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N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.

The ELL gene encodes an RNA polymerase II transcription factor that frequently undergoes translocation with the MLL gene in acute human myeloid leukemia. Here, we report that ELL can regulate cell proliferation and survival. In order to better understand the physiological role of the ELL protein, we have developed an ELL-inducible cell line. Cells expressing ELL were uniformly inhibited for growth by a loss of the G(1) population and an increase in the G(2)/M population. This decrease in cell growth is followed by the condensation of chromosomal DNA, activation of caspase 3, poly(ADP ribose) polymerase cleavage, and an increase in sub-G(1) population, which are all indicators of the process of programmed cell death. In support of the role of ELL in induction of cell death, expression of an ELL antisense RNA or addition of the caspase inhibitor ZVAD-fmk results in a reversal of ELL-mediated death. We have also demonstrated that the C-terminal domain of ELL, which is conserved among the ELL family of proteins that we have cloned (ELL, ELL2, and ELL3), is required for ELL's activity in the regulation of cell growth. These novel results indicate that ELL can regulate cell growth and survival and may explain how ELL translocations result in the development of human malignancies.

The human ELL gene, which is a frequent target for translocation in acute myeloid leukemia, was initially isolated from rat liver nuclei and found to be an RNA polymerase II elongation factor. Based on homology to ELL, we later cloned ELL2 and demonstrated that it can also increase the catalytic rate of transcription elongation by RNA polymerase II. To better understand the role of ELL proteins in the regulation of transcription by RNA polymerase II, we have initiated a search for proteins related to ELLs. In this report, we describe the molecular cloning, expression, and characterization of ELL3, a novel RNA polymerase II elongation factor approximately 50% similar to both ELL and ELL2. Our transcriptional studies have demonstrated that ELL3 can also increase the catalytic rate of transcription elongation by RNA polymerase II. The C-terminal domain of ELL, which we recently demonstrated to be required and sufficient for the immortalization of myeloid progenitor cells, shares strong similarities to the C-terminal domain of ELL3. ELL3 was localized by immunofluorescence to the nucleus of cells, and Northern analysis indicated that ELL3 is a testis-specific RNA polymerase II elongation factor.

The Wilms tumor suppressor protein, WT1, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of WT1 are beginning to be unraveled. It appears that physical interactions of other cellular proteins (p53 and par-4) with WT1 can modulate the function of WT1. Here, we report the identification and cloning of a novel WT1-interacting protein termed Ciao 1, a member of the WD40 family of proteins. Ciao 1 specifically interacts with WT1 both in vitro and in vivo. This interaction alters the mobility of a WT1.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by WT1. Ciao 1 does not inhibit binding of WT1 to its consensus nucleotide sequence and does not affect the repression activity of WT1. Thus, Ciao 1 appears to specifically modulate the transactivation activity of WT1 and may function to regulate the physiological functions of WT1 in cell growth and differentiation.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.