloquinazoline LBL1 Targets Nuclear Lamins.

Target identification of bioactive compounds is critical for understanding their mechanism of action. We previously discovered a novel pyrroloquinazoline compound LBL1 with significant anticancer activity. However, its molecular targets remain to be established. Herein, we developed a clickable photoaffinity probe based on LBL1. Using extensive chemical, biochemical, and cellular studies with this probe and LBL1, we found that LBL1 targets nuclear lamins, which are type V intermediate filament (IF) proteins. Further studies showed that LBL1 binds to the coiled-coil domain of lamin A. These results revealed that IF proteins can also be targeted with appropriate small molecules besides two other cytoskeletal proteins actin filaments and microtubules, providing a novel avenue to investigate lamin biology and a novel strategy to develop distinct anticancer therapies.

Design, synthesis and evaluation of antitumor acylated monoaminopyrroloquinazolines.

Pyrroloquinazoline is a privileged chemical scaffold with diverse biological activities. We recently described a series of N-3 acylated 1,3-diaminopyrroloquinazolines with potent anticancer activities. The N-1 primary amino group in 1,3-diaminopyrroloquinazoline is critical for its inhibitory activity against dihydrofolate reductase (DHFR). In order to design out this unnecessary DHFR inhibition activity and further expand the chemical space associated with pyrroloquinazoline, we removed the N-1 primary amino group. In this report, we describe our design and synthesis of a series of N-3 acylated monoaminopyrroloquinazolines. Biological evaluation of these compounds identified a naphthamide 4a as a potent anticancer agent (GI50=88-200nM), suggesting that removing the N-1 primary amino group in 1,3-diaminopyrroloquinazoline is a useful chemical modification that can be introduced to improve the anticancer activity.

Design, synthesis and biological evaluation of regioisomers of 666-15 as inhibitors of CREB-mediated gene transcription.

cAMP-response element binding protein (CREB) is a nuclear transcription factor that has been implicated in the pathogenesis and maintenance of various types of human cancers. Identification of small molecule inhibitors of CREB-mediated gene transcription has been pursued as a novel strategy for developing cancer therapeutics. We recently discovered a potent and cell-permeable CREB inhibitor called 666-15. 666-15 is a bisnaphthamide and has been shown to possess efficacious anti-breast cancer activity without toxicity in vivo. In this study, we designed and synthesized a series of analogs of 666-15 to probe the importance of regiochemistry in naphthalene ring B. Biological evaluations of these analogs demonstrated that the substitution pattern of the alkoxy and carboxamide in naphthalene ring B is very critical for maintaining potent CREB inhibition activity, suggesting that the unique bioactive conformation accessible in 666-15 is critically important.

Systemic Inhibition of CREB is Well-tolerated in vivo.

cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.

The chemistry and pharmacology of privileged pyrroloquinazolines.

The advent of next-generation sequencing (NGS) technology has plummeted the cost of whole genome sequencing, which has provided a long list of putative drug targets for a variety of diseases ranging from infectious diseases to cancers. The majority of these drug targets are still awaiting high-quality small molecule ligands to validate their therapeutic potential and track their druggability. Screening compound libraries based on privileged scaffolds is an efficient strategy to identify potential ligands to distinct biological targets. 7H-Pyrrolo[3,2-f]quinazoline (PQZ) is a potential privileged heterocyclic scaffold with diverse pharmacological properties. A number of biological targets have been identified for different derivatives of PQZ. This review summarized the synthetic strategies to access the chemical space associated with PQZ and discussed their unique biological profiles.

Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity.

Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB's transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 µM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.

Novel Type of Prodrug Activation through a Long-Range O,N-Acyl Transfer: A Case of Water-Soluble CREB Inhibitor.

CREB (cAMP response element binding protein) has been shown to play an important role in tumor initiation, progression, and metastasis. We discovered that naphthol AS-E, a cell-permeable CREB inhibitor, presented antiproliferative activity in a broad panel of cancer cell lines in vitro. However, it has limited aqueous solubility. In this report, we described a water-soluble inhibitor (compound 6) of CREB-mediated gene transcription with in vivo anticancer activity. Unexpectedly, compound 6 was found to be a prodrug of compound 12 necessitating an unprecedented long-range O,N-acyl transfer. The rate of this transfer was pH- and temperature-dependent. To the best of our knowledge, this is the first time to show that a long-range O,N-acyl transfer could be exploited as a prodrug activation strategy to improve aqueous solubility. This type of prodrug may be applicable to other structures with spatially arranged hydroxyl amide to improve their aqueous solubility.

Discovery of a Potent Anti-tumor Agent through Regioselective Mono-N-acylation of 7H-Pyrrolo[3,2-f]quinazoline-1,3-diamine.

7H-Pyrrolo[3,2-f]quinazoline-1,3-diamine (1) is a privileged chemical scaffold with significant biological activities. However, the currently accessible chemical space derived from 1 is rather limited. Here we expanded the chemical space related to 1 by developing efficient methods for regioselective monoacylation at N1 , N3 and N7 , respectively. With this novel methodology, a focused library of mono-N-acylated pyrroloquinazoline-1,3-diamines were prepared and screened for anti-breast cancer activity. The structure-activity relationship (SAR) results showed that N3 -acylated compounds were in general more potent than N1 -acylated compounds while N7 -acylation significantly reduced their solubility. Among the compounds evaluated, 7f possessed 8-fold more potent activity than 1 in MDA-MB-468 cells. More importantly, 7f was not toxic to normal human cells. These results suggest that 7f is a novel compound as a potential anti-breast cancer agent without harming normal cells.

Identification, synthesis and evaluation of substituted benzofurazans as inhibitors of CREB-mediated gene transcription.

Cyclic-AMP response-element binding protein (CREB) is a stimulus-activated transcription factor. Its transcription activity requires its binding with CREB-binding protein (CBP) after CREB is phosphorylated at Ser133. The domains involved for CREB-CBP interaction are kinase-inducible domain (KID) from CREB and KID-interacting domain (KIX) from CBP. Recent studies suggest that CREB is an attractive target for novel cancer therapeutics. To identify novel chemotypes as inhibitors of KIX-KID interaction, we screened the NCI-diversity set of compounds using a split renilla luciferase assay and identified 2-[(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio]pyridine 1-oxide (compound 1, NSC228155) as a potent inhibitor of KIX-KID interaction. However, compound 1 was not particularly selective against CREB-mediated gene transcription in living HEK 293T cells. Further structure-activityrelationship studies identified 4-aniline substituted nitrobenzofurazans with improved selectivity.

Synthesis and Evaluation of an O-Aminated Naphthol AS-E as a Prodrug of CREB-mediated Gene Transcription Inhibition.

An O-aminated naphthol AS-E was designed as a prodrug to achieve reductive activation and improved aqueous solubility. During the synthesis of this designed compound, a novel transformation from aryl triflates and ethyl acetohydroximate to oxazoles was discovered. Although the initially designed O-amino naphthol AS-E was not made successfully, the eventually synthesized O-tert-butylamino derivative was found to be biologically inactive, suggesting that reductive N-O cleavage in this compound was not facile due to unfavorable steric and electronic effects.